Oncogenic CD44 is required for pancreatic cancer cell tumorigenesis and CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy

Oncogenic CD44 is required for pancreatic cancer cell tumorigenesis and CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy | 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 Article Oncogenic CD44 is required for pancreatic cancer cell tumorigenesis and CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy Quansheng Zhou, Yuxi Liu, Mei Meng, Nana Zheng, Mengli Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3677039/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract CD44 is a cancer stem cell marker and is aberrantly overexpressed in cancer stem/progenitor cells of malignant tumors. Overexpression of CD44 promotes carcinogenesis and is closely associated with poor prognosis in cancer patients, including pancreatic cancer. However, CD44-targeted drug against pancreatic cancer is unavailable in the clinical setting, and the effect of CD44 gene knockout on pancreatic cancer has not yet been reported in the literature. In this study, we investigated the effect of CD44 gene knockout on pancreatic cancer cell tumorigenesis. We found that CD44 genetic disruption notably inhibited pancreatic cancer cell tumorigenesis, migration, and invasion; increased intracellular DNA damage, sensitized pancreatic cancer cells to the anticancer drug cisplatin; and also suppressed tumor growth in xenograft mice. Mechanistically, CD44 genetic disruption suppressed expression of multiple oncogenic genes; particularly, the levels of oncogenic X-inactive specific transcription (Xist) were reduced for 35-fold through diminishing promoter activity, unraveling a novel oncogenic CD44-Xist axis in cancer cells. Additionally, CD44 genetic disruption inhibited the tumorigenic AKT and ERK signaling pathways, and concurrently activated the tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of CD44 gene in pancreatic cancer and provide a new strategy for targeted pancreatic cancer therapy. Biological sciences/Cell biology Biological sciences/Stem cells/Cancer stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Pancreatic cancer is a high lethal malignant tumor and the five-year survival rate of the cancer is only 8% [ 1 ] . The incidence of pancreatic cancer is continuously increasing globally, and it is expected that by 2039 pancreatic cancer will be the second and fourth leading cancer-related deaths in the Western countries and developing countries, respectively [ 2 , 3 ] . Although great efforts have been made in fighting pancreatic cancer in recent decades, the five-year survival rate of the cancer only gains very minor improvement. The main reasons for the poor prognosis of pancreatic cancer patients are mainly shallow understanding the mechanism of the cancer, short of good druggable targets, and lack of effective therapeutics against the cancer in the clinical setting [ 3 – 6 ] . Thus, the mechanism of pancreatic cancer remains to be elucidated, the sensible targets and effective therapeutics for pancreatic cancer need to be explored [ 2 , 3 ] . Pancreatic cancer stem cells, with the molecular markers CD44+, CD133+, CD24+, and ESA+, play pivotal roles in pancreatic cancer initiation, angiogenesis, metastasis, drug resistance, and tumor recurrence [ 7 – 11 ] . Notably, the numbers of cancer stem cells increase up to 36% of the total tumor cells in pancreatic cancer tissues, belonging to the highest among all of the malignant tumors examined [ 10 ] . More importantly, the numbers of cancer stem cells are closely associated with the poor prognosis in pancreatic cancer patients [ 10 , 11 ] . Therefore, pancreatic cancer stem cells are reasonable target for cancer therapy. However, how to eliminate cancer stem cells has long been a scientific problem to be solved in the cancer research field [ 10 , 11 ] . CD44, a key cancer stem cell marker and a trans-membrane protein, is aberrantly overexpressed in cancer stem/progenitor cells, but is not or barely expressed in normal cells [ 12 , 13 ] . Overexpression of CD44 triggers cell epithelial–mesenchymal transition (EMT) [ 14 ] , enhances tumor cell uncontrolled proliferation [ 15 ] , stemness [ 15 – 17 ] , dissemination [ 16 , 17 ] , drug resistance [ 18 ] , and tumor microenvironment [ 19 ] . The CD44 gene possesses 20 exons which express distinct pattern between normal and tumor cells [ 20 – 22 ] . In normal cells, CD44 expresses as a standard form (CD44s) which consists of the CD44 exons 1–5 and exons 16–20, while the CD44 exons from 6 to 15 are removed by mRNA splicing enzyme during mRNA maturation; in tumor cells, a defect in mRNA splicing enzyme results in many CD44 exon splicing errors, and produces various tumor cell-specific CD44 variants (CD44v), including CD44v3-v10, CD44v6- v10, and so on [ 22 ] . CD44 variants gain constitutive oncogenic activity, robustly promote pancreatic cancer cell tumorigenesis, angiogenesis, invasion, and dissemination, therefore triggering pancreatic cancer initiation, progression, and metastasis [ 20 – 22 ] . Particularly, various types of CD44v are collectively overexpressed in 67%-93% of pancreatic cancer patients, but are absent in normal people, therefore CD44v is high tumor-specific in pancreatic cancer [ 20 – 22 ] . Accumulated evidence shows that aberrant overexpression of CD44v is closely associated with poor prognosis in pancreatic cancer patients [ 23 – 27 ] . Thus, D44 variants are excellent targets for cancer therapy. Various monoclonal antibodies against CD44v have been developed; unfortunately, the efficacy of CD44v monoclonal antibodies against pancreatic cancer is low in preclinical trails, because the CD44v specific domains are located nearby the cell membrane which exerts a steric hindrance to block the binding of antibodies to CD44v; so far, CD44v antibody has not been used as a drug for pancreatic cancer therapy in the clinical setting [ 28 ] . CD44s and CD44v are activated during carcinogenesis [ 23 – 27 ] . CD44 is a receptor of hyaluronic acid (HA) [ 29 ] . HA activates CD44 and downstream various tumorigenic genes and signaling pathways; notably, CD44 variants in cancer cells are constitutively activated in the tumor microenvironment [ 29 ] . CD44 variants robustly promote pancreatic cancer cell proliferation, migration, invasion, and dissemination [ 30 , 31 ] . However, how to effectively inhibit CD44-mediated carcinogenesis has long been a turf scientific problem to be solved. In light of that all of the CD44s and CD44v contain a translational initiation codon ATG at the exon 1, we think that we can capture all with one haul of the dragnet by disruption of CD44 exon 1 using CRISPR-Cas9 gene editing system. CRISPR/Cas9 technology has been recently used for gene knockout and genetic error correction, and has exhibited a great potentiality in targeted therapy for diseases [ 32 – 36 ] . However, the effect of CD44 gene knockout on pancreatic cancer has not yet been described in the literature. In the current investigation, we first established CD44 gene knockout pancreatic cancer cell lines using CRISPR/Cas9 technology, then studied CD44-mediated pancreatic cell tumorigenesis, and evaluated whether CD44 gene knockout is a good approach for targeted pancreatic cancer therapy. We found that CD44 gene knockout not only effectively reduced pancreatic cancer cell tumorigenesis, migration, invasion, but also increased intracellular DNA damage and sensitized pancreatic cancer cells to the anticancer drug cisplatin. Mechanistic studies revealed that CD44 gene knockout markedly inhibited expression of the 1290bp long noncoding RNA (LncRNA) X-inactive-specific transcript (Xist) [ 37 , 38 ] . Emerging evidence shows that overexpression of Xist plays an oncogenic role in triggering carcinogenesis, tumor progression, and metastasis [ 38 – 41 ] , and is closely associated with poor prognosis in cancer patients [ 42 – 49 ] . However, the method that effectively reduces the expression of oncogenic Xist in cancer has not been reported in the literature. We for the first time unravel a novel oncogenic CD44-Xist axis in cancer cells, and found a new strategy for effective reduction of Xist levels in cancer cells through CD44 gene knockout. Additionally, CD44 gene knockout suppressed tumorigenic AKT and ERK signaling pathways. Furthermore, CD44 gene knockout activated tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of CD44 in carcinogenesis of pancreatic cancer and provide a new strategy for targeted pancreatic cancer therapy. 2. Materials and methods 2.1. Materials Human pancreatic carcinoma cell line Panc-1 was obtained from American Tissue Culture Collection (Manassas, VA, USA). Human pancreatic carcinoma cell line PaTu8988 was from Cell Resource Center, Shanghai Academy of Life Sciences (Shanghai, China). DMEM was from Biological Industries (Corning, USA). The fetal bovine serum (FBS) and blasticidin were purchased from Thermo Scientific (GIBCO, USA). DNA ligase was purchased from Annoron (Beijing, China). The antibodies against CD44, Erk1/2, phosphorylated Erk1/2 (T202/Y204), AKT, phosphorylated AKT (T308), p53, phosphorylated p53, p38, and phosphorylated γH2AX were from Cell Signaling Technology (Boston, MA, USA). The antibodies against phosphorylated p38 (Thr180/Tyr182) was from Proteintech. β-actin antibody was purchased from Sigma (Darmstadt, Germany). DMEM was from Thermo Scientific (GIBCO, Gaithersburg, MD, USA). RNA reverse transcription kit was purchased from Vazyme. The Cas9-expression Lenti-Cas9-Blast vector and Lenti-guide-puro-IRES-GFP vector were purchased from Addgene (#52962, Shanghai, China). 2.2. Cell culture Human pancreatic cancer Panc1 and PaTu8988 cells were cultured in DMEM (high glucose) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2 as we previously reported [ 50 ] . 2.3. Establishment of CD44 gene knockout pancreatic cancer cells CRISPR/Cas9 technology was used to knock out CD44 gene in pancreatic cancer cells [ 51 ] . PaTu8988 and Panc1 cells were first transfected with Lenti-Cas9 cDNA-Blast-vector and stably selected with 10 µg/mL blasticidin. The sequences of single chain guide RNA (sgRNA) for targeting CD44 gene were selected from the CRISPR library using a CRISPR design tool ( http://www.crisprscan.org/ ). CD44 sgRNA1 (GCTACTT CAGACAACCACA) and sgRNA2 (CGCTACAGCATCTCTCGGA) were synthesized as oligonucleotide pairs by Synbio (Suzhou, China) and cloned into the Lenti-guide-puro-IRES-GFP vector. Cas9-overexpressed pancreatic cancer PaTu8988 and Panc1 cells were first infected with CD44-sgRNA1-puro-IRES-GFP-lentivirus, CD44-sgRNA2-puro-IRES-GFP-lentivirus and puro-IRES-GFP-lentivirus, respectively, then stably selected with puromycin. CD44 gene knockout PaTu8988 and Panc-1 cells were validated by RT-PCR, QT-PCR, and Western blotting as described below. 