YAP1 Inhibitors Enhance the Therapeutic Effect of Gemcitabine on PDCA by Inhibiting MSLN Expression, EMT, and Pancreatic Fibrosis

YAP1 Inhibitors Enhance the Therapeutic Effect of Gemcitabine on PDCA by Inhibiting MSLN Expression, EMT, and Pancreatic Fibrosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article YAP1 Inhibitors Enhance the Therapeutic Effect of Gemcitabine on PDCA by Inhibiting MSLN Expression, EMT, and Pancreatic Fibrosis Jili Hu, Xu Guo, Jia Wang, Xinming Li, Jian Zhou, Zhuoyin Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6589227/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with poor prognosis and limited response to gemcitabine-based chemotherapy. Chemoresistance in PDAC arises from both cancer-intrinsic mechanisms and extrinsic factors like stromal fibrosis. This study investigates the role of mesothelin (MSLN) and the YAP1 inhibitor TED-347 in modulating gemcitabine resistance. Elevated MSLN expression in PDAC correlates with advanced disease stages and poor prognosis. Mechanistically, MSLN promotes gemcitabine resistance by counteracting drug-induced apoptosis and upregulating ABCC1, a key drug efflux transporter. YAP1 transcriptionally activates MSLN by binding to its promoter, independent of the Canscript sequence. The YAP1 inhibitor TED-347 disrupts this interaction, reducing MSLN expression and suppressing PDAC cell migration, invasion, and epithelial-mesenchymal transition (EMT). In a mouse model, TED-347 combined with gemcitabine enhanced antitumor efficacy, reduced fibrosis, and increased gemcitabine sensitivity. Notably, TED-347 alleviated stromal fibrosis by inhibiting pancreatic stellate cell (PSC) activation, addressing a critical barrier to drug delivery. While gemcitabine itself induces fibrosis, TED-347 mitigates this effect, offering a dual therapeutic strategy. These findings highlight the YAP1-MSLN axis as a key driver of chemoresistance and fibrosis in PDAC, with TED-347 demonstrating potential to improve clinical outcomes by targeting both malignant and stromal components. This study provides a translational research framework for combining YAP1 inhibitors with chemotherapy to overcome resistance in PDAC. Pancreatic cancer Chemotherapy resistance MSLN Pancreatic fibrosis YAP1 inhibitors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Pancreatic Ductal Adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is an aggressively malignant tumor within the digestive system. Globally, there has been a concerning increase in both the incidence and mortality rates of PDAC, with the two rates being nearly identical [ 1 , 2 ] . Over the past four decades, advancements in treatment techniques and evolving concepts have led to a reduction in surgical complications and mortality associated with PDAC. Despite these improvements, the five-year survival rate for patients with PDAC remains stubbornly below 10% [ 3 ] . Chemotherapy, particularly gemcitabine-based regimens, is a pivotal component in the systemic management of PDAC [ 4 ] . However, the overall response rate remains suboptimal, primarily due to the chemoresistant nature of PDAC, which is intricately linked to the cancer cells themselves and the extensive fibrosis of the mesenchymal tissue [ 5 – 8 ] . The elusive mechanisms behind chemoresistance in PDAC pose a significant challenge in its treatment. The gemcitabine-based chemotherapy regimen is the preferred choice for treating PDAC [ 9 , 10 ] . Consequently, there is an urgent need for in-depth research to elucidate the mechanisms associated with chemotherapy resistance in PDAC, with the aim of enhancing the efficacy of chemotherapy in the current treatment landscape. Chemotherapy is a pivotal component in the systemic management of PDAC [ 4 ] . However, the overall response rate remains suboptimal, primarily due to the chemoresistant nature of PDAC which is intricately linked to the cancer cells themselves and the extensive fibrosis of the mesenchymal tissue [ 5 – 8 ] . The elusive mechanisms behind chemoresistance in PDAC pose a significant challenge in its treatment. The gemcitabine-based chemotherapy regimen is the preferred choice for treating PDAC [ 9 , 10 ] . Consequently, there is an urgent need for in-depth research to elucidate the mechanisms associated with chemotherapy resistance in PDAC, with the aim of enhancing the efficacy of chemotherapy in the current treatment landscape. Mesothelin (MSLN), a glycoprotein anchored to cell membranes via glycosylphosphatidylinositol, is typically present in low levels in the pleura and peritoneum but is markedly overexpressed in mesothelioma and ovarian cancer, where it fuels the invasive metastasis of these cancers [ 11 , 12 ] . Studies have indicated that patients with mesothelioma and colorectal cancer exhibiting high MSLN expression are more likely to develop resistance to chemotherapeutic drugs [ 12 , 13 ] . The MSLN-targeting antibody, Amatuximab, has been shown to enhance the sensitivity of PDAC cells to gemcitabine in murine models [ 14 ] . Our previous research revealed high MSLN expression in PDAC tissues [ 15 ] , and further research demonstrated that MSLN knockdown reverses epithelial-mesenchymal transition (EMT) in cancer cells, while its overexpression promotes EMT, suggesting a link between MSLN and chemoresistance [ 16 ] . Yes-associated protein 1 (YAP1), a pivotal transcriptional cofactor, forms complexes with transcription factors such as the transcriptionally enhanced associate domains (TEADs) within the nucleus to regulate gene transcription [ 17 , 18 ] . Evidence suggests that the YAP1-TEADs complex is implicated in the transcriptional regulation of MSLN in PDAC cells [ 19 , 20 ] . YAP1 engages with TEADs through an extensive planar interface [ 21 ] , and thus, disrupting this interaction could offer a novel therapeutic strategy for MSLN-targeted treatments in PDAC. The small molecule inhibitor TED-347 has been shown to rapidly and selectively form covalent bonds with specific cysteine residues within the deep hydrophobic palmitate-binding pocket of TEADs, thereby irreversibly inhibiting the YAP1-TEADs interaction and blocking their transcriptional activity over an extended period [ 22 , 23 ] . The potential of TED-347 to enhance the sensitivity of PDAC cells to gemcitabine remains an open question. It is known that PDAC can develop resistance to gemcitabine through epithelial-mesenchymal transition and pancreatic fibrosis [ 24 – 26 ] . Pancreatic stellate cells (PSCs), as the primary fibrogenic cells in the pancreas, are activated by tumor cells to secrete a substantial amount of extracellular matrix, which promotes pancreatic fibrosis and hinders the delivery of chemotherapeutic agents [ 27 ] . Reports indicate that YAP1 can stimulate the activation of PSCs, and the inhibition of YAP1 expression can reverse this activation [ 28 ] . This research provided a comprehensive investigation into the mechanisms through which MSLN confered gemcitabine resistance in PDAC, utilizing both in vitro and in vivo experimental approaches. The study revealed that the small molecule inhibitor TED-347, which homed in on the YAP1-TEAD1 interaction, can diminish MSLN expression in PDAC cells. This reduction in MSLN expression subsequently curbed cell migration, invasion, and EMT. Moreover, TED-347 was shown to mitigate fibrosis within PDAC tissues and to augment the therapeutic effectiveness of gemcitabine in the treatment of PDAC. Method 1. Database analysis The Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database [ 29 ] ( http://gepia2.cancer-pku.cn/index.html ) was utilized to evaluate the expression of MSLN in diverse malignancies. Additionally, the Tumor Immune Infiltration Evaluation Resource 2.0 (TIMER 2.0) [ 30 ] database ( https://timer.cistrome.org/ ) was employed to analyze MSLN expression in PDAC patients from the The Cancer Genome Atlas (TCGA) database, considering clinical parameters such as disease stage, gender, age, tumor purity, and prognosis through multifactorial regression analysis. Furthermore, the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org ) was used to investigate the expression levels of YAP1 and TEAD4 in PDAC. 2. Cell culture ASPC-1 cells with knockout of MSLN (AKO), Mia capa2 cells with overexpressing of MSLN(MOE) and their negative control (ANC and MNC respectively) were constructed previously [ 16 ] . ASPC-1, ANC, and AKO cells were cultured in RPMI-1640(Procell, Cat.#PM150110). PANC-1 were purchased from Procell (Wuhan, China). MOE, MNC and PANC-1 were cultured in DMEM (Procell, Cat.# PM150210). All media contained 10% FBS and 1% Penicillin-Streptomycin. 3.Annexin V-PE/7-AAD double-staining for cell apoptosis analysis Annexin V-PE/7-AAD Apoptosis Detection Kit (YEASEN, Cat.#40310ES20) was employed to detect gemcitabine-induced apoptosis in PDAC cells. Briefly, Cells were harvested and resuspended in 1 × binding buffer at a concentration of 1× 10 6 cells/mL, and subsequently incubated with Annexin V-PE and 7-AAD for 15 minutes in the dark at room temperature. Finally, the labeled cells were immediately assessed using a flow cytometer, and the data were processed using Flowjo v10 software. Cells negative for both PE and 7-AAD were classified as live cells, those positive for PE but negative for 7-AAD were identified as early apoptotic, and those positive for both PE and 7-AAD were categorized as late apoptotic or necrotic. 4.RNA extraction and quantitative real-time polymerase chain reaction (qPCR) Total RNA was extracted from PDAC cells using TRIzol reagent (Invitrogen, Cat# 15596026). The RNA concentration and quality were measured by NanoDrop 5000 spectrophotometer (BioTeke, China). Total 500 ng RNA from each sample was reverse transcribed in 20 µl total volume using the HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Cat.#R201-01). Gene expression of PDAC cells was measured using SupRealQ Purple Universal SYBR qPCR Master Mix (U+) (Vazyme, Cat.#Q412-02) and analyzed on the ABI 7500 RT PCR System (Life Technologies). Gene expression was normalized to GAPDH mRNA. Data are presented as fold-change in gene expression in each group relative to control group. Primer synthesis was completed by Shanghai Sangon Co., Ltd. (Table 1 ). Table 1 Primer sequence Gene name Forward primer (5'-3') Reverse primer (5'-3') hENT1/SLC29A1 AACTCTCAGCCCACCAATGAAAGC GAAGCAGACAGAGAAAGCCAGGAC AKT1 CAGGAGGAGGAGGAGATGGACTTC CCCAGCAGCTTCAGGTACTCAAAC CDKN1B GCTTGCCCGAGTTCTACTACAGAC ACCAAATGCGTGTCCTCAGAGTTAG BIRC5/survivin CAAGGACCACCGCATCTCTACATTC CCAAGTCTGGCTCGTTCTCAGTG ABCC1/MRP1 TGCACATTTGACGCTAGTG CACGATGCTGATGACCAT DCK GAGGGGACCCGCATCAAGAAAATC CCATCTGGCAACAGGTTCAGGAAC P53 GCCCATCCTCACCATCATCACAC GCACAAACACGCACCTCAAAGC CMYC AGCAGCGACTCTGAGGAGGAAC TCCAGCAGAAGGTGATCCAGACTC CDK6 GTGACCAGCAGCGGACAAATAAAAC ACGACCACTGAGGTTAGAGCCATC ABCG2 GCAGCAGGTCAGAGTGTGGTTTC ACTGAAGCCATGACAGCCAAGATG RRM1 AGCGGCTAATCCAATCCAGTTCAC CTGCTGTGTTCCTCTCCTTCTCTTC MDR1/ABCB1 TTGATTGACAGCTACAGCACGGAAG TTCTTCACCTCCAGGCTCAGTCC MCL1 GGGGCAGGATTGTGACTCTCATTTC CTAGCCAGTCCCGTTTTGTCCTTAC BCL2 TCGCCCTGTGGATGACTGAGTAC ACAGCCAGGAGAAATCAAACAGAGG Cyclin D2/CCND2 CTTCCGCAGTGCTCCTACTTCAAG TTCCTCACAGACCTCCAGCATCC BCL-xl/BCL2L1 GCTGGTGGTTGACTTTCTCTCCTAC TCTCCATCTCCGATTCAGTCCCTTC GAPDH TGACATCAAGAAGGTGGTGAAGCAG GTGTCGCTGTTGAAGTCAGAGGAG 5.Western blot Protein was extracted from PDAC cell lines and PSC using RIPA lysis buffer with proteinase inhibitor (Solarbio, Cat.# R0020). Protein concentration was measured by Omni-Rapid™ Rapid protein quantification kits (Yazyme, Cat.# ZJ103). A total of 20 µg of protein mixed with 5 × SDS loading buffer was loaded per lane, separated by 10% SDS-polyacrylamide gel electrophoresis. The primary antibodies used in this study includes YAP1 (1:1000 dilution, Cell Signaling, Cat.# 14074 ), MSLN (1:1000 dilution, Cell Signaling, Cat.#99966), Vimentin (1:1000 dilution, Cell Signaling, Cat.#5741 ), E-cadherin(1:1000 dilution, Cell Signaling, Cat.# 3195), α-SMA(1:1000 dilution, Abcam, Cat.#ab184705), COL1A1(1:5000 dilution, Abcam, Cat.#ab138492), COL3A1(1:1000 dilution, abcam, Cat.#ab184993) and GAPDH (1:1000 dilution, Servicebio, Cat.#GB15002-100). Secondary antibodies conjugated to Dylight 680(1:10000 dilution, Cell Signaling, Cat.#5470) or Dylight 800 (1:10000 dilution, Cell Signaling, cat.#5151) were used for detection. GAPDH was used as an internal loading control. Protein bands were visualized using the Odyssey® CLX two-color infrared laser imaging system (Li-cor, USA) and quantified by Image J software. 6. Cell viability assay and Calculation of combination index Cell viability was assessed using CCK-8 Cell Counting kit (Yeasen, Cat.#40203ES76). PANC-1 or APSC-1 cells were seeded into 96-well plate at a density of 2×10 4 cells per well and were treated with 100 µL of culture medium containing varying concentrations of TED-347 and gemcitabine. Three replicate wells were used for each treatment group. cells were then incubated with 10 µL of CCK-8 per well for 2 h, and the absorbance at 450 nm was measured with a microplate reader (Tecan Spark 10 M luminometer). The Combination Index (CI) TED-347 and gemcitabine was calculated by Compusyn software. As for the synergistic assay, the concentrations of cells treated by TED-347 and gemcitabine for 48 h were listed in Table 2 . The Chou-Talalay method and CompuSyn software were used to analyse the synergistic effect of the two drug combinations. The CI of Chou-Talalay was calculated from the data derived from monotherapy and combination treatment by CompuSyn software, and the CI value quantitatively defines additive effect (CI = 1), synergism (CI 1) in drug combinations. Table 2 CI value of the combined action of TED-347 and GEM in non-fixed proportion combination Dose TED-347(µM) Dose GEM(nM) Effect CI 1.25 0.09 0.341 0.21247 2.5 0.39 0.473 0.30910 5.0 1.56 0.555 0.52738 7.5 6.25 0.628 0.68968 10.0 25.0 0.624 0.95916 Table 3 Multivariate Cox analysis of prognosis of pancreatic cancer patients by TIMER 2.0 MSLN in PAAD (n = 179): Model: Survive (OS, EVENT) ~ ‘MSLN’ + Age + Gender + Race + Stage + Purity 165 patients with 89 dying ( 14 missing obs. ) Co ef HR se(co ef) 95%CI_l 95%CI_u z p Sig nif MSLN 0.17 1.185 0.057 1.06 1.325 2.99 0.003 ** Age 0.017 1.017 0.011 0.995 1.039 1.502 0.133 Gender male -0.247 0.781 0.217 0.51 1.195 -1.139 0.255 Race Black 0.344 1.411 0.742 0.33 6.035 0.464 0.643 Race White 0.557 1.746 0.475 0.688 4.432 1.172 0.241 Stage Stage2 0.357 1.43 0.439 0.604 3.383 0.814 0.416 Stage3 -0.422 0.656 1.094 0.077 5.598 -0.386 0.7 Stage4 -0.194 0.824 0.827 0.163 4.168 -0.234 0.815 Purity -0.661 0.516 0.42 0.227 1.176 -1.575 0.115 R square = 0.144 (max possible = 9.91e-01) Likelihood ratio test p = 2.25e-03 Wald test p = 1.08e-02 Score (log rank) test p = 6.89e-03 7. Wound-healing assay Cells migration was tested by a wound healing assay. Transfected cells were plated in 12-well dishes (5×10 4 cells/well), and incubated in RPMI-1640 medium (Procell, Cat.#PM150110) without FBS at 37°C, reaching a confluence of 80%. Then the cells were scratched across the surface of the well by a 10-µl pipette. After an incubation at 37°C of 12h and 24 h, the scratches were observed. 8. Transwell migration and invasion assay Transwell analysis was performed to assess the effects of TED-347 on the migration and invasion ability of PADC cells. For migration assay, PADC cells were precultured with a particular concentration of TED-347 for 48 h. A total of 2×10 4 cells were seeded in serum-free medium in the upper chamber (STEMCELL, Cat.#38023), and the lower chamber was filled with RPMI-1640 containing 10% FBS. After 48 h, the non-migrating cells on the upper chambers were carefully removed with a cotton swab, and migrated cells underside of the filter stained and counted in five different fields. For invasion assays, transwell inserts (STEMCELL, Cat.#38023) were pre-coated with Matrigel (Corning, Cat.#354234). After treatment with TED-347 for 48 h, 2 × 104 cells in 200 µL of serum-free medium were seeded in Matrigel-coated upper chambers. The lower chamber contained 750 µL of medium supplemented with 10% FBS. After 24 h of incubation, the invading cells on the lower surface of the membrane were stained with 0.1% crystal violet. The cells were then washed and the number of invading cells in the lower chamber was observed under a microscope. Finally, 5 fields per chamber were selected for cell counting at 20×magnification. The cell count in each field was analyzed using Image J software. 9. Immunofluorescence (IF) analysis Briefly, Cells for IF experiments were grown on coverslips to 70–80% confluence, washed in PBS and fixed in 4% paraformaldehyde. The cells were permeablized by adding TBST with 0.1% Triton for 5 min, blocked in 5% BSA and incubated with the primary antibody and secondary antibody in a humidified chamber. The coverslip is then mounted onto a slide for viewing using a mounting medium with DAPI. Immunofluorescence images were captured using a confocal microscope (Nikon TiE-A1 plus) using a 40× objective with fixed optical slice, laser power and detector/amplifier settings for all samples across each individual experiment to allow comparison. Cells were cultured on coverslips to 70–80% confluence, washed with PBS, and fixed in 4% paraformaldehyde. Cells were then permeabilized by incubating in TBST with 0.1% Triton X-100 for 5 min, followed by blocking in 5% BSA. Primary and secondary antibodies were applied sequentially in a humidified chamber. After incubation, the coverslip were mounted onto slide using a mounting medium containing DAPI to stain the nuclei. Immunofluorescence images were acquired using a confocal microscope (Nikon TiE-A1 plus) with a 40× objective. The optical slice, laser power, and detector/amplifier settings were fixed for all samples within each individual experiment, allowing for direct comparison between conditions. 10. Multiple-color immunofluorescence staining of tissues Paraffin-embedded sections of pancreatic cancer tissues were collected from 10 patients diagnosed with pancreatic ductal adenocarcinoma following surgery. These patients were treated at our hospital between 2018 and 2020. The study was approved by the Ethics Committee of Beijing Shijitan Hospital, Capital Medical University(sjtkyll-lx-2022(069)). In short, immunofluorescence staining was performed on PDAC patient tissues using the mouse-rabbit triple-label four-color kit (ImmunoWay Biotechnology, Cat.#RS0035) protocol to label the expression of YAP1(1:200 dilution, CST, Cat.#14074), TEAD1(1:200 dilution, CUSABIO, Cat.#CSB-PA023363DSR2HU) and TEAD4(1:200 dilution, CUSABIO, Cat.#CSB-PA618010LA01HU) in PDAC. According to the instructions, apply the diluted primary antibody solution onto the sample area, incubate overnight at 4 ℃, and then reheat to room temperature for 30 minutes. Then, incubate the tissue with HRP secondary antibody working solution under RT conditions for 30 minutes, and rinse the slices three times with TBST for 5 minutes each time. Next, cover the tissue with 1 × 100 uL dye signal amplification working solution under RT conditions for 10 minutes, and then rinse the slices twice with TBST for 3 minutes each time. Then repeat the above process and stain the same sections with different colored immunofluorescence dyes. Finally, observe the sections under a fluorescence microscope. 11. Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) The JASPAR CORE database was used to identify the top ten binding sites between YAP1 and the 2000 bp upstream and 100bp downstream genomic regions of the MSLN promoter (Table 4 .). Immunoprecipitated chromatin was analysed by qPCR using primers targeting the predicted binding sites (Table 5 .). The experiment was conducted according to the scheme of Chromatin immunoprecipitation kit (Beyotime, Cat.#P2078). Briefly, PADC cells were fixed in 1% final formaldehyde for 10 min at room temperature and quenched by glycine. After cell lysis, the chromatin was fragmented into 200–1000 bp by ultrasonic cell shredder (Qsonica, Q500-2 20). Protein-DNA complexes were immunoprecipitated by YAP1 antibody (1:50 dilution, Cell Signaling, Cat.# 14074) or anti-IgG antibody conjugated with Protein A + G Agarose/Salmon Sperm DNA mix on rotator, incubated overnight at 4°C. After washing, reversal of crosslink and DNA purification, input DNA was used as a template for conventional PCR assay using specific primers (listed in Table 6 .). Table 4 The upstream 2000pb to downstream 100bp sequence of human MSLN gene promoter >NC_000016.10:758734–760834 Homo sapiens chromosome 16, GRCh38.p14 Primary Assembly CTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAAGATGGTCTCGAT CTCCTGACCTCATGATCTGCCTGCCTCGGCCTCCAAAAGTGCTGGGATTACAGGCGTGA GTCACTGCGCCCGGCATTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGGGTCTTGCTCTG TTGCCCAGGCTGGAGTGCAGTGGCGTGATCTCGACTCACTGCAACCCCTACCTCCTGGG TTCAAGTGATTCTTCTGCCTCAGCCTCCTGAGTAACTAGGATTACAGGCATGTGCCACCA CGCCCAGTTAATTTTCATATTTTTAGTAGAAACGGGGTTCCTCCATGTTGGCCAGGCTGG TCTCGAACTCCTGACCTCAGGTGATCCGCCCACCTCGGCCTACCAAAGTGCTGGGATGA CAGGCGTGCGCCACCACACAGGGCCTCATTTAAAGATTCTTTATAGAGATGAGGTGTCA CTACGTTGCCCAGGCTGGTCTTGAACTTCTGGCCTCAACGGACCCTCCCGCATCGGCCT CCCACGGTGCTGGCTGAAGGGTGAGCCCAGGCTGGCCACAGCGCGTTTTCATCATTGTC CGCAGCTTGCAGTCGGCTGGTTCAGAGCTTAGCCGGGCACATGGGCCCCTCTGAGGCTC CTGTTCAGGGCTCAGCCGTGTCTACGGGGCCCTCCGAGCTCTTCCTGGACCCTCCTCGG CTCCTTCAGTTCAGGGCTCAGCTGGGCACGTGGGCGCCTCCTTGGCTCCTCCGGCTCAG GCCTCTCCTCGAGCTCTGGGCCCTGAGTATTCTGGGCTCCTTCCTTGGTTTCCTTTGGCC TTTGGCCGGGAAAGTTGTGGGTGTCCGTGGCAGCCTGGGCCACTTCACAGCCCCGCAG CCAACCTGCGGCTCCTTCAGAGCAAAGCTGTGTGGACACAAAGGGAACGCCACTCGG AGCTGGCCTCTCCCTTTACCTGTGAGTTCCCGCCCAAGCCGGCTGCCTTCTGTCCCCTC CCCAGAGCCCTTGGGTAACTGGTTTGCTACAAGAGTGTCTGGAATTTTTCAGTTGTTCT CTGCGGAAGGGAGTTTTTAAAAGGCCCTTAATCCCTTCTTGACATTTGTAAGTTGACGC TTACACCTGGCAGCCTTGCTGAATTCTGTGTGCGTGAAGGTCCGATTCCACCGCGAGTC ACGATAGAAAACCCACTCTGTGGAGAGACCAGAGATGACCGCCGCGCACACCTCCGT TCAGCACACAAACCTTTGCAGGTGTTCATAGCGGAGGCAGATTCCGTACTGGGGATAA GAGCTCACGACATGCTGGGAGGGGTTTCAGGGGCAGGGAGGGGGCTTCTGTGGCCCC AGGTCAGGAGGAGCAGCTCTTCCATCAGCAGCAGGCAGCCAGCCCAGATGCGTAGGG AAGACAGCTCCCACTTCGCCAGGCCAGAGAGCGCCCGGGGGCAGCTCTGTTCCAGTC GACCCTGCGAGAAAGGGGTGTGCGTGTGCCTGGAGCTGGGCCCCGTCCTGCCTCCCT GACCTGTGTGCTCCCACAGCCCTGAGACGGACGGCTCACAGCCTTGCGAGGCCCACA CTGCACTGGGGGTCAGGCTTGTGCTCCCGGGAGTCCTGTCTGGGCTGCGTGGCCACC ATCCAGAGCCTGCTGACCTGCGACTGGGGGGGCCAGTGCTCCCTGGGTTTCAGCACC TGAGAATCAGAGTGGGATCCCGTGAAACCTGGGCCCAGGCTCCCACCCACGCCCCAC ACCCACCCAGGGAAGCCATGAAACCTGGGCCCGGGCTCCTACACATGCCCCACACCC ACCCAGGGCAGCCGTGAAACCTGGGCCCGGGCTCCCACCCTCGCCCACCGAGGGCA GCTTTGCCTTCCTGGGCATCCCTCCTCCCCCAGGCCTGGCCCGCTGCCTGTCCAAGG CTCCTGTGCGGGGTCTCCACCCACACATTCCTGGGGCGTGAGGCGCCACCACTCCCT GCTGCCCCGGGCAAAGCCGTCATTTGTTCCCTTTGACGGCCCGGGAGGCTGCCAGGC TCTCCACCCCCACTTCCCAATTGAGGAAACCGAGGCAGAGGAGGCTCAGGTGTGGC CAATCA Table 5 Possible transcription factor binding sites of YAP1-TEADs predicted by JASPAR 2022 Matrix ID Name Score Relative score Sequence ID Start End Strand Predicted sequence MA0090.1 MA0090.1.TEAD1 15.025486 0.973801862 NC_000016.10:758734–760834 1949 1960 + CACATTCCTGGG MA0090.3 MA0090.3.TEAD1 14.489286 0.94454802 NC_000016.10:758734–760834 1948 1960 + ACACATTCCTGGG MA0809.2 MA0809.2.TEAD4 13.52531 0.948052879 NC_000016.10:758734–760834 1948 1959 + ACACATTCCTGG MA0090.3 MA0090.3.TEAD1 12.748998 0.91341974 NC_000016.10:758734–760834 1044 1056 - AAAAATTCCAGAC MA0809.1 MA0809.1.TEAD4 12.132757 0.979330593 NC_000016.10:758734–760834 1949 1958 + CACATTCCTG MA0809.2 MA0809.2.TEAD4 12.009904 0.918816513 NC_000016.10:758734–760834 1045 1056 - AAAAATTCCAGA MA0090.2 MA0090.2.TEAD1 11.853291 0.966348763 NC_000016.10:758734–760834 1949 1958 + CACATTCCTG MA0809.1 MA0809.1.TEAD4 10.621073 0.946984752 NC_000016.10:758734–760834 1046 1055 - AAAATTCCAG MA0090.1 MA0090.1.TEAD1 9.284123 0.827026447 NC_000016.10:758734–760834 1873 1884 + TGCCTTCCTGGG MA0090.2 MA0090.2.TEAD1 9.028421 0.908144731 NC_000016.10:758734–760834 1046 1055 - AAAATTCCAG Table 6 ChIP-qPCR primers Primer name Primer sequence (5’-3’) Destination site PCR product size ChIP 1045–1056 F gagcccttgggtaactggtttg 1045–1056 114bp ChIP 1045–1056 R gcgtcaacttacaaatgtcaagaagg ChIP 1948–1959 F gctgcctgtccaaggctcc 1948–1960 94bp ChIP 1948–1959 R tgacggctttgcccggg ChIP 1145–1154 F ccttcttgacatttgtaagttgacgc 1145–1154 133bp ChIP 1145–1154 R tgcgcggcggtcatctct GAPDH promoter F tactagcggttttacgggcg GAPDH promoter region 166bp GAPDH promoter R tcgaacaggaggagcagagagcga 12. Animal experiment The animal study was approved by the Animal Experimentation Ethics Committee of Beijing shijitan Hospital, Capital Medical University (sjtkyll-lx-2022(069)). Twenty male BALB/c nude mice (4 weeks old) were obtained from the Laboratory Animal Center of Beijing Shijitan Hospital, Capital Medical University. Mice were randomly assigned to four groups, with five mice in each group. To establish a subcutaneous model, ASPC-1 cells with were resuspended in PBS at a concentration of 10 7 cells/100 µl. Each mouse was subcutaneously injected in the right flank with 100 µl of cell suspension to induce tumor formation. When the tumor sizes reached 100 mm 3 , the mice were injected weekly with 50mg/kg gemcitabine hydrochloride (Chiata Tianqiong) or 20mg/kg TED-347 (MedChemExpress, Cat.# 2378626-29-8). Tumor sizes were measured weekly, and the volumes (in cubic millimeters) were calculated according to the following formula: width 2 × length × 0.5. Length and width refer to the longest and shortest diameters, respectively. Six weeks after the first injection, the mice were euthanized, and the tumors were excised. Tumor tissues were harvested for further processing. A 4‑mm portion of each tumor was fixed with paraffin. The hearts, livers, spleens, lungs and kidneys of mice in each group were fixed with paraformaldehyde, embedded in paraffin, and subjected to hematoxylin and eosin (H&E) staining using HE staining kit (Solarbio, Cat.#G1120). 13. Modified Masson staining and scoring Tissues were fixed in 4% paraformaldehyde and then slowly frozen and thawed 2–3 times repeatedly. Subsequent steps were performed according to the standard Masson’s trichrome staining protocol. The area percentage of blue fibres was analysed by image J software and used as an indicator to assess the degree of fibrosis. 14. Enzymatic extraction of mouse pancreatic stellate cells After 4–6 weeks of C57bl/6 (Purchased from Beijing Viton Lever Ltd.) mouse execution, the pancreas was carefully excised, with any excess adipose tissue removed. The pancreas was washed 3 times with pre-cooled PBS for 5 min. Following this, the tissues were trimmed and digested with 0.25% trypsin (Procell, Cat.#PB180226) for 30 min at 37°C before terminating the digestion with 10% FBS in DMEM medium(Procell, Cat.#PM150210). The adherent cells were then purified using differential attachment and identified. 15. Statistical analyses All of the experimental data were repeated at least three times and expressed as the mean ± SD. Student’s t-test was used to compare differences between two groups. P values < 0.05 were considered to be statistically significant. Result 1. High expression of MSLN in PDAC correlates positively with tumor stage and negatively with patient prognosis. Analysis of MSLN expression across pan-cancer database from TCGA showed that MSLN was highly expressed in a variety of tumor tissues, including pancreatic, ovarian and colon cancer ( Figure.1a ). Similarly, TCGA database and GETx database analyses showed that MSLN was highly expressed in cancer tissues at the transcriptional level in 179 PDAC cases and 171 normal pancreatic tissues (Fig. 1 b). Furthermore, HPA database analyses showed that MSLN was highly expressed in PDAC tissues at the protein level (Fig. 1 d,e). Analyses of the relationship between MSLN expression and PDAC tumor staging indicated a positive correlation between High expression of MSLN and advanced ( Figure.1f ). To assess the prognostic impact of MSLN, overall survival (OS) and disease-free survival (DFS) of pancreatic cancer patients were analyzed by GEPIA2, stratifying patients based on high and low MSLN expression levels. Patients were stratified into quartiles based on MSLN expression levels, with the top 25% representing the highest expression and the bottom 25% representing the lowest. Additionally, comparisons were made between the top 25% and the bottom 25%, as well as between the top 25% and the bottom 75% of patients. The cut-off values for high and low MSLN expression were adjusted to ensure accurate stratification. Kaplan-Meier survival curves showed that patients with high MSLN expression exhibited significantly improved OS and DFS, while those with low MSLN expression had poorer outcomes (Fig. 1 g-j). Further analysis using the TIMER 2.0 database, which incorporated clinical variables such as age, gender, race, tumor stage and tumor purity, revealed that MSLN expression was significantly associated with prognosis of pancreatic cancer patients. High expression of MSLN was inversely correlated with patient survival, highlighting its potential as a negative prognostic marker in pancreatic cancer (Table 3 ). 2. High MSLN expression confers resistance to gemcitabine-induced early apoptosis of pancreatic cancer cells. The impact of gemcitabine on apoptosis induction in PDAC cells, in relation to levels of MSLN expression, is illustrated in Fig. 2 . pre-established MSLN-knockout PDAC cell lines (MSLN-knockout ASPC1 cells: AKO; Negative-control ASPC1 cells: ANC) [ 16 ] was employed to investigate this relationship by Annexin V-PE/7-AAD double staining flow cytometry. After 48 h of treatment with 80 µM gemcitabine (a concentration that is fivefold the IC50 value), both ANC and AKO exhibited significant apoptosis induction (Fig. 2 a). No significant difference was observed in early or late apoptosis between ANC and AKO in PDAC cells untreated with gemcitabine. However, AKO displayed a significantly higher early apoptosis rate, compared to ANC, with no significant difference observed in late apoptosis (Fig. 2 b-d). This suggests that high MSLN expression significantly confers resistance to gemcitabine-induced early apoptosis in pancreatic cancer cells, and high MSLN expression may contribute to gemcitabine resistance by inhibiting early apoptosis signaling. 3. ABCC1 as a key gene in MSLN- Mediated gemcitabine resistance in pancreatic cancer cells. Database visualization and analysis revealed the correlation between MSLN expression levels of mRNA levels of several gemcitabine resistance-related genes, for gemcitabine resistance-related molecules, including ABCB1 [ 31 ] , ABCC1 [ 32 ] , ABCG2 [ 33 ] , AKT1 [ 34 ] , BCL2 [ 35 ] , BCL2L1 [ 35 ] , BIRC5 [ 36 ] , CCND2 [ 37 ] , CDK6 [ 38 ] , CDKN1B [ 39 ] , DCK [ 40 ] , MCL1 [ 41 ] , MYC [ 42 ] , RRM1 [ 43 ] , SLC29A1 [ 24 ] , and TP53 [ 44 ] , as depicted in Fig. 3 a. Positive correlations with MSLN expression were observed for ABCC1, AKT1, BCL2L1, and BIRC5, while negative correlations were noted for ABCB1, ABCG2, BCL2, CCND2, CDK6, and DCK. Genes with no significant correlation included CDKN1B, MCL1, MYC, RRM1, SLC29A1, and TP53. qPCR analysis of pancreatic cancer cells with MSLN knockdown or overexpression demonstrated that MSLN reduction led to a decrease in ABCB1, ABCC1, and ABCG2, whereas MSLN overexpression resulted in their elevation (Fig. 3 b, c). Further analysis indicates that ABCC1 is the only gene consistently correlated with MSLN expression, suggesting that ABCC1 is a critical gene driving MSLN-mediated gemcitabine resistance in pancreatic cancer cells. 4. The YAP1 inhibitor TED-347 can suppress MSLN expression in PDAC cells and enhance their sensitivity to gemcitabine. The effect of TED-347 on MSLN expression in PDAC cells is shown in Figs. 4 a-d. After treatment with various concentrations of TED-347 for 48 hours, Western blot analysis revealed a significant and dose-dependent reduction in MSLN expression in both ASPC-1 and PANC-1.These findings suggests that the YAP1 inhibitor TED-347 can effectively inhibit MSLN expression in PDAC cells. The impact of the combination of TED-347 with gemcitabine on the viability of PDAC cells is depicted in Figs. 4 e-g. The results indicate that treatment with various concentrations of TED-347 and gemcitabine for 48 h, either individually or in combination, resulted in significantly lower optical density (OD) values in the CCK-8 assay for both ASPC-1 and PANC-1 cells. The combination treatment exhibited a more potent inhibitory effect on pancreatic cancer cell viability compared to gemcitabine alone. Furthermore, CI calculations, using the CompuSyn software, revealed that all CI values for the drug combinations were less than 1 (Table 2 – 2 ), suggesting a synergistic interaction between TED-347 and gemcitabine. The influence of TED-347 on the gemcitabine resistance gene ABCC1 in PDAC cells is shown in Fig. 4 h. qPCR results revealed that the expression levels of ABCC1 were significantly reduced in both ASPC-1 and PANC-1 cells treated with TED-347 compared to the control group, indicating that TED-347 can enhance the sensitivity of PDAC cells to gemcitabine. 5. TED-347 inhibits the migration, invasion, and EMT of PDAC cells. The impact of TED-347 on the migration, invasion, and EMT of ASPC-1 and PANC-1 is depicted in Fig. 5 . Wound-healing assays were performed to assess the horizontal migration ability of PDAC cells after treatment with various concentrations of TED-347. The results demonstrated that at 0, 12, and 24 hours post-scratch, the wound healing rate in both ASPC-1 and PANC-1 cells treated with 5µM and 10µM TED-347 was significantly reduced compared to the control group. Notably, higher concentrations of TED-347 led to a more pronounced inhibitory effect (Fig. 5 a, b). Transwell migration and invasion assays were used to evaluate the vertical migration and invasion abilities of PDAC cells after treatment with different concentrations of TED-347. These findings indicate that TED-347 effectively inhibits vertical migration (Fig. 5 c-e) and invasion (Fig. 5 f-h) of pancreatic cancer cells, with the extent of inhibition intensifying in a dose-dependent manner. Our previous investigations have established that MSLN can facilitate the EMT in PDAC cells. In a complementary experiment, with fluorescent signal acquisition standardized, we utilized immunofluorescence to assess the expression of EMT markers, E-cadherin and N-cadherin, in PDAC cells. The findings revealed that, in MSLN-knockdown ASPC-1 cells, there was an elevation in E-cadherin expression and a reduction in N-cadherin expression when compared to both the control and vector groups ( Figure S1 a ). Subsequently, we explored the alterations in EMT markers within ASPC-1 and PANC-1 cells following treatment with TED-347. The data indicated that, in the presence of TED-347, there was an upregulation of E-cadherin and a downregulation of Vimentin compared to the control group, implying that TED-347 is capable of inhibiting EMT in PDAC cells (Figs. 6 c-g). 6. The YAP1-TEAD1 complex binds to the MSLN promoter region to regulate MSLN expression. Transcription factor binding sequences of MSLN were obtained from the NCBI website (Fig. 7 a and Table 4 ). The top 10 transcription factor binding sites with the highest matching scores were predicted using the JASPAR website, as shown in Table 5 . These binding sites were primarily located in the regions [1045, 1056], [1145, 1154], and [1949, 1960]. Three pairs of PCR primers were designed on these regions (Fig. 7 b, Table 6 ). The effectiveness of ultrasonic DNA fragmentation was assessed using agarose gel electrophoresis, showing that cellular genomic DNA was fragmented into sizes ranging from 200-1000bp (Fig. 7 c). ChIP assays was performed using an anti-YAP1 antibody to isolate YAP1-bound transcription factors and DNA. ChIP products served as templates for PCR amplification, followed by gel electrophoresis to detect the DNA corresponding to the three pairs of ChIP-qPCR primers, enabling the evaluation of YAP1 binding sites. The analysis identified the [1045, 1056] region as the site with the strongest YAP1 binding (Fig. 7 d). Subsequent ChIP-qPCR experiments focused on the enrichment of this site. Furthermore, enrichment multiples of the YAP1 antibody in ASPC-1 cells, which highly express MSLN, was found a statistically significant increasing compared to the IgG control antibody. This findings indicated that in ASPC-1 cells with high MSLN expression, YAP1-TEADs can bind to the MSLN promoter region (Fig. 7 e). Given that the top 10 sites predicted by JASPAR were predominantly associated with TEAD1 and TEAD4, we employed multiplex immunofluorescence to detect the expression of YAP1, TEAD1, TEAD4 in PDAC tissue. The results revealed that YAP1 was expressed in both PDAC tissue and the surrounding stroma, with a higher expression in cancer epithelial cells compared to the tumor stroma. TEAD1 expression was specifically localized to the nuclei of ductal cells, whereas TEAD4 expression was nearly absent in both PDAC tissue and stroma. These results were further corroborated by data from HPA database (Figs. 7 g, h). Collectively, these findings suggest that YAP1 preferentially binds to the TEAD1 rather than TEAD4 in PDAC. 7. The combination of TED-347 and gemcitabine effectively inhibits tumor growth in mice. The impact of the combination of TED-347 and gemcitabine on the growth of subcutaneous pancreatic tumors in nude mice is depicted in Fig. 8 . Tumor-bearing mice were prepared and treated according to the schematic (Fig. 8 a). Mice were assigned into four groups based on treatment: GEM-/TED-347-, GEM+/TED-347-, GEM-/TED-347+, and GEM+/TED-347 + groups. Tumor volumes in the GEM+/TED-347-, GEM-/TED-347+, and GEM-/TED-347- groups increased steadily, with no significant differences among the three groups, suggesting that neither gemcitabine nor TED-347 alone was effective in inhibiting tumor growth (Fig. 8 b, c). In contrast, the combined treatment group (GEM+/TED-347+) showed a marked reduced in average tumor volume, with a statistically significant difference, compared to the other groups, indicating that the combination therapy effectively suppressed tumor growth (Fig. 8 e). Concurrently, body weight fluctuations were observed in the treated mice, with both gemcitabine and TED-347 affecting weight, though the body weight of the treatment groups was significantly lower than that of the control group. However, no statistically significant difference in body weight was found among the three treatment groups (Fig. 8 d). Morphological examination of heart, liver, spleen, lung, and kidney tissues from mice in all treatment group was conducted using H&E staining. There was no significant abnormalities observed in the appearance or histological structure and tissue morphology of organs in the GEM+/TED-, GEM-/TED+, GEM+/TED+, and the negative control GEM-/TED- groups, indicating that TED-347 did not adversely affect the organ structure or histological in nude mice ( Figure S2 ). 