Inhibition of TFF3 Synergizes with c-MET Inhibitors to Decrease the CSC-like Phenotype and Metastatic Burden in ER+HER2+ Mammary Carcinoma

Inhibition of TFF3 Synergizes with c-MET Inhibitors to Decrease the CSC-like Phenotype and Metastatic Burden in ER+HER2+ Mammary Carcinoma | 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 Inhibition of TFF3 Synergizes with c-MET Inhibitors to Decrease the CSC-like Phenotype and Metastatic Burden in ER+HER2+ Mammary Carcinoma Peter Lobie, Chuyu He, Xuejuan Wang, Yi-Shiou Chiou, Basappa Basappa, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4982898/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract The interaction between HER2 and ERα signaling pathways contributes to resistance to anti-estrogen and HER2-targeted therapies, presenting substantial treatment challenges in ER-positive (ER+) HER2-positive (HER2+) mammary carcinoma (MC). Trefoil Factor-3 (TFF3) has been reported to mediate resistance to both anti-estrogen and anti-HER2 targeted therapies in ER+ and ER+HER2+ MC, respectively. Herein, the function and mechanism of TFF3 in ER+HER2+ MC was delineated; and novel combinatorial therapeutic strategies were identified. Elevated expression of TFF3 promoted the oncogenicity of ER+HER2+ MC cells, including enhanced cell proliferation, survival, anchorage-independent growth, 3D growth, cancer stem cell-like (CSC-like) phenotype, invasion, migration, and xenograft growth. Targeting TFF3 with an interfering RNA plasmid or a small-molecule inhibitor (AMPC) inhibited these oncogenic characteristics, highlighting the therapeutic potential of targeting TFF3 in ER+HER2+ MC. Furthermore, a high-throughput combinatorial anti-cancer compound library screening revealed that AMPC preferentially synergized with receptor tyrosine kinase c-MET inhibitors (c-METis) to reduce cell survival and the CSC-like phenotype. The combination of AMPC and c-METis also synergistically suppressed in vivo growth of ER+HER2+ MC cell-derived xenografts and abrogated lung metastasis. Mechanistically, TFF3 was observed to activate c-MET signaling through a positive-feedback loop to enhance the CSC-like phenotype of ER+HER2+ MC. Therefore, proof of concept is provided herein that antagonizing of TFF3 is a promising therapeutic strategy in combination with c-MET inhibition for the treatment of ER+HER2+ MC. Biological sciences/Cancer/Breast cancer Biological sciences/Cancer/Cancer therapy/Targeted therapies ER+ HER2 + mammary carcinoma TFF3 AMPC c-MET inhibitors combination therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Mammary carcinoma (MC) is a heterogeneous disease comprising multiple subtypes with unique biological characteristics that influence responses to treatment modalities and clinical outcomes[1]. Human epidermal growth factor receptor 2 (HER2) gene amplification occurs in 13–22% of MC cases, characterized by increased proliferation, tumor growth and metastatic potential[2, 3], contributing to aggressive behavior with an unfavorable clinical prognosis. Approximately 60–70% of HER2+ MC cases also express estrogen receptor-α (ERα)[3]. This molecular overlap defines the ER+HER2+ MC subtype, representing approximately 10% of MC cases[4]. Research has demonstrated that administration of combined anti-estrogen and HER2-targeted therapy to ER+HER2+ MC patients significantly improved overall response rate (ORR), clinical benefit rate (CBR), and progression-free survival (PFS) compared to anti-estrogen monotherapy[5]. However, reports have identified a complex bidirectional crosstalk between the ER and HER2 pathways, an intrinsic interaction that impedes therapeutic effectiveness[3, 4]. Furthermore, current treatment approaches for ER+HER2+ MC have limited therapeutic efficacy due to the constraints of anti-estrogen and HER2-targeted therapies and lack of predictive markers for therapeutic resistance[4, 6]. These challenges in ER+HER2+ MC necessitate the exploration of targeted therapies that utilize novel regulatory mechanisms. Trefoil factor 3 (TFF3) is a 59 amino acid peptide initially observed to be secreted by intestinal goblet cells and reported to effect mucosal repair in the gastrointestinal tract[7]. Recent findings have suggested that the biological role of TFF3 extends beyond mucosal protection, and is also associated with oncogenic progression in different types of cancer[7]. TFF3 expression is elevated and often associated with prognosis in gastric[8], colorectal[9], hepatocellular[10], thyroid[11], lung[12], pancreatic[13], prostate[14], cervical[15], endometrial[16], and ER+ mammary carcinomas[17-21]. Functionally, TFF3 has been reported to interact with CXCR4/7[22], CD147[23], CRP-ductin/DMBT1 gp340 [24] , PAR2[25], and LINGO2[26]. In addition, TFF3 enhances the activation of HER1-4[18], c-MET[18], IGFR1[18], and c-SRC[18, 19], and increases the expression of multiple WNT ligands[13], thereby promoting downstream signaling pathways including p44/42 MAPK[9], NF-κB[27], PI3K/AKT[28, 29] STAT3[15, 16], and WNT[13] to promote cancer cell survival, proliferation, a cancer stem cell like (CSC-like) phenotype, invasion, migration and metastasis[7, 30]. In ER+HER2+ MC, a bidirectional crosstalk between ER and HER2 contributes to resistance against both anti-estrogens and HER2-targeted therapies[3, 31], with TFF3 implicated in this resistance mechanism[17, 18, 20, 32]. As an estrogen-responsive gene[32], TFF3 is positively correlated with ER+ status (at least in Caucasian MC patient cohorts), and its high expression is associated with a poor prognosis in ER+ MC patients following endocrine therapy[17, 32-34]. Furthermore, TFF3 has been demonstrated to enhance ERα transcriptional activity in MC, leading to the stimulation of estrogen-independent proliferation and reduced responsiveness to anti-estrogens[17, 20, 32]. Specifically, TFF3 is elevated in MC resistant to tamoxifen[17, 20] and aromatase inhibition[35]. Conversely, TFF3 depletion or inhibition resensitizes resistant cells to the respective anti-estrogen treatment[17, 20, 35]. As indicated above, a previous study has suggested bidirectional functional cross-regulation between HER2 and TFF3, partially independent of ERα in ER+HER2+ MC[18]. Specifically, TFF3 expression is decreased by HER2 activation and increased upon trastuzumab inhibition of HER2; and TFF3 is functionally involved in mediating acquired trastuzumab resistance in ER+HER2+ MC[18]. Given that the dysregulation of ER and HER pathways in MC can be the result of the activation of downstream signaling pathways including p44/42 MAPK and PI3K/AKT[36] which are promoted by TFF3, the reported high TFF3 expression[19] and its role in ER and HER2 signaling [17, 18, 20, 32], targeting TFF3 offers a potential therapeutic approach for ER+HER2+ MC to overcome the resistance of anti-estrogens and/or HER2-targeted therapies and improve clinical outcomes for patients. As a transmembrane receptor tyrosine kinase (RTK), c-MET has been identified as a receptor for hepatocyte growth factor (HGF) and documented to be increased in expression and activity in MC[37, 38]. c-MET amplification plays a crucial role in in MC mediating cellular responses including proliferation, migration, and invasion[38], demonstrating a function in maintaining an oncogenic phenotype. The co-expression and activation of c-MET with other RTKs holds prognostic importance in MC, lung carcinoma and glioma[39]. Furthermore, the interplay between c-MET and other RTKs significantly impacts resistance to anti-RTK treatments, which is a critical concern in MC therapy[38, 40]. Considering that targeted therapies often face challenges such as the development of resistance, combinations that target complimentary pathways or potential escape mechanisms appear to be more effective than single or sequential therapy[41]. Herein, novel combinatorial therapy strategies were investigated to identify potential strategies that may improve treatment outcomes in ER+HER2+ MC. High-throughput screening of 247 anticancer compounds in combination with a small molecule inhibitor of TFF3 (named AMPC) was performed in MDA-MB-361 and BT474 cells. The results demonstrated that targeting TFF3 not only enhances the sensitivity of cells to protein tyrosine kinase inhibitors but also prevents the feedback mechanisms consequent to utilization of these inhibitors, thereby achieving superior synergistic efficacy. Among these agents, c-MET inhibitors exhibited high synergy with TFF3 inhibition. The synergistic actions of the drug combinations in the treatment of ER+HER2+ MC in vitro and ex vivo as well as in vivo , using orthotopic ER+HER2+ MC xenograft models was demonstrated. These results indicated that a combination inhibition of TFF3 and c-MET inhibition may serve as a novel therapeutic strategy for ER+HER2+ MC patients. Methods 1 Cell culture and transfection MDA-MB-361 and BT474 cell lines were obtained from BeNa Culture Collection Co. Ltd (Suzhou, China) and Procell Life Science & Technology Co. Ltd (Wuhan, China), respectively. MDA-MB-361 cells were cultured at 37 °C in a CO 2 -free incubator with L15 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (Thermo Fisher Scientific, Waltham, MA, USA). BT474 cells were maintained at 37 °C in a 5% CO 2 incubator with RPMI-1640 medium, supplemented with 20% FBS, 1% P/S, 2 mM L-glutamine, and 10 ng/mL insulin. All experiments were performed in the respective media containing 2%FBS. As previous studies reported[17], MDA-MB-361 and BT474 cells underwent stable transfection with the pIRESneo3-TFF3 plasmid to force the expression of TFF3, facilitated by Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Simultaneously, cells were transfected with the pIRESneo3-vector plasmid as respectively empty control. G418 selection was applied at a concentration of 400 µg/mL for 4 weeks. Moreover, MDA-MB-361 cells underwent transfection with a shRNA plasmid targeting TFF3 to generate stable TFF3-depleted cells, employing a control shRNA plasmid for comparison. Hygromycin B selection was utilized at a concentration of 200 µg/mL for 4 weeks[18]. These established cell lines are denoted as MDA-MB-361-TFF3, MDA-MB-361-VEC, BT474-TFF3, BT474-VEC, MDA-MB-361-shTFF3, and MDA-MB-361-shCtrl cells. For transient RNA interference, MDA-MB-361 and BT474 cells were transfected with plasmids containing scrambled siRNA, siMET #1, or siMET #2 sourced from GENEWIZ (Suzhou, China). The specifications for siMET #1 and siMET #2 employed in the study were as following, siMET #1 5'-AGAAUGUCAUCAUUCAGGCTT-3' siMET #2 5'-UACUCAGCAACCUUCUGAAGGTT-3' 2 Reagents The 247 anti-cancer compounds library (Cambridge Cancer Compound Library, Catalog No. L2300) utilized in this study was acquired from SelleckChem (Houston, TX, USA). Additionally, three c-MET inhibitors, namely Cabozantinib, SU11274, and PHA-665752 were sourced from SelleckChem (Houston, TX, USA). 3 Western blot (WB) analysis Radio Immunoprecipitation Assay (RIPA) Lysis buffer was employed for cell lysis, followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) to fractionate the proteins within the cell lysate. WB analysis was carried out following established protocols[42]. Immunoblots were developed using a Gel Imager System (Bio-Rad, USA). Details of the antibodies utilized are provided in Supplementary information 11. 4 Cell function assays and Enzyme-Linked immunosorbent assay (ELISA) The monolayer cell viability, Annexin-V/Propidium Iodide (PI) staining, CASPASE 3/7 activity, foci formation, 3D-Matrigel growth, and spheroid formation assays were conducted according to established protocols[43, 44]. For the monolayer cell viability assay, 3000 cells were seeded in 2% serum medium in 96-well plate and incubated for 6 days, using the AlamarBlue assay reagent (BioChip, Beijing, China). To analyze apoptotic cell death, cells were seeded at 50-70% confluency in a 6-cm dish and allowed to adhere overnight before treatment. After a 3-day drug treatment period, the cells were harvested and resuspended in 400 μL of Annexin V binding buffer per sample. Subsequently, 5 μL of Annexin V-Alexa Fluor 488 staining solution was added, followed by a 15-minute incubation period in the absence of light. 5 μL of 7-AAD staining solution was then introduced, followed by a subsequent 5-minute incubation. Samples were analyzed within 30 minutes using a Cytoflex Flow Cytometry system (Beckman Coulter, CA, USA). For CASPASE 3/7 activity assay, cells were seeded at a density of 2×10 4 cells per well in a 96-well plate and evaluated 3 days post-seeding or drug treatment using the Caspase-Glo ® 3/7 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. For foci formation assay, 3000 cells were plated into 24-well plates in the medium supplemented with 2% FBS at 37 °C for 2 weeks until foci formation. Then, the foci were fixed with formalin and stained with crystal violet (Sigma-Aldrich, MO, USA). Survival fraction was measured by eluting the crystal violet with methanol and absorbance was detected at 595 nm. 3D-Matrigel assays were performed in 48-well plates coated with Matrigel (Corning, MA, USA) and allowed to solidify for 30 minutes. 2000 cells were then plated to the pre-coated plates in the medium supplemented with 2% FBS and 4% Matrigel for 12 days. Medium was refreshed every 3 days. AlamarBlue (BioChip, Beijing, China) was used to determine cell viability at the end of the experiment. In spheroid formation assay, 1000 cells were seeded in a 24-well ultralow attachment plate (Corning, MA, USA) culturing with serum-free medium supplemented with P/S, 10 ng/mL recombinant human basic FGF, 20 ng/ml recombinant human EGF, 2% B27, and 5 μg/ml bovine insulin. Mammosphere formation was observed under a microscope (Olypus, Tokyo, Japan) after 12 days of seeding. Mammospheres with diameter exceeding 50 μm were counted. The concentration of secreted TFF3 from cells and serum TFF3 levels were determined using the Quantikine ® ELISA Human TFF3 Immunoassay kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol. Cells were plated at a density of 3×10 5 cells in a 6 cm dish and cultured 600 μL of serum-free medium for 2 days. Subsequently, the supernatant was collected as the sample. Furthermore, a fresh blood sample was obtained from the cardiac puncture of a euthanized mice and subjected to centrifugation at 4°C for 10 minutes at 3000 rpm to isolate the serum, which was then stored at -80°C for subsequent analyses. 5 ALDEFLUOR assay The ALDEFLUOR assay was performed using the ALDEFLUOR assay kit (STEMCELL Technologies, Vancouver, Canada) in accordance with the manufacturer’s instructions. Cells were seeded at 50-70% confluency in 6-well plate and were exposed to the respective drugs for 3 days. The ALDEFLOUR activity was assessed via fluorescence-activated cell sorting (FACS) analysis. Re-suspended cells were exposed to the ALDEFLUOR substrate (BAAA, BODIPY®-aminoacetaldehyde) to identify the ALDH1-positive population, while the baseline fluorescence was established using a specific ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB). 