2.4. Western blotting Equal amounts of protein were loaded in each lane and resolved by SDS-PAGE with Tris-glycine running buffer. Separated proteins were then transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk and incubated with primary antibodies to either CD44, or H2AX, or AKT, or ERK, or p53 or p38 at 4°C overnight, followed by incubation with HRP-coupled secondary antibody for 1 h at room temperature. Blots were visualized using enhanced chemiluminescence detection reagents and exposed to X-ray film as we described previously [ 52 ] . 2.5. RT-PCR and real time quantitative PCR Total RNAs were extracted from pancreatic cancer cells using Trizol (Vazyme, China). Next, 1 µg of RNA was used to synthesize the complementary DNA (cDNA) by reverse transcriptase (Vazyme, China). The resulting complementary cDNA was used for PCR analysis. RT-PCR was performed in each tube with 20 µL PCR reaction. The PCR reaction contained an initial denaturation at 94°C for 3 min, followed by 22–35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. PCR products were analyzed by 1% agarose gel. Quantitative real time PCR (QT-PCR) was performed as we previously reported [ 53 ] . Gene expression levels in pancreatic cancer cells were normalized to the house-keeping gene β-actin. Reactions were performed in triplicate with ABI QuantStudio6 Q6 (Applied Biosystems, USA). 2.6. Cell migration assay Pancreatic cancer cells were seeded in 6-well plates at a density of 5 × 10 5 cells per well, and incubated with 2 mL of DMEM supplemented with 10% FBS. When cells were grown to confluence, the cell monolayer on the bottom of the 6-well plate was scraped with a 200 µL plastic pipette tip (time set as 0 h). PBS was used to remove floating cells. Subsequently, the cells were incubated in fresh DMEM supplemented with 2% FBS for 24 h and imaged. The numbers of migrated cells were counted under a light microscope as we previously described [ 54 ] . 2.7. Cell invasion assay Pancreatic cancer cell invasion was measured using a cell transwell assay [ 55 ] . The upper chamber of transwell was first coated with 10% Matrigel at 37˚C for 30 min, then 2 × 10 4 cells were suspended in serum-free medium and seeded in the upper chamber of the transwell. Subsequently, the DMEM medium containing 20% FBS was added to the lower chambers of the transwell. After 24 hours of incubation, cells on the upper membrane surface were first scraped off with a cotton swab, then the invaded cells in the sub-membrane surface of the upper transwell were stained and counted under a microscope as we described before [ 54 ] . 2.8. Luciferase assay for measurement of gene promoter activity The genomic DNA fragment of the 5' flanking promoter region of the Xist gene was cloned into the luciferase reporter pGL4.17 vector. Pancreatic cancer cells were transfected with pGL4.17- Xist promoter construct and control pGL4.17, respectively, using Lipofectamine® 2000 reagent (Invitrogen) and incubated at 37°C for 48 h. The luciferase activity in cell lysates was measured using the Dual-Luciferase Reporter assay kit (Promega, Madison, WI, USA) as we previously described [ 55 ] . 2.9. Tumor xenograft in mice Tumor xenograft in mice was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University as we previously reported [ 52 – 54 ] . The 8-week-old female NOD-SCID mice (18–22 g) were randomly divided into two groups (n = 8). Each mouse was subcutaneously injected with 1 × 10 7 either Panc1-CD44 KO cells or control CD44-expressing Panc1 cells in 200 µL PBS on the back. The weight of each mouse was encoded every other day and the tumor volume was monitored using a digital caliper. The tumor volume was calculated according to the following formula: tumor volume = 0.5 × length × width 2 . After 67 days of xenograft, the mice were imaged, the tumors and main organs were collected, weighted, and statistically analyzed. 2.10. Statistical analysis All results are presented as the mean ± SD. The experiments were repeated in triplicate. Differences between the two groups were assessed by one-way ANOVA using GraphPad Prism 5. Statistical comparisons were performed using Student’s t-test. The significance of differences is indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. 3. Results 3.1. Establishment and verification of CD44 gene knockout pancreatic cancer cells. In light of that CD44 gene knockout in pancreatic cells has not yet been reported in the literature, we used the CRISPR/Cas9 technology to establish CD44 knockout pancreatic cancer cell lines. In the CRISPR/Cas9 system, CD44 sgRNAs direct Cas9 protein to the complementary strand of CD44 genomic DNA to achieve CD44 gene knockout in pancreatic cancer cells (Fig. 1 A). To fulfil this aim, we first screened the sgRNA library and obtained two CD44 sgRNAs with the highest score for targeting CD44 gene, named as CD44-sgRNA1 (GCTACTTCAGACAACCACA) and CD44-sgRNA2 (CGCTACAGCATCTCTCGGA), respectively. These two CD44-sgRNAs were synthesized and cloned into a Lenti-puro-IRES-GFP vector. Next, CD44-expressing pancreatic cancer Panc1 and PaTu8988 cell lines were first transfected with Cas9-vector, then the Cas-9-overexpressed cells were infected with Lenti-puro-CD44 sgRNA1-IRES-GFP, Lenti-puro-CD44sgRNA2-IRES-GFP, and Lenti-control sgRNA-puro-IRES-GFP, respectively (Fig. 1 A). Western blotting showed that both control sgRNA and CD44-sgRNA1 did not significantly alter CD44 expression levels in these two pancreatic cancer cell lines (Fig. 1 B- 1 E). By contrast, CD44-sgRNA2 almost completely abolished CD44 expression in PaTu8988 (Fig. 1 B, 1 C) and Panc1 (Fig. 1 D, 1 E) cells. Additionally, immunofluorescence showed that CD44-sgRNA2 markedly reduced CD44 levels in PaTu8988 (Fig. 1 F) and Panc1 (Fig. 1 G) cells. These data indicate that CD44 is knocked out in these pancreatic cancer cell lines (referred as CD44-KO cells). 3.2. CD44 genetic disruption markedly inhibits pancreatic cancer cell tumorigenesis, migration, and invasion. Next, we investigated the effect of CD44 gene knockout on pancreatic cancer cell tumorigenesis, migration, and invasion. Colony formation assay showed that the colony numbers in CD44-KO Panc1 cells were significantly reduced compared to that of control CD44-expressing Panc1 cells (Fig. 2 A, 2 B), suggesting that CD44 gene knockout diminishes pancreatic cancer cell tumorigenesis. In light of that overexpression of CD44 enhances tumor cell stemness [ 16 , 17 ] , we studied whether CD44 gene knockout reduced pancreatic cancer cell stemness using cell sphere formation assay, a method for accessing cell stemness. The result showed that the spheres formed by CD44 -KO Panc1 cells were much smaller than that of control CD44-expressing Panc1 cells (Fig. 2 C, 2 D), indicating that CD44 gene knockout decreases pancreatic cancer cell stemness. When CD44 -KO and control CD44-expressing Panc1 cells were subcutaneously injected into NOD-SCID mice (n = 8/group), the tumor growth rate in the mice transplanted with CD44 -KO Panc1 cells was significantly lower than that of control mice transplanted with CD44-expressing Panc1 cells (Fig. 2 E, 2 F); meanwhile, the tumor weight in the CD44 -KO group was lighter than that of the control group (Fig. 2 G, 2 H). Together, these data indicate that CD44 gene knockout significantly reduces pancreatic cancer cell tumorigenesis and stemness, and inhibits tumor growth in the xenograft mice. In light of that pancreatic cancer cells have high motility that results in cancer metastasis [ 17 ] , we investigated the effect of CD44 gene knockout on pancreatic cancer cell migration and invasion. Cell migration assay showed that CD44 gene knockout reduced the migration of pancreatic cancer PaTu8988 (Fig. 3 A, 3 B) and Panc1 (Fig. 3 C, 3 D) cells for 3.2-fold. Cell invasion assay showed that CD44 gene knockout notably inhibited invasion of Panc1 (Fig. 3 E, 3 F) and PaTu8988 (Fig. 3 G, 3 H) cells. In short, these data indicate that CD44 gene knockout effectively inhibits pancreatic cancer cell migration and invasion. 3.3. CD44 genetic disruption induces tumor cell DNA damage and sensitizes pancreatic cancer cells to the anti-cancer drug cisplatin Cisplatin, a DNA damage inducer, is a first line anti-cancer drug and has been widely used in cancer therapy. Unfortunately, cisplatin has a toxic effect on bone marrow and intestine epithelial cells at effective dosages, resulting in side effects in cancer patients; furthermore, pancreatic cancer cells rapidly become cisplatin resistance, thereby reducing anticancer efficacy of the drug [ 18 ] . Therefore, how to sensitize cisplatin-induced DNA damage in pancreatic cancer cells, and how to reduce cisplatin drug resistance become critical scientific problems to be solved. In light of that CD44 overexpression increases cancer cell drug resistance [ 18 ] , we explored whether CD44 gene knockout affects DNA damage and changes cisplatin sensitivity in pancreatic cancer cells using the DNA damage marker γH2AX via Western blotting and immunofluorescence. To our surprise, γH2AX protein levels in CD44 -KO Panc1 cells were significantly higher than that of control cells even in the absence of cisplatin (Fig. 4 A, 4 B, 4 F), suggesting that CD44 gene knockout along induces DNA damage in pancreatic cancer cells, and CD44 has a novel function in regulation of DNA damage in cancer cells. More interestingly, CD44 gene knockout further increased γH2AX protein levels in Panc1 cells in the presence of cisplatin (Fig. 4 C, 4 D, 4 E, 4 G), suggesting that CD44 gene knockout sensitizes pancreatic cancer cells to the anti-cancer drug cisplatin. 3.4. Molecular mechanism underlying CD44 gene knockout-mediated inhibition of pancreatic cancer cell tumorigenesis 3.4.1. CD44 genetic disruption markedly reduces expression of oncogenic Xist in pancreatic cancer cells. We utilized mRNA-Seq to analyze gene expression profiles in CD44 gene knockout vs control CD44-expressing pancreatic cancer Panc1 cells. The results showed that CD44 gene knockout significantly changed expression levels of 734 genes (> 2-fold, P < 0.05); among them 544 genes were up-regulated, while 190 genes were down-regulated (Fig. 5 A, 5 B). Next, we focused on analysis of the top differentially expressed oncogenic genes. Strikingly, we found that expression levels of the X-inactive specific transcription (Xist), an emerging oncogenic long non-coding RNA with 1290bp [ 37 , 38 ] , was markedly reduced for 43.6-fold in CD44 gene knockout pancreatic Pan1 cells compared to that of control CD44-expressing Panc1 cells (Fig. 5 C). Meanwhile, the expression of other oncogenic genes, such as SFTA1P, UGT8, ZNF93, CHRM1, SMO, TRIP6, and LTBR, were also significantly decreased (Fig. 5 D). In light of that overexpression of Xist in tumor cells triggers tumorigenesis and cancer progression [ 37 – 41 ] , we designed three pairs of PCR primers to further confirm CD44 gene knockout-mediated reduction of Xist expression in pancreatic cancer cells (Fig. 5 E). RT-PCR showed that CD44 gene knockout almost completely inhibited Xist expression in pancreatic cancer cells (Fig. 5 F). Additionally, QT-PCR indicated that CD44 gene knockout markedly reduced Xist levels for 35-fold in pancreatic cancer cells (Fig. 5 G). In light of the effect of CD44 on Xist expression has not been described in the literature, we investigated the mechanism underlying CD44 gene knockout-mediated reduction of Xist expression by analysis of gene promoter activity. A Xist genomic DNA fragment (530 bp) in the promoter region of the gene was first cloned into the gene transcription reporter vector pGL4.17 (Fig. 5 H), then pancreatic cancer Panc1 cells were transfected with Xist gene promoter DNA fragment-pGL4.17 and control pGL4.17, respectively. Luciferase assay showed that the promoter activity in CD44 gene knockout Panc1 cells was significantly reduced as compared with that of control CD44-expressing Panc1 cells (Fig. 5 I), suggesting CD44 gene knockout inhibits Xist gene transcription. 