8. Gemcitabine has been shown to promote fibrosis in PDAC tissue, and TED-347 can alleviate this fibrosis. Subsequently, due to the difficulty in distinguishing between cytoplasm and intercellular fibrosis staining in routine Masson staining of PDAC tissue (Fig. 9 d,e), we immersed the tissue in 4% paraformaldehyde and subjected it to repeated slow freezing and thawing 2–3 times at -20°C. After this process, when we performed Masson staining again, the cytoplasm automatically detached, leaving only a small amount "squeezed" around the cell membrane, making the blue-stained fibrillar proteins between cells more visible ( Fig. 9 a ) . Masson staining results revealed that compared to the control group (GEM-/TED-347-), the gemcitabine monotherapy group (GEM+/TED-347-) exhibited significantly increased fibrosis in PDAC. The fibrosis levels in the TED-347 monotherapy group were comparable to the control group, while the combination therapy group (GEM+/TED-347+) had a lighter degree of PDAC fibrosis than the gemcitabine monotherapy group. This indicates that gemcitabine exacerbates fibrosis in mouse PDAC tissue, and TED-347 can mitigate gemcitabine-induced fibrosis in PDAC tissue. 9. TED-347 down-regulated the expression of collagen in PSCs by inhibiting the binding of YAP1 and TEAD1 to reduce the fibrosis of PDAC. Under phase-contrast microscopy, PSCs isolated on the third day appeared polygonal with minimal cell extension, a low nucleus-to-cytoplasm ratio, and visible refractile lipid droplets within the cytoplasm. By the seventh day, PSCs exhibited accelerated proliferation, increased cell extension, a higher nucleus-to-cytoplasm ratio, and the disappearance of refractile lipid droplets in the cytoplasm (Fig. 10 a). Immunofluorescence analysis confirmed that the third-generation PSCs expressed vimentin and α-SMA, with nearly 100% cellular purity (Fig. 10 b). All-trans retinoic acid (ATRA) is well-established to revert activated PSCs into a quiescent state, inhibiting the expression of α-SMA, vimentin, COL1A1, and COL3A1 in PSCs, and restoring the formation of lipid droplets within the cytoplasm, which is a well-recognized model for PSCs deactivation [ 45 – 48 ] . In this study, PSCs were treated with serum-free medium containing 20µM ATRA or 5µM TED-347, while the control group was treated with DMSO. After 48 hours, immunoblotting was used to detect the expression levels of YAP1, α-SMA, vimentin, COL1A1, and COL3A1 in PSCs. As with ATRA, TED-347 significantly inhibited the expression of COL1A1 and COL3A1 in PSCs. Notably, TED-347 treatment led to a compensatory increase in YAP1 expression. While both ATRA and TED-347 treatments resulted in a reduction of vimentin and α-SMA levels, no statistically significant difference was observed after normalization across the three groups (Fig. 10 c, d). Discussion PDAC is among the most aggressive tumors, with the majority of patients presenting at an advanced stage, beyond the reach of surgical intervention. For those fortunate enough to undergo surgery, the risk of recurrence and metastasis remains high postoperatively [ 1 ] . In the current landscape of treatment, chemotherapy-based systemic therapy holds a pivotal position in the management of PDAC [ 4 ] . Our research delves into the roles of MSLN in PDAC cells, EMT, and PSCs within the stroma. We have demonstrated that the YAP1 inhibitor TED-347 can effectively inhibit the expression of MSLN in PDAC cells and reduce fibrosis in PDAC tissues, which in turn enhances the therapeutic effects of gemcitabine. Furthermore, our study investigates the effects of YAP1 inhibitors on PDAC cells and stromal fibrosis, as well as the underlying mechanisms associated with chemoresistance in PDAC. In this study, we employed bioinformatics analysis to further clarify that MSLN is highly expressed at both the transcriptional and protein levels in PDAC tissues, consistent with our previous findings that PDAC tissues in patients exhibit high MSLN expression [ 10 ] . The high expression of MSLN is positively correlated with poor prognosis in PDAC patients and is an independent prognostic factor affecting PDAC patients, providing a theoretical basis for potential MSLN-targeted therapy in PDAC. Given that high MSLN expression is positively correlated with poor prognosis in PDAC patients, we hypothesize that high MSLN expression is also related to chemoresistance in PDAC. Similarly, patients with mesothelioma and colorectal cancer who have high MSLN expression are prone to chemoresistance to chemotherapy drugs [ 49 , 50 ] . The MSLN antibody amatuximab can increase the sensitivity of human PDAC cells to gemcitabine in xenograft mouse models [ 14 , 51 ] . In our published study, we employed CCK8 assays to evaluate the viability of PDAC cells with MSLN either knocked out or overexpressed, thereby determining the IC50 for gemcitabine. We found that PDAC cells overexpressing MSLN displayed a diminished response to gemcitabine, whereas cells with MSLN knocked out demonstrated heightened sensitivity to the chemotherapy agent. This finding underscores the relationship between MSLN and the chemoresistance of PDAC cells to gemcitabine. To further clarify the role of MSLN in gemcitabine chemoresistance in PDAC cells, this study also explored the relationship between MSLN and apoptosis in PDAC cells as well as their connection to chemoresistance. Apoptosis is an active, programmed necrosis of cells, a continuous process of cell death. Gemcitabine induces apoptosis in PDAC cells through two mechanisms: one is by inhibiting DNA synthesis; the other is by inhibiting further synthesis of DNA strands [ 52 ] . Using double staining flow cytometry to measure apoptosis rates, we observed that PDAC cells with MSLN knockout exhibited an elevated rate of early apoptosis. Conversely, high levels of MSLN expression were found to counteract the early apoptotic effects of gemcitabine in these cells. This suggests that elevated MSLN expression may contribute to gemcitabine resistance in PDAC by inhibiting the early apoptosis that would otherwise be triggered by the chemotherapy drug. To further elucidate the key genes and potential molecular mechanisms by which MSLN promotes gemcitabine resistance in PDAC cells, we conducted a literature review and identified a subset of genes associated with gemcitabine chemoresistance in PDAC, including ABCB1, ABCC1, ABCG2, AKT1, BCL2, BCL2L1, BIRC5, CCND2, CDK6, CDKN1B, DCK, MCL1, MYC, RRM1, SLC29A1, and TP53. We analyzed the correlation between MSLN and the expression of these gemcitabine resistance-related molecules in the PDAC TCGA database, and then employed qPCR to detect the expression levels of gemcitabine resistance-related molecules in PDAC cells with MSLN knockdown or overexpression. Interestingly, our study revealed that the expression level of ABCC1 was correlated with MSLN expression in both MSLN-knockdown and overexpressing PDAC cells, suggesting that ABCC1 may be a key gene in MSLN-mediated promotion of gemcitabine resistance in PDAC cells. This finding partially explains the molecular mechanism underlying the chemoresistance of PDAC cells with high MSLN expression to gemcitabine chemotherapy. ABCC1, also known as multidrug resistance protein 1 (MRP1), is a member of the ATP-binding cassette transporter superfamily [ 53 ] . In PDAC [ 54 ] , cholangiocarcinoma [ 55 ] , bladder cance [ 56 ] , ovarian cancer [ 57 ] , and various other malignancies, ABCC1 plays a crucial role in the development of chemoresistance to gemcitabine. Therefore, it is imperative to delve deeper into the possibility of targeting MSLN for the treatment of PDAC, starting with its role in promoting gemcitabine resistance. Kern et al. [ 19 , 20 , 58 ] identified an enhancer within the Canscript sequence of the MSLN promoter that is capable of recruiting TEAD1. It remains unclear whether inhibiting the YAP1-TEAD1 interaction can suppress the expression of MSLN in PDAC cells. Studies have shown that in PDAC cell lines with high MSLN expression, such as ASPC-1 cells, interfering with the expression of YAP1 or TEAD1 can inhibit the expression of MSLN. Conversely, in cells with low MSLN expression, such as MiaCaPa-2 cells, overexpressing YAP1 or TEAD1 does not induce MSLN expression [ 20 ] . Currently, aside from gene-editing techniques [ 59 , 60 ] or directly targeting MSLN mRNA [ 61 , 62 ] , there is still a significant gap in the literature regarding the transcriptional regulation of MSLN. In this study, we treated ASPC-1 and PANC-1 PDAC cells, which express MSLN, with varying concentrations of TED-347 and assessed MSLN expression levels using protein immunoblotting after treatment. We found that TED-347 could suppress the expression of MSLN in PDAC cells, with higher concentrations of TED-347 leading to reduced MSLN expression within the cells. This suggests that TED-347 can inhibit the transcription of MSLN by disrupting the interaction between YAP1 and TEADs. Therefore, targeting the YAP-TEAD interaction to suppress MSLN may represent a novel and effective therapeutic approach for cancer treatment. To further elucidate which TEADs transcription factors YAP1 interacts with and the specific binding sites within the promoter region, we conducted ChIP assays on the PDAC cell line ASPC-1. Our results revealed that in ASPC-1 cells, which highly express MSLN, the YAP1 antibody significantly enriched specific sequences within the MSLN promoter. Concurrently, by aligning the MSLN promoter sequence using SnapGene software, particularly the Canscript sequence [1949, 1960] (5'-CCACCCACACATTCCTGG-3'), we observed that the YAP1 antibody precipitated very few DNA fragments at positions [1949, 1960], with fluorescence intensity on the gel image nearly identical to that of the negative control. In contrast, a marked enrichment was noted at position [1045, 1056]. Thus, our data support that the YAP1-TEAD1 binding site is not within the Canscript sequence but at a location further from the transcription start site. Furthermore, we examined the co-expression of YAP1, TEAD1, and TEAD4 in PDAC tissue and stroma through multicolor immunofluorescence, revealing that TEAD1 is specifically expressed in PDAC tissue, while TEAD4 is virtually not expressed, which may explain TEAD1's involvement in MSLN transcription in PDAC. Additionally, we noted that YAP1 and TEAD1 are relatively more expressed in PDAC tissue and stroma, with minimal expression in non-cancerous tissue. The differences between cancerous tissue, stroma, and adjacent non-cancerous tissue lay the groundwork for our subsequent research into PDAC fibrosis. Most current research supports that YAP1-TEADs can promote tumor cell migration, invasion [ 28 ] , tumor stemness, and EMT [ 63 ] ; inhibiting YAP1 expression can suppress the malignant tendencies of PDAC. Our previous findings indicate that MSLN also promotes the proliferation, invasion, and EMT of PDAC cells, and knocking out MSLN can reverse the malignant characteristics of PDAC cell lines [ 16 ] . PDAC may acquire resistance to gemcitabine, a phenomenon that can be mediated by EMT and the development of pancreatic fibrosis [ 76 , 77 ] . Consequently, the inhibitory effects of TED-347 on the invasion, migration, and EMT of PDAC cells provide a partial explanation for how TED-347 might enhance the sensitivity of these cells to gemcitabine. Of course, molecules that share similar functions with MSLN, such as SGLT2 [ 64 ] , PAF1 [ 65 ] , and Neuromedin U [ 66 ] , are also associated with YAP1. The inhibitory effects of TED-347 on the proliferation, invasion, and EMT of PDAC cannot be entirely attributed to its suppression of MSLN expression. However, regardless, the relationship between YAP1 and MSLN is intimately connected in the development of PDAC, suggesting that targeting the expression of YAP1 or MSLN for the treatment of PDAC may hold broad therapeutic potential. Dense fibrotic desmoplasia is a hallmark of PDAC, characterized by substantial deposition of cellular stroma within the tumor tissue. This stroma serves a dual role: on one hand, it forms a physical barrier that impedes the recruitment of immune cells, such as T cells, to the tumor centre, thereby weakening the anti-tumor immune response [ 67 ] ; on the other hand, it leads to vascular collapse and hypoperfusion, creating an ischaemic and hypoxic tumor microenvironment that hinders the effective delivery of chemotherapy drugs to the cancerous tissue [ 67 ] . Studies have indicated that PDAC tissues overexpress YAP1, which correlates positively with the degree of pancreatic fibrosis [ 28 ] , and that inhibiting pancreatic fibrosis can enhance the therapeutic efficacy of gemcitabine on tumors [ 68 , 69 ] . Therefore, inhibiting pancreatic fibrosis is one of the crucial strategies in the treatment of PDAC. Our study demonstrated in vitro that TED-347 can suppress the secretion of collagen by PSCs, suggesting that TED-347, by inhibiting the binding of YAP1 to TEADs, downregulates the expression of collagen in PSCs, inhibits PSC activation, and alleviates fibrosis in mouse PDAC tissues. During the transition of activated PSCs to a quiescent state, the expression of YAP1 shifts from the nucleus to the cytoplasm [ 28 ] , a process that aligns with the characteristics of YAP1 as an auxiliary transcription factor. The irreversible binding of YAP1 to TEADs, inhibited by TED-347, also leads to a compensatory increase in YAP1 expression. Research has shown that ATRA can induce the transition of activated PSCs to a quiescent state, resulting in a decrease or disappearance of the expression of markers of PSC activation, such as VIM and α-SMA [ 28 ] . In our study, although ATRA and TED-347 could inhibit the secretion of collagen by PSCs and suppress PSC activation, there were no significant changes in the markers VIM and α-SMA, possibly due to the short intervention time of ATRA and TED-347 on PSCs. To exclude the impact of PSC proliferation on the experimental results, we intervened with PSCs for only 48 hours, significantly shorter than the intervention times reported in the literature, which range from one week to longer [ 48 , 70 , 71 ] . Perhaps if we extended the intervention time to over a week, we might obtain similar results. However, it is noteworthy that both TED-347 and ATRA can inhibit the expression of collagen by PSCs even before completely reversing PSC activation, indicating that both drugs have a role in reducing PDAC fibrosis. Additionally, our study was limited to mouse PSCs and did not combine clinical human PDAC specimens to investigate whether inhibiting the binding of YAP1 to TEADs can suppress the activation of fibroblasts in tumors, which is a certain limitation. Chemotherapy, while targeting the destruction or inhibition of PDAC cell growth, can also induce fibrosis within the PDAC tissue [ 72 , 73 ] . However, due to the short overall survival period of PDAC patients, particularly those in advanced stages, it is challenging to detect chemoresistance caused by chemotherapy-induced pancreatic fibrosis [ 74 ] , a concern that has not yet garnered sufficient attention [ 75 , 76 ] . Our study, through Masson's staining, observed that gemcitabine promotes fibrosis in mouse PDAC tissue, aligning with the research findings of Kim DK et al. [ 77 ] , which indicate that gemcitabine can enhance fibrosis in mouse orthotopically transplanted PDAC tissue during chemotherapy. From the perspective of inhibiting PDAC tissue fibrosis with TED-347, our study also provides a good explanation for the superior tumor suppression effects observed with the combination of TED-347 and gemcitabine. The results of this study suggest that, in addition to chemoresistance caused by PDAC fibrosis, we should also focus on the fibrosis induced by gemcitabine itself, leading to chemoresistance, thereby enhancing our understanding of chemotherapy for PDAC. The combination of PDAC chemotherapy with anti-fibrotic therapy may be one of the future research directions for PDAC chemotherapy [ 68 , 78 ] . In conclusion, this research initiative, pivoting on the link between YAP1 inhibitors and resistance to gemcitabine in PDAC, delves into the interplay and underlying mechanisms among PDAC MSLN, EMT in PDAC, activation of PSCs, fibrosis associated with PDAC, and chemoresistance. Through an array of in vivo and in vitro experiments, coupled with clinical studies on PDAC patients, this work sheds new light on the avenues for chemotherapy and targeted therapeutic strategies in PDAC. Conclusion High expression of MSLN in PDAC tissues indicates a poor prognosis. MSLN may contribute to chemoresistance by promoting resistance to apoptosis in PDAC cells and upregulating ABCC1. TED-347, a YAP1 inhibitor, has demonstrated the ability to reduce MSLN expression and curb the migration, invasion, and EMT in PDAC cells. Additionally, it mitigates PDAC fibrosis, which in turn boosts the effectiveness of gemcitabine in PDAC treatment. Abbreviations ATP binding cassette subfamily C member 1 (ABCC1); Chromatin immunoprecipitation (ChIP); Combination Index (CI); Disease-free survival (DFS); E-cadherin (E-CAD); epithelial-mesenchymal transition (EMT); Gene Expression Profiling Interactive Analysis 2 (GEPIA2); Mesothelin (MSLN); Overall survival (OS); Pancreatic Ductal Adenocarcinoma (PDAC); Pancreatic stellate cells (PSCs); The Cancer Genome Atlas (TCGA); Transcriptionally enhanced associate domains (TEADs); Tumor Immune Infiltration Evaluation Resource 2.0 (TIMER 2.0); Vimentin (VIM); Yes associated protein 1 (YAP1) Declarations Author Contributions The manuscript was prepared through contributions of all authors. The contribution of the authors to the manuscript is as follows: Conceptualization, Jili Hu, and Bin Zhu; Data curation, Jili Hu and Jia Wang; Formal analysis, Jili Hu and Jia Wang; Methodology, Jili Hu, Xu Guo, Xinming Li, Jia Wang, Buhe Amin and Wenlong Zhai; Project administration, Bin Zhu; Resources, Jiawei Xu, Bin Zhu; Software, Jili Hu, Xu Guo, Xinming Li, Zhuoyin Wang and Jian Zhou; Supervision, Buhe Amin and Bin Zhu; Validation, Zhuoyin Wang and Nengwei Zhang; Visualization, Jian Zhou and Qing Fan; Writing – original draft, Jili Hu; Writing–review & editing, Jili Hu and Bin Zhu. Data availability statement All data used in this study have been included in the manuscript. Funding This work was supported by Henan Province medical science and technology research plan joint construction project [grant number: LHGJ20240213], and the Chunhui Project Foundation of the Education Department of China (Grant No. HZKY20220055). 