6 Real time quantitative polymerase chain reaction (qPCR) analysis Tissue samples were first rinsed with sterile saline and subsequently lysed in TRIzol (Sigma-Aldrich, MO, USA) for subsequent RNA extraction. Total RNA extraction followed established protocols, which included DNase I treatment, conversion of total RNA to complementary DNA (cDNA), PCR, and qPCR assays, conducted as previously described[45]. The qPCR procedure was carried out according to the outlined methodology[45]. The primers employed for qPCR were as follows, hHPRT1 forward: 5'-TTCCTTGGTCAGGCAGTATAATCC-3' hHPRT1 reverse: 5'-AGTCTGGCTTATATCCAACACTTCG-3' mgapdh forward: 5'-CTCACTCAAGATTGTCAGCAATG-3' mgapdh reverse: 5'-CACATTGGGGGTAGGAACAC-3′ 7 Co-immunoprecipitation (Co-IP) assay Whole-cell lysates were obtained by extracting cells with a cell lysis buffer containing protease inhibitor cocktail (TargetMol, Shanghai, China). Subsequently, the normalized lysates were incubated overnight at 4 °C with gentle agitation after adding 5 µg of primary or anti-IgG antibodies. The lysate–antibody mixture was then incubated with magnetic protein G DynabeadsTM (Thermo Fisher Scientific, Waltham, MA, USA) and washed thrice with protein binding buffer (composed of 150 mM NaCl, 20 mM Tris pH 8.0, 1% NP-40, and supplemented with protease and phosphatase inhibitors). The immunoprecipitant was eluted in sample buffer containing 1% β-mercaptoethanol and subjected to SDS-PAGE immunoblotting. Details of the antibodies used are listed in Supplementary information 11. 8 Xenografts All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Laboratory Animal Centre of Peking University Shenzhen Graduate School (Certificate number: YW) and the “Ethical Development no. 9 (year 2020)” from Tsinghua Shenzhen International Graduate School as previously described[20]. Specific pathogen free (SPF) female BALB/c athymic nude mice were obtained from Guangdong Vital River Laboratory Animal Technology Co. (Foshan, Guangdong, China) and housed in a SPF animal facility with ad libitum access to clean water and food. After a one-week acclimation period, eight mice were randomly assigned to each subgroup. Mice were subcutaneously implanted with 0.72 mg 90-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, FL, USA) at the neck back. Following a 3-day interval, 1 × 10 7 MDA-MB-361 cells were orthotopically implanted into the right fourth mammary fat pad of mice to establish xenograft model. Animal weight and xenograft volume were monitored daily. The xenograft volume was calculated using formula 0.52 × (length × width 2 )[19]. Once the xenograft volumes reached approximately 80-100 mm 3 , xenograft bearing mice were intraperitoneally administered with vehicle (1% DMSO / 10% PEG400 in distilled saline), 20 mg/kg AMPC or intragastrically administered with 60 mg/kg Cabozantinib (SelleckChem, Houston, TX, USA). The mice were sacrificed after a two-week treatment period. 9 Immunohistochemistry (IHC) staining and TUNEL assays IHC staining was conducted employing the labeled streptavidin-biotin-peroxidase complex method[13]. TUNEL assay was performed by using TUNEL Assay Kit (Abcam, Waltham, MA, USA), as previously described[13, 21]. Subsequent scoring of IHC staining was performed utilizing the immunoreactive score (IRS) method[46]. Two independent researchers, blinded to the clinical data, meticulously assessed and validated the staining results. Details regarding the antibodies employed are provided in Supplementary information 11. 10 Statistical analysis Graphical representations and statistical analyses were performed utilizing GraphPad Prism 9 (GraphPad Software, Inc., CA, USA). The statistical significance between two groups was assessed using a two-tailed unpaired Student’s t-test, while analysis of variance (ANOVA) was employed for comparisons among multiple treatment groups. Statistical significance thresholds were set at * P < 0.05, ** P < 0.01, and *** P < 0.001. Data conforming to a normal distribution were presented as mean ± standard deviation (SD). Results 1 Forced expression of TFF3 enhances oncogenic behavior in ER+HER2+ MC cells To elucidate the functions of TFF3 in ER+HER2+ MC, MDA-MB-361 and BT474 cells stably transfected with TFF3 cDNA were generated and validated through western blot analysis (Supplementary information 1A, 2A). Both MDA-MB-361 and BT474 cells with forced expression of TFF3 demonstrated a significant increase in cell proliferation, as observed by total cell number (Fig. 1A and Supplementary information 2B) and BrdU incorporation assays (Fig. 1B and Supplementary information 2C), compared to the respective vector-transfected ER+HER+ MC cell lines. Additionally, ER+HER+ MC cells stably transfected with TFF3 demonstrated a significant decrease in CASPASE 3/7 activity (Fig. 1C and Supplementary information 2D) and reduced apoptotic cell death, particularly in the late stage of apoptosis (Fig. 1D and Supplementary information 1B, 2E), compared to their respective vector transfected counterparts. Loss of contact inhibition and anchorage-independent growth is a key hallmark of oncogenic transformation and cancer progression[47]. In order to examine and assess this characteristic of cancer cells in vitro , foci formation assays were performed. MDA-MB-361-TFF3 and BT474-TFF3 cells exhibited enhanced anchorage-independent growth as compared to MDA-MB-361-VEC and BT474-VEC cells (Fig. 1E and Supplementary information 2F). In 3D-Matrigel culture, which more closely mimics in vivo conditions, ER+HER2+ MC cells with forced expression of TFF3 demonstrated higher viability, characterized by increased number of live cells and reduced number of dead cells compared to their vector-transfected counterparts (Fig. 1F and Supplementary information 2G). These results demonstrated that TFF3 promotes proliferation, cell survival and anchorage-independent growth in ER+HER2+ MC cells in vitro and promotes cancer cell colony growth ex vivo . Cancer cell migration and invasion are pivotal for cancer progression, and directly contribute to metastatic dissemination[47]. Transwell assays were conducted to assess the effect of forced expression of TFF3 on the migratory and invasive capacities of ER+HER2+ MC cells. The results demonstrated that forced expression of TFF3 in MDA-MB-361 cells led to increased migration and invasion compared to vector-transfected cells (Supplementary information 1C, 1D). Similarly, in BT474 cells, forced expression of TFF3 resulted in increased migration and invasion (Supplementary information 2H, 2I). CSCs play a crucial role in MC progression, possessing the ability for self-renewal and tumor initiating capacity[48]. Prior studies have reported elevated TFF3 expression in breast cancer stem cells (BCSCs)[18, 20]. Herein, the effect of TFF3 on CSC-like phenotype of ER+HER2+ MC cells was assessed. Spheroid formation assays revealed that the number of spheroids in MDA-MB-361 and BT474 cells with forced expression of TFF3 were higher in comparison to their respective vector-transfected counterparts (Fig. 1G and Supplementary information 2J). Consistent results were observed by ALDEFLUOR assay, which demonstrated that MDA-MB-361 and BT474 cells with forced expression of TFF3 exhibited a significantly higher population of ALDH1-positive cells compared to the vector transfected control (Fig. 1H and Supplementary information 1E, 2K). Hence, TFF3 promotes the CSC-like phenotype in ER+HER2+ MC cells. Subsequently, the impact of TFF3 on ER+HER2+ MC growth in vivo was assessed in a MDA-MB-361 xenograft model. Consistent with the in vitro findings, MDA-MB-361 xenografts generated by cells with forced expression of TFF3 exhibited significant increases in both volume and weight compared to vector transfected cells at the end of the experiment (Fig. 1I, 1J). Further analysis of xenograft specimens by IHC revealed a higher proportion of MKI67-positive cells (Fig. 1K), indicative of increased proliferation, and a reduction in apoptosis indicated by TUNEL analysis, in the xenografts generated by MDA-MB-361 cells with forced expression of TFF3, compared to the vector transfected cell generated xenografts (Fig. 1L). These results indicate that forced expression of TFF3 promotes in vivo growth of MDA-MB-361 cells. Hence, the forced expression of TFF3 augmented the oncogenicity of ER+HER2+ MC cells both in vitro , ex vivo and in vivo . 2 Depletion of TFF3 suppresses oncogenic behaviors in ER+HER2+ MC cells A MDA-MB-361 cell model with depleted TFF3 expression was generated by a shRNA-based approach (designed as MDA-MB-361-shTFF3) and validated through western blot analysis (Supplementary information 3A). Resulting functional consequences were investigated by using approaches similar to those used to examine the consequences of forced expression of TFF3 (Fig. 1, Supplementary information 1-2). TFF3-depleted MDA-MB-361 cells exhibited a significant decrease in total cell number (Fig. 2A) and BrdU incorporation (Fig. 2B). Additionally, depleted expression of TFF3 significantly increased CASPASE 3/7 activity and promoted apoptosis (Fig. 2C, 2D and Supplementary information 3B). TFF3-depleted MDA-MB-361 cells demonstrated markedly reduced foci formation (Fig. 2E). Similarly, in 3D-Matrigel culture, MDA-MB-361-shTFF3 cells formed fewer colonies and demonstrated reduced viability, characterized by a higher number of dead cells and fewer live cells as compared to the vector control cells (Fig. 2F). The depletion of TFF3 significantly reduced migration and invasion of MDA-MB-361 cells (Supplementary information 3C, 3D). Additionally, spheroid formation assays indicated that TFF3-depleted MDA-MB-361 cells formed significantly fewer spheroids compared to the control vector cells (Fig. 2G). Consistently, ALDEFLUOR assay results revealed a significantly lower population of ALDH1-positive cells in TFF3-depleted MDA-MB-361 cells (Fig. 2H and Supplementary information 3E). The in vivo growth of TFF3-depleted MDA-MB-361 cells was also evaluated. The results demonstrated that TFF3 depleted MDA-MB-361 cell generated xenografts exhibited significantly reduced growth in volume compared to the control vector xenografts, a finding further confirmed by the lower xenograft weights at the end of the experiment (Fig. 2I, 2J). IHC and TUNEL analysis additionally revealed a decreased proportion of MKI67-positive cells, and higher apoptosis in the TFF3-depleted xenografts compared to the control vector cell generated xenografts (Fig. 2K, 2L). Thus, the depletion of TFF3 decreased oncogenicity of ER+HER2+ MC cells both in vitro , ex vivo and in vivo . 3 AMPC inhibited the oncogenicity of ER+HER2+ MC cells in vitro and ex vivo TFF3 has been reported to form homodimers via the seventh cysteine (Cys57) residue, and the homodimeric form of TFF3 plays a critical role in inhibiting apoptosis[7, 30]. Recently, a small-molecule inhibitor named AMPC ( 2-amino-4-(4-(6-fluoro-5-methylpyridin-3-yl)phenyl)-5-oxo-4H,5H-pyrano [3,2-c]chromene-3-carbonitrile ) was developed that interfered with dimerization of TFF3 via the Cys57 residue[30]. AMPC leads to the rapid degradation of the monomeric form of TFF3, thereby diminishing TFF3-mediated signaling pathways crucial for cancer cell survival[9, 12, 13, 20, 30]. Given the observed oncogenic functions of TFF3 in ER+HER2+ MC cells, AMPC was employed to investigate the effect and therapeutic potential of small molecule mediated inhibition of TFF3 in ER+HER2+ MC cells. The IC 50 value of AMPC in MDA-MB-361 (3.206 ± 0.757 µM) and BT474 cells (2.268 ± 0.605 µM) was determined using total cell number assays (Supplementary information 4A). Consistent with the effect of siRNA mediated depletion of TFF3, inhibition of TFF3 by AMPC decreased the BrdU incorporation of MDA-MB-361 and BT474 cells, and in a dose-dependent manner (Fig. 3A). Concomitantly, CASPASE-3/7 activity increased with increasing concentrations of AMPC in both MDA-MB-361 and BT474 cell lines (Fig. 3B). Moreover, the proportion of cells undergoing early and late apoptosis rose with increasing AMPC concentrations in the respective cell lines (Fig. 3C and Supplementary information 4B). In addition, AMPC dose dependently inhibited MDA-MB-361 and BT474 cell anchorage-independent growth, as demonstrated by foci formation assays (Fig 3D and Supplementary information 4C). Similarly, the viability of 3D-Matrigel colonies formed by MDA-MB-361 and BT474 cells exhibited a decrement relative to the increasing dose of AMPC. This was evidenced by a discernible decrease in green fluorescence (indicating live cells) and a concomitant increase in red fluorescence (indicating dead cells), as illustrated in Fig 3E and Supplementary information 4D. Furthermore, the effect of AMPC on the CSC-like phenotype of MDA-MB-361 and BT474 cells were further examined. Increasing concentrations of AMPC led to a dose-responsive decrease in the size and number of spheroids in both MDA-MB-361 and BT474 cell lines (Fig 3F and Supplementary information 4E). Consistently, it was observed that AMPC dose dependently decreased the percentage of the ALDH1-positive cell population in both MDA-MB-361 and BT474 cells (Fig 3G and Supplementary information 4F). These findings indicated that pharmacological inhibition of TFF3 reduced cell viability by suppressing proliferation and inducing apoptosis, and that AMPC is a potent and effective inhibitor of cell proliferation, survival, oncogenicity, 3D-growth, and CSC-like behavior in ER+HER2+ MC cells. 4 c-MET inhibitors (c-METis) identified as the most synergistic compounds in combination with AMPC to decrease ER+HER2+ MC cell viability Combination therapy improves therapeutic efficacy compared to single-drug treatment by enhancing cytotoxicity and reducing the development of drug resistance in cancer cells[49]. To explore the therapeutic potential of AMPC-based combinations for the treatment of ER+HER2+ MC cells, the Cambridge Anti-Cancer Compound Library was screened in combination with varying concentrations of AMPC (0, 5, 10 or 20 µM) in MDA-MB-361 and BT474 cells, revealing inhibition of cell viability (Fig. 4A). This comprehensive library comprises 247 anti-cancer compounds as illustrated in Fig. 4A. Combination index (CI) analysis[50] revealed that 107 compounds exhibited synergy with AMPC in MDA-MB-361 cells, whereas 79 compounds showed synergy in BT474 cells. Among these, 40 compounds demonstrated synergistic effects with AMPC in both cell lines with detailed information listed in Supplementary information 5. The prominent pathways for the 40 synergistic compounds included protein tyrosine kinase, DNA damage, endocrinology & hormones, cell cycle, cytoskeletal signaling, and epigenetics. Notably, the protein tyrosine kinase pathway was targeted by 7 out of the 40 compounds, comprising 17.5% of all synergistic compounds and ranking first among them (Fig 4B). Given the role of TFF3 in activating RTKs in ER+HER2+ MC[18], the 7 identified compounds in the protein tyrosine kinase pathway primarily targeted four distinct RTKs, c-MET, EGFR, VEGFR, and c-KIT (Fig 4C). Further scatterplot regression analysis of CI values for compounds demonstrating synergy with AMPC in MDA-MB-361 and BT474 cells revealed that the combination of two c-MET inhibitors, PHA-665752 and SU11274, with AMPC exhibited the highest synergy, characterized by low CI values in both MDA-MB-361 and BT474 cells (Fig 4D). These findings indicated a synergistic effect of c-MET inhibitors in combination with AMPC in decreasing viability of ER+HER2+ MC cells. 