3.4.2. CD44 genetic disruption inhibits tumorigenic signaling pathway and activates the tumor-suppressive signaling pathway in pancreatic cancer cells. According to the RNA-Seq results as mentioned above, we conducted KEGG signaling pathway analysis for accessing the effect of CD44 gene knockout on cell signaling in pancreatic cancer cells. The result showed that CD44 gene knockout affected multiple signaling pathways, including DNA repair, PI3K-AKT, Hedgehog, Ras, MAPK, and Wnt (Fig. 6 A). Western blotting showed that CD44 gene knockout significantly reduced the levels of phosphorylated ERK and phosphorylated AKT proteins (Fig. 6 B- 6 D); particularly, the 60 kDa band of phosphorylated AKT protein was markedly diminished (Fig. 6 B). These data suggest that CD44 gene knockout inhibits oncogenic AKT and ERK signaling pathways in pancreatic cancer. On other hand, CD44 gene knockout increased levels of tumor-suppressive phosphorylated p53 protein (Fig. 6 E, 6 F) and phosphorylated 38 protein (Fig. 6 E, 6 G) in pancreatic cancer cells, indicating that CD44 gene knockout activates the tumor-suppressive p38-p53 signaling pathway in the cancer cells. In addition, CD44 gene knockout notably elevated levels of the DNA damage marker γH2AX protein (Fig. 6 E, 6 H), indicating that CD44 gene knockout induces DNA breakage in pancreatic cancer cells even in the absence DNA damage inducer. These data together with the data that CD44 gene knockout sensitizes pancreatic cancer cells to the anti-cancer drug cisplatin as mentioned above (Fig. 4 ) imply that CD44 regulates DNA damage in cancer cells, and suggest that CD44 gene knockout may reduce cisplatin drug resistance in pancreatic cancer. Collectively, we found that CD44 gene knockout inhibited pancreatic cancer cell tumorigenesis, migration, invasion, and tumor growth in xenograft mice. Additionally, CD44 gene knockout induced pancreatic cancer cell DNA damage and sensitized the cancer cells to the anti-cancer drug cisplatin. Mechanistically, CD44 gene knockout inhibited the expression of oncogenic Xist and various other oncogenes, activated tumorigenic ERK and AKT signaling pathways, and simultaneously escalated the tumor-suppressive p38-p53 signaling pathway in pancreatic cancer cells (Fig. 7 ). Our findings highlight the critical role of CD44 in pancreatic cancer, and provides a new strategy for targeted pancreatic cancer therapy. 4. Discussion Although great efforts have been made in fighting pancreatic cancer in recent decades, the cancer still poses a serious threat to human life and health [ 1 , 2 ] . The main reasons for the tragedy are that our understanding the mechanism of pancreatic cancer is shallow, and the effective drug against the cancer is unavailable in the clinical setting [ 3 – 6 ] . In the current study, we found that CD44 gene knockout effectively inhibited pancreatic cancer cell tumorigenesis, migration, and invasion mainly through down-regulating oncogenic Xist, suppressing tumorigenic AKT and ERK signaling pathways, and activating the tumor-suppressive p38-p53 signaling pathway. Our findings indicate that CRISPR/Cas9-mediated CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy. The tumor tissues of pancreatic cancer are rich in cancer stem cells which trigger tumor initiation, progression, metastasis, drug resistance, recurrence. Additionally, the numbers of cancer stem cells in the tumor tissues are closely associated with poor prognosis in pancreatic cancer patients [ 7 – 11 ] . Thus, cancer stem cells are a good target for cancer therapy. However, the approach that effectively removals cancer stem cells in malignant tumors is limited, and the therapy that targets pancreatic cancer stem cells is absent in the clinical setting [ 3 ] . In this study, we demonstrated in concept that CD44 gene knockout reduced pancreatic cancer cell stemness and tumorigenesis. Thereby, CD44 gene knockout is a new approach for eliminating pancreatic cancer stem cells and raising anti-pancreatic cancer efficacy. In pancreatic cancer, overexpression of CD44, particularly in aberrant expression of various CD44 variants, is closely associated with poor prognosis in cancer patients [ 23 – 27 ] . However, effective CD44-targeted drug against pancreatic cancer is not available in the clinical setting. As mentioned before, the mRNA splicing error causes production of multiple CD44 variants; notably, many CD44 variants are constitutively activated in the tumor microenvironment, and these active CD44 variants escalate multiple downstream oncogenic genes and tumorigenic signaling pathways, therefore triggering pancreatic cancer initiation and progression [ 20 – 22 ] . Whereas, there is no effective therapy for CD44- and CD44 variants-targeted pancreatic cancer therapy at present, and CD44 gene knockout in pancreatic cancer has not been reported in the literature. In the current study, we found that CD44 gene knockout effectively inhibited pancreatic cancer cell tumorigenesis, migration, invasion, and tumor growth in xenograft mice. Our findings indicate that CD44 and CD44 variants are sensible targets for targeted pancreatic cancer therapy; thus, CD44 gene knockout catches all the gang of CD44s and numerous CD44 variants and is a new strategy for treatment of pancreatic cancer. Xist is abnormally overexpressed in various types of malignant tumors, including pancreatic cancer [ 38 – 41 ] . In pancreatic cancer, Xist upregulates expression of oncogenic EGFR, YAP, ZEB1, TGF-β, Notch1, and Notch2, promotes pancreatic cancer cell proliferation, migration, and invasion, and triggers the initiation and development of many diseases, including cancer [ 42 – 49 , 56 ] . However, the method that effectively inhibits expression of oncogenic Xist in cancer cells has not yet been reported in the literature. In this study, we found that CD44 gene knockout markedly suppressed Xist expression in pancreatic cancer cells, suggesting that CD44 controls Xist expression in cancer cells. Accordingly, we propose that there is a new oncogenic CD44-Xist signaling axis in cancer. To our knowledge, this is the first time for unraveling the novel CD44-Xist signaling axis in malignant tumor. Our findings provide a new approach for reducing oncogenic Xist expression in cancer cells, and offer a platform for developing novel therapy for inhibition of oncogenic CD44-Xist axis in malignant tumors. The tumorigenic AKT and ERK are abnormally activated in pancreatic cancer, while tumor-suppressive p53 is usually deficient in malignant tumor. In the current study, we found that CD44 gene knockout not only suppressed tumorigenic AKT and ERK signaling pathways, but also concurrently activated the tumor-suppressive p38-p53 signaling pathway. Additionally, CD44 gene knockout induced DNA damage in pancreatic cancer cells and increased the sensibility of pancreatic cancer cells to the anti-cancer drug cisplatin, suggesting that CD44 gene knockout reduces drug resistance and increase cisplatin-mediated anti-pancreatic cancer efficacy. CRISPR/Cas9 technology has recently been used in preclinical and primary clinical trials for treatment of diseases [ 51 , 57 , 58 ] , and has shown promising therapeutic results [ 59 , 60 ] . In this study, we found that CD44-sgRNA2 (CGCTACAGCATCTCTC GGA) and CRISPR-Cas9-mediated CD44 genomic disruption inhibited pancreatic cancer cell tumorigenesis. Our study demonstrated in concept that CD44 gene knockout is a potential approach for targeted pancreatic cancer therapy. In the future, CD44sgRNA2 and Cas9 can be packed in the HA-liposome nanoparticles as described in recent studies [ 51 , 57 , 58 ] . In this way, the HA-liposome nanoparticles-CD44sgRNA2 and Cas9 can bind to cancer stem/progenitor cells surface, and deliver CD44sgRNA2 and Cas9 into the cancer cells, consequently disrupting CD44 gene in vivo and inhibiting pancreatic cancer effectively. Our data suggest that CRISPR/Cas9-mediated CD44 gene knockout is a new approach for targeted treatment of pancreatic cancer. In conclusion, CD44 gene knockout notably inhibited pancreatic cancer cell tumorigenesis, migration, invasion, increased intracellular DNA damage, sensitized pancreatic cancer cells to the anticancer drug cisplatin, and suppressed tumor growth in xenograft mice. CD44 gene knockout markedly reduced expression of oncogenic Xist, suppressed tumorigenic AKT and ERK signaling pathways, and concurrently activated the key tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of CD44 in pancreatic cancer pathogenesis and provide a new strategy for targeted pancreatic cancer therapy. Declarations Data Availability Statement All other relevant data are available upon request. Statements and Declarations: There are no statements and declarations. Competing Interests: The authors declare no conflict of interest. Acknowledgments: None. Author contributions Y.L. and M.M.: data curation, formal analysis, funding acquisition, investigation, methodology, writing-original draft; M.Z., N.Z, Y.C., X. Li., X.S., Y.C., Z. X.: data curation, investigation, methodology. Q.Z. and P.X.: Conceptualization, funding acquisition, writing and editing of the manuscript. Ethics statement All experimental protocols were approved by the Suchow University Animal Care Committee and were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 2011). Funding This study was supported by grants from the National Natural Science Foundation of China (Grants No.81772535, No.81902647, No.82073225); National Clinical Research Center for Hematologic Diseases (Grant No. 2020ZKMB04); A project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin 2023; 73(1): 17–48. http://doi.org/10.3322/caac.21763 . Yu J, Yang X, He W, Ye W. Burden of pancreatic cancer along with attributable risk factors in Europe between 1990 and 2019, and projections until 2039. Int J Cancer 2021; 149(5): 993–1001. http://doi.org/10.1002/ijc.33617 . 