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Philip, P.A., et al., Molecular Characterization of KRAS Wild-type Tumors in Patients with Pancreatic Adenocarcinoma. Clin Cancer Res, 2022. 28 (12): p. 2704-2714. Goulart, M.R., et al., Pentraxin 3 is a stromally-derived biomarker for detection of pancreatic ductal adenocarcinoma. NPJ Precis Oncol, 2021. 5 (1): p. 61. Sarper, M., et al., ATRA modulates mechanical activation of TGF-beta by pancreatic stellate cells. Sci Rep, 2016. 6 : p. 27639. Han, X., et al., Reversal of pancreatic desmoplasia by re-educating stellate cells with a tumour microenvironment-activated nanosystem. Nat Commun, 2018. 9 (1): p. 3390. McCarroll, J.A., et al., Vitamin A inhibits pancreatic stellate cell activation: implications for treatment of pancreatic fibrosis. Gut, 2006. 55 (1): p. 79-89. Rozengurt, E., J. Sinnett-Smith, and G. Eibl, Yes-associated protein (YAP) in pancreatic cancer: at the epicenter of a targetable signaling network associated with patient survival. 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Yang, J., et al., Enhanced gemcitabine cytotoxicity with knockdown of multidrug resistance protein genes in human cholangiocarcinoma cell lines. J Gastroenterol Hepatol, 2021. 36 (4): p. 1103-1109. Li, B., D. Xie, and H. Zhang, Long non-coding RNA GHET1 contributes to chemotherapeutic resistance to Gemcitabine in bladder cancer. Cancer Chemother Pharmacol, 2019. 84 (1): p. 187-194. Haque, A., et al., Interaction Analysis of MRP1 with Anticancer Drugs Used in Ovarian Cancer: In Silico Approach. Life (Basel), 2022. 12 (3). Ren, Y.R., et al., Unbiased discovery of interactions at a control locus driving expression of the cancer-specific therapeutic and diagnostic target, mesothelin. J Proteome Res, 2012. 11 (11): p. 5301-10. Avula, L.R., et al., Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases. Mol Cancer Res, 2020. 18 (2): p. 229-239. Nunes, M., et al., Mesothelin Expression Is Not Associated with the Presence of Cancer Stem Cell Markers SOX2 and ALDH1 in Ovarian Cancer. Int J Mol Sci, 2022. 23 (3). Bharadwaj, U., et al., Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis, 2011. 32 (7): p. 1013-24. He, X., et al., Mesothelin promotes epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells. Mol Cancer, 2017. 16 (1): p. 63. Thompson, B.J., YAP/TAZ: Drivers of Tumor Growth, Metastasis, and Resistance to Therapy. Bioessays, 2020. 42 (5): p. e1900162. Ren, D., et al., SGLT2 promotes pancreatic cancer progression by activating the Hippo signaling pathway via the hnRNPK-YAP1 axis. Cancer Lett, 2021. 519 : p. 277-288. Nimmakayala, R.K., et al., PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer. Cell Death Dis, 2022. 13 (10): p. 839. Yoo, W., et al., The YAP1-NMU Axis Is Associated with Pancreatic Cancer Progression and Poor Outcome: Identification of a Novel Diagnostic Biomarker and Therapeutic Target. Cancers (Basel), 2019. 11 (10). Apte, M.V., et al., A starring role for stellate cells in the pancreatic cancer microenvironment. Gastroenterology, 2013. 144 (6): p. 1210-9. Sherman, M.H., et al., Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy. Cell, 2014. 159 (1): p. 80-93. Hwang, H.J., et al., Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells. J Exp Clin Cancer Res, 2019. 38 (1): p. 258. Guan, J., et al., Retinoic acid inhibits pancreatic cancer cell migration and EMT through the downregulation of IL-6 in cancer associated fibroblast cells. Cancer Lett, 2014. 345 (1): p. 132-9. 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Supplementary Files Suppplementaryfigures.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 May, 2025 Reviewers invited by journal 12 May, 2025 Editor assigned by journal 06 May, 2025 First submitted to journal 04 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6589227","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455552454,"identity":"5879e61d-59a2-410a-9c51-c42435c6605a","order_by":0,"name":"Jili 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University","correspondingAuthor":false,"prefix":"","firstName":"Buhe","middleName":"","lastName":"Amin","suffix":""},{"id":455552462,"identity":"ae8ef989-944e-4717-bf44-2c1688b343d3","order_by":8,"name":"Nengwei Zhang","email":"","orcid":"","institution":"Beijing Shijitan Hospital Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nengwei","middleName":"","lastName":"Zhang","suffix":""},{"id":455552463,"identity":"cae6ad97-fb7f-4bff-9cde-c6502d1e97a2","order_by":9,"name":"Wenlong Zhai","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Wenlong","middleName":"","lastName":"Zhai","suffix":""},{"id":455552464,"identity":"a52a9dc5-bdc8-417c-b1c0-ad69b0f65f1a","order_by":10,"name":"Jiawei Xu","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jiawei","middleName":"","lastName":"Xu","suffix":""},{"id":455552465,"identity":"0e3954a9-3d8a-4413-a864-2d8c38501430","order_by":11,"name":"Bin Zhu","email":"","orcid":"","institution":"Beijing Shijitan Hospital Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2025-05-04 15:43:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6589227/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6589227/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82863307,"identity":"46fc808e-dfcf-4d3d-a6b7-450c3503006e","added_by":"auto","created_at":"2025-05-16 07:23:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":609487,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of Mesothelin (MSLN) expression in pancreatic cancer (PDAC) and its prognostic implications based on database bioinformatics. a: The expression of MSLN in PDAC in TCGA database was analyzed through GAPIA2 online tool. Each point represented one sample, with red indicating tumor samples and green indicating paired normal tissues from GETx. b: Prognosis analysis of MSLN in solid tumor. The solid box indicates that MSLN expression significantly affects patient prognosis. Red denotes poor prognosis associated with high MSLN expression, while blue indicates better prognosis for patients with high MSLN expression. c: Box diagram of MSLN expression in pancreatic and adjacent pancreatic tissues and normal pancreatic tissues in GETx database, * indicating p\u0026lt;0.05. d and e: Immunohistochemical images of MSLN expression in pancreatic cancer and normal tissue from the HPA database. f: GAPIA2 analyzed the relationship between MSLN and PDAC staging. g,h,i,j: Survival analysis of pancreatic cancer patients stratified by MSLN expression levels. Panels g and h represent OS and DFS for the top 25% and bottom 75% of MSLN expression levels, while i and j represent OS and DFS for the top 25% and bottom 25% of MSLN expression.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/ee7691b49b439126c4fe692d.png"},{"id":82862736,"identity":"24e3be3a-04ab-45fe-8c83-49006b6101cf","added_by":"auto","created_at":"2025-05-16 07:15:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":283474,"visible":true,"origin":"","legend":"\u003cp\u003eMSLN partially resists gemcitabine-induced early apoptosis in pancreatic cancer cells. a: Scatter plots illustrate cell apoptosis in ANC and AKO following treatment with varying concentrations of gemcitabine. Q1 represents necrotic cells or cell debris due to mechanical injury (PE-/7-AAD+), Q2 represents the late apoptotic and dead cell populations (PE+/7-AAD+), Q3 represents the early apoptotic cell population (PE+/7-AAD-), and Q4 represents the viable cell population (PE-/7-AAD-). b, c, and d: Bar graphs represent the mean ± standard deviation of the proportion of cells in each phase of apoptosis from three independent. The P-values for the differences between corresponding groups were calculated using a t-test, with ns indicating no significant difference, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.005, and ****P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/498edd2239649fc8f4a4346f.png"},{"id":82862738,"identity":"98e37707-6bb6-4b27-9153-461136ed9c4b","added_by":"auto","created_at":"2025-05-16 07:15:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":549162,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of molecules associated with MSLN-mediated gemcitabine resistance in PDAC cells. a: The correlation between MSLN expression and gemcitabine resistance-related genes reported in existing literature was analysed using the TIMER2.0 online tool within the TCGA-PDAC database. b, c: Validation of mRNA levels of gemcitabine resistance-related genes before and after MSLN knockout and overexpression by qPCR. Among these, ABCC1 consistently showed a similar trend in both database analysis and qPCR validation results.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/cee8b43e0e641665c87f9694.png"},{"id":82862720,"identity":"3a9fd845-8e43-4943-a2a4-d1b641d49298","added_by":"auto","created_at":"2025-05-16 07:14:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":285691,"visible":true,"origin":"","legend":"\u003cp\u003eTED-347 inhibits the expression of MSLN in PDAC cells and enhances their sensitivity to gemcitabine. a-d: Immunoblot analysis of MSLN expression in ASPC-1 and PANC-1 cells treated with TED-347. e: The CCK8 assay to detect the cell viability of ASPC-1 and PANC-1 cells following treatment with 5μM TED-347 alone or in combination with various concentrations of gemcitabine. f, g: CCK8 assay to evaluate the cellular viability of ASPC-1 and PANC-1 cells treated with varying concentration ratios of gemcitabine and TED-347. h: qPCR analysis of ABCC1 gene expression in ASPC-1 and PANC-1 cells before and after treatment with 5μM TED-347.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/d51aaa082c4abfa12276c2ee.png"},{"id":82862717,"identity":"f779efc0-1ea7-4c50-87fd-31500a225508","added_by":"auto","created_at":"2025-05-16 07:14:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":523013,"visible":true,"origin":"","legend":"\u003cp\u003eTED-347 inhibits the migration and invasion of pancreatic cancer cells. a, b: The effect of various concentrations of TED-347 on the wound healing of ASPC-1 and PANC-1 cells at 0, 12, and 24 h post-scratch. c: The impact of various concentrations of TED-347 on the vertical migration ability of ASPC-1 and PANC-1 cells. d, e: Quantification of the migration ability of ASPC-1 and PANC-1 cells treated with various concentrations of TED-347 after data normalization. f: The influence of various concentrations of TED-347 on the invasive ability of ASPC-1 and PANC-1 cells. g, h: Quantification of the invasive ability of ASPC-1 and PANC-1 cells treated with various concentrations of TED-347 after data normalization.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/1f45ad8366a323558a1b28e1.png"},{"id":82862721,"identity":"d38af158-8486-460d-ae2a-067ad5cdeb2f","added_by":"auto","created_at":"2025-05-16 07:14:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":118088,"visible":true,"origin":"","legend":"\u003cp\u003eTED-347 inhibits EMT in PDAC cells. a, b: Western blot analysis of the expression of EMT markers E-cadherin (E-CAD) and Vimentin (VIM) in ASPC-1 and PANC-1 cells treated with TED-347. D1, D2, D3 and T1, T2, T3 represent the repeats of three independent experiments with TED-347. c-f: Quantification of the expression of EMT markers in ASPC-1 and PANC-1 cells treated with TED-347. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/b0ad646b3cb19f286d9b4bc7.png"},{"id":82862748,"identity":"5a022870-564a-4532-b63d-b016f457632b","added_by":"auto","created_at":"2025-05-16 07:15:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":562984,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism by which TED-347 suppresses MSLN expression. a: A schematic representation of the MSLN gene structure. b: Predicted transcription factor binding sites within the MSLN promoter region, as identified by JASPAR. Three pairs of PCR primers were designed to amplify the target sites based on the top 10 transcription factor recognition sites with the highest scores. c: DNA fragments prepared using an ultrasonic disruptor under various conditions, followed by gel electrophoresis to assess the size of the DNA fragments. d: PCR results obtained using three pairs of ChIP-PCR primers, with the ChIP product as the template. The intensity of the bands reflects the quantity of the DNA fragments, with GAPDH mRNA and GAPDH promoter primers serving as negative and positive controls, respectively. e: Quantification of the enrichment capacity of the YAP1 antibody for specific sequences in the promoter region using the enrichment multiple method. f: Multiplex immunofluorescence validation of the expression of YAP1/TEAD1/TEAD4 in human PDAC tissue.g, h: Expression of YAP1 and TEAD4 in PDAC tissue within the HPA database.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/89ee7c4ee8fe1575f090a706.png"},{"id":82862729,"identity":"73d75eba-f6cf-42b0-af22-44cdc763f77c","added_by":"auto","created_at":"2025-05-16 07:15:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":383544,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of TED-347 and gemcitabine significantly inhibits tumor growth in nude mice. a: A schematic overview of the animal experiment procedure. Mice began receiving treatments on the 10th day following tumor inoculation, with TED-347 and gemcitabine administered once a week, and tumor size and mouse body weight measured every 3-4 days. b: Mice were euthanized at the 6th week. The changes of tumor volume and weight in each treatment group were observed. c: A curve showing the change in subcutaneous tumor volume in nude mice over time with treatment. d: A curve illustrating the change in body weight of nude mice over time with treatment. e: A bar chart analysis of the average tumor weight at the end of the experiment for each treatment group.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/ff331011de713b91a909d9b8.png"},{"id":82862756,"identity":"335c54ea-a3e2-4b72-95b0-0344796d256b","added_by":"auto","created_at":"2025-05-16 07:15:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1060695,"visible":true,"origin":"","legend":"\u003cp\u003eTED-347 alleviates gemcitabine-induced fibrosis in PDAC. a: Masson's staining of PDAC tissue from various treatment groups at different magnifications. b: Quantitative analysis of key parameters processed by Image J software for image analysis. c: Calculation of fibrillar protein coverage area using Image J software. d, e: Masson's staining images of tissue not subjected to repeated freezing and thawing.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/28f2255a843febedc2f91746.png"},{"id":82863303,"identity":"37a1a78e-494e-4261-aad4-eceaef1289c9","added_by":"auto","created_at":"2025-05-16 07:22:59","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":427233,"visible":true,"origin":"","legend":"\u003cp\u003eTED-347 inhibits the expression of collagen in PSCs. a: Phase-contrast microscopy images showing the morphological characteristics of PSCs extracted on days 3 and 7, with a scale bar of 200μm. b: Cellular immunofluorescence was used to identify PSCs, with green fluorescence indicating Alexa Fluor 488-labelled Vimentin, red fluorescence indicating Alexa Fluor 594-labelled α-SMA, and blue fluorescence representing DAPI-stained nuclei, with a scale bar of 100μm. c: Western blot was used to detect the effects of ARTA and TED-347 on COL3A1, COL1A1, YAP1, Vimentin and α-SMA expression of PSCs. d-h: Quantification of the expression of PSCs markers in PSCs treated with ARTA and TED-347. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/636eebfa01d854d0800f7f29.png"},{"id":82864173,"identity":"4fa79f60-d5a9-44a1-a2ee-a55397627aa4","added_by":"auto","created_at":"2025-05-16 07:31:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6474985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/d50a3ee7-3f91-46eb-88fe-af1454e672f1.pdf"},{"id":82862722,"identity":"e4d38b75-537c-48c3-8a8d-ed97514b9c1b","added_by":"auto","created_at":"2025-05-16 07:14:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1758292,"visible":true,"origin":"","legend":"","description":"","filename":"Suppplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6589227/v1/0c68d06f4002aca08464567f.docx"}],"financialInterests":"","formattedTitle":"YAP1 Inhibitors Enhance the Therapeutic Effect of Gemcitabine on PDCA by Inhibiting MSLN Expression, EMT, and Pancreatic Fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic Ductal Adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is an aggressively malignant tumor within the digestive system. Globally, there has been a concerning increase in both the incidence and mortality rates of PDAC, with the two rates being nearly identical\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Over the past four decades, advancements in treatment techniques and evolving concepts have led to a reduction in surgical complications and mortality associated with PDAC. Despite these improvements, the five-year survival rate for patients with PDAC remains stubbornly below 10%\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChemotherapy, particularly gemcitabine-based regimens, is a pivotal component in the systemic management of PDAC\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, the overall response rate remains suboptimal, primarily due to the chemoresistant nature of PDAC, which is intricately linked to the cancer cells themselves and the extensive fibrosis of the mesenchymal tissue\u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The elusive mechanisms behind chemoresistance in PDAC pose a significant challenge in its treatment. The gemcitabine-based chemotherapy regimen is the preferred choice for treating PDAC\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Consequently, there is an urgent need for in-depth research to elucidate the mechanisms associated with chemotherapy resistance in PDAC, with the aim of enhancing the efficacy of chemotherapy in the current treatment landscape. Chemotherapy is a pivotal component in the systemic management of PDAC\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, the overall response rate remains suboptimal, primarily due to the chemoresistant nature of PDAC which is intricately linked to the cancer cells themselves and the extensive fibrosis of the mesenchymal tissue\u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The elusive mechanisms behind chemoresistance in PDAC pose a significant challenge in its treatment. The gemcitabine-based chemotherapy regimen is the preferred choice for treating PDAC\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Consequently, there is an urgent need for in-depth research to elucidate the mechanisms associated with chemotherapy resistance in PDAC, with the aim of enhancing the efficacy of chemotherapy in the current treatment landscape.