5 AMPC synergizes with c-METis to reduce ER+HER2+ MC cell survival and growth in vitro and ex vivo To further substantiate the synergistic effects of AMPC and c-METis identified through high-throughput screening of the anti-cancer compound library, the pharmacological inhibition of TFF3 by AMPC in combination with three c-METis was further evaluated in MDA-MB-361 and BT474 cells by total cell number assay (Fig. 5A). Cabozantinib, an FDA-approved c-METi not included in the Cambridge Anti-Cancer Compound Library (Supplementary information 6A), alongside the identified PHA-665752 and SU11274, were chosen for continued investigation. The combinatorial treatments of AMPC and c-METis exhibited synergistic effects, as demonstrated by the Chou-Talalay method and 3D zip synergy analysis in both cell lines (Fig. 5B, 5C). Subsequently, combination treatment of AMPC (2.5 μM) - c-METis significantly increased the efficacy of c-METis compared to c-METi treatment alone in MDA-MB-361 and BT474 cells, as demonstrated by dose-response analysis (Fig. 5D). Specifically, AMPC reduced IC 50 values of Cabozantinib, SU11274, and PHA-665752 by approximately 10-fold, 5-fold, and 30-fold, respectively, in MDA-MB-361 cells (Fig. 5D). Similarly, in BT474 cells, AMPC notably decreased the IC 50 values of Cabozantinib, SU11274 and PHA-665752 by approximately 10-fold, 10-fold and 20-fold, respectively (Fig. 5D). These findings underscore the synergistic potential of combining AMPC with c-METis to decrease cell survival in ER+HER2+ MC cells. Furthermore, the effect of the combinatorial treatment on cell proliferation and apoptosis was investigated. The results indicated that single-agent AMPC or c-METis significantly inhibited proliferative capability compared to vehicle treatment in both MDA-MB-361 and BT474 cells (Supplementary information 7A). Importantly, the combined AMPC-c-METi treatments further amplified the inhibitory effect observed with the single treatments. Additionally, single-agent AMPC or c-METis promoted CASPASE 3/7 activity compared to vehicle treatment in both cell lines, and combined AMPC-c-METi treatments further enhanced this effect (Supplementary information 7B). This observation was consistent with an increase in apoptosis, as evidenced by elevated populations of early and late apoptotic cells upon single-agent and combined AMPC-c-METi treatments in both cell lines (Supplementary information 7C). Subsequently, the effect of combined AMPC-c-METi treatments on foci forming capacity and 3D growth of MDA-MB-361 and BT474 cells was evaluated. Foci formation assays demonstrated that single treatments with AMPC or c-METis significantly reduced colony formation and viability compared to the vehicle control. Notably, the combined treatment of AMPC and c-METis further augmented these effects observed with the individual treatments (Fig. 5E and Supplementary information 7D). Similarly, in 3D Matrigel culture, single treatments led to a substantial reduction in the number and size of colonies, decreased cell viability, increased numbers of dead cells, and fewer live cells compared to the vehicle control. These effects were more pronounced with the combined treatments (Fig. 5F and Supplementary information 7E). Collectively, these findings indicate that AMPC synergizes with c-MET inhibitors to significantly reduce the survival and growth of ER+HER2+ MC cells in vitro and ex vivo . 6 AMPC synergizes with c-METis to suppress CSC-like phenotype in ER+HER2+ MC Cells in vitro To assess the effect of the combinatorial treatment on the migration and invasion of ER+ HER2+ MC cells, the migratory and invasive capacities were evaluated following single treatments with AMPC, c-METis, and their combination in MDA-MB-361 and BT474 cells. The findings revealed that single-agent treatments notably suppressed both the migratory and invasive abilities of the cells compared to the control treatment (Fig. 6A and Supplementary information 8A). The combined AMPC-c-METi treatments exhibited an augmented inhibition effect beyond that of the single-agent treatments, suggesting a synergistic enhancement in restraining the migratory and invasive capacities of the ER+HER2+ MC cells. There exists a positive relationship between the size of the subpopulation of MC CSCs and migration and invasion[51]. Hence, the spheroid formation and ALDEFFLOUR activity assays were conducted to explore the effect of AMPC and c-METis combined treatment on CSC-like behavior in ER+HER2+ MC cells. The results from the spheroid formation assay indicated that single treatments with AMPC or c-METis significantly reduced the number of spheroids compared to vehicle treatment, and the combined AMPC-c-METi treatments further diminished spheroid formation in both ER+HER2+ MC cell lines (Fig. 6B and Supplementary information 8B). Consistently, the percentage of the ALDH1-positive cell population decreased after treatment with either AMPC or c-METis alone compared to the vehicle control (Fig. 6C and Supplementary information 8C). Notably, the combined AMPC-c-METi treatments resulted in a significant reduction in the ALDH1-positive cell population compared to c-METi treatment alone (Fig. 6C and Supplementary information 8C). Subsequently, the mechanistic basis underlying synergistic effects of AMPC and c-METis was further analyzed using western blot analysis. Single treatments with either AMPC or c-METis, decreased TFF3 expressoion and the p-c-MET Y1234/1235 /c-MET ratio in MDA-MB-361 and BT474 cells; and combined AMPC and c-METi treatments further significantly reduced TFF3 expression and the p-c-MET Y1234/1235 /c-MET ratio compared to single agent treatment of ER+HER2+ MC cells (Fig. 6D and Supplementary information 8D). Previous studies have shown that CSCs express elevated levels of genes related to migration and invasion, such as OCT4, BMI1, SOX2, ALDH1A1, and CD44 in MC[52-55]. Diminished expression of OCT4, BMI1, SOX2, ALDH1A1, and CD44 proteins were observed in MDA-MB-361 and BT474 cells treated with AMPC or c-METis, in comparison to vehicle-treated cells (Fig. 6D and Supplementary information 8D). These reductions were further magnified following combined AMPC-c-METi treatments (Fig. 6D and Supplementary information 8D). Thus, the combined treatment of AMPC with c-METis demonstrated significant potential to synergistically inhibit the CSC-like phenotype of ER+HER2+ MC cells. 7 AMPC synergizes with c-MET inhibition to suppresses the MDA-MB-361 xenograft growth and lung metastasis Building on the synergistic effects observed in vitro and ex vivo , the efficacy of the combined treatment of AMPC and c-MET inhibition was further evaluated in vivo . The xenograft model was established by orthotopically implanting MDA-MB-361 cells into the right fourth mammary fat pad of female mice. Cabozantinib was selected for the in vivo experiments as it is the FDA-approved drug for c-MET inhibition, and has been utilized in patients with ER+ MC or HER2+ MC for two Phase II clinical trials[56, 57]. Mice bearing MDA-MB-361 xenografts were randomized into four treatment groups (n = 8) to receive either vehicle (V), AMPC (A), Cabozantinib (C), or a combination of AMPC and Cabozantinib (A+C). Each treatment was administered for a duration of two weeks. Throughout the treatment period, animal weights and xenograft volumes were measured daily. Upon completion of the treatment regimen, major organs, including the spleen, lungs, and liver, were collected post-mortem. There was no significant difference in body weight or major organ weights among the four treatment groups, indicating that the treatments were well-tolerated by the mice (Supplementary information 9A, 9B). Daily assessments of xenograft volumes revealed that single-agent treatments with either AMPC or Cabozantinib significantly reduced the volumes of MDA-MB-361 xenografts compared to the vehicle-treated control. Notably, the combination treatment with AMPC and Cabozantinib resulted in an even greater reduction in xenograft volumes compared to either single-agent treatment alone (Fig. 7A). Consistent results were observed with xenograft weight (Fig. 7B) and resected xenografts (Fig. 7C). Moreover, as demonstrated by xenograft burden change and mRECIST analysis (Fig. 7D and Supplementary information 9C), whereas single-agent treatments with AMPC or Cabozantinib failed to achieve complete or partial responses in MDA-MB-361 xenografts, a proportion of xenografts treated with AMPC (37.5%) or Cabozantinib (12.5%) exhibited stable disease (mSD), contrasting with xenografts treated with the vehicle (0.0%), which showed progression (mPD). Remarkably, xenografts treated with the combination of AMPC and Cabozantinib displayed a partial response (mPR) in 12.5% of cases and mSD in 87.5% of cases. Furthermore, IHC and TUNEL analyses revealed a decreased proportion of MKI67-positive cells, and a higher incidence of apoptosis in the MDA-MB-361 xenograft specimens treated with single-agent treatments compared to those treated with the vehicle control (Supplementary information 9D, 9E). These effects were further enhanced in specimens treated with the combination of AMPC and Cabozantinib. Therefore, these results collectively provide evidence of the increased effectiveness of the combination treatment in suppressing MDA-MB-361 xenograft growth and halting disease progression. To further investigate the effects of the combined treatment on CSCs in vivo , IHC coupled with IRS score analysis was conducted on xenograft specimens. The results showed a decrease in TFF3 protein levels and concurrent decreased phosphorylation of c-MET in xenograft tissues after treatment with single-agent AMPC or Cabozantinib. The decreased phosphorylation of c-MET was significantly amplified when the AMPC-Cabozantinib combination was administered (Fig. 7E and Supplementary information 9F). Moreover, the diminished expression of TFF3 was validated by analyzing the relative levels of serum TFF3 in comparison to xenograft weight (Fig. 7F). Subsequently, the effect of combined targeting on CSC markers in the xenograft specimens was conducted. A significant decrease in the expression of CSC markers OCT4, BMI1, SOX2, ALDH1A1, and CD44 in xenograft samples from the groups treated with single drugs compared to the vehicle-treated control group was observed (Fig. 7G and Supplementary information 9G). The group receiving the combination treatment demonstrated a further significant suppression of the expression of these markers in comparison to the individual drug treatments. Next, the potential metastatic dissemination of MDA-MB-361 cells from the xenograft site to major organs was determined. Metastasis was first assessed in lung tissue sections using hematoxylin-eosin (H&E) staining (Fig. 7H and Supplementary information 9H). The H&E results revealed that there was reduced incidence of lung metastasis in both single-agent AMPC (6/8) and Cabozantinib (5/8) treated groups compared to the vehicle-treated group (7/8). Notably, the incidence of lung metastasis was dramatically reduced in the combined AMPC-Cabozantinib treated group (2/8). Quantification of metastatic nodules in the lungs further corroborated this reduction (Fig. 7H). Moreover, the human HPRT gene ( hHPRT ) was utilized to distinguish the metastatic burden of cells of human origin, as previously reported[44, 58]. The relative expression of hHPRT to m gapdh verified a significantly decreased metastatic burden in both single-agent AMPC and Cabozantinib treated groups compared to the vehicle-treated group (Fig. 7I). The reduced metastatic burdens were further confirmed through IHC and IRS score analysis of hHPRT protein in lungs from the different treatment groups (Fig. 7J). Collectively, these findings demonstrate the enhanced efficacy of the combined treatment in mitigating micrometastatic spread. In summation, combined treatment employing AMPC and Cabozantinib as a therapeutic strategy effectively controlled the growth of primary xenografts and reduced the tendency for lung metastasis in ER+HER2+ MC xenograft model. 8 TFF3 enhances c-MET signaling through a positive feedback loop to enhance the CSC-like phenotype of ER+ HER2+ MC A previous study has reported that the forced expression of TFF3 enhanced various RTK activities in ER+HER2+ MC, including c-MET, suggesting a potential regulatory relationship between TFF3 and c-MET[18]. To delineate this potential relationship, the phosphorylation of c-MET was assessed in MDA-MB-361 and BT474 cells. Western blot analysis revealed that the phosphorylation levels of c-MET at Tyrosine 1234/1235 were significantly elevated in MDA-MB-361 cells with forced expression of TFF3 compared to the vector transfected control (Fig. 8A). Conversely, the c-MET phosphorylation levels were markedly decreased in MDA-MB-361 cells with TFF3 depletion compared to control vector cells (Fig. 8A). Moreover, pharmacological inhibition of TFF3 by AMPC induced a dose-dependent decrease in c-MET phosphorylation at Tyrosine 1234/1235 in MDA-MB-361 and BT474 cells (Fig. 8B). Given the intricate interplay previously reported between HER2 and c-MET[59, 60], alongside their shared attributes as tyrosine receptor kinases and the findings from combination experiments herein demonstrating the suppressive effect of c-METis on TFF3 expression both in vitro and in vivo (Fig. 6D, 7E); whether there is a regulatory association between TFF3 and c-MET was therefore further investigated. It was observed that the expression of TFF3 was markedly suppressed in MDA-MB-361 and BT474 cells following c-MET depletion using two independent siRNAs targeting c-MET (siMET#1 and siMET2#2), when compared to the respective scrambled siRNA transfected cells (Fig. 8C and Supplementary information 10A). Furthermore, the expression of TFF3 exhibited a dose-dependent reduction upon treatment with c-METis in both cell lines (Fig. 8D). Since c-MET also appears to be functionally downstream of TFF3 activated pathways, Co-IP assays were conducted to investigate whether TFF3 itself may be an alternate ligand for c-MET. However, no association of TFF3 to c-MET was observed in MDA-MB-361 cells, whereas the reported c-MET ligand HGF demonstrated interaction between c-MET and HFG as a positive control (Fig. 8E and Supplementary information 10B). Therefore, the phosphorylation of c-MET enhanced by TFF3 may occur indirectly, as for other RTKs[26, 61, 62]. The synergistic inhibition of TFF3 by AMPC combined with c-METis effectively decreased CSC-like phenotype in ER+HER2+ MC cells both in vitro and in vivo (Fig. 6C and Supplementary information 7G). To further explore the effect of the TFF3-c-MET pathway on CSC-like behavior, ER+HER2+ MC cells with forced expression of TFF3 and with c-MET depletion were examined by western blot and ALDEFLUOR assays. The elevated population of ALDH1-positive cells observed in cells with forced expression of TFF3 was significantly reduced following c-MET depletion compared to the scrambled siRNA transfected cells (Fig. 8F and Supplementary information 10C). Western blot analyses demonstrated that the increased expression of CSC markers BMI1, SOX2, and ALDH1A1 in both MDA-MB-361 and BT474 cells with forced expression of TFF3 was mitigated upon c-MET depletion (Fig. 8G). Collectively, it is apparent that c-MET positively regulates its own signaling through TFF3 with consequent enhancement of CSC-like phenotype in ER+HER2+ MC cells. Discussion Despite significant advances in treatments for both HER2+ and ER+ MC, and the resultant survival benefits for affected patients, ER+HER2+ MC remains an underrepresented subgroup lacking sufficient tailored therapeutic options due to its distinct characteristics from either HER2+ or ER+ MC[63, 64]. Over the past decade, TFF3 has emerged as a promising therapeutic target due to its promotory role in cancer progression, including colorectal, hepatocellular, lung, pancreatic, prostate, cervical, endometrial, and ER+ mammary carcinomas[9, 10, 12-21]. Furthermore, inhibition