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Safety and efficacy of CRISPR-based non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells in patients with relapsed or refractory Non-Hodgkin's lymphoma: a first-in-human phase I study. EClinicalMedicine 2023; 60: 102010. http://doi.org/10.1016/j.eclinm.2023.102010 . Additional Declarations There is NO conflict of interest to disclose. Cite Share Download PDF Status: Posted Version 1 posted 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-3677039","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":265352564,"identity":"7be331b9-9eae-4641-b817-160b3f0f7b7e","order_by":0,"name":"Quansheng Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACCTBpA8QJQMxGvJY00rUcJkGL/OzmYxJv/py3Nzie/IDhQ9lhBv7ZDfi1MM45liY5t+124oYzzwwYZ5w7zCBx5wB+LcwSOWbSvA23EwxuJBgw87YdZjCQSMCvhQ2khefPOXuDG+kfmP8So4UHrIXtAOOGGzkGzIzEaJGQSEu2nNuWnDjzzJuCgz3n0nkkbhDQIj8j+eCNN3/s7PmOp2988KPMWo5/BgEtENdB6QNIbCK1jIJRMApGwSjACgCaNEHEjJ9lNwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4712-0731","institution":"Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Soochow University, Jiangsu","correspondingAuthor":true,"prefix":"","firstName":"Quansheng","middleName":"","lastName":"Zhou","suffix":""},{"id":265352565,"identity":"fab711e3-975e-4d48-981f-b41a94730374","order_by":1,"name":"Yuxi Liu","email":"","orcid":"","institution":"Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Soochow University, Jiangsu","correspondingAuthor":false,"prefix":"","firstName":"Yuxi","middleName":"","lastName":"Liu","suffix":""},{"id":265352566,"identity":"a53abd84-e24a-4478-a073-43a39fe28397","order_by":2,"name":"Mei Meng","email":"","orcid":"","institution":"Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Soochow University, Jiangsu","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Meng","suffix":""},{"id":265352567,"identity":"02006eb1-9459-4ca6-910a-9b9617c68859","order_by":3,"name":"Nana Zheng","email":"","orcid":"","institution":"Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Soochow University, Jiangsu","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Zheng","suffix":""},{"id":265352568,"identity":"08d4523c-df9a-4b77-a187-3a1965d57517","order_by":4,"name":"Mengli Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mengli","middleName":"","lastName":"Zhang","suffix":""},{"id":265352569,"identity":"adc80c9d-beb6-4b79-844e-d6f0eb47ae06","order_by":5,"name":"Yu Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Chen","suffix":""},{"id":265352570,"identity":"9a3dcf81-40f2-460f-9972-2e85b046fb17","order_by":6,"name":"Juntao Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Juntao","middleName":"","lastName":"Liu","suffix":""},{"id":265352571,"identity":"4b3d2236-1981-469d-836e-536a0cfd03ad","order_by":7,"name":"Xu Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Li","suffix":""},{"id":265352572,"identity":"9056cb46-5c61-48f3-80c6-d18597701ecb","order_by":8,"name":"Xiaoxiao Song","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxiao","middleName":"","lastName":"Song","suffix":""},{"id":265352573,"identity":"4e7283fe-d54d-412a-b676-7eb2f3d0e32b","order_by":9,"name":"Peng Xu","email":"","orcid":"","institution":"Jiangsu Institute of Hematology","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2023-11-28 14:16:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3677039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3677039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49326076,"identity":"cdcbcbb7-a54e-4322-b687-53cbf1717d32","added_by":"auto","created_at":"2024-01-08 17:29:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003etablishment and verification of CD44\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene knockout pancreatic cancer cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCas9-cDNA and CD44 single chair guide RNAs (sgRNAs) were cloned into lentivirus vector, and the infectious lentiviruses were prepared as described in the method section. Cas9-positive pancreatic cancer PaTu8988 and Panc1 cells were infected with either CD44-sgRNA1 lentivirus, or CD44-sgRNA2 lentivirus, or control lentivirus, respectively (A). \u003cem\u003eCD44\u003c/em\u003e gene knockout in the cells was verified by Western Blotting (B-E), immunofluorescence staining, and confocal microscope analysis (F, G, 600×), red:CD44-positive, blue: DAPI, Merge: both CD44 and DAPI. Ctrl: CD44-expressing cells, CD44-sgRNA1: CD44 gene single chain guide RNA1, CD44-sgRNA2: CD44 gene single chain guide RNA2. Notably, CD44-sgRNA2 and CRISPR-Cas9 almost completely knocked out CD44 in pancreatic cancer cells; accordingly, CD44-sgRNA2-mediated CD44 knockout pancreatic cells were named as CD44-KO. Data in Figure1B-1E are shown as the mean ± SD of three independent replicates. ****p＜0.0001 in an unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/6a938d2e0cd34805150ebefc.jpg"},{"id":49326073,"identity":"9afdf315-6922-4843-926a-05cbc90a8509","added_by":"auto","created_at":"2024-01-08 17:29:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCD44 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene knockout effectively inhibits pancreatic cancer cell tumorigenesis and reduces tumor cell stemness.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumorigenic capability of \u003cem\u003eCD44\u003c/em\u003e gene knockout (CD44-KO) and control CD44-expressing (Ctrl) pancreatic cancer Panc1 cells was measured by the colony formation assay (2A, 2B). The stemness of CD44-KO and Ctrl Panc1 cells was accessed by the cell spherical formation assay (2C, 2D). Additionally, the effect of\u003cem\u003e CD44\u003c/em\u003e gene knockout on tumor growth \u003cem\u003ein vivo\u003c/em\u003e was conducted in xenograft mice as described in the method section. The tumor volumes in CD44-KO and Ctrl groups of xenograft mice were measured every other day (2E, 2F). Finally, the tumors in CD44-KO and Ctrl groups were collected, photographed (2G), weighted, and statistically analyzed (2H). Data in Figure 2A-2D are shown as the mean ± SD of three independent replicates. **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/e35a47b4af99561bce86d7df.jpg"},{"id":49326074,"identity":"83c15dd9-94cc-44f5-ab71-0d079c462a22","added_by":"auto","created_at":"2024-01-08 17:29:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCD44 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene knockout inhibits pancreatic cancer cell migration and invasion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePancreatic cancer PaTu8988 and Panc1 cells were cultured in six-well plates for a wound-healing assay. The cell monolayer was photographed at 0h and 24h of the assay, respectively (A, C), and the number of migrated cells was counted and statistically analyzed (B, D). In the cell invasion assay, PaTu8988 and Panc1 cells were cultured in the Transwell chambers pre-coated with 10% Matrigel and incubated for 24h, the invaded cells were stained by Giemsa staining and photographed under an orthostatic microscope (200×), and the number of invaded cells was counted and statistically analyzed (E-H). Data are shown as the mean ± SD of three independent replicates. *P\u0026lt;0.05 , ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/50e020d8c9088f1d4897a20c.jpg"},{"id":49326075,"identity":"ef511c58-0542-4cbb-93bc-120a2e02a600","added_by":"auto","created_at":"2024-01-08 17:29:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCD44 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene knockout induces DNA damage and enhances cisplatin-mediated DNA cleavage in pancreatic cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA damage in pancreatic cells was accessed by measurement of the protein level of γ-H2AX, a widely used DNA breakage marker, with Western blotting (A). CD44-KO and CD44-expressing Panc1 cells (Ctrl) were incubated with the anti-cancer drug cisplatin at concentrations of 0-5 μM. CD44-KO induced an increase in γ-H2AX levels even in the absence of cisplatin (B) and further significantly enhanced\u003cstrong\u003e \u003c/strong\u003ecisplatin-induced DNA damage in pancreatic cancer cells (C-E). Additionally, CD44-KO and CD44-expressing Panc1 cells were treated with 0 μM and 5 μM cisplatin for 48 h, respectively. After immunofluorescence staining with γ-H2AX specific antibody, the cells were imaged with confocal microscope (4F, 4G, 800×); red: γ-H2AX, blue: DAPI, Merge: γ-H2AX and DAPI. Data in the Figure 4A-4E are shown as the mean ± SD of three independent replicates. **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/ab491b2025d26c670de032a8.jpg"},{"id":49326079,"identity":"c9df7c6a-59ff-4bd3-b7da-b4b922f49663","added_by":"auto","created_at":"2024-01-08 17:29:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCD44 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene knockout markedly down-regulates expression of oncogenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXist\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and various other tumorigenic genes in pancreatic cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene expression profile in three independent sets of CD44-KO and control CD44-expressing Panc1 cells was analyzed by RNA-Seq (A), and a volcano map of gene expression profile was depictured in Figure 5B, red: up-regulated genes; green: down-regulated genes; black: genes that did not significantly change (A,B). RNA-Seq showed that\u003cem\u003e CD44\u003c/em\u003e gene knockout markedly inhibited expression of oncogenic Xist (C); meanwhile, expression levels of various other tumorigenic genes in CD44-KO Panc1 cells were also significantly reduced (D). To further confirm \u003cem\u003eCD44\u003c/em\u003e gene knockout-mediated Xist expression, three Xist primers (P1, P2, and P3) were designed at the indicated position of the long non-coding RNA Xist (19260 bp) as depictured in Figure 5E. Xist levels in CD44-KO and control CD44-expressing Panc1 cells were detected by RT-PCR (F) and real time quantitative PCR (G), respectively. To study the effect of CD44 on \u003cem\u003eXist\u003c/em\u003e gene transcription, a promoter region DNA fragment (530 bp) of \u003cem\u003eXist\u003c/em\u003e gene was cloned into the pGL-4.17-luciferase vector (H), and the construct was used to transfect CD44-KO and CD44-expressing Panc1 cells, respectively. Luciferase activity in these cells was determined and statistically analyzed (I). Data are shown as the mean ± SD of three independent replicates. **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/1fc613f35d86cc903ffc7ff1.jpg"},{"id":49327355,"identity":"097d4304-b4f9-49b4-b1d5-3316dbc4902d","added_by":"auto","created_at":"2024-01-08 17:37:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":143742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCD44 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene knockout inhibits tumorigenic ERK and AKT signaling