\u003c/p\u003e \u003cp\u003eMesothelin (MSLN), a glycoprotein anchored to cell membranes via glycosylphosphatidylinositol, is typically present in low levels in the pleura and peritoneum but is markedly overexpressed in mesothelioma and ovarian cancer, where it fuels the invasive metastasis of these cancers\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Studies have indicated that patients with mesothelioma and colorectal cancer exhibiting high MSLN expression are more likely to develop resistance to chemotherapeutic drugs\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The MSLN-targeting antibody, Amatuximab, has been shown to enhance the sensitivity of PDAC cells to gemcitabine in murine models\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Our previous research revealed high MSLN expression in PDAC tissues\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, and further research demonstrated that MSLN knockdown reverses epithelial-mesenchymal transition (EMT) in cancer cells, while its overexpression promotes EMT, suggesting a link between MSLN and chemoresistance\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eYes-associated protein 1 (YAP1), a pivotal transcriptional cofactor, forms complexes with transcription factors such as the transcriptionally enhanced associate domains (TEADs) within the nucleus to regulate gene transcription \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Evidence suggests that the YAP1-TEADs complex is implicated in the transcriptional regulation of MSLN in PDAC cells\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. YAP1 engages with TEADs through an extensive planar interface\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, and thus, disrupting this interaction could offer a novel therapeutic strategy for MSLN-targeted treatments in PDAC. The small molecule inhibitor TED-347 has been shown to rapidly and selectively form covalent bonds with specific cysteine residues within the deep hydrophobic palmitate-binding pocket of TEADs, thereby irreversibly inhibiting the YAP1-TEADs interaction and blocking their transcriptional activity over an extended period \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The potential of TED-347 to enhance the sensitivity of PDAC cells to gemcitabine remains an open question. It is known that PDAC can develop resistance to gemcitabine through epithelial-mesenchymal transition and pancreatic fibrosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e–\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Pancreatic stellate cells (PSCs), as the primary fibrogenic cells in the pancreas, are activated by tumor cells to secrete a substantial amount of extracellular matrix, which promotes pancreatic fibrosis and hinders the delivery of chemotherapeutic agents\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Reports indicate that YAP1 can stimulate the activation of PSCs, and the inhibition of YAP1 expression can reverse this activation\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis research provided a comprehensive investigation into the mechanisms through which MSLN confered gemcitabine resistance in PDAC, utilizing both in vitro and in vivo experimental approaches. The study revealed that the small molecule inhibitor TED-347, which homed in on the YAP1-TEAD1 interaction, can diminish MSLN expression in PDAC cells. This reduction in MSLN expression subsequently curbed cell migration, invasion, and EMT. Moreover, TED-347 was shown to mitigate fibrosis within PDAC tissues and to augment the therapeutic effectiveness of gemcitabine in the treatment of PDAC.\u003c/p\u003e "},{"header":"Method","content":"\u003ch3\u003e1. Database analysis\u003c/h3\u003e\u003cp\u003eThe Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia2.cancer-pku.cn/index.html\u003c/span\u003e\u003cspan address=\"http://gepia2.cancer-pku.cn/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to evaluate the expression of MSLN in diverse malignancies. Additionally, the Tumor Immune Infiltration Evaluation Resource 2.0 (TIMER 2.0)\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://timer.cistrome.org/\u003c/span\u003e\u003cspan address=\"https://timer.cistrome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to analyze MSLN expression in PDAC patients from the The Cancer Genome Atlas (TCGA) database, considering clinical parameters such as disease stage, gender, age, tumor purity, and prognosis through multifactorial regression analysis. Furthermore, the Human Protein Atlas (HPA) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to investigate the expression levels of YAP1 and TEAD4 in PDAC.\u003c/p\u003e\u003ch2\u003e2. Cell culture\u003c/h2\u003e\u003cp\u003eASPC-1 cells with knockout of MSLN (AKO), Mia capa2 cells with overexpressing of MSLN(MOE) and their negative control (ANC and MNC respectively) were constructed previously \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. ASPC-1, ANC, and AKO cells were cultured in RPMI-1640(Procell, Cat.#PM150110). PANC-1 were purchased from Procell (Wuhan, China). MOE, MNC and PANC-1 were cultured in DMEM (Procell, Cat.# PM150210). All media contained 10% FBS and 1% Penicillin-Streptomycin.\u003c/p\u003e\u003ch3\u003e3.Annexin V-PE/7-AAD double-staining for cell apoptosis analysis\u003c/h3\u003e\u003cp\u003eAnnexin V-PE/7-AAD Apoptosis Detection Kit (YEASEN, Cat.#40310ES20) was employed to detect gemcitabine-induced apoptosis in PDAC cells. Briefly, Cells were harvested and resuspended in 1 × binding buffer at a concentration of 1× 10\u003csup\u003e6\u003c/sup\u003e cells/mL, and subsequently incubated with Annexin V-PE and 7-AAD for 15 minutes in the dark at room temperature. Finally, the labeled cells were immediately assessed using a flow cytometer, and the data were processed using Flowjo v10 software. Cells negative for both PE and 7-AAD were classified as live cells, those positive for PE but negative for 7-AAD were identified as early apoptotic, and those positive for both PE and 7-AAD were categorized as late apoptotic or necrotic.\u003c/p\u003e\u003ch3\u003e4.RNA extraction and quantitative real-time polymerase chain reaction (qPCR)\u003c/h3\u003e\u003cp\u003eTotal RNA was extracted from PDAC cells using TRIzol reagent (Invitrogen, Cat# 15596026). The RNA concentration and quality were measured by NanoDrop 5000 spectrophotometer (BioTeke, China). Total 500 ng RNA from each sample was reverse transcribed in 20 µl total volume using the HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Cat.#R201-01).\u003c/p\u003e\u003cp\u003eGene expression of PDAC cells was measured using SupRealQ Purple Universal SYBR qPCR Master Mix (U+) (Vazyme, Cat.#Q412-02) and analyzed on the ABI 7500 RT PCR System (Life Technologies). Gene expression was normalized to GAPDH mRNA. Data are presented as fold-change in gene expression in each group relative to control group. Primer synthesis was completed by Shanghai Sangon Co., Ltd. (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequence\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward primer (5'-3')\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse primer (5'-3')\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ehENT1/SLC29A1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAACTCTCAGCCCACCAATGAAAGC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAAGCAGACAGAGAAAGCCAGGAC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAKT1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAGGAGGAGGAGGAGATGGACTTC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCCAGCAGCTTCAGGTACTCAAAC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDKN1B\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCTTGCCCGAGTTCTACTACAGAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACCAAATGCGTGTCCTCAGAGTTAG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBIRC5/survivin\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAAGGACCACCGCATCTCTACATTC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCAAGTCTGGCTCGTTCTCAGTG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABCC1/MRP1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGCACATTTGACGCTAGTG\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACGATGCTGATGACCAT\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDCK\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAGGGGACCCGCATCAAGAAAATC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCATCTGGCAACAGGTTCAGGAAC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP53\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCCCATCCTCACCATCATCACAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCACAAACACGCACCTCAAAGC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMYC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGCAGCGACTCTGAGGAGGAAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCCAGCAGAAGGTGATCCAGACTC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDK6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGACCAGCAGCGGACAAATAAAAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACGACCACTGAGGTTAGAGCCATC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABCG2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCAGCAGGTCAGAGTGTGGTTTC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACTGAAGCCATGACAGCCAAGATG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRRM1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGCGGCTAATCCAATCCAGTTCAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTGCTGTGTTCCTCTCCTTCTCTTC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDR1/ABCB1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTGATTGACAGCTACAGCACGGAAG\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTCTTCACCTCCAGGCTCAGTCC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCL1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGGCAGGATTGTGACTCTCATTTC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTAGCCAGTCCCGTTTTGTCCTTAC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCL2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCGCCCTGTGGATGACTGAGTAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACAGCCAGGAGAAATCAAACAGAGG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclin D2/CCND2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTTCCGCAGTGCTCCTACTTCAAG\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTCCTCACAGACCTCCAGCATCC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCL-xl/BCL2L1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCTGGTGGTTGACTTTCTCTCCTAC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCTCCATCTCCGATTCAGTCCCTTC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGACATCAAGAAGGTGGTGAAGCAG\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTGTCGCTGTTGAAGTCAGAGGAG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch3\u003e5.Western blot\u003c/h3\u003e\u003cp\u003eProtein was extracted from PDAC cell lines and PSC using RIPA lysis buffer with proteinase inhibitor (Solarbio, Cat.# R0020). Protein concentration was measured by Omni-Rapid™ Rapid protein quantification kits (Yazyme, Cat.# ZJ103). A total of 20 µg of protein mixed with 5 × SDS loading buffer was loaded per lane, separated by 10% SDS-polyacrylamide gel electrophoresis. The primary antibodies used in this study includes YAP1 (1:1000 dilution, Cell Signaling, Cat.# 14074 ), MSLN (1:1000 dilution, Cell Signaling, Cat.#99966), Vimentin (1:1000 dilution, Cell Signaling, Cat.#5741 ), E-cadherin(1:1000 dilution, Cell Signaling, Cat.# 3195), α-SMA(1:1000 dilution, Abcam, Cat.#ab184705), COL1A1(1:5000 dilution, Abcam, Cat.#ab138492), COL3A1(1:1000 dilution, abcam, Cat.#ab184993) and GAPDH (1:1000 dilution, Servicebio, Cat.#GB15002-100). Secondary antibodies conjugated to Dylight 680(1:10000 dilution, Cell Signaling, Cat.#5470) or Dylight 800 (1:10000 dilution, Cell Signaling, cat.#5151) were used for detection. GAPDH was used as an internal loading control. Protein bands were visualized using the Odyssey® CLX two-color infrared laser imaging system (Li-cor, USA) and quantified by Image J software.\u003c/p\u003e\u003ch3\u003e6. Cell viability assay and Calculation of combination index\u003c/h3\u003e\u003cp\u003eCell viability was assessed using CCK-8 Cell Counting kit (Yeasen, Cat.#40203ES76). PANC-1 or APSC-1 cells were seeded into 96-well plate at a density of 2×10\u003csup\u003e4\u003c/sup\u003e cells per well and were treated with 100 µL of culture medium containing varying concentrations of TED-347 and gemcitabine. Three replicate wells were used for each treatment group. cells were then incubated with 10 µL of CCK-8 per well for 2 h, and the absorbance at 450 nm was measured with a microplate reader (Tecan Spark 10 M luminometer). The Combination Index (CI) TED-347 and gemcitabine was calculated by Compusyn software. As for the synergistic assay, the concentrations of cells treated by TED-347 and gemcitabine for 48 h were listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The Chou-Talalay method and CompuSyn software were used to analyse the synergistic effect of the two drug combinations. The CI of Chou-Talalay was calculated from the data derived from monotherapy and combination treatment by CompuSyn software, and the CI value quantitatively defines additive effect (CI = 1), synergism (CI \u0026lt; 1), and antagonism (CI \u0026gt; 1) in drug combinations.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCI value of the combined action of TED-347 and GEM in non-fixed proportion combination\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose TED-347(µM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDose GEM(nM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEffect\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.341\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.21247\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.473\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.30910\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.555\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.52738\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.628\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.68968\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.624\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.95916\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultivariate Cox analysis of prognosis of pancreatic cancer patients by TIMER 2.0\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e \u003cp\u003eMSLN in PAAD (n = 179):\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e \u003cp\u003eModel: Survive (OS, EVENT) ~ ‘MSLN’ + Age + Gender + Race + Stage + Purity\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e \u003cp\u003e165 patients with 89 dying ( 14 missing obs. )\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCo ef\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ese(co ef)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95%CI_l\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e95%CI_u\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ez\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eSig nif\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMSLN\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.185\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.057\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1.325\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.99\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.017\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.995\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1.039\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.502\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eGender male\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.247\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.781\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1.195\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-1.139\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.255\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eRace Black\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.344\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.411\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.742\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e6.035\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.464\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.643\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eRace White\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.557\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.746\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.475\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.688\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e4.432\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.172\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.241\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStage2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.357\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.439\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.604\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e3.383\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.814\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.416\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStage3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.422\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.656\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e1.094\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e5.598\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-0.386\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStage4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.194\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.824\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.827\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.163\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e4.168\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-0.234\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.815\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePurity\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.661\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.516\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.227\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1.176\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-1.575\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.115\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e \u003cp\u003eR square = 0.144 (max possible = 9.91e-01)\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eLikelihood ratio test p = 2.25e-03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eWald test p = 1.08e-02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c12\" namest=\"c9\"\u003e \u003cp\u003eScore (log rank) test p = 6.89e-03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003e7. Wound-healing assay\u003c/h2\u003e\u003cp\u003eCells migration was tested by a wound healing assay. Transfected cells were plated in 12-well dishes (5×10\u003csup\u003e4\u003c/sup\u003e cells/well), and incubated in RPMI-1640 medium (Procell, Cat.#PM150110) without FBS at 37°C, reaching a confluence of 80%. Then the cells were scratched across the surface of the well by a 10-µl pipette. After an incubation at 37°C of 12h and 24 h, the scratches were observed.\u003c/p\u003e\u003ch3\u003e8. Transwell migration and invasion assay\u003c/h3\u003e\u003cp\u003eTranswell analysis was performed to assess the effects of TED-347 on the migration and invasion ability of PADC cells.