of TFF3 has been shown to enhance the efficacy of ionizing radiation, Gemicitabine, Taxanes and MEK1/2 inhibitors, and to overcome resistance to 5-FU, anti-estrogen and HER2-targeted therapy[9, 12, 18]. Previous studies have reported that the pharmacological inhibition of TFF3 modulates the PI3K/AKT, MAPK, WNT and JAK/STAT3 signaling pathways in carcinoma cells[9, 12, 13, 20, 30]. Consistently, this study revealed that TFF3 possesses an oncogenic role in ER+HER2+ MC and demonstrated that TFF3 inhibition enhances the effectiveness of inhibition of c-MET. These findings suggest TFF3 as a novel promising target for combination therapeutic strategies in ER+HER2+ MC. CSCs are a critical subpopulation of tumor-initiating cells implicated in cancer relapse, metastasis, and resistance to radiotherapy and chemotherapy[65]. Breast cancer stem cells (BCSCs) were initially recognized based on the relative expression of CD44 and CD24[66]. CD44, CD24, and aldehyde dehydrogenase-1 (ALDH1) are now widely used as biomarkers for identifying BCSC characteristics[67]. One of the defining characteristics of ER+HER2+ MC is the presence of ALDH1+ epithelial BCSCs, which is associated with poor clinical prognosis[3]. Moreover, BCSCs have been implicated in the failure of endocrine therapy, chemotherapy, radiotherapy, and immunotherapy in MC treatment, ultimately promoting relapse[68]. Recently, several studies have reported that TFF3 promotes CSC-like phenotype in pancreatic, colorectal, hepatocellular, lung, cervical, and ER+ mammary carcinoma[10, 13, 15, 18-20, 69]. In ER+ MC patients, expression and activation of c-MET is significantly higher in metastatic sites than in primary sites[70]. Furthermore, c-MET expression has been strongly correlated with CSC markers, ALDH1A3 and CD133 in MC[71]. High c-MET expression and its activation are also suggested to be involved in the promotion of ALDH1A3 gene expression in the basal-like type of MC[71]. This investigation delineated the role of TFF3 in enhancing CSC-like phenotype in ER+HER2+ MC cells, as evidenced by heightened ALDH1 activity and increased spheroid formation capacity (Fig. 1G, 1H). Additionally, this study revealed that inhibiting TFF3 reduced CSC-like phenotype, and dual inhibition of TFF3 and c-MET led to a further reduction, suggesting a potential mechanism for the decreased metastatic burden observed with combinatorial therapy of AMPC and c-METis in ER+HER2+ MC. Intriguingly, the high-throughput anti-cancer compound screening assays demonstrated that TFF3 inhibition by AMPC synergized most effectively with compounds targeting four distinct RTKs, EGFR, VEGFR, c-KIT, and c-MET in MDA-MB-361 and BT474 cells. These RTKs have all been implicated in oncogenic progression and are potential targets for cancer therapy. Inhibiting EGFR, VEGFR, and c-MET, whether by single drugs or drug combinations, has proven beneficial by halting cell growth, proliferation and metastasis[72, 73] although resistance ultimately develops. Mechanistically, it has been previously shown that TFF3 can competitively bind with LINGO2 to disrupt EGFR-LINGO2 complexes, leading to the release of EGFR activity[74]. Additionally, targeting TFF3 with AMPC resulted in decreased EGFR activity in ER+MC cells[30]. The capacity of TFF3 to enhance the activation of the EGFR further indicates the importance of TFF3 as a therapeutic target in that TFF3 may modulate other RTKs through its involvement in multiple signaling pathways, including p44/42 MAPK[9], PI3K/AKT[28, 29] and STAT3[15, 16]. Given the reported functions of TFF3 in modulating RTK-mediated cellular functions[18, 30], including the data herein, it may be thus reasoned that RTK inhibition in cancer will be rendered more efficacious by TFF3 depletion or inhibition. To date, no FDA-approved c-MET inhibitor exists for MC, however clinical investigations are currently underway to assess the effectiveness of c-MET-targeted therapies in MC patients. Cabozantinib is a multi-kinase inhibitor targeting c-MET, VEGFR1-3, RET, AXL, FLT3, and c-KIT[72]. In a single-arm Phase II study recruited patients with ER+ MC and bone metastases treated with daily Cabozantinib (NCT01441947) demonstrated efficacy of Cabozantinib[57]. The clinical benefits of Cabozantinib were also explored in ER+ MC and HER2+ MC patients with brain metastases (NCT02260531) in a Phase II trial[75]. Clinical trials involving c-MET-targeted medications in MC have exhibited varied outcomes suggesting that a combination strategy with c-MET inhibition in ER+HER2+ MC may be more useful. In ER+HER2+ MC, increased expression of TFF3 has been implicated in trastuzumab-resistance, activating both the HER family of tyrosine kinases and crosstalk partners, including c-MET[18]. Moreover, crosstalk of c-MET signaling pathways with ER and HER2 signaling pathways has been reported[76, 77]. Stephen et al. reported the increased expression of c-MET along with a marked increase in the migratory and invasive capacity of Fulvestrant-resistant MC cells; and observed that increased expression of c-MET in endocrine therapy-resistant epithelial MC cells promoted cancer progression[76]. Furthermore, David et al. reported that c-MET is frequently co-expressed with HER2 in HER2+ MC and contributes to trastuzumab resistance of HER2+ MC cells through sustained AKT activation; whereas the loss of c-MET function, either through RNA interference–mediated depletion or small molecule–mediated inhibition, significantly improves the response to trastuzumab[77]. c-MET enhances the activation of PI3K/AKT and p44/42 MAPK signaling, two downstream signaling pathways also enhanced by TFF3[7], and which display heightened activity in ER+HER2+ MC and in lymph node metastases of this MC subtype[7, 72, 78]. This may explain the lack of a complete response observed for xenograft growth and lung metastasis in xenograft models with the combination treatment of AMPC and Cabozantinib (Fig. 7A and Supplementary information 9H). However, given the observed efficacy, it may be postulated that prolonging the treatment duration or optimizing the dosage/dose intensity may markedly enhance the therapeutic response. Additionally, supplementing with a third drug, such as selective estrogen receptor modulators (SERMs) or HER2 targeting agents, might provide a more efficacious approach to treating ER+HER2+ MC and improving patient outcomes. Consistent with a previous study for HER2 [18], it is herein hypothesized that c-MET regulates its own signaling; although through a positive feedback loop by TFF3 in ER+HER2+ MC cell lines. However, co-immunoprecipitation assays failed to demonstrate direct binding of TFF3 and c-MET (Fig. 8E). This is consistent with other RTKs, as TFF3-stimulated EGFR activation was achieved without direct binding or colocalization of TFF3 and EGFR[61, 62]. A possible mechanism through which TFF3 activates c-MET in ER+HER2+ MC is activation via crosstalk pathways including HER2, as TFF3 has been previously shown to activate HER2, a previously demonstrated heterodimeric partner of c-MET[18, 79, 80]. There may also exist a mechanism analogous to TFF3-LINGO2-EGFR[26] in which TFF3 sequesters proteins interacting with c-MET that inhibit its activation. The investigations herein highlight the pivotal role of TFF3 in the oncogenicity of ER+HER2+ MC cells and disease progression. This study therefore enhances the understanding of HER+ER+ MC progression by delineating the bidirectional control mechanisms involving c-MET and TFF3 in ER+HER2+ MC cells. TFF3 enhanced phosphorylation of c-MET and c-MET signaling was abrogated by TFF3 inhibition or depletion. Furthermore, TFF3 expression was increased by c-MET activation, and decreased by inhibition of c-MET. Hence, the complex molecular landscape of this subtype has been further clarified by the delineation of the interactions of TFF3 and c-MET signaling pathways, offering insight into the potential amelioration of targeted therapy for ER+HER2+ MC. The collective evidence suggests that targeting TFF3 and c-MET represents a promising and potentially efficacious treatment approach for addressing the unique challenges posed by ER+HER2+ MC. Declarations Acknowledgements This research was supported by the National Natural Science Foundation of China (82172618 and 82102768), China; Guangdong Basic and Applied Basic Research Foundation (2020A1515111064), China; the Shenzhen Key Laboratory of Innovative Oncotherapeutics (ZDSYS20200820165400003) (Shenzhen Science and Technology Innovation Commission), China; Universities Stable Funding Key Projects (WDZC20200821150704001), China; China Postdoctoral Science Foundation (2022M721894), China; Overseas Research Cooperation Project (HW2020008) (Tsinghua Shenzhen International Graduate School), China and The Shenzhen Bay Laboratory, Oncotherapeutics (21310031), China. Conflict of Interest The authors declare the following competing interests: P.E.L. have previously consulted for Perseis Therapeutics Ltd. P.E.L. are named on PCT application numbers WO 2006/69253 and WO 2008/042435 and US provisional application number 61/059558 and derivatives thereof. V.P., B.B., and P.E.L. are named as inventors on PCT application WO/2018/226155 (PCT/SG2018/050277), Compounds, As Inhibitors of TFF3 Dimerization, Methods and Applications Thereof (and derivatives thereof including US Patent 11,141,402). P.E.L. is an equity holder in Sinotar Pharmaceuticals Ltd which currently holds PCT/SG2018/050277 and derivatives thereof including issued US Patent no. 11,141,402. All other authors have no competing interests to declare. Availability of Data and Materials The data sets used in this study are available from the corresponding author on reasonable request. Author Contributions Peter E. Lobie, Vijay Pandey and Chuyu He designed the study; Basappa synthesized AMPC. Chuyu He, Xuejuan Wang, Yi-Shiou Chiou performed the experiments; Chuyu He, Xuejuan Wang, Yi-Shiou Chiou, Tao Zhu, Vijay Pandey and Peter E. Lobie analyzed the data; Chuyu He, Vijay Pandey and Peter E. Lobie wrote the manuscript. All authors read and approved the final manuscript. 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Additional Declarations (Not answered) Supplementary Files chuyuMS202408Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 08 Oct, 2024 Review # 2 received at journal 04 Oct, 2024 Review # 1 received at journal 28 Sep, 2024 Reviewer # 2 agreed at journal 17 Sep, 2024 Reviewer # 1 agreed at journal 16 Sep, 2024 Reviewers invited by journal 08 Sep, 2024 Submission checks completed at journal 28 Aug, 2024 First submitted to journal 27 Aug, 2024 Editor assigned by journal 27 Aug, 2024 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. 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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-4982898","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":351102778,"identity":"66aee45f-f0cf-41cc-8f40-999972f2460f","order_by":0,"name":"Peter Lobie","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-8445-184X","institution":"Tsinghua University","correspondingAuthor":true,"prefix":"","firstName":"Peter","middleName":"","lastName":"Lobie","suffix":""},{"id":351102779,"identity":"43ff4daf-54db-479d-819b-ef82b90a6593","order_by":1,"name":"Chuyu He","email":"","orcid":"https://orcid.org/0000-0001-6232-4594","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Chuyu","middleName":"","lastName":"He","suffix":""},{"id":351102780,"identity":"db316ad7-ec46-413c-91f6-d2a2364c26a7","order_by":2,"name":"Xuejuan Wang","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Xuejuan","middleName":"","lastName":"Wang","suffix":""},{"id":351102781,"identity":"6822ab09-c6a0-4770-b24c-5d46df34ac49","order_by":3,"name":"Yi-Shiou Chiou","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Shiou","middleName":"","lastName":"Chiou","suffix":""},{"id":351102782,"identity":"7ee85b37-ab2d-4887-9823-506a48d024c4","order_by":4,"name":"Basappa Basappa","email":"","orcid":"https://orcid.org/0000-0002-8844-468X","institution":"University of Mysore","correspondingAuthor":false,"prefix":"","firstName":"Basappa","middleName":"","lastName":"Basappa","suffix":""},{"id":351102783,"identity":"6b705f29-68d8-4d15-a341-3fbfdca4677a","order_by":5,"name":"tao Zhu","email":"","orcid":"https://orcid.org/0000-0003-4130-1144","institution":"University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"tao","middleName":"","lastName":"Zhu","suffix":""},{"id":351102784,"identity":"06c7f60b-cd36-4179-94e6-380820f49982","order_by":6,"name":"Vijay Pandey","email":"","orcid":"https://orcid.org/0000-0001-8704-0694","institution":"Tsinghua-Berkeley Shenzhen Institute and Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Vijay","middleName":"","lastName":"Pandey","suffix":""}],"badges":[],"createdAt":"2024-08-27 08:41:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4982898/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4982898/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07387-5","type":"published","date":"2025-02-07T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66323713,"identity":"7522e039-788d-4912-a698-28054df4fca3","added_by":"auto","created_at":"2024-10-10 12:26:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1227332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eForced expression of TFF3 enhances oncogenic behaviors in MDA-MB-361 cells.\u003c/strong\u003e MDA-MB-361 cells were stably transfected with \u003cem\u003epIRESneo3-TFF3\u003c/em\u003e (designated MDA-MB-361-TFF3 / TFF3) or \u003cem\u003epIRESneo3-vector\u003c/em\u003e (MDA-MB-361-VEC / VEC) plasmid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Total cell number counting. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e BrdU incorporation assay was performed after 12 hours of serum deprivation. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e CASPASE 3/7 activity was assessed after 12 hours of serum deprivation. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Apoptotic cell death was determined after 12 hours of serum deprivation by Annexin-V/PI. The percentages of cells in early or late apoptotic phase were analyzed. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Foci formation was assessed in MDA-MB-361-VEC and MDA-MB-361-TFF3 cells cultured in monolayer at low cell density for 14 days. Foci were stained by crystal violet and fold change in survival fraction was measured by eluting the crystal violet with methanol and detecting absorbance at 595 nm. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Microscopic visualization of LIVE/DEAD\u003csup\u003eTM\u003c/sup\u003e Cell Imaging kit-stained colonies was conducted after 12 days of cell culturing in medium containing 2% FBS and 4% Matrigel. The bright-field images were displayed on the left, while the merged images of live and dead cells were on the right, with red indicating dead colonies and green indicating live colonies. Scale bars, 100 μm. Fold change in cell viability of 3D colonies was determined by AlamarBlue (BioChip, Beijing, China). Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Spheroid formation assay was performed in MDA-MB-361-VEC and MDA-MB-361-TFF3 cells seeded in ultralow attachment plates and cultured in spheroid growth media for 12 days. Scale bar, 50 μm. The spheroids with diameters greater than 50 μm in each well were counted. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e ALDEFLUOR activity was measured after 12 hours of serum deprivation. The cells were then harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population. DEAB was used to establish the baseline fluorescence. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e xenograft formation was evaluated by xenograft volumes (mm\u003csup\u003e3\u003c/sup\u003e) every day until the 36 days. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u003c/strong\u003e Xenograft weight of each group in all animals after scarification. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK.\u003c/strong\u003e Representative micrographs and IRS of IHC staining for MKI67 in the indicated xenografts. Scale bar, 20 μm. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL.\u003c/strong\u003e Representative micrographs and IRS of TUNEL in the indicated xenografts. Scale bar, 20 μm. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"chuyuMSFIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/57674ac1689fbfc3ef3fa53e.png"},{"id":66325047,"identity":"c068dfc8-7f3a-4f0e-a61c-0cdb861cfd39","added_by":"auto","created_at":"2024-10-10 12:42:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1218318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepletion of TFF3 suppresses oncogenic behaviors in MDA-MB-361 cells.\u003c/strong\u003e MDA-MB-361 cells were stably transfected with \u003cem\u003epSilencersiTFF3\u003c/em\u003e (designated MDA-MB-361-shTFF3 / shTFF3) or empty vector (MDA-MB-361-shCtrl / shCtrl) plasmid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Total cell number counting. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e BrdU incorporation assay was performed after 12 hours of serum deprivation. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e CASPASE 3/7 activity was assessed after 12 hours of serum deprivation. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Apoptotic cell death was determined after 12 hours of serum deprivation by Annexin-V/PI. The percentages of cells in early or late apoptotic phase were analyzed. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Foci formation was assessed in MDA-MB-361-shCtrl and MDA-MB-361-shTFF3 cells cultured in monolayer at low cell density for 14 days. Foci were stained by crystal violet, and fold change in survival fraction was measured by absorbance at 595 nm. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Microscopic visualization of LIVE/DEAD\u003csup\u003eTM\u003c/sup\u003e Cell Imaging kit-stained colonies was conducted after 12 days of cell culturing in medium containing 2% FBS and 4% Matrigel. The bright-field images were displayed on the left, while the merged images of live and dead cells were on the right, with red indicating dead colonies and green indicating live colonies. Scale bars, 100 μm. fold change in cell viability of 3D colonies was determined by AlamarBlue (BioChip, Beijing, China). Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Spheroid formation assay was performed in MDA-MB-361-VEC and MDA-MB-361-TFF3 cells seeded in ultralow attachment plates and cultured in spheroid growth media for 12 days. Scale bar, 50 μm. The spheroids with diameters greater than 50 μm in each well were counted. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e ALDEFLUOR activity was measured after 12 hours of serum deprivation. The cells were then harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population. DEAB was used to establish the baseline fluorescence. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e xenograft formation was evaluated by xenograft volumes (mm\u003csup\u003e3\u003c/sup\u003e) every day until the 36 days. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u003c/strong\u003e Xenograft weight of each group in all animals after scarification. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK.\u003c/strong\u003e Representative micrographs and IRS of IHC staining for MKI67 in the indicated xenografts. Scale bar, 20 μm. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL.\u003c/strong\u003e Representative micrographs and IRS of TUNEL in the indicated xenografts. Scale bar, 20 μm. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 6). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"chuyuMSFIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/cbaff242d543db2ed5461d6c.png"},{"id":66323910,"identity":"4455f0ec-8cab-40a5-82b0-7cb7e981ca95","added_by":"auto","created_at":"2024-10-10 12:34:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1294007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMPC inhibited the oncogenicity of ER+ HER2+ MC cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e BrdU incorporation assays in MDA-MB-361 and BT474 cells treated with 0, 2.5, 5 or 10 µM AMPC for 3 days. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eCASPASE 3/7 activities were determined after 3 days of AMPC (0, 2.5, 5 or 10 µM) treatment. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Apoptotic cell death of MDA-MB-361 and BT474 cells was determined after 2 days of AMPC treatment (0, 2.5, 5 or 10 µM) by Annexin-V/PI. The percentages of early apoptotic and late apoptotic between are shown. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Foci formation was conducted with MDA-MB-361 and BT474 cells treated with 0, 2.5, 5 or 10 µM AMPC in monolayer culture at low cell density for 14 days. Foci were stained by crystal violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Microscopic visualization of colonies stained with the LIVE/DEAD™ Cell Imaging Kit after 12 days of AMPC treatment (0, 2.5, 5 or 10 µM) of MDA-MB-361 and BT474 cells cultured in medium containing 2% FBS and 4% Matrigel after 3 days of pre-culture. The bright-field images were shown above, and the merged images of live (green) and dead (red) colonies were shown below. Scale bars, 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Spheroid formation assay. MDA-MB-361 and BT474 cells were seeded in ultralow attachment plates and cultured in spheroid growth media for 12 days with AMPC treatment (0, 2.5, 5 or 10 µM). Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e ALDEFLUOR activity was measured after 3 days of AMPC treatment (0, 2.5, 5 or 10 µM). MDA-MB-361 and BT474 cells were then harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population. DEAB was used to establish the baseline fluorescence. Data are expressed as mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"chuyuMSFIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/f1e25ccfd473c14e138f9a41.png"},{"id":66323715,"identity":"b02bc566-420d-433f-bcc9-c176233ce5d0","added_by":"auto","created_at":"2024-10-10 12:26:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":816740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ec-METis are identified as the most synergistic compounds in combination with AMPC to decrease ER+HER2+ MC cell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Heatmap plot (https://hiplot.com.cn/) depicted the cell viability of MDA-MB-361 and BT474 cells post-treatment as Fraction affected (Fa) (Scale: Blue to Red). Fa was calculated as 1 - cell viability. The cell viability of cells with the combination’s treatment of AMPC (0, 5, 10 or 20 µM) with 247 compounds for 3 days was assessed by AlamarBlue (BioChip, Beijing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Synergistic pathways with AMPC were statistically analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Heatmap plot depicted the combination index (CI) of MDA-MB-361 and BT474 cells on the protein tyrosine kinase pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eCI analysis was conducted on compounds exhibiting synergistic effect with AMPC in both MDA-MB-361 and BT474 cells. CI was calculated by bliss independence method (Bliss CI=(E\u003csub\u003eA\u003c/sub\u003e+E\u003csub\u003eB\u003c/sub\u003e-E\u003csub\u003eA\u003c/sub\u003e×E\u003csub\u003eB\u003c/sub\u003e)/E\u003csub\u003eAB\u003c/sub\u003e). CI\u0026lt;1 indicated a synergistic effect. The red box highlighted the compounds that exhibited the high synergy with AMPC in both MDA-MB-361 and BT474 cells.\u003c/p\u003e","description":"","filename":"chuyuMSFIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/b0fefe2fa160a0aa4fd7092e.png"},{"id":66323720,"identity":"e6e822e8-f61a-441c-aa76-36a0b75903bc","added_by":"auto","created_at":"2024-10-10 12:26:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2176816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e MDA-MB-361 and BT474 cells were treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P) and the combination in medium containing 2% FBS and 4% Matrigel for 12 days after 3 days of pre-culture. Bright-field images showed the colonies, while merged images depicted Live/Dead staining, with red indicating dead colonies and green indicating live colonies. Scale bars, 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAMPC synergizes with c-METis to reduce ER+HER2+ MC cell survival and growth \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e MDA-MB-361 and BT474 cells were treated with the varying concentration (0.01, 0.1, 1, 10 or 100 μM) of AMPC (A) and Cabozantinib (C), SU11274 (S), or PHA-665752 (P) for 6 days. The survival fraction (SF) in response to AMPC (A) was assessed using a total cell number counting assay. Data are represented as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e CI was determined using the Chou-Talalay method, where CI \u0026lt; 1 indicated synergy, CI = 1 indicated additivity, CI \u0026gt; 1 indicated antagonism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eSynergy scores were calculated using bliss synergy analysis (www.synergyfinder.com), where synergy score\u0026gt; 0 indicated synergy, synergy score \u0026lt;0 indicated antagonism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Dose-response analysis of the shift in IC\u003csub\u003e50\u003c/sub\u003e of Cabozantinib (C), SU11274 (S), or PHA-665752 (P) in MDA-MB-361 and BT474 cells after co-treatment with AMPC (2.5 μM) was conducted with total cell number assay. The arrow indicats the fold reduction in IC\u003csub\u003e50\u003c/sub\u003e of the respective c-METis in the presence of AMPC. Data are represented as means ± SD (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Foci formation was performed on MDA-MB-361 and BT474 cells treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P) and the combination in monolayer culture at low cell density for 14 days. Foci colonies were stained by crystal violet.\u003c/p\u003e","description":"","filename":"chuyuMSFIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/9b3bc6916695ce5e255d7340.png"},{"id":66323913,"identity":"d0571ac1-4a63-4d88-b782-d9e17359a2d2","added_by":"auto","created_at":"2024-10-10 12:34:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":716074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMPC synergizes with c-METis to suppress CSC-like phenotype in ER+HER2+ MC Cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Cell migration and invasion assay was conducted on MDA-MB-361 and BT474 cells treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P) or the combination. Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e MDA-MB-361 and BT474 cells were seeded in ultralow attachment plates and cultured in spheroid growth media, and treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination for 12 days. The spheroids with diameters greater than 50 μm in each well were counted. Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eALDEFLUOR activity was measured after 3 days of treatment with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination. MDA-MB-361 and BT474 cells were then harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population, withDEAB used to establish the baseline fluorescence. Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Western blot analysis was conducted to assess the level of TFF3, c-MET and CSC related proteins in MDA-MB-361 and BT474 cells following treatment with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination for 3 days. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are indicated on the left.\u003c/p\u003e","description":"","filename":"chuyuMSFIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/8bcb4c6c73200cbc348a4feb.png"},{"id":66323718,"identity":"01ae640f-9f64-4269-ba1a-77ff6e56f6f6","added_by":"auto","created_at":"2024-10-10 12:26:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2038356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMPC synergizes with a c-METi to suppress the MDA-MB-361 xenograft growth and lung metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eThe volumes of\u003cstrong\u003e \u003c/strong\u003eMDA-MB-361 xenografts (mm\u003csup\u003e3\u003c/sup\u003e) were measured every day and calculated using the formula: 0.52 × length × [width]\u003csup\u003e2\u003c/sup\u003e for the treatments with vehicle (V), 20 mg/kg AMPC (A), 60 mg/kg Cabozantinib (C) and the combination of AMPC and Cabozantinib (A+C). Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 8). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Resected MDA-MB-361 xenografts of each treatment group at the termination of the experiments (\u003cem\u003en\u003c/em\u003e = 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Weights of resected MDA-MB-361-derived xenografts were determined at the termination of the experiments.\u003cstrong\u003e \u003c/strong\u003eData are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 8). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Xenograft burden change of MDA-MB-361 xenografts for each treatment group was calculated after sacrifice at the termination of the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Representative micrographs of IHC staining for TFF3 and c-MET in the indicated xenografts. Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eELISA analysis was conducted to measure serum levels of human TFF3 derived from xenografts.\u003cstrong\u003e \u003c/strong\u003eData are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 8). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eIRS score of ALDH1A1, SOX2, BMI1, and OCT4 was assessed. Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 8). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. \u003c/strong\u003eH\u0026amp;E staining and quantitative measurement of micrometastatic nodules were performed in lungs of MDA-MB-361 xenograft model. Scale bar, 20 μm.\u003cstrong\u003e \u003c/strong\u003eData are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 8). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003emRNA expression of human HPRT gene in the lungs at the termination of the experiments was detected. The dotted line represented the baseline expression of the human HPRT gene in mice without intravenous injection of MDA-MB-361 cells. Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 8). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u003c/strong\u003e Representative micrographs of IHC staining for HPRT were showed in the indicated lungs. Scale bar, 20 μm.\u003c/p\u003e","description":"","filename":"chuyuMSFIG7.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/92221f17d3dff17b84f95d76.png"},{"id":66323912,"identity":"0cbec1aa-d15f-46cf-8e2f-481564a4d2a5","added_by":"auto","created_at":"2024-10-10 12:34:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":855129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTFF3 enhances c-MET signaling through a positive feedback loop to enhance the CSC-like phenotype of ER+ HER2+ MC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Western blot analysis was conducted to assess the level of c-MET and c-MET phosphorylation at Y1234/1235 in MDA-MB-361 cells with TFF3 forced expression (V \u0026amp; T) and depletion (sh \u0026amp; shT). β-ACTIN was used as input control. The sizes of detected protein blots in kDa are on the left.