pathways, but enhances tumor-suppressive p38-p53 signaling axis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKEGG signaling pathway analysis showed that \u003cem\u003eCD44 \u003c/em\u003egene knockout affected multiple signaling pathways in pancreatic cancer cells (A), the large circle indicates highly enriched genes, while the small circle indicates lowly enriched genes in signaling pathways; notably, the DAN repair regulatory genes in the non-homologous end-joining were largely enriched (A). Western blotting showed that \u003cem\u003eCD44 \u003c/em\u003egene knockout reduced the levels of phosphorylated ERK (B, C) and phosphorylated AKT (B, D), but elevated the levels of phosphorylated p53 (E, F), phosphorylated p38 (E, G), and the DNA-breakage marker γ-H2AX (E, H). Data are shown as the mean ± SD of three independent replicates. **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/228cf9dfae3ba0bde5bc9151.jpg"},{"id":49326077,"identity":"cd099f1d-6dd4-44d1-a497-9dcd55779429","added_by":"auto","created_at":"2024-01-08 17:29:59","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eD44\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene knockout by CRISPR/Cas9 inhibits pancreatic cancer cell tumorigenesis and is a new strategy for targeted pancreatic cancer therapy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCD44 \u003c/em\u003egene knockout inhibits pancreatic cancer cell tumorigenesis, migration, and invasion through downregulating oncogenic \u003cem\u003eXist\u003c/em\u003e and various other tumorigenic genes, suppressing tumorigenic AKT and ERK signaling pathways in pancreatic cancer cells. On the other hand, \u003cem\u003eCD44 \u003c/em\u003egene knockout activates tumor-suppressive p38-p53 signaling pathway, induces DNA damage, and enhances cisplatin-mediated DNA brokerage in pancreatic cancer cells. These data\u003cstrong\u003e \u003c/strong\u003esuggest that \u003cem\u003eCD44\u003c/em\u003e gene knockout is a new strategy for targeted pancreatic cancer therapy.\u003c/p\u003e","description":"","filename":"image7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/237be28c72bb778458cf511d.jpg"},{"id":50046524,"identity":"451fa896-32d2-40ab-bce9-45cd6e5fd619","added_by":"auto","created_at":"2024-01-23 16:02:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1350104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3677039/v1/8eb6864b-7142-4005-a67d-0d45ee81ee47.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Oncogenic CD44 is required for pancreatic cancer cell tumorigenesis and CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePancreatic cancer is a high lethal malignant tumor and the five-year survival rate of the cancer is only 8%\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The incidence of pancreatic cancer is continuously increasing globally, and it is expected that by 2039 pancreatic cancer will be the second and fourth leading cancer-related deaths in the Western countries and developing countries, respectively\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Although great efforts have been made in fighting pancreatic cancer in recent decades, the five-year survival rate of the cancer only gains very minor improvement. The main reasons for the poor prognosis of pancreatic cancer patients are mainly shallow understanding the mechanism of the cancer, short of good druggable targets, and lack of effective therapeutics against the cancer in the clinical setting\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Thus, the mechanism of pancreatic cancer remains to be elucidated, the sensible targets and effective therapeutics for pancreatic cancer need to be explored\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePancreatic cancer stem cells, with the molecular markers CD44+, CD133+, CD24+, and ESA+, play pivotal roles in pancreatic cancer initiation, angiogenesis, metastasis, drug resistance, and tumor recurrence\u003csup\u003e[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Notably, the numbers of cancer stem cells increase up to 36% of the total tumor cells in pancreatic cancer tissues, belonging to the highest among all of the malignant tumors examined\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. More importantly, the numbers of cancer stem cells are closely associated with the poor prognosis in pancreatic cancer patients\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, pancreatic cancer stem cells are reasonable target for cancer therapy. However, how to eliminate cancer stem cells has long been a scientific problem to be solved in the cancer research field\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCD44, a key cancer stem cell marker and a trans-membrane protein, is aberrantly overexpressed in cancer stem/progenitor cells, but is not or barely expressed in normal cells\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Overexpression of CD44 triggers cell epithelial\u0026ndash;mesenchymal transition (EMT)\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, enhances tumor cell uncontrolled proliferation\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, stemness\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, dissemination\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, drug resistance\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and tumor microenvironment\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The \u003cem\u003eCD44\u003c/em\u003e gene possesses 20 exons which express distinct pattern between normal and tumor cells\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In normal cells, CD44 expresses as a standard form (CD44s) which consists of the CD44 exons 1\u0026ndash;5 and exons 16\u0026ndash;20, while the CD44 exons from 6 to 15 are removed by mRNA splicing enzyme during mRNA maturation; in tumor cells, a defect in mRNA splicing enzyme results in many CD44 exon splicing errors, and produces various tumor cell-specific CD44 variants (CD44v), including CD44v3-v10, CD44v6- v10, and so on\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. CD44 variants gain constitutive oncogenic activity, robustly promote pancreatic cancer cell tumorigenesis, angiogenesis, invasion, and dissemination, therefore triggering pancreatic cancer initiation, progression, and metastasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Particularly, various types of CD44v are collectively overexpressed in 67%-93% of pancreatic cancer patients, but are absent in normal people, therefore CD44v is high tumor-specific in pancreatic cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Accumulated evidence shows that aberrant overexpression of CD44v is closely associated with poor prognosis in pancreatic cancer patients\u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Thus, D44 variants are excellent targets for cancer therapy. Various monoclonal antibodies against CD44v have been developed; unfortunately, the efficacy of CD44v monoclonal antibodies against pancreatic cancer is low in preclinical trails, because the CD44v specific domains are located nearby the cell membrane which exerts a steric hindrance to block the binding of antibodies to CD44v; so far, CD44v antibody has not been used as a drug for pancreatic cancer therapy in the clinical setting\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCD44s and CD44v are activated during carcinogenesis\u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. CD44 is a receptor of hyaluronic acid (HA) \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. HA activates CD44 and downstream various tumorigenic genes and signaling pathways; notably, CD44 variants in cancer cells are constitutively activated in the tumor microenvironment\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. CD44 variants robustly promote pancreatic cancer cell proliferation, migration, invasion, and dissemination\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. However, how to effectively inhibit CD44-mediated carcinogenesis has long been a turf scientific problem to be solved. In light of that all of the CD44s and CD44v contain a translational initiation codon ATG at the exon 1, we think that we can capture all with one haul of the dragnet by disruption of CD44 exon 1 using CRISPR-Cas9 gene editing system. CRISPR/Cas9 technology has been recently used for gene knockout and genetic error correction, and has exhibited a great potentiality in targeted therapy for diseases\u003csup\u003e[\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. However, the effect of \u003cem\u003eCD44\u003c/em\u003e gene knockout on pancreatic cancer has not yet been described in the literature.\u003c/p\u003e \u003cp\u003eIn the current investigation, we first established \u003cem\u003eCD44\u003c/em\u003e gene knockout pancreatic cancer cell lines using CRISPR/Cas9 technology, then studied CD44-mediated pancreatic cell tumorigenesis, and evaluated whether \u003cem\u003eCD44\u003c/em\u003e gene knockout is a good approach for targeted pancreatic cancer therapy. We found that \u003cem\u003eCD44\u003c/em\u003e gene knockout not only effectively reduced pancreatic cancer cell tumorigenesis, migration, invasion, but also increased intracellular DNA damage and sensitized pancreatic cancer cells to the anticancer drug cisplatin. Mechanistic studies revealed that \u003cem\u003eCD44\u003c/em\u003e gene knockout markedly inhibited expression of the 1290bp long noncoding RNA (LncRNA) X-inactive-specific transcript (Xist)\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence shows that overexpression of Xist plays an oncogenic role in triggering carcinogenesis, tumor progression, and metastasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, and is closely associated with poor prognosis in cancer patients\u003csup\u003e[\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46 CR47 CR48\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. However, the method that effectively reduces the expression of oncogenic Xist in cancer has not been reported in the literature. We for the first time unravel a novel oncogenic CD44-Xist axis in cancer cells, and found a new strategy for effective reduction of Xist levels in cancer cells through \u003cem\u003eCD44\u003c/em\u003e gene knockout. Additionally, \u003cem\u003eCD44\u003c/em\u003e gene knockout suppressed tumorigenic AKT and ERK signaling pathways. Furthermore, \u003cem\u003eCD44\u003c/em\u003e gene knockout activated tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of CD44 in carcinogenesis of pancreatic cancer and provide a new strategy for targeted pancreatic cancer therapy.