\u003c/p\u003e\u003cp\u003eFor migration assay, PADC cells were precultured with a particular concentration of TED-347 for 48 h. A total of 2×10\u003csup\u003e4\u003c/sup\u003e cells were seeded in serum-free medium in the upper chamber (STEMCELL, Cat.#38023), and the lower chamber was filled with RPMI-1640 containing 10% FBS. After 48 h, the non-migrating cells on the upper chambers were carefully removed with a cotton swab, and migrated cells underside of the filter stained and counted in five different fields.\u003c/p\u003e\u003cp\u003eFor invasion assays, transwell inserts (STEMCELL, Cat.#38023) were pre-coated with Matrigel (Corning, Cat.#354234). After treatment with TED-347 for 48 h, 2 × 104 cells in 200 µL of serum-free medium were seeded in Matrigel-coated upper chambers. The lower chamber contained 750 µL of medium supplemented with 10% FBS. After 24 h of incubation, the invading cells on the lower surface of the membrane were stained with 0.1% crystal violet. The cells were then washed and the number of invading cells in the lower chamber was observed under a microscope. Finally, 5 fields per chamber were selected for cell counting at 20×magnification. The cell count in each field was analyzed using Image J software.\u003c/p\u003e\u003ch3\u003e9. Immunofluorescence (IF) analysis\u003c/h3\u003e\u003cp\u003eBriefly, Cells for IF experiments were grown on coverslips to 70–80% confluence, washed in PBS and fixed in 4% paraformaldehyde. The cells were permeablized by adding TBST with 0.1% Triton for 5 min, blocked in 5% BSA and incubated with the primary antibody and secondary antibody in a humidified chamber. The coverslip is then mounted onto a slide for viewing using a mounting medium with DAPI. Immunofluorescence images were captured using a confocal microscope (Nikon TiE-A1 plus) using a 40× objective with fixed optical slice, laser power and detector/amplifier settings for all samples across each individual experiment to allow comparison.\u003c/p\u003e\u003cp\u003eCells were cultured on coverslips to 70–80% confluence, washed with PBS, and fixed in 4% paraformaldehyde. Cells were then permeabilized by incubating in TBST with 0.1% Triton X-100 for 5 min, followed by blocking in 5% BSA. Primary and secondary antibodies were applied sequentially in a humidified chamber. After incubation, the coverslip were mounted onto slide using a mounting medium containing DAPI to stain the nuclei. Immunofluorescence images were acquired using a confocal microscope (Nikon TiE-A1 plus) with a 40× objective. The optical slice, laser power, and detector/amplifier settings were fixed for all samples within each individual experiment, allowing for direct comparison between conditions.\u003c/p\u003e\u003ch2\u003e10. Multiple-color immunofluorescence staining of tissues\u003c/h2\u003e\u003cp\u003eParaffin-embedded sections of pancreatic cancer tissues were collected from 10 patients diagnosed with pancreatic ductal adenocarcinoma following surgery. These patients were treated at our hospital between 2018 and 2020. The study was approved by the Ethics Committee of Beijing Shijitan Hospital, Capital Medical University(sjtkyll-lx-2022(069)). In short, immunofluorescence staining was performed on PDAC patient tissues using the mouse-rabbit triple-label four-color kit (ImmunoWay Biotechnology, Cat.#RS0035) protocol to label the expression of YAP1(1:200 dilution, CST, Cat.#14074), TEAD1(1:200 dilution, CUSABIO, Cat.#CSB-PA023363DSR2HU) and TEAD4(1:200 dilution, CUSABIO, Cat.#CSB-PA618010LA01HU) in PDAC. According to the instructions, apply the diluted primary antibody solution onto the sample area, incubate overnight at 4 ℃, and then reheat to room temperature for 30 minutes. Then, incubate the tissue with HRP secondary antibody working solution under RT conditions for 30 minutes, and rinse the slices three times with TBST for 5 minutes each time. Next, cover the tissue with 1 × 100 uL dye signal amplification working solution under RT conditions for 10 minutes, and then rinse the slices twice with TBST for 3 minutes each time. Then repeat the above process and stain the same sections with different colored immunofluorescence dyes. Finally, observe the sections under a fluorescence microscope.\u003c/p\u003e\u003ch2\u003e11. Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR)\u003c/h2\u003e\u003cp\u003eThe JASPAR CORE database was used to identify the top ten binding sites between YAP1 and the 2000 bp upstream and 100bp downstream genomic regions of the MSLN promoter (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e.). Immunoprecipitated chromatin was analysed by qPCR using primers targeting the predicted binding sites (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e.). The experiment was conducted according to the scheme of Chromatin immunoprecipitation kit (Beyotime, Cat.#P2078). Briefly, PADC cells were fixed in 1% final formaldehyde for 10 min at room temperature and quenched by glycine. After cell lysis, the chromatin was fragmented into 200–1000 bp by ultrasonic cell shredder (Qsonica, Q500-2 20). Protein-DNA complexes were immunoprecipitated by YAP1 antibody (1:50 dilution, Cell Signaling, Cat.# 14074) or anti-IgG antibody conjugated with Protein A + G Agarose/Salmon Sperm DNA mix on rotator, incubated overnight at 4°C. After washing, reversal of crosslink and DNA purification, input DNA was used as a template for conventional PCR assay using specific primers (listed in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e.).\u003c/p\u003e\u003ctable id=\"Tab3\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe upstream 2000pb to downstream 100bp sequence of human MSLN gene promoter\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026gt;NC_000016.10:758734–760834 Homo sapiens chromosome 16, GRCh38.p14 Primary Assembly\u003c/p\u003e\n \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAAGATGGTCTCGAT\u003cbr\u003eCTCCTGACCTCATGATCTGCCTGCCTCGGCCTCCAAAAGTGCTGGGATTACAGGCGTGA\u003cbr\u003eGTCACTGCGCCCGGCATTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGGGTCTTGCTCTG\u003cbr\u003eTTGCCCAGGCTGGAGTGCAGTGGCGTGATCTCGACTCACTGCAACCCCTACCTCCTGGG\u003cbr\u003eTTCAAGTGATTCTTCTGCCTCAGCCTCCTGAGTAACTAGGATTACAGGCATGTGCCACCA\u003cbr\u003eCGCCCAGTTAATTTTCATATTTTTAGTAGAAACGGGGTTCCTCCATGTTGGCCAGGCTGG\u003cbr\u003eTCTCGAACTCCTGACCTCAGGTGATCCGCCCACCTCGGCCTACCAAAGTGCTGGGATGA\u003cbr\u003eCAGGCGTGCGCCACCACACAGGGCCTCATTTAAAGATTCTTTATAGAGATGAGGTGTCA\u003cbr\u003eCTACGTTGCCCAGGCTGGTCTTGAACTTCTGGCCTCAACGGACCCTCCCGCATCGGCCT\u003cbr\u003eCCCACGGTGCTGGCTGAAGGGTGAGCCCAGGCTGGCCACAGCGCGTTTTCATCATTGTC\u003cbr\u003eCGCAGCTTGCAGTCGGCTGGTTCAGAGCTTAGCCGGGCACATGGGCCCCTCTGAGGCTC\u003cbr\u003eCTGTTCAGGGCTCAGCCGTGTCTACGGGGCCCTCCGAGCTCTTCCTGGACCCTCCTCGG\u003cbr\u003eCTCCTTCAGTTCAGGGCTCAGCTGGGCACGTGGGCGCCTCCTTGGCTCCTCCGGCTCAG\u003cbr\u003eGCCTCTCCTCGAGCTCTGGGCCCTGAGTATTCTGGGCTCCTTCCTTGGTTTCCTTTGGCC\u003cbr\u003eTTTGGCCGGGAAAGTTGTGGGTGTCCGTGGCAGCCTGGGCCACTTCACAGCCCCGCAG\u003cbr\u003eCCAACCTGCGGCTCCTTCAGAGCAAAGCTGTGTGGACACAAAGGGAACGCCACTCGG\u003cbr\u003eAGCTGGCCTCTCCCTTTACCTGTGAGTTCCCGCCCAAGCCGGCTGCCTTCTGTCCCCTC\u003cbr\u003eCCCAGAGCCCTTGGGTAACTGGTTTGCTACAAGAGTGTCTGGAATTTTTCAGTTGTTCT\u003cbr\u003eCTGCGGAAGGGAGTTTTTAAAAGGCCCTTAATCCCTTCTTGACATTTGTAAGTTGACGC\u003cbr\u003eTTACACCTGGCAGCCTTGCTGAATTCTGTGTGCGTGAAGGTCCGATTCCACCGCGAGTC\u003cbr\u003eACGATAGAAAACCCACTCTGTGGAGAGACCAGAGATGACCGCCGCGCACACCTCCGT\u003cbr\u003eTCAGCACACAAACCTTTGCAGGTGTTCATAGCGGAGGCAGATTCCGTACTGGGGATAA\u003cbr\u003eGAGCTCACGACATGCTGGGAGGGGTTTCAGGGGCAGGGAGGGGGCTTCTGTGGCCCC\u003cbr\u003eAGGTCAGGAGGAGCAGCTCTTCCATCAGCAGCAGGCAGCCAGCCCAGATGCGTAGGG\u003cbr\u003eAAGACAGCTCCCACTTCGCCAGGCCAGAGAGCGCCCGGGGGCAGCTCTGTTCCAGTC\u003cbr\u003eGACCCTGCGAGAAAGGGGTGTGCGTGTGCCTGGAGCTGGGCCCCGTCCTGCCTCCCT\u003cbr\u003eGACCTGTGTGCTCCCACAGCCCTGAGACGGACGGCTCACAGCCTTGCGAGGCCCACA\u003cbr\u003eCTGCACTGGGGGTCAGGCTTGTGCTCCCGGGAGTCCTGTCTGGGCTGCGTGGCCACC\u003cbr\u003eATCCAGAGCCTGCTGACCTGCGACTGGGGGGGCCAGTGCTCCCTGGGTTTCAGCACC\u003cbr\u003eTGAGAATCAGAGTGGGATCCCGTGAAACCTGGGCCCAGGCTCCCACCCACGCCCCAC\u003cbr\u003eACCCACCCAGGGAAGCCATGAAACCTGGGCCCGGGCTCCTACACATGCCCCACACCC\u003cbr\u003eACCCAGGGCAGCCGTGAAACCTGGGCCCGGGCTCCCACCCTCGCCCACCGAGGGCA\u003cbr\u003eGCTTTGCCTTCCTGGGCATCCCTCCTCCCCCAGGCCTGGCCCGCTGCCTGTCCAAGG\u003cbr\u003eCTCCTGTGCGGGGTCTCCACCCACACATTCCTGGGGCGTGAGGCGCCACCACTCCCT\u003cbr\u003eGCTGCCCCGGGCAAAGCCGTCATTTGTTCCCTTTGACGGCCCGGGAGGCTGCCAGGC\u003cbr\u003eTCTCCACCCCCACTTCCCAATTGAGGAAACCGAGGCAGAGGAGGCTCAGGTGTGGC\u003cbr\u003eCAATCA\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePossible transcription factor binding sites of YAP1-TEADs predicted by JASPAR 2022\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMatrix ID\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRelative score\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSequence ID\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStart\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEnd\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eStrand\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePredicted sequence\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0090.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0090.1.TEAD1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.025486\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.973801862\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1949\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1960\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCACATTCCTGGG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0090.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0090.3.TEAD1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.489286\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.94454802\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1948\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1960\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eACACATTCCTGGG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0809.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0809.2.TEAD4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.52531\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.948052879\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1948\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1959\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eACACATTCCTGG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0090.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0090.3.TEAD1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.748998\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.91341974\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1044\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1056\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAAAAATTCCAGAC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0809.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0809.1.TEAD4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.132757\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.979330593\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1949\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1958\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCACATTCCTG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0809.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0809.2.TEAD4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.009904\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.918816513\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1045\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1056\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAAAAATTCCAGA\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0090.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0090.2.TEAD1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.853291\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.966348763\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1949\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1958\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCACATTCCTG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0809.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0809.1.TEAD4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.621073\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.946984752\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1046\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1055\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAAAATTCCAG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0090.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0090.1.TEAD1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.284123\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.827026447\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1873\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1884\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTGCCTTCCTGGG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMA0090.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA0090.2.TEAD1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.028421\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.908144731\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC_000016.10:758734–760834\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1046\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1055\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAAAATTCCAG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChIP-qPCR primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer sequence (5’-3’)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDestination site\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePCR product size\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChIP 1045–1056 F\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egagcccttgggtaactggtttg\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1045–1056\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e114bp\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChIP 1045–1056 R\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egcgtcaacttacaaatgtcaagaagg\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChIP 1948–1959 F\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egctgcctgtccaaggctcc\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1948–1960\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e94bp\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChIP 1948–1959 R\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etgacggctttgcccggg\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChIP 1145–1154 F\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eccttcttgacatttgtaagttgacgc\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1145–1154\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e133bp\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChIP 1145–1154 R\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etgcgcggcggtcatctct\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH promoter F\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etactagcggttttacgggcg\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGAPDH promoter region\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e166bp\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH promoter R\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etcgaacaggaggagcagagagcga\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003e12. Animal experiment\u003c/h2\u003e\u003cp\u003e The animal study was approved by the Animal Experimentation Ethics Committee of Beijing shijitan Hospital, Capital Medical University (sjtkyll-lx-2022(069)). Twenty male BALB/c nude mice (4 weeks old) were obtained from the Laboratory Animal Center of Beijing Shijitan Hospital, Capital Medical University. Mice were randomly assigned to four groups, with five mice in each group. To establish a subcutaneous model, ASPC-1 cells with were resuspended in PBS at a concentration of 10\u003csup\u003e7\u003c/sup\u003e cells/100 µl. Each mouse was subcutaneously injected in the right flank with 100 µl of cell suspension to induce tumor formation. When the tumor sizes reached 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were injected weekly with 50mg/kg gemcitabine hydrochloride (Chiata Tianqiong) or 20mg/kg TED-347 (MedChemExpress, Cat.# 2378626-29-8). Tumor sizes were measured weekly, and the volumes (in cubic millimeters) were calculated according to the following formula: width\u003csup\u003e2\u003c/sup\u003e × length × 0.5. Length and width refer to the longest and shortest diameters, respectively. Six weeks after the first injection, the mice were euthanized, and the tumors were excised. Tumor tissues were harvested for further processing. A 4‑mm portion of each tumor was fixed with paraffin. The hearts, livers, spleens, lungs and kidneys of mice in each group were fixed with paraformaldehyde, embedded in paraffin, and subjected to hematoxylin and eosin (H\u0026amp;E) staining using HE staining kit (Solarbio, Cat.#G1120).\u003c/p\u003e\u003ch2\u003e13. Modified Masson staining and scoring\u003c/h2\u003e\u003cp\u003eTissues were fixed in 4% paraformaldehyde and then slowly frozen and thawed 2–3 times repeatedly. Subsequent steps were performed according to the standard Masson’s trichrome staining protocol. The area percentage of blue fibres was analysed by image J software and used as an indicator to assess the degree of fibrosis.\u003c/p\u003e\u003ch2\u003e14. Enzymatic extraction of mouse pancreatic stellate cells\u003c/h2\u003e\u003cp\u003eAfter 4–6 weeks of C57bl/6 (Purchased from Beijing Viton Lever Ltd.) mouse execution, the pancreas was carefully excised, with any excess adipose tissue removed. The pancreas was washed 3 times with pre-cooled PBS for 5 min. Following this, the tissues were trimmed and digested with 0.25% trypsin (Procell, Cat.#PB180226) for 30 min at 37°C before terminating the digestion with 10% FBS in DMEM medium(Procell, Cat.#PM150210). The adherent cells were then purified using differential attachment and identified.\u003c/p\u003e\u003ch2\u003e15. Statistical analyses\u003c/h2\u003e\u003cp\u003eAll of the experimental data were repeated at least three times and expressed as the mean ± SD. Student’s t-test was used to compare differences between two groups. \u003cem\u003eP\u003c/em\u003e values \u0026lt; 0.05 were considered to be statistically significant.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e \u003cb\u003e1. High expression of MSLN in PDAC correlates positively with tumor stage and negatively with patient prognosis.