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Western blot analysis was conducted to assess the level of c-MET and c-MET phosphorylation at Y1234/1235 in MDA-MB-361 and BT474 cells with AMPC (0, 0.1, 0.5, 1, 5 or 10 μM) treatment for 3 days. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Western blot analysis was performed to assess the level of TFF3 in MDA-MB-361 and BT474 cells transfected with scrambled siRNA, siMET#1 or siMET#2 plasmid. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Western blot analysis was performed to assess the level of TFF3 in MDA-MB-361 and BT474 cells treated with Cabozantinib (0, 0.02, 0.1, 0.2, 1 or 2 μM), SU11274 (0, 0.02, 0.1, 0.2, 1 or 2 μM) or PHA-665752 (0, 0.04, 0.2, 0.4, 2 or 4 μM) for 3 days. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eA potentialc-MET/TFF3 interaction in MDA-MB-361 cells was investigated by immunoprecipitation (IP) and immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e MDA-MB-361 and BT474 cells transfected with scrambled siRNA, siMET#1 or siMET#2 plasmid were harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population. DEAB was used to establish the baseline fluorescence. Data are expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Western blot analysis was utilized to examine the level of CSC-related proteins in MDA-MB-361 and BT474 cells transfected with scrambled siRNA, siMET#1 or siMET#2 plasmid. β-ACTIN was used as input control. The sizes of detected protein blots in kDa are shown on the left.\u003c/p\u003e","description":"","filename":"chuyuMSFIG8.png","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/2ba977d94350f87ac3768a49.png"},{"id":75695548,"identity":"fbda6af9-3e47-448c-b3cd-787de7b9ad14","added_by":"auto","created_at":"2025-02-07 08:10:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12723217,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/cff78fac-3bbf-456c-b969-2c93d297f19d.pdf"},{"id":66323721,"identity":"067f6db6-0c84-4041-806b-9ac522c294ac","added_by":"auto","created_at":"2024-10-10 12:26:35","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":5058631,"visible":true,"origin":"","legend":"","description":"","filename":"chuyuMS202408Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4982898/v1/3171eb8464ca54ebcc0feb9b.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Inhibition of TFF3 Synergizes with c-MET Inhibitors to Decrease the CSC-like Phenotype and Metastatic Burden in ER+HER2+ Mammary Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMammary carcinoma (MC) is a heterogeneous disease comprising multiple subtypes with unique biological characteristics that influence responses to treatment modalities and clinical outcomes[1]. Human epidermal growth factor receptor 2 (HER2) gene amplification occurs in 13\u0026ndash;22% of MC cases, characterized by increased proliferation, tumor growth and metastatic potential[2, 3], contributing to aggressive behavior with an unfavorable clinical prognosis. Approximately 60\u0026ndash;70% of HER2+ MC cases also express estrogen receptor-\u0026alpha; (ER\u0026alpha;)[3]. This molecular overlap defines the ER+HER2+ MC subtype, representing approximately 10% of MC cases[4]. Research has demonstrated that administration of combined anti-estrogen and HER2-targeted therapy to ER+HER2+ MC patients significantly improved overall response rate (ORR), clinical benefit rate (CBR), and progression-free survival (PFS) compared to anti-estrogen monotherapy[5]. However, reports have identified a complex bidirectional crosstalk between the ER and HER2 pathways, an intrinsic interaction that impedes therapeutic effectiveness[3, 4]. Furthermore, current treatment approaches for ER+HER2+ MC have limited therapeutic efficacy due to the constraints of anti-estrogen and HER2-targeted therapies and lack of predictive markers for therapeutic resistance[4, 6]. These challenges in ER+HER2+ MC necessitate the exploration of targeted therapies that utilize novel regulatory mechanisms.\u003c/p\u003e\n\u003cp\u003eTrefoil factor 3 (TFF3) is a 59 amino acid peptide initially observed to be secreted by intestinal goblet cells and reported to effect mucosal repair in the gastrointestinal tract[7]. Recent findings have suggested that the biological role of TFF3 extends beyond mucosal protection, and is also associated with oncogenic progression in different types of cancer[7]. TFF3 expression is elevated and often associated with prognosis in gastric[8], colorectal[9], hepatocellular[10], thyroid[11], lung[12], pancreatic[13], prostate[14], cervical[15], endometrial[16], and ER+ mammary carcinomas[17-21]. Functionally, TFF3 has been reported to interact with CXCR4/7[22], CD147[23], CRP-ductin/DMBT1\u003csup\u003egp340\u003c/sup\u003e\u003csup\u003e[24]\u003c/sup\u003e, PAR2[25], and LINGO2[26]. In addition, TFF3 enhances the activation of HER1-4[18], c-MET[18], IGFR1[18], and c-SRC[18, 19], and increases the expression of multiple WNT ligands[13], thereby promoting downstream signaling pathways including p44/42 MAPK[9], NF-\u0026kappa;B[27], PI3K/AKT[28, 29] STAT3[15, 16], and WNT[13] to promote cancer cell survival, proliferation, a cancer stem cell like (CSC-like) phenotype, invasion, migration and metastasis[7, 30]. In ER+HER2+ MC, a bidirectional crosstalk between ER and HER2 contributes to resistance against both anti-estrogens and HER2-targeted therapies[3, 31], with TFF3 implicated in this resistance mechanism[17, 18, 20, 32]. As an estrogen-responsive gene[32], TFF3 is positively correlated with ER+ status (at least in Caucasian MC patient cohorts), and its high expression is associated with a poor prognosis in ER+ MC patients following endocrine therapy[17, 32-34]. Furthermore, TFF3 has been demonstrated to enhance ER\u0026alpha; transcriptional activity in MC, leading to the stimulation of estrogen-independent proliferation and reduced responsiveness to anti-estrogens[17, 20, 32]. Specifically, TFF3 is elevated in MC resistant to tamoxifen[17, 20] and aromatase inhibition[35]. Conversely, TFF3 depletion or inhibition resensitizes resistant cells to the respective anti-estrogen treatment[17, 20, 35]. As indicated above, a previous study has suggested bidirectional functional cross-regulation between HER2 and TFF3, partially independent of ER\u0026alpha; in ER+HER2+ MC[18]. Specifically, TFF3 expression is decreased by HER2 activation and increased upon trastuzumab inhibition of HER2; and TFF3 is functionally involved in mediating acquired trastuzumab resistance in ER+HER2+ MC[18]. Given that the dysregulation of ER and HER pathways in MC can be the result of the activation of downstream signaling pathways including p44/42 MAPK and PI3K/AKT[36] which are promoted by TFF3, the reported high TFF3 expression[19] and its role in ER and HER2 signaling [17, 18, 20, 32], targeting TFF3 offers a potential therapeutic approach for ER+HER2+ MC to overcome the resistance of anti-estrogens and/or HER2-targeted therapies and improve clinical outcomes for patients.\u003c/p\u003e\n\u003cp\u003eAs a transmembrane receptor tyrosine kinase (RTK), c-MET has been identified as a receptor for hepatocyte growth factor (HGF) and documented to be increased in expression and activity in MC[37, 38]. c-MET amplification plays a crucial role in in MC mediating cellular responses including proliferation, migration, and invasion[38], demonstrating a function in maintaining an oncogenic phenotype. The co-expression and activation of c-MET with other RTKs holds prognostic importance in MC, lung carcinoma and glioma[39]. Furthermore, the interplay between c-MET and other RTKs significantly impacts resistance to anti-RTK treatments, which is a critical concern in MC therapy[38, 40].\u003c/p\u003e\n\u003cp\u003eConsidering that targeted therapies often face challenges such as the development of resistance, combinations that target complimentary pathways or potential escape mechanisms appear to be more effective than single or sequential therapy[41]. Herein, novel combinatorial therapy strategies were investigated to identify potential strategies that may improve treatment outcomes in ER+HER2+ MC. High-throughput screening of 247 anticancer compounds in combination with a small molecule inhibitor of TFF3 (named AMPC) was performed in MDA-MB-361 and BT474 cells. The results demonstrated that targeting TFF3 not only enhances the sensitivity of cells to protein tyrosine kinase inhibitors but also prevents the feedback mechanisms consequent to utilization of these inhibitors, thereby achieving superior synergistic efficacy. Among these agents, c-MET inhibitors exhibited high synergy with TFF3 inhibition. The synergistic actions of the drug combinations in the treatment of ER+HER2+ MC \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e as well as \u003cem\u003ein vivo\u003c/em\u003e, using orthotopic ER+HER2+ MC xenograft models was demonstrated. These results indicated that a combination inhibition of TFF3 and c-MET inhibition may serve as a novel therapeutic strategy for ER+HER2+ MC patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003e1 Cell culture and transfection\u003c/h3\u003e\n\u003cp\u003eMDA-MB-361 and BT474 cell lines were obtained from BeNa Culture Collection Co. Ltd (Suzhou, China) and Procell Life Science \u0026amp; Technology Co. Ltd (Wuhan, China), respectively. MDA-MB-361 cells were cultured at 37 °C in a CO\u003csub\u003e2\u003c/sub\u003e-free incubator with L15 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (Thermo Fisher Scientific, Waltham, MA, USA). BT474 cells were maintained at 37 °C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator with RPMI-1640 medium, supplemented with 20% FBS, 1% P/S, 2 mM L-glutamine, and 10 ng/mL insulin. All experiments were performed in the respective media containing 2%FBS.\u003c/p\u003e\n\u003cp\u003eAs previous studies reported[17], MDA-MB-361 and BT474 cells underwent stable transfection with the \u003cem\u003epIRESneo3-TFF3 plasmid\u003c/em\u003e to force the expression of TFF3, facilitated by Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Simultaneously, cells were transfected with the \u003cem\u003epIRESneo3-vector plasmid\u003c/em\u003e as respectively empty control. G418 selection was applied at a concentration of 400 µg/mL for 4 weeks. Moreover, MDA-MB-361 cells underwent transfection with a \u003cem\u003eshRNA plasmid\u003c/em\u003e targeting TFF3 to generate stable TFF3-depleted cells, employing a control \u003cem\u003eshRNA plasmid\u0026nbsp;\u003c/em\u003efor comparison. Hygromycin B selection was utilized at a concentration of 200 µg/mL for 4 weeks[18]. These established cell lines are denoted as MDA-MB-361-TFF3, MDA-MB-361-VEC, BT474-TFF3, BT474-VEC, MDA-MB-361-shTFF3, and MDA-MB-361-shCtrl cells. For transient RNA interference, MDA-MB-361 and BT474 cells were transfected with plasmids containing scrambled siRNA, siMET #1, or siMET #2 sourced from GENEWIZ (Suzhou, China). The specifications for siMET #1 and siMET #2 employed in the study were as following,\u003c/p\u003e\n\u003cp\u003esiMET #1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5'-AGAAUGUCAUCAUUCAGGCTT-3'\u003c/p\u003e\n\u003cp\u003esiMET #2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5'-UACUCAGCAACCUUCUGAAGGTT-3'\u003c/p\u003e\n\u003ch3\u003e2 Reagents\u003c/h3\u003e\n\u003cp\u003eThe 247 anti-cancer compounds library (Cambridge Cancer Compound Library, Catalog No. L2300) utilized in this study was acquired from SelleckChem (Houston, TX, USA). Additionally, three c-MET inhibitors, namely Cabozantinib, SU11274, and PHA-665752 were sourced from SelleckChem (Houston, TX, USA).\u003c/p\u003e\n\u003ch3\u003e3 Western blot (WB) analysis\u003c/h3\u003e\n\u003cp\u003eRadio Immunoprecipitation Assay (RIPA) Lysis buffer was employed for cell lysis, followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) to fractionate the proteins within the cell lysate. WB analysis was carried out following established protocols[42]. Immunoblots were developed using a Gel Imager System (Bio-Rad, USA). Details of the antibodies utilized are provided in Supplementary information 11.\u003c/p\u003e\n\u003ch3\u003e4 Cell function assays and Enzyme-Linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eThe monolayer cell viability, Annexin-V/Propidium Iodide (PI) staining, CASPASE 3/7 activity, foci formation, 3D-Matrigel growth, and spheroid formation assays were conducted according to established protocols[43, 44]. For the monolayer cell viability assay, 3000 cells were seeded in 2% serum medium in 96-well plate and incubated for 6 days, using the AlamarBlue assay reagent (BioChip, Beijing, China). To analyze apoptotic cell death, cells were seeded at 50-70% confluency in a 6-cm dish and allowed to adhere overnight before treatment. After a 3-day drug treatment period, the cells were harvested and resuspended in 400 μL of Annexin V binding buffer per sample. Subsequently, 5 μL of Annexin V-Alexa Fluor 488 staining solution was added, followed by a 15-minute incubation period in the absence of light. 5 μL of 7-AAD staining solution was then introduced, followed by a subsequent 5-minute incubation. Samples were analyzed within 30 minutes using a Cytoflex Flow Cytometry system (Beckman Coulter, CA, USA). For CASPASE 3/7 activity assay, cells were seeded at a density of 2×10\u003csup\u003e4\u003c/sup\u003e cells per well in a 96-well plate and evaluated 3 days post-seeding or drug treatment using the Caspase-Glo\u003csup\u003e®\u003c/sup\u003e 3/7 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. For foci formation assay, 3000 cells were plated into 24-well plates in the medium supplemented with 2% FBS at 37 °C for 2 weeks until foci formation. Then, the foci were fixed with formalin and stained with crystal violet (Sigma-Aldrich, MO, USA). Survival fraction was measured by eluting the crystal violet with methanol and absorbance was detected at 595 nm. 3D-Matrigel assays were performed in 48-well plates coated with Matrigel (Corning, MA, USA) and allowed to solidify for 30 minutes. 2000 cells were then plated to the pre-coated plates in the medium supplemented with 2% FBS and 4% Matrigel for 12 days. Medium was refreshed every 3 days. AlamarBlue (BioChip, Beijing, China) was used to determine cell viability at the end of the experiment. In spheroid formation assay, 1000 cells were seeded in a 24-well ultralow attachment plate (Corning, MA, USA) culturing with serum-free medium supplemented with P/S, 10 ng/mL recombinant human basic FGF, 20 ng/ml recombinant human EGF, 2% B27, and 5 μg/ml bovine insulin. Mammosphere formation was observed under a microscope (Olypus, Tokyo, Japan) after 12 days of seeding. Mammospheres with diameter exceeding 50 μm were counted.\u003c/p\u003e\n\u003cp\u003eThe concentration of secreted TFF3 from cells and serum TFF3 levels were determined using the Quantikine\u003csup\u003e®\u003c/sup\u003e ELISA Human TFF3 Immunoassay kit (R\u0026amp;D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol. Cells were plated at a density of 3×10\u003csup\u003e5\u003c/sup\u003e cells in a 6 cm dish and cultured 600 μL of serum-free medium for 2 days. Subsequently, the supernatant was collected as the sample. \u0026nbsp;Furthermore, a fresh blood sample was obtained from the cardiac puncture of a euthanized mice and subjected to centrifugation at 4°C for 10 minutes at 3000 rpm to isolate the serum, which was then stored at -80°C for subsequent analyses.