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eHuman pancreatic carcinoma cell line Panc-1 was obtained from American Tissue Culture Collection (Manassas, VA, USA). Human pancreatic carcinoma cell line PaTu8988 was from Cell Resource Center, Shanghai Academy of Life Sciences (Shanghai, China). DMEM was from Biological Industries (Corning, USA). The fetal bovine serum (FBS) and blasticidin were purchased from Thermo Scientific (GIBCO, USA). DNA ligase was purchased from Annoron (Beijing, China). The antibodies against CD44, Erk1/2, phosphorylated Erk1/2 (T202/Y204), AKT, phosphorylated AKT (T308), p53, phosphorylated p53, p38, and phosphorylated γH2AX were from Cell Signaling Technology (Boston, MA, USA). The antibodies against phosphorylated p38 (Thr180/Tyr182) was from Proteintech. β-actin antibody was purchased from Sigma (Darmstadt, Germany). DMEM was from Thermo Scientific (GIBCO, Gaithersburg, MD, USA). RNA reverse transcription kit was purchased from Vazyme. The Cas9-expression Lenti-Cas9-Blast vector and Lenti-guide-puro-IRES-GFP vector were purchased from Addgene (#52962, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e \u003cp\u003eHuman pancreatic cancer Panc1 and PaTu8988 cells were cultured in DMEM (high glucose) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37\u0026deg;C in a humidified atmosphere of 5% CO2 as we previously reported\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Establishment of \u003cem\u003eCD44\u003c/em\u003e gene knockout pancreatic cancer cells\u003c/h2\u003e \u003cp\u003eCRISPR/Cas9 technology was used to knock out \u003cem\u003eCD44\u003c/em\u003e gene in pancreatic cancer cells\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. PaTu8988 and Panc1 cells were first transfected with Lenti-Cas9 cDNA-Blast-vector and stably selected with 10 \u0026micro;g/mL blasticidin. The sequences of single chain guide RNA (sgRNA) for targeting CD44 gene were selected from the CRISPR library using a CRISPR design tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.crisprscan.org/\u003c/span\u003e\u003cspan address=\"http://www.crisprscan.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). CD44 sgRNA1 (GCTACTT CAGACAACCACA) and sgRNA2 (CGCTACAGCATCTCTCGGA) were synthesized as oligonucleotide pairs by Synbio (Suzhou, China) and cloned into the Lenti-guide-puro-IRES-GFP vector. Cas9-overexpressed pancreatic cancer PaTu8988 and Panc1 cells were first infected with CD44-sgRNA1-puro-IRES-GFP-lentivirus, CD44-sgRNA2-puro-IRES-GFP-lentivirus and puro-IRES-GFP-lentivirus, respectively, then stably selected with puromycin. \u003cem\u003eCD44\u003c/em\u003e gene knockout PaTu8988 and Panc-1 cells were validated by RT-PCR, QT-PCR, and Western blotting as described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Western blotting\u003c/h2\u003e \u003cp\u003eEqual amounts of protein were loaded in each lane and resolved by SDS-PAGE with Tris-glycine running buffer. Separated proteins were then transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk and incubated with primary antibodies to either CD44, or H2AX, or AKT, or ERK, or p53 or p38 at 4\u0026deg;C overnight, followed by incubation with HRP-coupled secondary antibody for 1 h at room temperature. Blots were visualized using enhanced chemiluminescence detection reagents and exposed to X-ray film as we described previously\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. RT-PCR and real time quantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNAs were extracted from pancreatic cancer cells using Trizol (Vazyme, China). Next, 1 \u0026micro;g of RNA was used to synthesize the complementary DNA (cDNA) by reverse transcriptase (Vazyme, China). The resulting complementary cDNA was used for PCR analysis. RT-PCR was performed in each tube with 20 \u0026micro;L PCR reaction. The PCR reaction contained an initial denaturation at 94\u0026deg;C for 3 min, followed by 22\u0026ndash;35 cycles of denaturation at 94\u0026deg;C for 30 s, annealing at 60\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 1 min. PCR products were analyzed by 1% agarose gel. Quantitative real time PCR (QT-PCR) was performed as we previously reported\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Gene expression levels in pancreatic cancer cells were normalized to the house-keeping gene β-actin. Reactions were performed in triplicate with ABI QuantStudio6 Q6 (Applied Biosystems, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell migration assay\u003c/h2\u003e \u003cp\u003ePancreatic cancer cells were seeded in 6-well plates at a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well, and incubated with 2 mL of DMEM supplemented with 10% FBS. When cells were grown to confluence, the cell monolayer on the bottom of the 6-well plate was scraped with a 200 \u0026micro;L plastic pipette tip (time set as 0 h). PBS was used to remove floating cells. Subsequently, the cells were incubated in fresh DMEM supplemented with 2% FBS for 24 h and imaged. The numbers of migrated cells were counted under a light microscope as we previously described\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cell invasion assay\u003c/h2\u003e \u003cp\u003ePancreatic cancer cell invasion was measured using a cell transwell assay\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. The upper chamber of transwell was first coated with 10% Matrigel at 37˚C for 30 min, then 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were suspended in serum-free medium and seeded in the upper chamber of the transwell. Subsequently, the DMEM medium containing 20% FBS was added to the lower chambers of the transwell. After 24 hours of incubation, cells on the upper membrane surface were first scraped off with a cotton swab, then the invaded cells in the sub-membrane surface of the upper transwell were stained and counted under a microscope as we described before\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Luciferase assay for measurement of gene promoter activity\u003c/h2\u003e \u003cp\u003eThe genomic DNA fragment of the 5' flanking promoter region of the \u003cem\u003eXist\u003c/em\u003e gene was cloned into the luciferase reporter pGL4.17 vector. Pancreatic cancer cells were transfected with pGL4.17-\u003cem\u003eXist\u003c/em\u003e promoter construct and control pGL4.17, respectively, using Lipofectamine\u0026reg; 2000 reagent (Invitrogen) and incubated at 37\u0026deg;C for 48 h. The luciferase activity in cell lysates was measured using the Dual-Luciferase Reporter assay kit (Promega, Madison, WI, USA) as we previously described \u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Tumor xenograft in mice\u003c/h2\u003e \u003cp\u003eTumor xenograft in mice was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University as we previously reported\u003csup\u003e[\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. The 8-week-old female NOD-SCID mice (18\u0026ndash;22 g) were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;8). Each mouse was subcutaneously injected with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e either Panc1-CD44 KO cells or control CD44-expressing Panc1 cells in 200 \u0026micro;L PBS on the back. The weight of each mouse was encoded every other day and the tumor volume was monitored using a digital caliper. The tumor volume was calculated according to the following formula: tumor volume\u0026thinsp;=\u0026thinsp;0.5 \u0026times; length \u0026times; width\u003csup\u003e2\u003c/sup\u003e. After 67 days of xenograft, the mice were imaged, the tumors and main organs were collected, weighted, and statistically analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The experiments were repeated in triplicate. Differences between the two groups were assessed by one-way ANOVA using GraphPad Prism 5. Statistical comparisons were performed using Student\u0026rsquo;s t-test. The significance of differences is indicated as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1. Establishment and verification of\u003c/b\u003e \u003cb\u003eCD44\u003c/b\u003e \u003cb\u003egene knockout pancreatic cancer cells.\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn light of that \u003cem\u003eCD44\u003c/em\u003e gene knockout in pancreatic cells has not yet been reported in the literature, we used the CRISPR/Cas9 technology to establish \u003cem\u003eCD44\u003c/em\u003e knockout pancreatic cancer cell lines. In the CRISPR/Cas9 system, CD44 sgRNAs direct Cas9 protein to the complementary strand of \u003cem\u003eCD44\u003c/em\u003e genomic DNA to achieve \u003cem\u003eCD44\u003c/em\u003e gene knockout in pancreatic cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To fulfil this aim, we first screened the sgRNA library and obtained two CD44 sgRNAs with the highest score for targeting \u003cem\u003eCD44\u003c/em\u003e gene, named as CD44-sgRNA1 (GCTACTTCAGACAACCACA) and CD44-sgRNA2 (CGCTACAGCATCTCTCGGA), respectively. These two CD44-sgRNAs were synthesized and cloned into a Lenti-puro-IRES-GFP vector. Next, CD44-expressing pancreatic cancer Panc1 and PaTu8988 cell lines were first transfected with Cas9-vector, then the Cas-9-overexpressed cells were infected with Lenti-puro-CD44 sgRNA1-IRES-GFP, Lenti-puro-CD44sgRNA2-IRES-GFP, and Lenti-control sgRNA-puro-IRES-GFP, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blotting showed that both control sgRNA and CD44-sgRNA1 did not significantly alter CD44 expression levels in these two pancreatic cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). By contrast, CD44-sgRNA2 almost completely abolished CD44 expression in PaTu8988 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and Panc1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) cells. Additionally, immunofluorescence showed that CD44-sgRNA2 markedly reduced CD44 levels in PaTu8988 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and Panc1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) cells. These data indicate that CD44 is knocked out in these pancreatic cancer cell lines (referred as CD44-KO cells).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. \u003cem\u003eCD44\u003c/em\u003e genetic disruption markedly inhibits pancreatic cancer cell tumorigenesis, migration, and invasion.\u003c/h2\u003e \u003cp\u003eNext, we investigated the effect of \u003cem\u003eCD44\u003c/em\u003e gene knockout on pancreatic cancer cell tumorigenesis, migration, and invasion. Colony formation assay showed that the colony numbers in CD44-KO Panc1 cells were significantly reduced compared to that of control CD44-expressing Panc1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting that \u003cem\u003eCD44\u003c/em\u003e gene knockout diminishes pancreatic cancer cell tumorigenesis.\u003c/p\u003e \u003cp\u003eIn light of that overexpression of CD44 enhances tumor cell stemness\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, we studied whether \u003cem\u003eCD44\u003c/em\u003e gene knockout reduced pancreatic cancer cell stemness using cell sphere formation assay, a method for accessing cell stemness. The result showed that the spheres formed by \u003cem\u003eCD44\u003c/em\u003e-KO Panc1 cells were much smaller than that of control CD44-expressing Panc1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that \u003cem\u003eCD44\u003c/em\u003e gene knockout decreases pancreatic cancer cell stemness.