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eAnalysis of MSLN expression across pan-cancer database from TCGA showed that MSLN was highly expressed in a variety of tumor tissues, including pancreatic, ovarian and colon cancer (\u003cb\u003eFigure.1a\u003c/b\u003e). Similarly, TCGA database and GETx database analyses showed that MSLN was highly expressed in cancer tissues at the transcriptional level in 179 PDAC cases and 171 normal pancreatic tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Furthermore, HPA database analyses showed that MSLN was highly expressed in PDAC tissues at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e). Analyses of the relationship between MSLN expression and PDAC tumor staging indicated a positive correlation between High expression of MSLN and advanced (\u003cb\u003eFigure.1f\u003c/b\u003e). To assess the prognostic impact of MSLN, overall survival (OS) and disease-free survival (DFS) of pancreatic cancer patients were analyzed by GEPIA2, stratifying patients based on high and low MSLN expression levels. Patients were stratified into quartiles based on MSLN expression levels, with the top 25% representing the highest expression and the bottom 25% representing the lowest. Additionally, comparisons were made between the top 25% and the bottom 25%, as well as between the top 25% and the bottom 75% of patients. The cut-off values for high and low MSLN expression were adjusted to ensure accurate stratification. Kaplan-Meier survival curves showed that patients with high MSLN expression exhibited significantly improved OS and DFS, while those with low MSLN expression had poorer outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-j). Further analysis using the TIMER 2.0 database, which incorporated clinical variables such as age, gender, race, tumor stage and tumor purity, revealed that MSLN expression was significantly associated with prognosis of pancreatic cancer patients. High expression of MSLN was inversely correlated with patient survival, highlighting its potential as a negative prognostic marker in pancreatic cancer (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003e2. High MSLN expression confers resistance to gemcitabine-induced early apoptosis of pancreatic cancer cells.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe impact of gemcitabine on apoptosis induction in PDAC cells, in relation to levels of MSLN expression, is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. pre-established MSLN-knockout PDAC cell lines (MSLN-knockout ASPC1 cells: AKO; Negative-control ASPC1 cells: ANC) \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e was employed to investigate this relationship by Annexin V-PE/7-AAD double staining flow cytometry. After 48 h of treatment with 80 µM gemcitabine (a concentration that is fivefold the IC50 value), both ANC and AKO exhibited significant apoptosis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). No significant difference was observed in early or late apoptosis between ANC and AKO in PDAC cells untreated with gemcitabine. However, AKO displayed a significantly higher early apoptosis rate, compared to ANC, with no significant difference observed in late apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). This suggests that high MSLN expression significantly confers resistance to gemcitabine-induced early apoptosis in pancreatic cancer cells, and high MSLN expression may contribute to gemcitabine resistance by inhibiting early apoptosis signaling.\u003c/p\u003e\u003cp\u003e \u003cb\u003e3. ABCC1 as a key gene in MSLN- Mediated gemcitabine resistance in pancreatic cancer cells.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eDatabase visualization and analysis revealed the correlation between MSLN expression levels of mRNA levels of several gemcitabine resistance-related genes, for gemcitabine resistance-related molecules, including ABCB1\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, ABCC1\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, ABCG2\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, AKT1\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, BCL2\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, BCL2L1\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, BIRC5\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, CCND2\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, CDK6 \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, CDKN1B \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, DCK\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, MCL1\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, MYC\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, RRM1\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e, SLC29A1\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and TP53\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. Positive correlations with MSLN expression were observed for ABCC1, AKT1, BCL2L1, and BIRC5, while negative correlations were noted for ABCB1, ABCG2, BCL2, CCND2, CDK6, and DCK. Genes with no significant correlation included CDKN1B, MCL1, MYC, RRM1, SLC29A1, and TP53. qPCR analysis of pancreatic cancer cells with MSLN knockdown or overexpression demonstrated that MSLN reduction led to a decrease in ABCB1, ABCC1, and ABCG2, whereas MSLN overexpression resulted in their elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). Further analysis indicates that ABCC1 is the only gene consistently correlated with MSLN expression, suggesting that ABCC1 is a critical gene driving MSLN-mediated gemcitabine resistance in pancreatic cancer cells.\u003c/p\u003e\u003cp\u003e \u003cb\u003e4. The YAP1 inhibitor TED-347 can suppress MSLN expression in PDAC cells and enhance their sensitivity to gemcitabine.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe effect of TED-347 on MSLN expression in PDAC cells is shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-d. After treatment with various concentrations of TED-347 for 48 hours, Western blot analysis revealed a significant and dose-dependent reduction in MSLN expression in both ASPC-1 and PANC-1.These findings suggests that the YAP1 inhibitor TED-347 can effectively inhibit MSLN expression in PDAC cells.\u003c/p\u003e\u003cp\u003eThe impact of the combination of TED-347 with gemcitabine on the viability of PDAC cells is depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-g. The results indicate that treatment with various concentrations of TED-347 and gemcitabine for 48 h, either individually or in combination, resulted in significantly lower optical density (OD) values in the CCK-8 assay for both ASPC-1 and PANC-1 cells. The combination treatment exhibited a more potent inhibitory effect on pancreatic cancer cell viability compared to gemcitabine alone. Furthermore, CI calculations, using the CompuSyn software, revealed that all CI values for the drug combinations were less than 1 (Table\u0026nbsp;\u0026lt;link rid=\"tb2\"\u0026gt;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u0026lt;/link\u0026gt;\u003c/span\u003e–\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting a synergistic interaction between TED-347 and gemcitabine.\u003c/p\u003e\u003cp\u003eThe influence of TED-347 on the gemcitabine resistance gene ABCC1 in PDAC cells is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh. qPCR results revealed that the expression levels of ABCC1 were significantly reduced in both ASPC-1 and PANC-1 cells treated with TED-347 compared to the control group, indicating that TED-347 can enhance the sensitivity of PDAC cells to gemcitabine.\u003c/p\u003e\u003cp\u003e \u003cb\u003e5. TED-347 inhibits the migration, invasion, and EMT of PDAC cells.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe impact of TED-347 on the migration, invasion, and EMT of ASPC-1 and PANC-1 is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Wound-healing assays were performed to assess the horizontal migration ability of PDAC cells after treatment with various concentrations of TED-347. The results demonstrated that at 0, 12, and 24 hours post-scratch, the wound healing rate in both ASPC-1 and PANC-1 cells treated with 5µM and 10µM TED-347 was significantly reduced compared to the control group. Notably, higher concentrations of TED-347 led to a more pronounced inhibitory effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Transwell migration and invasion assays were used to evaluate the vertical migration and invasion abilities of PDAC cells after treatment with different concentrations of TED-347. These findings indicate that TED-347 effectively inhibits vertical migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e) and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-h) of pancreatic cancer cells, with the extent of inhibition intensifying in a dose-dependent manner.\u003c/p\u003e\u003cp\u003eOur previous investigations have established that MSLN can facilitate the EMT in PDAC cells. In a complementary experiment, with fluorescent signal acquisition standardized, we utilized immunofluorescence to assess the expression of EMT markers, E-cadherin and N-cadherin, in PDAC cells. The findings revealed that, in MSLN-knockdown ASPC-1 cells, there was an elevation in E-cadherin expression and a reduction in N-cadherin expression when compared to both the control and vector groups (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e). Subsequently, we explored the alterations in EMT markers within ASPC-1 and PANC-1 cells following treatment with TED-347. The data indicated that, in the presence of TED-347, there was an upregulation of E-cadherin and a downregulation of Vimentin compared to the control group, implying that TED-347 is capable of inhibiting EMT in PDAC cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-g).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003e6. The YAP1-TEAD1 complex binds to the MSLN promoter region to regulate MSLN expression.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTranscription factor binding sequences of MSLN were obtained from the NCBI website (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The top 10 transcription factor binding sites with the highest matching scores were predicted using the JASPAR website, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e. These binding sites were primarily located in the regions [1045, 1056], [1145, 1154], and [1949, 1960]. Three pairs of PCR primers were designed on these regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The effectiveness of ultrasonic DNA fragmentation was assessed using agarose gel electrophoresis, showing that cellular genomic DNA was fragmented into sizes ranging from 200-1000bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). ChIP assays was performed using an anti-YAP1 antibody to isolate YAP1-bound transcription factors and DNA. ChIP products served as templates for PCR amplification, followed by gel electrophoresis to detect the DNA corresponding to the three pairs of ChIP-qPCR primers, enabling the evaluation of YAP1 binding sites. The analysis identified the [1045, 1056] region as the site with the strongest YAP1 binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Subsequent ChIP-qPCR experiments focused on the enrichment of this site. Furthermore, enrichment multiples of the YAP1 antibody in ASPC-1 cells, which highly express MSLN, was found a statistically significant increasing compared to the IgG control antibody. This findings indicated that in ASPC-1 cells with high MSLN expression, YAP1-TEADs can bind to the MSLN promoter region (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003eGiven that the top 10 sites predicted by JASPAR were predominantly associated with TEAD1 and TEAD4, we employed multiplex immunofluorescence to detect the expression of YAP1, TEAD1, TEAD4 in PDAC tissue. The results revealed that YAP1 was expressed in both PDAC tissue and the surrounding stroma, with a higher expression in cancer epithelial cells compared to the tumor stroma. TEAD1 expression was specifically localized to the nuclei of ductal cells, whereas TEAD4 expression was nearly absent in both PDAC tissue and stroma. These results were further corroborated by data from HPA database (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, h). Collectively, these findings suggest that YAP1 preferentially binds to the TEAD1 rather than TEAD4 in PDAC.\u003c/p\u003e\u003cp\u003e \u003cb\u003e7. The combination of TED-347 and gemcitabine effectively inhibits tumor growth in mice.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe impact of the combination of TED-347 and gemcitabine on the growth of subcutaneous pancreatic tumors in nude mice is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Tumor-bearing mice were prepared and treated according to the schematic (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Mice were assigned into four groups based on treatment: GEM-/TED-347-, GEM+/TED-347-, GEM-/TED-347+, and GEM+/TED-347 + groups. Tumor volumes in the GEM+/TED-347-, GEM-/TED-347+, and GEM-/TED-347- groups increased steadily, with no significant differences among the three groups, suggesting that neither gemcitabine nor TED-347 alone was effective in inhibiting tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c). In contrast, the combined treatment group (GEM+/TED-347+) showed a marked reduced in average tumor volume, with a statistically significant difference, compared to the other groups, indicating that the combination therapy effectively suppressed tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). Concurrently, body weight fluctuations were observed in the treated mice, with both gemcitabine and TED-347 affecting weight, though the body weight of the treatment groups was significantly lower than that of the control group. However, no statistically significant difference in body weight was found among the three treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eMorphological examination of heart, liver, spleen, lung, and kidney tissues from mice in all treatment group was conducted using H\u0026amp;E staining. There was no significant abnormalities observed in the appearance or histological structure and tissue morphology of organs in the GEM+/TED-, GEM-/TED+, GEM+/TED+, and the negative control GEM-/TED- groups, indicating that TED-347 did not adversely affect the organ structure or histological in nude mice (\u003cb\u003eFigure S2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003e8. Gemcitabine has been shown to promote fibrosis in PDAC tissue, and TED-347 can alleviate this fibrosis.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eSubsequently, due to the difficulty in distinguishing between cytoplasm and intercellular fibrosis staining in routine Masson staining of PDAC tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed,e), we immersed the tissue in 4% paraformaldehyde and subjected it to repeated slow freezing and thawing 2–3 times at -20°C. After this process, when we performed Masson staining again, the cytoplasm automatically detached, leaving only a small amount \"squeezed\" around the cell membrane, making the blue-stained fibrillar proteins between cells more visible \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Masson staining results revealed that compared to the control group (GEM-/TED-347-), the gemcitabine monotherapy group (GEM+/TED-347-) exhibited significantly increased fibrosis in PDAC. The fibrosis levels in the TED-347 monotherapy group were comparable to the control group, while the combination therapy group (GEM+/TED-347+) had a lighter degree of PDAC fibrosis than the gemcitabine monotherapy group. This indicates that gemcitabine exacerbates fibrosis in mouse PDAC tissue, and TED-347 can mitigate gemcitabine-induced fibrosis in PDAC tissue.\u003c/p\u003e\u003cp\u003e \u003cb\u003e9. TED-347 down-regulated the expression of collagen in PSCs by inhibiting the binding of YAP1 and TEAD1 to reduce the fibrosis of PDAC.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eUnder phase-contrast microscopy, PSCs isolated on the third day appeared polygonal with minimal cell extension, a low nucleus-to-cytoplasm ratio, and visible refractile lipid droplets within the cytoplasm. By the seventh day, PSCs exhibited accelerated proliferation, increased cell extension, a higher nucleus-to-cytoplasm ratio, and the disappearance of refractile lipid droplets in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). Immunofluorescence analysis confirmed that the third-generation PSCs expressed vimentin and α-SMA, with nearly 100% cellular purity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eAll-trans retinoic acid (ATRA) is well-established to revert activated PSCs into a quiescent state, inhibiting the expression of α-SMA, vimentin, COL1A1, and COL3A1 in PSCs, and restoring the formation of lipid droplets within the cytoplasm, which is a well-recognized model for PSCs deactivation \u003csup\u003e[\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e–\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. In this study, PSCs were treated with serum-free medium containing 20µM ATRA or 5µM TED-347, while the control group was treated with DMSO. After 48 hours, immunoblotting was used to detect the expression levels of YAP1, α-SMA, vimentin, COL1A1, and COL3A1 in PSCs. As with ATRA, TED-347 significantly inhibited the expression of COL1A1 and COL3A1 in PSCs. Notably, TED-347 treatment led to a compensatory increase in YAP1 expression. While both ATRA and TED-347 treatments resulted in a reduction of vimentin and α-SMA levels, no statistically significant difference was observed after normalization across the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec, d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePDAC is among the most aggressive tumors, with the majority of patients presenting at an advanced stage, beyond the reach of surgical intervention. For those fortunate enough to undergo surgery, the risk of recurrence and metastasis remains high postoperatively \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In the current landscape of treatment, chemotherapy-based systemic therapy holds a pivotal position in the management of PDAC \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Our research delves into the roles of MSLN in PDAC cells, EMT, and PSCs within the stroma. We have demonstrated that the YAP1 inhibitor TED-347 can effectively inhibit the expression of MSLN in PDAC cells and reduce fibrosis in PDAC tissues, which in turn enhances the therapeutic effects of gemcitabine. Furthermore, our study investigates the effects of YAP1 inhibitors on PDAC cells and stromal fibrosis, as well as the underlying mechanisms associated with chemoresistance in PDAC.