\u003c/p\u003e\n\u003ch3\u003e5 ALDEFLUOR assay\u003c/h3\u003e\n\u003cp\u003eThe ALDEFLUOR assay was performed using the ALDEFLUOR assay kit (STEMCELL Technologies, Vancouver, Canada) in accordance with the manufacturer’s instructions. Cells were seeded at 50-70% confluency in 6-well plate and were exposed to the respective drugs for 3 days. The ALDEFLOUR activity was assessed via fluorescence-activated cell sorting (FACS) analysis. Re-suspended cells were exposed to the ALDEFLUOR substrate (BAAA, BODIPY®-aminoacetaldehyde) to identify the ALDH1-positive population, while the baseline fluorescence was established using a specific ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB).\u003c/p\u003e\n\u003ch3\u003e6 Real time quantitative polymerase chain reaction (qPCR) analysis\u003c/h3\u003e\n\u003cp\u003eTissue samples were first rinsed with sterile saline and subsequently lysed in TRIzol (Sigma-Aldrich, MO, USA) for subsequent RNA extraction. Total RNA extraction followed established protocols, which included DNase I treatment, conversion of total RNA to complementary DNA (cDNA), PCR, and qPCR assays, conducted as previously described[45]. The qPCR procedure was carried out according to the outlined methodology[45]. The primers employed for qPCR were as follows,\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ehHPRT1\u003c/em\u003e forward: 5'-TTCCTTGGTCAGGCAGTATAATCC-3'\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ehHPRT1\u003c/em\u003e reverse: 5'-AGTCTGGCTTATATCCAACACTTCG-3'\u003c/p\u003e\n\u003cp\u003e\u003cem\u003emgapdh\u003c/em\u003e forward: 5'-CTCACTCAAGATTGTCAGCAATG-3'\u003c/p\u003e\n\u003cp\u003e\u003cem\u003emgapdh\u003c/em\u003e reverse: 5'-CACATTGGGGGTAGGAACAC-3′\u003c/p\u003e\n\u003ch3\u003e7 Co-immunoprecipitation (Co-IP) assay\u003c/h3\u003e\n\u003cp\u003eWhole-cell lysates were obtained by extracting cells with a cell lysis buffer containing protease inhibitor cocktail (TargetMol, Shanghai, China). Subsequently, the normalized lysates were incubated overnight at 4 °C with gentle agitation after adding 5 µg of primary or anti-IgG antibodies. The lysate–antibody mixture was then incubated with magnetic protein G DynabeadsTM (Thermo Fisher Scientific, Waltham, MA, USA) and washed thrice with protein binding buffer (composed of 150 mM NaCl, 20 mM Tris pH 8.0, 1% NP-40, and supplemented with protease and phosphatase inhibitors). The immunoprecipitant was eluted in sample buffer containing 1% β-mercaptoethanol and subjected to SDS-PAGE immunoblotting. Details of the antibodies used are listed in Supplementary information 11.\u003c/p\u003e\n\u003ch3\u003e8 Xenografts\u003c/h3\u003e\n\u003cp\u003eAll animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Laboratory Animal Centre of Peking University Shenzhen Graduate School (Certificate number: YW) and the “Ethical Development no. 9 (year 2020)” from Tsinghua Shenzhen International Graduate School as previously described[20]. Specific pathogen free (SPF) female BALB/c athymic nude mice were obtained from Guangdong Vital River Laboratory Animal Technology Co. (Foshan, Guangdong, China) and housed in a SPF animal facility with ad libitum access to clean water and food. After a one-week acclimation period, eight mice were randomly assigned to each subgroup. Mice were subcutaneously implanted with 0.72 mg 90-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, FL, USA) at the neck back. Following a 3-day interval, 1 × 10\u003csup\u003e7\u003c/sup\u003e MDA-MB-361 cells were orthotopically implanted into the right fourth mammary fat pad of mice to establish xenograft model. Animal weight and xenograft volume were monitored daily. The xenograft volume was calculated using formula 0.52 × (length × width\u003csup\u003e2\u003c/sup\u003e)[19]. Once the xenograft volumes reached approximately 80-100 mm\u003csup\u003e3\u003c/sup\u003e, xenograft bearing mice were intraperitoneally administered with vehicle (1% DMSO / 10% PEG400 in distilled saline), 20 mg/kg AMPC or intragastrically administered with 60 mg/kg Cabozantinib (SelleckChem, Houston, TX, USA). The mice were sacrificed after a two-week treatment period.\u003c/p\u003e\n\u003ch3\u003e9 Immunohistochemistry (IHC) staining and TUNEL assays\u003c/h3\u003e\n\u003cp\u003eIHC staining was conducted employing the labeled streptavidin-biotin-peroxidase complex method[13]. TUNEL assay was performed by using TUNEL Assay Kit (Abcam, Waltham, MA, USA), as previously described[13, 21]. Subsequent scoring of IHC staining was performed utilizing the immunoreactive score (IRS) method[46]. Two independent researchers, blinded to the clinical data, meticulously assessed and validated the staining results. Details regarding the antibodies employed are provided in Supplementary information 11.\u003c/p\u003e\n\u003ch3\u003e10 Statistical analysis\u003c/h3\u003e\n\u003cp\u003eGraphical representations and statistical analyses were performed utilizing GraphPad Prism 9 (GraphPad Software, Inc., CA, USA). The statistical significance between two groups was assessed using a two-tailed unpaired Student’s t-test, while analysis of variance (ANOVA) was employed for comparisons among multiple treatment groups. Statistical significance thresholds were set at *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Data conforming to a normal distribution were presented as mean ± standard deviation (SD).\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003e1 Forced expression of TFF3 enhances oncogenic behavior in ER+HER2+ MC cells\u003c/h3\u003e\n\u003cp\u003eTo elucidate the functions of TFF3 in ER+HER2+ MC, MDA-MB-361 and BT474 cells stably transfected with TFF3 cDNA were generated and validated through western blot analysis (Supplementary information 1A, 2A). Both MDA-MB-361 and BT474 cells with forced expression of TFF3 demonstrated a significant increase in cell proliferation, as observed by total cell number (Fig. 1A and Supplementary information 2B) and BrdU incorporation assays (Fig. 1B and Supplementary information 2C), compared to the respective vector-transfected ER+HER+ MC cell lines. Additionally, ER+HER+ MC cells stably transfected with TFF3 demonstrated a significant decrease in CASPASE 3/7 activity (Fig. 1C and Supplementary information 2D) and reduced apoptotic cell death, particularly in the late stage of apoptosis (Fig. 1D and Supplementary information 1B, 2E), compared to their respective vector transfected counterparts.\u003c/p\u003e\n\u003cp\u003eLoss of contact inhibition and anchorage-independent growth is a key hallmark of oncogenic transformation and cancer progression[47]. In order to examine and assess this characteristic of cancer cells \u003cem\u003ein vitro\u003c/em\u003e, foci formation assays were performed. MDA-MB-361-TFF3 and BT474-TFF3 cells exhibited enhanced anchorage-independent growth as compared to MDA-MB-361-VEC and BT474-VEC cells (Fig. 1E and Supplementary information 2F). In 3D-Matrigel culture, which more closely mimics \u003cem\u003ein vivo\u003c/em\u003e conditions, ER+HER2+ MC cells with forced expression of TFF3 demonstrated higher viability, characterized by increased number of live cells and reduced number of dead cells compared to their vector-transfected counterparts (Fig. 1F and Supplementary information 2G). These results demonstrated that TFF3 promotes proliferation, cell survival and anchorage-independent growth in ER+HER2+ MC cells \u003cem\u003ein vitro\u003c/em\u003e and promotes cancer cell colony growth \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eCancer cell migration and invasion are pivotal for cancer progression, and directly contribute to metastatic dissemination[47]. Transwell assays were conducted to assess the effect of forced expression of TFF3 on the migratory and invasive capacities of ER+HER2+ MC cells. The results demonstrated that forced expression of TFF3 in MDA-MB-361 cells led to increased migration and invasion compared to vector-transfected cells (Supplementary information 1C, 1D). Similarly, in BT474 cells, forced expression of TFF3 resulted in increased migration and invasion (Supplementary information 2H, 2I).\u003c/p\u003e\n\u003cp\u003eCSCs play a crucial role in MC progression, possessing the ability for self-renewal and tumor initiating capacity[48]. Prior studies have reported elevated TFF3 expression in breast cancer stem cells (BCSCs)[18, 20]. Herein, the effect of TFF3 on CSC-like phenotype of ER+HER2+ MC cells was assessed. Spheroid formation assays revealed that the number of spheroids in MDA-MB-361 and BT474 cells with forced expression of TFF3 were higher in comparison to their respective vector-transfected counterparts (Fig. 1G and Supplementary information 2J). Consistent results were observed by ALDEFLUOR assay, which demonstrated that MDA-MB-361 and BT474 cells with forced expression of TFF3 exhibited a significantly higher population of ALDH1-positive cells compared to the vector transfected control (Fig. 1H and Supplementary information 1E, 2K). Hence, TFF3 promotes the CSC-like phenotype in ER+HER2+ MC cells.\u003c/p\u003e\n\u003cp\u003eSubsequently, the impact of TFF3 on ER+HER2+ MC growth \u003cem\u003ein vivo\u003c/em\u003e was assessed in a MDA-MB-361 xenograft model. Consistent with the \u003cem\u003ein vitro\u003c/em\u003e findings, MDA-MB-361 xenografts generated by cells with forced expression of TFF3 exhibited significant increases in both volume and weight compared to vector transfected cells at the end of the experiment (Fig. 1I, 1J). Further analysis of xenograft specimens by IHC revealed a higher proportion of MKI67-positive cells (Fig. 1K), indicative of increased proliferation, and a reduction in apoptosis indicated by TUNEL analysis, in the xenografts generated by MDA-MB-361 cells with forced expression of TFF3, compared to the vector transfected cell generated xenografts (Fig. 1L). These results indicate that forced expression of TFF3 promotes \u003cem\u003ein vivo\u003c/em\u003e growth of MDA-MB-361 cells. Hence, the forced expression of TFF3 augmented the oncogenicity of ER+HER2+ MC cells both \u003cem\u003ein vitro\u003c/em\u003e,\u003cem\u003e\u0026nbsp;ex vivo\u0026nbsp;\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003e2 Depletion of TFF3 suppresses oncogenic behaviors in ER+HER2+ MC cells\u003c/h3\u003e\n\u003cp\u003eA MDA-MB-361 cell model with depleted TFF3 expression was generated by a shRNA-based approach (designed as MDA-MB-361-shTFF3) and validated through western blot analysis (Supplementary information 3A). Resulting functional consequences were investigated by using approaches similar to those used to examine the consequences of forced expression of TFF3 (Fig. 1, Supplementary information 1-2). TFF3-depleted MDA-MB-361 cells exhibited a significant decrease in total cell number (Fig. 2A) and BrdU incorporation (Fig. 2B). Additionally, depleted expression of TFF3 significantly increased CASPASE 3/7 activity and promoted apoptosis (Fig. 2C, 2D and Supplementary information 3B). TFF3-depleted MDA-MB-361 cells demonstrated markedly reduced foci formation (Fig. 2E). Similarly, in 3D-Matrigel culture, MDA-MB-361-shTFF3 cells formed fewer colonies and demonstrated reduced viability, characterized by a higher number of dead cells and fewer live cells as compared to the vector control cells (Fig. 2F). The depletion of TFF3 significantly reduced migration and invasion of MDA-MB-361 cells (Supplementary information 3C, 3D). Additionally, spheroid formation assays indicated that TFF3-depleted MDA-MB-361 cells formed significantly fewer spheroids compared to the control vector cells (Fig. 2G). Consistently, ALDEFLUOR assay results revealed a significantly lower population of ALDH1-positive cells in TFF3-depleted MDA-MB-361 cells (Fig. 2H and Supplementary information 3E).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e growth of TFF3-depleted MDA-MB-361 cells was also evaluated. The results demonstrated that TFF3 depleted MDA-MB-361 cell generated xenografts exhibited significantly reduced growth in volume compared to the control vector xenografts, a finding further confirmed by the lower xenograft weights at the end of the experiment (Fig. 2I, 2J). IHC and TUNEL analysis additionally revealed a decreased proportion of MKI67-positive cells, and higher apoptosis in the TFF3-depleted xenografts compared to the control vector cell generated xenografts (Fig. 2K, 2L). Thus, the depletion of TFF3 decreased oncogenicity of ER+HER2+ MC cells both \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003eex vivo\u0026nbsp;\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003e3 AMPC inhibited the oncogenicity of ER+HER2+ MC cells \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;ex vivo\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eTFF3 has been reported to form homodimers via the seventh cysteine (Cys57) residue, and the homodimeric form of TFF3 plays a critical role in inhibiting apoptosis[7, 30]. Recently, a small-molecule inhibitor named AMPC (\u003cem\u003e2-amino-4-(4-(6-fluoro-5-methylpyridin-3-yl)phenyl)-5-oxo-4H,5H-pyrano [3,2-c]chromene-3-carbonitrile\u003c/em\u003e) was developed that interfered with dimerization of TFF3 via the Cys57 residue[30]. AMPC leads to the rapid degradation of the monomeric form of TFF3, thereby diminishing TFF3-mediated signaling pathways crucial for cancer cell survival[9, 12, 13, 20, 30]. Given the observed oncogenic functions of TFF3 in ER+HER2+ MC cells, AMPC was employed to investigate the effect and therapeutic potential of small molecule mediated inhibition of TFF3 in ER+HER2+ MC cells.\u003c/p\u003e\n\u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e value of AMPC in MDA-MB-361 (3.206 ± 0.757 µM) and BT474 cells (2.268 ± 0.605 µM) was determined using total cell number assays (Supplementary information 4A). Consistent with the effect of siRNA mediated depletion of TFF3, inhibition of TFF3 by AMPC decreased the BrdU incorporation of MDA-MB-361 and BT474 cells, and in a dose-dependent manner (Fig. 3A). Concomitantly, CASPASE-3/7 activity increased with increasing concentrations of AMPC in both MDA-MB-361 and BT474 cell lines (Fig. 3B). Moreover, the proportion of cells undergoing early and late apoptosis rose with increasing AMPC concentrations in the respective cell lines (Fig. 3C and Supplementary information 4B). In addition, AMPC dose dependently inhibited MDA-MB-361 and BT474 cell anchorage-independent growth, as demonstrated by foci formation assays (Fig 3D and Supplementary information 4C). Similarly, the viability of 3D-Matrigel colonies formed by MDA-MB-361 and BT474 cells exhibited a decrement relative to the increasing dose of AMPC. This was evidenced by a discernible decrease in green fluorescence (indicating live cells) and a concomitant increase in red fluorescence (indicating dead cells), as illustrated in Fig 3E and Supplementary information 4D.\u003c/p\u003e\n\u003cp\u003eFurthermore, the effect of AMPC on the CSC-like phenotype of MDA-MB-361 and BT474 cells were further examined. Increasing concentrations of AMPC led to a dose-responsive decrease in the size and number of spheroids in both MDA-MB-361 and BT474 cell lines (Fig 3F and Supplementary information 4E). Consistently, it was observed that AMPC dose dependently decreased the percentage of the ALDH1-positive cell population in both MDA-MB-361 and BT474 cells (Fig 3G and Supplementary information 4F). These findings indicated that pharmacological inhibition of TFF3 reduced cell viability by suppressing proliferation and inducing apoptosis, and that AMPC is a potent and effective inhibitor of cell proliferation, survival, oncogenicity, 3D-growth, and CSC-like behavior in ER+HER2+ MC cells.