\u003c/p\u003e \u003cp\u003eWhen \u003cem\u003eCD44\u003c/em\u003e-KO and control CD44-expressing Panc1 cells were subcutaneously injected into NOD-SCID mice (n\u0026thinsp;=\u0026thinsp;8/group), the tumor growth rate in the mice transplanted with \u003cem\u003eCD44\u003c/em\u003e-KO Panc1 cells was significantly lower than that of control mice transplanted with CD44-expressing Panc1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF); meanwhile, the tumor weight in the \u003cem\u003eCD44\u003c/em\u003e-KO group was lighter than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Together, these data indicate that \u003cem\u003eCD44\u003c/em\u003e gene knockout significantly reduces pancreatic cancer cell tumorigenesis and stemness, and inhibits tumor growth in the xenograft mice.\u003c/p\u003e \u003cp\u003eIn light of that pancreatic cancer cells have high motility that results in cancer metastasis\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, we investigated the effect of \u003cem\u003eCD44\u003c/em\u003e gene knockout on pancreatic cancer cell migration and invasion. Cell migration assay showed that \u003cem\u003eCD44\u003c/em\u003e gene knockout reduced the migration of pancreatic cancer PaTu8988 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and Panc1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) cells for 3.2-fold. Cell invasion assay showed that \u003cem\u003eCD44\u003c/em\u003e gene knockout notably inhibited invasion of Panc1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and PaTu8988 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) cells. In short, these data indicate that \u003cem\u003eCD44\u003c/em\u003e gene knockout effectively inhibits pancreatic cancer cell migration and invasion.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3.\u003c/b\u003e \u003cb\u003eCD44\u003c/b\u003e \u003cb\u003egenetic disruption induces tumor cell DNA damage and sensitizes pancreatic cancer cells to the anti-cancer drug cisplatin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCisplatin, a DNA damage inducer, is a first line anti-cancer drug and has been widely used in cancer therapy. Unfortunately, cisplatin has a toxic effect on bone marrow and intestine epithelial cells at effective dosages, resulting in side effects in cancer patients; furthermore, pancreatic cancer cells rapidly become cisplatin resistance, thereby reducing anticancer efficacy of the drug\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Therefore, how to sensitize cisplatin-induced DNA damage in pancreatic cancer cells, and how to reduce cisplatin drug resistance become critical scientific problems to be solved. In light of that CD44 overexpression increases cancer cell drug resistance\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, we explored whether \u003cem\u003eCD44\u003c/em\u003e gene knockout affects DNA damage and changes cisplatin sensitivity in pancreatic cancer cells using the DNA damage marker γH2AX via Western blotting and immunofluorescence. To our surprise, γH2AX protein levels in \u003cem\u003eCD44\u003c/em\u003e-KO Panc1 cells were significantly higher than that of control cells even in the absence of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), suggesting that \u003cem\u003eCD44\u003c/em\u003e gene knockout along induces DNA damage in pancreatic cancer cells, and CD44 has a novel function in regulation of DNA damage in cancer cells. More interestingly, \u003cem\u003eCD44\u003c/em\u003e gene knockout further increased γH2AX protein levels in Panc1 cells in the presence of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), suggesting that \u003cem\u003eCD44\u003c/em\u003e gene knockout sensitizes pancreatic cancer cells to the anti-cancer drug cisplatin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.4. Molecular mechanism underlying\u003c/b\u003e \u003cb\u003eCD44\u003c/b\u003e \u003cb\u003egene knockout-mediated inhibition of pancreatic cancer cell tumorigenesis\u003c/b\u003e\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e3.4.1. CD44 genetic disruption markedly reduces expression of oncogenic Xist in pancreatic cancer cells.\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe utilized mRNA-Seq to analyze gene expression profiles in \u003cem\u003eCD44\u003c/em\u003e gene knockout \u003cem\u003evs\u003c/em\u003e control CD44-expressing pancreatic cancer Panc1 cells. The results showed that \u003cem\u003eCD44\u003c/em\u003e gene knockout significantly changed expression levels of 734 genes (\u0026gt;\u0026thinsp;2-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); among them 544 genes were up-regulated, while 190 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Next, we focused on analysis of the top differentially expressed oncogenic genes. Strikingly, we found that expression levels of the X-inactive specific transcription (Xist), an emerging oncogenic long non-coding RNA with 1290bp\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, was markedly reduced for 43.6-fold in \u003cem\u003eCD44\u003c/em\u003e gene knockout pancreatic Pan1 cells compared to that of control CD44-expressing Panc1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Meanwhile, the expression of other oncogenic genes, such as SFTA1P, UGT8, ZNF93, CHRM1, SMO, TRIP6, and LTBR, were also significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn light of that overexpression of Xist in tumor cells triggers tumorigenesis and cancer progression\u003csup\u003e[\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, we designed three pairs of PCR primers to further confirm \u003cem\u003eCD44\u003c/em\u003e gene knockout-mediated reduction of Xist expression in pancreatic cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). RT-PCR showed that \u003cem\u003eCD44\u003c/em\u003e gene knockout almost completely inhibited Xist expression in pancreatic cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Additionally, QT-PCR indicated that \u003cem\u003eCD44\u003c/em\u003e gene knockout markedly reduced Xist levels for 35-fold in pancreatic cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eIn light of the effect of CD44 on Xist expression has not been described in the literature, we investigated the mechanism underlying \u003cem\u003eCD44\u003c/em\u003e gene knockout-mediated reduction of Xist expression by analysis of gene promoter activity. A \u003cem\u003eXist\u003c/em\u003e genomic DNA fragment (530 bp) in the promoter region of the gene was first cloned into the gene transcription reporter vector pGL4.17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), then pancreatic cancer Panc1 cells were transfected with \u003cem\u003eXist\u003c/em\u003e gene promoter DNA fragment-pGL4.17 and control pGL4.17, respectively. Luciferase assay showed that the promoter activity in \u003cem\u003eCD44\u003c/em\u003e gene knockout Panc1 cells was significantly reduced as compared with that of control CD44-expressing Panc1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), suggesting \u003cem\u003eCD44\u003c/em\u003e gene knockout inhibits \u003cem\u003eXist\u003c/em\u003e gene transcription.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4.2. CD44 genetic disruption inhibits tumorigenic signaling pathway and activates the tumor-suppressive signaling pathway in pancreatic cancer cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAccording to the RNA-Seq results as mentioned above, we conducted KEGG signaling pathway analysis for accessing the effect of \u003cem\u003eCD44\u003c/em\u003e gene knockout on cell signaling in pancreatic cancer cells. The result showed that \u003cem\u003eCD44\u003c/em\u003e gene knockout affected multiple signaling pathways, including DNA repair, PI3K-AKT, Hedgehog, Ras, MAPK, and Wnt (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Western blotting showed that \u003cem\u003eCD44\u003c/em\u003e gene knockout significantly reduced the levels of phosphorylated ERK and phosphorylated AKT proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD); particularly, the 60 kDa band of phosphorylated AKT protein was markedly diminished (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These data suggest that \u003cem\u003eCD44\u003c/em\u003e gene knockout inhibits oncogenic AKT and ERK signaling pathways in pancreatic cancer.\u003c/p\u003e \u003cp\u003eOn other hand, \u003cem\u003eCD44\u003c/em\u003e gene knockout increased levels of tumor-suppressive phosphorylated p53 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) and phosphorylated 38 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) in pancreatic cancer cells, indicating that CD44 gene knockout activates the tumor-suppressive p38-p53 signaling pathway in the cancer cells.\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003eCD44\u003c/em\u003e gene knockout notably elevated levels of the DNA damage marker γH2AX protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), indicating that \u003cem\u003eCD44\u003c/em\u003e gene knockout induces DNA breakage in pancreatic cancer cells even in the absence DNA damage inducer. These data together with the data that \u003cem\u003eCD44\u003c/em\u003e gene knockout sensitizes pancreatic cancer cells to the anti-cancer drug cisplatin as mentioned above (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) imply that CD44 regulates DNA damage in cancer cells, and suggest that \u003cem\u003eCD44\u003c/em\u003e gene knockout may reduce cisplatin drug resistance in pancreatic cancer.\u003c/p\u003e \u003cp\u003eCollectively, we found that \u003cem\u003eCD44\u003c/em\u003e gene knockout inhibited pancreatic cancer cell tumorigenesis, migration, invasion, and tumor growth in xenograft mice. Additionally, \u003cem\u003eCD44\u003c/em\u003e gene knockout induced pancreatic cancer cell DNA damage and sensitized the cancer cells to the anti-cancer drug cisplatin. Mechanistically, \u003cem\u003eCD44\u003c/em\u003e gene knockout inhibited the expression of oncogenic Xist and various other oncogenes, activated tumorigenic ERK and AKT signaling pathways, and simultaneously escalated the tumor-suppressive p38-p53 signaling pathway in pancreatic cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our findings highlight the critical role of CD44 in pancreatic cancer, and provides a new strategy for targeted pancreatic cancer therapy.