\u003c/p\u003e \u003cp\u003eIn this study, we employed bioinformatics analysis to further clarify that MSLN is highly expressed at both the transcriptional and protein levels in PDAC tissues, consistent with our previous findings that PDAC tissues in patients exhibit high MSLN expression\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The high expression of MSLN is positively correlated with poor prognosis in PDAC patients and is an independent prognostic factor affecting PDAC patients, providing a theoretical basis for potential MSLN-targeted therapy in PDAC. Given that high MSLN expression is positively correlated with poor prognosis in PDAC patients, we hypothesize that high MSLN expression is also related to chemoresistance in PDAC. Similarly, patients with mesothelioma and colorectal cancer who have high MSLN expression are prone to chemoresistance to chemotherapy drugs\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. The MSLN antibody amatuximab can increase the sensitivity of human PDAC cells to gemcitabine in xenograft mouse models \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. In our published study, we employed CCK8 assays to evaluate the viability of PDAC cells with MSLN either knocked out or overexpressed, thereby determining the IC50 for gemcitabine. We found that PDAC cells overexpressing MSLN displayed a diminished response to gemcitabine, whereas cells with MSLN knocked out demonstrated heightened sensitivity to the chemotherapy agent. This finding underscores the relationship between MSLN and the chemoresistance of PDAC cells to gemcitabine. To further clarify the role of MSLN in gemcitabine chemoresistance in PDAC cells, this study also explored the relationship between MSLN and apoptosis in PDAC cells as well as their connection to chemoresistance. Apoptosis is an active, programmed necrosis of cells, a continuous process of cell death. Gemcitabine induces apoptosis in PDAC cells through two mechanisms: one is by inhibiting DNA synthesis; the other is by inhibiting further synthesis of DNA strands\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Using double staining flow cytometry to measure apoptosis rates, we observed that PDAC cells with MSLN knockout exhibited an elevated rate of early apoptosis. Conversely, high levels of MSLN expression were found to counteract the early apoptotic effects of gemcitabine in these cells. This suggests that elevated MSLN expression may contribute to gemcitabine resistance in PDAC by inhibiting the early apoptosis that would otherwise be triggered by the chemotherapy drug.\u003c/p\u003e \u003cp\u003eTo further elucidate the key genes and potential molecular mechanisms by which MSLN promotes gemcitabine resistance in PDAC cells, we conducted a literature review and identified a subset of genes associated with gemcitabine chemoresistance in PDAC, including ABCB1, ABCC1, ABCG2, AKT1, BCL2, BCL2L1, BIRC5, CCND2, CDK6, CDKN1B, DCK, MCL1, MYC, RRM1, SLC29A1, and TP53. We analyzed the correlation between MSLN and the expression of these gemcitabine resistance-related molecules in the PDAC TCGA database, and then employed qPCR to detect the expression levels of gemcitabine resistance-related molecules in PDAC cells with MSLN knockdown or overexpression. Interestingly, our study revealed that the expression level of ABCC1 was correlated with MSLN expression in both MSLN-knockdown and overexpressing PDAC cells, suggesting that ABCC1 may be a key gene in MSLN-mediated promotion of gemcitabine resistance in PDAC cells. This finding partially explains the molecular mechanism underlying the chemoresistance of PDAC cells with high MSLN expression to gemcitabine chemotherapy. ABCC1, also known as multidrug resistance protein 1 (MRP1), is a member of the ATP-binding cassette transporter superfamily\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. In PDAC\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e, cholangiocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e, bladder cance\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e, ovarian cancer\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e, and various other malignancies, ABCC1 plays a crucial role in the development of chemoresistance to gemcitabine. Therefore, it is imperative to delve deeper into the possibility of targeting MSLN for the treatment of PDAC, starting with its role in promoting gemcitabine resistance.\u003c/p\u003e \u003cp\u003eKern et al.\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e identified an enhancer within the Canscript sequence of the MSLN promoter that is capable of recruiting TEAD1. It remains unclear whether inhibiting the YAP1-TEAD1 interaction can suppress the expression of MSLN in PDAC cells. Studies have shown that in PDAC cell lines with high MSLN expression, such as ASPC-1 cells, interfering with the expression of YAP1 or TEAD1 can inhibit the expression of MSLN. Conversely, in cells with low MSLN expression, such as MiaCaPa-2 cells, overexpressing YAP1 or TEAD1 does not induce MSLN expression\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Currently, aside from gene-editing techniques\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e or directly targeting MSLN mRNA\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, there is still a significant gap in the literature regarding the transcriptional regulation of MSLN. In this study, we treated ASPC-1 and PANC-1 PDAC cells, which express MSLN, with varying concentrations of TED-347 and assessed MSLN expression levels using protein immunoblotting after treatment. We found that TED-347 could suppress the expression of MSLN in PDAC cells, with higher concentrations of TED-347 leading to reduced MSLN expression within the cells. This suggests that TED-347 can inhibit the transcription of MSLN by disrupting the interaction between YAP1 and TEADs. Therefore, targeting the YAP-TEAD interaction to suppress MSLN may represent a novel and effective therapeutic approach for cancer treatment.\u003c/p\u003e \u003cp\u003eTo further elucidate which TEADs transcription factors YAP1 interacts with and the specific binding sites within the promoter region, we conducted ChIP assays on the PDAC cell line ASPC-1. Our results revealed that in ASPC-1 cells, which highly express MSLN, the YAP1 antibody significantly enriched specific sequences within the MSLN promoter. Concurrently, by aligning the MSLN promoter sequence using SnapGene software, particularly the Canscript sequence [1949, 1960] (5'-CCACCCACACATTCCTGG-3'), we observed that the YAP1 antibody precipitated very few DNA fragments at positions [1949, 1960], with fluorescence intensity on the gel image nearly identical to that of the negative control. In contrast, a marked enrichment was noted at position [1045, 1056]. Thus, our data support that the YAP1-TEAD1 binding site is not within the Canscript sequence but at a location further from the transcription start site. Furthermore, we examined the co-expression of YAP1, TEAD1, and TEAD4 in PDAC tissue and stroma through multicolor immunofluorescence, revealing that TEAD1 is specifically expressed in PDAC tissue, while TEAD4 is virtually not expressed, which may explain TEAD1's involvement in MSLN transcription in PDAC. Additionally, we noted that YAP1 and TEAD1 are relatively more expressed in PDAC tissue and stroma, with minimal expression in non-cancerous tissue. The differences between cancerous tissue, stroma, and adjacent non-cancerous tissue lay the groundwork for our subsequent research into PDAC fibrosis.\u003c/p\u003e \u003cp\u003eMost current research supports that YAP1-TEADs can promote tumor cell migration, invasion\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, tumor stemness, and EMT\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e; inhibiting YAP1 expression can suppress the malignant tendencies of PDAC. Our previous findings indicate that MSLN also promotes the proliferation, invasion, and EMT of PDAC cells, and knocking out MSLN can reverse the malignant characteristics of PDAC cell lines\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. PDAC may acquire resistance to gemcitabine, a phenomenon that can be mediated by EMT and the development of pancreatic fibrosis \u003csup\u003e[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]\u003c/sup\u003e. Consequently, the inhibitory effects of TED-347 on the invasion, migration, and EMT of PDAC cells provide a partial explanation for how TED-347 might enhance the sensitivity of these cells to gemcitabine. Of course, molecules that share similar functions with MSLN, such as SGLT2\u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e, PAF1\u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e, and Neuromedin U\u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e, are also associated with YAP1. The inhibitory effects of TED-347 on the proliferation, invasion, and EMT of PDAC cannot be entirely attributed to its suppression of MSLN expression. However, regardless, the relationship between YAP1 and MSLN is intimately connected in the development of PDAC, suggesting that targeting the expression of YAP1 or MSLN for the treatment of PDAC may hold broad therapeutic potential.\u003c/p\u003e \u003cp\u003eDense fibrotic desmoplasia is a hallmark of PDAC, characterized by substantial deposition of cellular stroma within the tumor tissue. This stroma serves a dual role: on one hand, it forms a physical barrier that impedes the recruitment of immune cells, such as T cells, to the tumor centre, thereby weakening the anti-tumor immune response\u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e; on the other hand, it leads to vascular collapse and hypoperfusion, creating an ischaemic and hypoxic tumor microenvironment that hinders the effective delivery of chemotherapy drugs to the cancerous tissue\u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e. Studies have indicated that PDAC tissues overexpress YAP1, which correlates positively with the degree of pancreatic fibrosis\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and that inhibiting pancreatic fibrosis can enhance the therapeutic efficacy of gemcitabine on tumors \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e. Therefore, inhibiting pancreatic fibrosis is one of the crucial strategies in the treatment of PDAC. Our study demonstrated in vitro that TED-347 can suppress the secretion of collagen by PSCs, suggesting that TED-347, by inhibiting the binding of YAP1 to TEADs, downregulates the expression of collagen in PSCs, inhibits PSC activation, and alleviates fibrosis in mouse PDAC tissues.\u003c/p\u003e \u003cp\u003eDuring the transition of activated PSCs to a quiescent state, the expression of YAP1 shifts from the nucleus to the cytoplasm\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, a process that aligns with the characteristics of YAP1 as an auxiliary transcription factor. The irreversible binding of YAP1 to TEADs, inhibited by TED-347, also leads to a compensatory increase in YAP1 expression. Research has shown that ATRA can induce the transition of activated PSCs to a quiescent state, resulting in a decrease or disappearance of the expression of markers of PSC activation, such as VIM and α-SMA\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In our study, although ATRA and TED-347 could inhibit the secretion of collagen by PSCs and suppress PSC activation, there were no significant changes in the markers VIM and α-SMA, possibly due to the short intervention time of ATRA and TED-347 on PSCs. To exclude the impact of PSC proliferation on the experimental results, we intervened with PSCs for only 48 hours, significantly shorter than the intervention times reported in the literature, which range from one week to longer\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/sup\u003e. Perhaps if we extended the intervention time to over a week, we might obtain similar results. However, it is noteworthy that both TED-347 and ATRA can inhibit the expression of collagen by PSCs even before completely reversing PSC activation, indicating that both drugs have a role in reducing PDAC fibrosis. Additionally, our study was limited to mouse PSCs and did not combine clinical human PDAC specimens to investigate whether inhibiting the binding of YAP1 to TEADs can suppress the activation of fibroblasts in tumors, which is a certain limitation.\u003c/p\u003e \u003cp\u003eChemotherapy, while targeting the destruction or inhibition of PDAC cell growth, can also induce fibrosis within the PDAC tissue\u003csup\u003e[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/sup\u003e. However, due to the short overall survival period of PDAC patients, particularly those in advanced stages, it is challenging to detect chemoresistance caused by chemotherapy-induced pancreatic fibrosis \u003csup\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e, a concern that has not yet garnered sufficient attention \u003csup\u003e[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]\u003c/sup\u003e. Our study, through Masson's staining, observed that gemcitabine promotes fibrosis in mouse PDAC tissue, aligning with the research findings of Kim DK et al. \u003csup\u003e[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]\u003c/sup\u003e, which indicate that gemcitabine can enhance fibrosis in mouse orthotopically transplanted PDAC tissue during chemotherapy. From the perspective of inhibiting PDAC tissue fibrosis with TED-347, our study also provides a good explanation for the superior tumor suppression effects observed with the combination of TED-347 and gemcitabine. The results of this study suggest that, in addition to chemoresistance caused by PDAC fibrosis, we should also focus on the fibrosis induced by gemcitabine itself, leading to chemoresistance, thereby enhancing our understanding of chemotherapy for PDAC. The combination of PDAC chemotherapy with anti-fibrotic therapy may be one of the future research directions for PDAC chemotherapy \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, this research initiative, pivoting on the link between YAP1 inhibitors and resistance to gemcitabine in PDAC, delves into the interplay and underlying mechanisms among PDAC MSLN, EMT in PDAC, activation of PSCs, fibrosis associated with PDAC, and chemoresistance. Through an array of in vivo and in vitro experiments, coupled with clinical studies on PDAC patients, this work sheds new light on the avenues for chemotherapy and targeted therapeutic strategies in PDAC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHigh expression of MSLN in PDAC tissues indicates a poor prognosis. MSLN may contribute to chemoresistance by promoting resistance to apoptosis in PDAC cells and upregulating ABCC1. TED-347, a YAP1 inhibitor, has demonstrated the ability to reduce MSLN expression and curb the migration, invasion, and EMT in PDAC cells. Additionally, it mitigates PDAC fibrosis, which in turn boosts the effectiveness of gemcitabine in PDAC treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATP binding cassette subfamily C member 1 (ABCC1); Chromatin immunoprecipitation (ChIP); Combination Index (CI); Disease-free survival (DFS); E-cadherin (E-CAD); epithelial-mesenchymal transition (EMT); Gene Expression Profiling Interactive Analysis 2 (GEPIA2); Mesothelin (MSLN); Overall survival (OS); Pancreatic Ductal Adenocarcinoma (PDAC); Pancreatic stellate cells (PSCs); The Cancer Genome Atlas (TCGA); Transcriptionally enhanced associate domains (TEADs); Tumor Immune Infiltration Evaluation Resource 2.0 (TIMER 2.0); Vimentin (VIM); Yes associated protein 1 (YAP1)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eThe manuscript was prepared through contributions of all authors. The contribution of the authors to the manuscript is as follows:\u0026nbsp;Conceptualization, Jili Hu, and Bin Zhu; Data curation, Jili Hu and Jia Wang; Formal analysis, Jili Hu and Jia Wang; Methodology, Jili Hu, Xu Guo, Xinming Li, Jia Wang, Buhe Amin and Wenlong Zhai; Project administration, Bin Zhu; Resources, Jiawei Xu, Bin Zhu; Software, Jili Hu, Xu Guo, Xinming Li, Zhuoyin Wang and Jian Zhou; Supervision, Buhe Amin and Bin Zhu; Validation, Zhuoyin Wang and Nengwei Zhang; Visualization, Jian Zhou and Qing Fan; Writing \u0026ndash; original draft, Jili Hu; Writing\u0026ndash;review \u0026amp; editing, Jili Hu and Bin Zhu.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll data used in this study have been included in the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported\u0026nbsp;by Henan Province medical science and technology research plan joint construction project [grant\u0026nbsp;number: LHGJ20240213], and the Chunhui Project Foundation of the Education Department of China (Grant No. HZKY20220055).\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eWe thank the Central Laboratory of Beijing Shijitan Hospital, Capital Medical University (CMU) and\u0026nbsp;Peking University Health Science Center.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeclaration of interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoreide, K., et al., \u003cem\u003ePancreatic cancer.\u003c/em\u003e Eur J Surg Oncol, 2023. \u003cstrong\u003e49\u003c/strong\u003e(2): p. 521-525.\u003c/li\u003e\n\u003cli\u003eHu, Z.I. and E.M. 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1700-1712.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pancreatic cancer, Chemotherapy resistance, MSLN, Pancreatic fibrosis, YAP1 inhibitors","lastPublishedDoi":"10.21203/rs.3.rs-6589227/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6589227/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with poor prognosis and limited response to gemcitabine-based chemotherapy. Chemoresistance in PDAC arises from both cancer-intrinsic mechanisms and extrinsic factors like stromal fibrosis. This study investigates the role of mesothelin (MSLN) and the YAP1 inhibitor TED-347 in modulating gemcitabine resistance. Elevated MSLN expression in PDAC correlates with advanced disease stages and poor prognosis. Mechanistically, MSLN promotes gemcitabine resistance by counteracting drug-induced apoptosis and upregulating ABCC1, a key drug efflux transporter. YAP1 transcriptionally activates MSLN by binding to its promoter, independent of the Canscript sequence. The YAP1 inhibitor TED-347 disrupts this interaction, reducing MSLN expression and suppressing PDAC cell migration, invasion, and epithelial-mesenchymal transition (EMT). In a mouse model, TED-347 combined with gemcitabine enhanced antitumor efficacy, reduced fibrosis, and increased gemcitabine sensitivity. Notably, TED-347 alleviated stromal fibrosis by inhibiting pancreatic stellate cell (PSC) activation, addressing a critical barrier to drug delivery. While gemcitabine itself induces fibrosis, TED-347 mitigates this effect, offering a dual therapeutic strategy. These findings highlight the YAP1-MSLN axis as a key driver of chemoresistance and fibrosis in PDAC, with TED-347 demonstrating potential to improve clinical outcomes by targeting both malignant and stromal components. This study provides a translational research framework for combining YAP1 inhibitors with chemotherapy to overcome resistance in PDAC.\u003c/p\u003e","manuscriptTitle":"YAP1 Inhibitors Enhance the Therapeutic Effect of Gemcitabine on PDCA by Inhibiting MSLN Expression, EMT, and Pancreatic Fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 07:14:53","doi":"10.21203/rs.3.rs-6589227/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-18T00:16:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-12T19:04:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T16:46:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-05-04T11:41:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd38257c-6ad0-4e94-9d1a-c0f903465bc1","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-16T07:14:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 07:14:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6589227","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6589227","identity":"rs-6589227","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}