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAlthough great efforts have been made in fighting pancreatic cancer in recent decades, the cancer still poses a serious threat to human life and health\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The main reasons for the tragedy are that our understanding the mechanism of pancreatic cancer is shallow, and the effective drug against the cancer is unavailable in the clinical setting\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In the current study, we found that \u003cem\u003eCD44\u003c/em\u003e gene knockout effectively inhibited pancreatic cancer cell tumorigenesis, migration, and invasion mainly through down-regulating oncogenic Xist, suppressing tumorigenic AKT and ERK signaling pathways, and activating the tumor-suppressive p38-p53 signaling pathway. Our findings indicate that CRISPR/Cas9-mediated \u003cem\u003eCD44\u003c/em\u003e gene knockout is a new strategy for targeted pancreatic cancer therapy.\u003c/p\u003e \u003cp\u003eThe tumor tissues of pancreatic cancer are rich in cancer stem cells which trigger tumor initiation, progression, metastasis, drug resistance, recurrence. Additionally, the numbers of cancer stem cells in the tumor tissues are closely associated with poor prognosis in pancreatic cancer patients\u003csup\u003e[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Thus, cancer stem cells are a good target for cancer therapy. However, the approach that effectively removals cancer stem cells in malignant tumors is limited, and the therapy that targets pancreatic cancer stem cells is absent in the clinical setting\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In this study, we demonstrated in concept that \u003cem\u003eCD44\u003c/em\u003e gene knockout reduced pancreatic cancer cell stemness and tumorigenesis. Thereby, \u003cem\u003eCD44\u003c/em\u003e gene knockout is a new approach for eliminating pancreatic cancer stem cells and raising anti-pancreatic cancer efficacy.\u003c/p\u003e \u003cp\u003eIn pancreatic cancer, overexpression of CD44, particularly in aberrant expression of various CD44 variants, is closely associated with poor prognosis in cancer patients\u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. However, effective CD44-targeted drug against pancreatic cancer is not available in the clinical setting. As mentioned before, the mRNA splicing error causes production of multiple CD44 variants; notably, many CD44 variants are constitutively activated in the tumor microenvironment, and these active CD44 variants escalate multiple downstream oncogenic genes and tumorigenic signaling pathways, therefore triggering pancreatic cancer initiation and progression\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Whereas, there is no effective therapy for CD44- and CD44 variants-targeted pancreatic cancer therapy at present, and \u003cem\u003eCD44\u003c/em\u003e gene knockout in pancreatic cancer has not been reported in the literature. In the current study, we found that \u003cem\u003eCD44\u003c/em\u003e gene knockout effectively inhibited pancreatic cancer cell tumorigenesis, migration, invasion, and tumor growth in xenograft mice. Our findings indicate that CD44 and CD44 variants are sensible targets for targeted pancreatic cancer therapy; thus, \u003cem\u003eCD44\u003c/em\u003e gene knockout catches all the gang of CD44s and numerous CD44 variants and is a new strategy for treatment of pancreatic cancer.\u003c/p\u003e \u003cp\u003eXist is abnormally overexpressed in various types of malignant tumors, including pancreatic cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. In pancreatic cancer, Xist upregulates expression of oncogenic EGFR, YAP, ZEB1, TGF-β, Notch1, and Notch2, promotes pancreatic cancer cell proliferation, migration, and invasion, and triggers the initiation and development of many diseases, including cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46 CR47 CR48\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. However, the method that effectively inhibits expression of oncogenic Xist in cancer cells has not yet been reported in the literature. In this study, we found that \u003cem\u003eCD44\u003c/em\u003e gene knockout markedly suppressed Xist expression in pancreatic cancer cells, suggesting that CD44 controls Xist expression in cancer cells. Accordingly, we propose that there is a new oncogenic CD44-Xist signaling axis in cancer. To our knowledge, this is the first time for unraveling the novel CD44-Xist signaling axis in malignant tumor. Our findings provide a new approach for reducing oncogenic Xist expression in cancer cells, and offer a platform for developing novel therapy for inhibition of oncogenic CD44-Xist axis in malignant tumors.\u003c/p\u003e \u003cp\u003eThe tumorigenic AKT and ERK are abnormally activated in pancreatic cancer, while tumor-suppressive p53 is usually deficient in malignant tumor. In the current study, we found that \u003cem\u003eCD44\u003c/em\u003e gene knockout not only suppressed tumorigenic AKT and ERK signaling pathways, but also concurrently activated the tumor-suppressive p38-p53 signaling pathway. Additionally, \u003cem\u003eCD44\u003c/em\u003e gene knockout induced DNA damage in pancreatic cancer cells and increased the sensibility of pancreatic cancer cells to the anti-cancer drug cisplatin, suggesting that \u003cem\u003eCD44\u003c/em\u003e gene knockout reduces drug resistance and increase cisplatin-mediated anti-pancreatic cancer efficacy.\u003c/p\u003e \u003cp\u003eCRISPR/Cas9 technology has recently been used in preclinical and primary clinical trials for treatment of diseases\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e, and has shown promising therapeutic results\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. In this study, we found that CD44-sgRNA2 (CGCTACAGCATCTCTC GGA) and CRISPR-Cas9-mediated \u003cem\u003eCD44\u003c/em\u003e genomic disruption inhibited pancreatic cancer cell tumorigenesis. Our study demonstrated in concept that \u003cem\u003eCD44\u003c/em\u003e gene knockout is a potential approach for targeted pancreatic cancer therapy. In the future, CD44sgRNA2 and Cas9 can be packed in the HA-liposome nanoparticles as described in recent studies\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. In this way, the HA-liposome nanoparticles-CD44sgRNA2 and Cas9 can bind to cancer stem/progenitor cells surface, and deliver CD44sgRNA2 and Cas9 into the cancer cells, consequently disrupting \u003cem\u003eCD44\u003c/em\u003e gene \u003cem\u003ein vivo\u003c/em\u003e and inhibiting pancreatic cancer effectively. Our data suggest that CRISPR/Cas9-mediated \u003cem\u003eCD44\u003c/em\u003e gene knockout is a new approach for targeted treatment of pancreatic cancer.\u003c/p\u003e \u003cp\u003eIn conclusion, \u003cem\u003eCD44\u003c/em\u003e gene knockout notably inhibited pancreatic cancer cell tumorigenesis, migration, invasion, increased intracellular DNA damage, sensitized pancreatic cancer cells to the anticancer drug cisplatin, and suppressed tumor growth in xenograft mice. \u003cem\u003eCD44\u003c/em\u003e gene knockout markedly reduced expression of oncogenic Xist, suppressed tumorigenic AKT and ERK signaling pathways, and concurrently activated the key tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of CD44 in pancreatic cancer pathogenesis and provide a new strategy for targeted pancreatic cancer therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll other relevant data are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations: \u003c/strong\u003eThere are no statements and declarations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments: \u003c/strong\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L. and M.M.: data curation, formal analysis, funding acquisition, investigation, methodology, writing-original draft; M.Z., N.Z, Y.C., X. Li., X.S., Y.C., Z. X.: data curation, investigation, methodology. Q.Z. and P.X.: Conceptualization, funding acquisition, writing and editing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the Suchow University Animal Care Committee and were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (Grants No.81772535, No.81902647, No.82073225); National Clinical Research Center for Hematologic Diseases (Grant No. 2020ZKMB04); A project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. 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Safety and efficacy of CRISPR-based non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells in patients with relapsed or refractory Non-Hodgkin's lymphoma: a first-in-human phase I study. EClinicalMedicine 2023; 60: 102010.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1016/j.eclinm.2023.102010\u003c/span\u003e\u003cspan address=\"10.1016/j.eclinm.2023.102010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3677039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3677039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCD44 is a cancer stem cell marker and is aberrantly overexpressed in cancer stem/progenitor cells of malignant tumors. Overexpression of CD44 promotes carcinogenesis and is closely associated with poor prognosis in cancer patients, including pancreatic cancer. However, CD44-targeted drug against pancreatic cancer is unavailable in the clinical setting, and the effect of \u003cem\u003eCD44\u003c/em\u003e gene knockout on pancreatic cancer has not yet been reported in the literature. In this study, we investigated the effect of \u003cem\u003eCD44\u003c/em\u003e gene knockout on pancreatic cancer cell tumorigenesis. We found that \u003cem\u003eCD44\u003c/em\u003e genetic disruption notably inhibited pancreatic cancer cell tumorigenesis, migration, and invasion; increased intracellular DNA damage, sensitized pancreatic cancer cells to the anticancer drug cisplatin; and also suppressed tumor growth in xenograft mice. Mechanistically, \u003cem\u003eCD44\u003c/em\u003e genetic disruption suppressed expression of multiple oncogenic genes; particularly, the levels of oncogenic X-inactive specific transcription (Xist) were reduced for 35-fold through diminishing promoter activity, unraveling a novel oncogenic CD44-Xist axis in cancer cells. Additionally, \u003cem\u003eCD44\u003c/em\u003e genetic disruption inhibited the tumorigenic AKT and ERK signaling pathways, and concurrently activated the tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of \u003cem\u003eCD44\u003c/em\u003e gene in pancreatic cancer and provide a new strategy for targeted pancreatic cancer therapy.\u003c/p\u003e","manuscriptTitle":"Oncogenic CD44 is required for pancreatic cancer cell tumorigenesis and CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 17:29:54","doi":"10.21203/rs.3.rs-3677039/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93b979d5-b2e9-403b-b673-5fc6687f1b79","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27967722,"name":"Biological sciences/Cell biology"},{"id":27967723,"name":"Biological sciences/Stem cells/Cancer stem cells"}],"tags":[],"updatedAt":"2024-01-23T15:54:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 17:29:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3677039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3677039","identity":"rs-3677039","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}