\u003c/p\u003e\n\u003ch3\u003e4 c-MET inhibitors (c-METis) identified as the most synergistic compounds in combination with AMPC to decrease ER+HER2+ MC cell viability\u003c/h3\u003e\n\u003cp\u003eCombination therapy improves therapeutic efficacy compared to single-drug treatment by enhancing cytotoxicity and reducing the development of drug resistance in cancer cells[49]. To explore the therapeutic potential of AMPC-based combinations for the treatment of ER+HER2+ MC cells, the Cambridge Anti-Cancer Compound Library was screened in combination with varying concentrations of AMPC (0, 5, 10 or 20 µM) in MDA-MB-361 and BT474 cells, revealing inhibition of cell viability (Fig. 4A). This comprehensive library comprises 247 anti-cancer compounds as illustrated in Fig. 4A. Combination index (CI) analysis[50] revealed that 107 compounds exhibited synergy with AMPC in MDA-MB-361 cells, whereas 79 compounds showed synergy in BT474 cells. Among these, 40 compounds demonstrated synergistic effects with AMPC in both cell lines with detailed information listed in Supplementary information 5. The prominent pathways for the 40 synergistic compounds included protein tyrosine kinase, DNA damage, endocrinology \u0026amp; hormones, cell cycle, cytoskeletal signaling, and epigenetics. Notably, the protein tyrosine kinase pathway was targeted by 7 out of the 40 compounds, comprising 17.5% of all synergistic compounds and ranking first among them (Fig 4B). Given the role of TFF3 in activating RTKs in ER+HER2+ MC[18], the 7 identified compounds in the protein tyrosine kinase pathway primarily targeted four distinct RTKs, c-MET, EGFR, VEGFR, and c-KIT (Fig 4C). Further scatterplot regression analysis of CI values for compounds demonstrating synergy with AMPC in MDA-MB-361 and BT474 cells revealed that the combination of two c-MET inhibitors, PHA-665752 and SU11274, with AMPC exhibited the highest synergy, characterized by low CI values in both MDA-MB-361 and BT474 cells (Fig 4D). These findings indicated a synergistic effect of c-MET inhibitors in combination with AMPC in decreasing viability of ER+HER2+ MC cells.\u003c/p\u003e\n\u003ch3\u003e5 AMPC synergizes with c-METis to reduce ER+HER2+ MC cell survival and growth \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eTo further substantiate the synergistic effects of AMPC and c-METis identified through high-throughput screening of the anti-cancer compound library, the pharmacological inhibition of TFF3 by AMPC in combination with three c-METis was further evaluated in MDA-MB-361 and BT474 cells by total cell number assay (Fig. 5A). Cabozantinib, an FDA-approved c-METi not included in the Cambridge Anti-Cancer Compound Library (Supplementary information 6A), alongside the identified PHA-665752 and SU11274, were chosen for continued investigation. The combinatorial treatments of AMPC and c-METis exhibited synergistic effects, as demonstrated by the Chou-Talalay method and 3D zip synergy analysis in both cell lines (Fig. 5B, 5C). Subsequently, combination treatment of AMPC (2.5 μM) - c-METis significantly increased the efficacy of c-METis compared to c-METi treatment alone in MDA-MB-361 and BT474 cells, as demonstrated by dose-response analysis (Fig. 5D). Specifically, AMPC reduced IC\u003csub\u003e50\u003c/sub\u003e values of Cabozantinib, SU11274, and PHA-665752 by approximately 10-fold, 5-fold, and 30-fold, respectively, in MDA-MB-361 cells (Fig. 5D). Similarly, in BT474 cells, AMPC notably decreased the IC\u003csub\u003e50\u003c/sub\u003e values of Cabozantinib, SU11274 and PHA-665752 by approximately 10-fold, 10-fold and 20-fold, respectively (Fig. 5D). These findings underscore the synergistic potential of combining AMPC with c-METis to decrease cell survival in ER+HER2+ MC cells.\u003c/p\u003e\n\u003cp\u003eFurthermore, the effect of the combinatorial treatment on cell proliferation and apoptosis was investigated. The results indicated that single-agent AMPC or c-METis significantly inhibited proliferative capability compared to vehicle treatment in both MDA-MB-361 and BT474 cells (Supplementary information 7A). Importantly, the combined AMPC-c-METi treatments further amplified the inhibitory effect observed with the single treatments. Additionally, single-agent AMPC or c-METis promoted CASPASE 3/7 activity compared to vehicle treatment in both cell lines, and combined AMPC-c-METi treatments further enhanced this effect (Supplementary information 7B). This observation was consistent with an increase in apoptosis, as evidenced by elevated populations of early and late apoptotic cells upon single-agent and combined AMPC-c-METi treatments in both cell lines (Supplementary information 7C).\u003c/p\u003e\n\u003cp\u003eSubsequently, the effect of combined AMPC-c-METi treatments on foci forming capacity and 3D growth of MDA-MB-361 and BT474 cells was evaluated. Foci formation assays demonstrated that single treatments with AMPC or c-METis significantly reduced colony formation and viability compared to the vehicle control. Notably, the combined treatment of AMPC and c-METis further augmented these effects observed with the individual treatments (Fig. 5E and Supplementary information 7D). Similarly, in 3D Matrigel culture, single treatments led to a substantial reduction in the number and size of colonies, decreased cell viability, increased numbers of dead cells, and fewer live cells compared to the vehicle control. These effects were more pronounced with the combined treatments (Fig. 5F and Supplementary information 7E). Collectively, these findings indicate that AMPC synergizes with c-MET inhibitors to significantly reduce the survival and growth of ER+HER2+ MC cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003e6 AMPC synergizes with c-METis to suppress CSC-like phenotype in ER+HER2+ MC Cells \u003cem\u003ein vitro\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eTo assess the effect of the combinatorial treatment on the migration and invasion of ER+ HER2+ MC cells, the migratory and invasive capacities were evaluated following single treatments with AMPC, c-METis, and their combination in MDA-MB-361 and BT474 cells. The findings revealed that single-agent treatments notably suppressed both the migratory and invasive abilities of the cells compared to the control treatment (Fig. 6A and Supplementary information 8A). The combined AMPC-c-METi treatments exhibited an augmented inhibition effect beyond that of the single-agent treatments, suggesting a synergistic enhancement in restraining the migratory and invasive capacities of the ER+HER2+ MC cells.\u003c/p\u003e\n\u003cp\u003eThere exists a positive relationship between the size of the subpopulation of MC CSCs and migration and invasion[51]. Hence, the spheroid formation and ALDEFFLOUR activity assays were conducted to explore the effect of AMPC and c-METis combined treatment on CSC-like behavior in ER+HER2+ MC cells. The results from the spheroid formation assay indicated that single treatments with AMPC or c-METis significantly reduced the number of spheroids compared to vehicle treatment, and the combined AMPC-c-METi treatments further diminished spheroid formation in both ER+HER2+ MC cell lines (Fig. 6B and Supplementary information 8B). Consistently, the percentage of the ALDH1-positive cell population decreased after treatment with either AMPC or c-METis alone compared to the vehicle control (Fig. 6C and Supplementary information 8C). Notably, the combined AMPC-c-METi treatments resulted in a significant reduction in the ALDH1-positive cell population compared to c-METi treatment alone (Fig. 6C and Supplementary information 8C).\u003c/p\u003e\n\u003cp\u003eSubsequently, the mechanistic basis underlying synergistic effects of AMPC and c-METis was further analyzed using western blot analysis. Single treatments with either AMPC or c-METis, decreased TFF3 expressoion and the p-c-MET\u003csup\u003eY1234/1235\u003c/sup\u003e/c-MET ratio in MDA-MB-361 and BT474 cells; and combined AMPC and c-METi treatments further significantly reduced TFF3 expression and the p-c-MET\u003csup\u003eY1234/1235\u003c/sup\u003e/c-MET ratio compared to single agent treatment of ER+HER2+ MC cells (Fig. 6D and Supplementary information 8D). Previous studies have shown that CSCs express elevated levels of genes related to migration and invasion, such as OCT4, BMI1, SOX2, ALDH1A1, and CD44 in MC[52-55]. Diminished expression of OCT4, BMI1, SOX2, ALDH1A1, and CD44 proteins were observed in MDA-MB-361 and BT474 cells treated with AMPC or c-METis, in comparison to vehicle-treated cells (Fig. 6D and Supplementary information 8D). These reductions were further magnified following combined AMPC-c-METi treatments (Fig. 6D and Supplementary information 8D). Thus, the combined treatment of AMPC with c-METis demonstrated significant potential to synergistically inhibit the CSC-like phenotype of ER+HER2+ MC cells.\u003c/p\u003e\n\u003ch3\u003e7 AMPC synergizes with c-MET inhibition to suppresses the MDA-MB-361 xenograft growth and lung metastasis\u003c/h3\u003e\n\u003cp\u003eBuilding on the synergistic effects observed \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;ex vivo\u003c/em\u003e, the efficacy of the combined treatment of AMPC and c-MET inhibition was further evaluated \u003cem\u003ein vivo\u003c/em\u003e. The xenograft model was established by orthotopically implanting MDA-MB-361 cells into the right fourth mammary fat pad of female mice. Cabozantinib was selected for the \u003cem\u003ein vivo\u003c/em\u003e experiments as it is the FDA-approved drug for c-MET inhibition, and has been utilized in patients with ER+ MC or HER2+ MC for two Phase II clinical trials[56, 57]. Mice bearing MDA-MB-361 xenografts were randomized into four treatment groups (n = 8) to receive either vehicle (V), AMPC (A), Cabozantinib (C), or a combination of AMPC and Cabozantinib (A+C). Each treatment was administered for a duration of two weeks. Throughout the treatment period, animal weights and xenograft volumes were measured daily. Upon completion of the treatment regimen, major organs, including the spleen, lungs, and liver, were collected post-mortem. There was no significant difference in body weight or major organ weights among the four treatment groups, indicating that the treatments were well-tolerated by the mice (Supplementary information 9A, 9B).\u003c/p\u003e\n\u003cp\u003eDaily assessments of xenograft volumes revealed that single-agent treatments with either AMPC or Cabozantinib significantly reduced the volumes of MDA-MB-361 xenografts compared to the vehicle-treated control. Notably, the combination treatment with AMPC and Cabozantinib resulted in an even greater reduction in xenograft volumes compared to either single-agent treatment alone (Fig. 7A). Consistent results were observed with xenograft weight (Fig. 7B) and resected xenografts (Fig. 7C). Moreover, as demonstrated by xenograft burden change and mRECIST analysis (Fig. 7D and Supplementary information 9C), whereas single-agent treatments with AMPC or Cabozantinib failed to achieve complete or partial responses in MDA-MB-361 xenografts, a proportion of xenografts treated with AMPC (37.5%) or Cabozantinib (12.5%) exhibited stable disease (mSD), contrasting with xenografts treated with the vehicle (0.0%), which showed progression (mPD). Remarkably, xenografts treated with the combination of AMPC and Cabozantinib displayed a partial response (mPR) in 12.5% of cases and mSD in 87.5% of cases. Furthermore, IHC and TUNEL analyses revealed a decreased proportion of MKI67-positive cells, and a higher incidence of apoptosis in the MDA-MB-361 xenograft specimens treated with single-agent treatments compared to those treated with the vehicle control (Supplementary information 9D, 9E). These effects were further enhanced in specimens treated with the combination of AMPC and Cabozantinib. Therefore, these results collectively provide evidence of the increased effectiveness of the combination treatment in suppressing MDA-MB-361 xenograft growth and halting disease progression.\u003c/p\u003e\n\u003cp\u003eTo further investigate the effects of the combined treatment on CSCs \u003cem\u003ein vivo\u003c/em\u003e, IHC coupled with IRS score analysis was conducted on xenograft specimens. The results showed a decrease in TFF3 protein levels and concurrent decreased phosphorylation of c-MET in xenograft tissues after treatment with single-agent AMPC or Cabozantinib. The decreased phosphorylation of c-MET was significantly amplified when the AMPC-Cabozantinib combination was administered (Fig. 7E and Supplementary information 9F). Moreover, the diminished expression of TFF3 was validated by analyzing the relative levels of serum TFF3 in comparison to xenograft weight (Fig. 7F). Subsequently, the effect of combined targeting on CSC markers in the xenograft specimens was conducted. A significant decrease in the expression of CSC markers OCT4, BMI1, SOX2, ALDH1A1, and CD44 in xenograft samples from the groups treated with single drugs compared to the vehicle-treated control group was observed (Fig. 7G and Supplementary information 9G). The group receiving the combination treatment demonstrated a further significant suppression of the expression of these markers in comparison to the individual drug treatments.\u003c/p\u003e\n\u003cp\u003eNext, the potential metastatic dissemination of MDA-MB-361 cells from the xenograft site to major organs was determined. Metastasis was first assessed in lung tissue sections using hematoxylin-eosin (H\u0026amp;E) staining (Fig. 7H and Supplementary information 9H). The H\u0026amp;E results revealed that there was reduced incidence of lung metastasis in both single-agent AMPC (6/8) and Cabozantinib (5/8) treated groups compared to the vehicle-treated group (7/8). Notably, the incidence of lung metastasis was dramatically reduced in the combined AMPC-Cabozantinib treated group (2/8). Quantification of metastatic nodules in the lungs further corroborated this reduction (Fig. 7H). Moreover, the human HPRT gene (\u003cem\u003ehHPRT\u003c/em\u003e) was utilized to distinguish the metastatic burden of cells of human origin, as previously reported[44, 58]. The relative expression of \u003cem\u003ehHPRT\u003c/em\u003e to \u003cem\u003em\u003c/em\u003e\u003cem\u003egapdh\u003c/em\u003e verified a significantly decreased metastatic burden in both single-agent AMPC and Cabozantinib treated groups compared to the vehicle-treated group (Fig. 7I). The reduced metastatic burdens were further confirmed through IHC and IRS score analysis of hHPRT protein in lungs from the different treatment groups (Fig. 7J). Collectively, these findings demonstrate the enhanced efficacy of the combined treatment in mitigating micrometastatic spread.\u003c/p\u003e\n\u003cp\u003eIn summation, combined treatment employing AMPC and Cabozantinib as a therapeutic strategy effectively controlled the growth of primary xenografts and reduced the tendency for lung metastasis in ER+HER2+ MC xenograft model.\u003c/p\u003e\n\u003ch3\u003e8 TFF3 enhances c-MET signaling through a positive feedback loop to enhance the CSC-like phenotype of ER+ HER2+ MC\u003c/h3\u003e\n\u003cp\u003eA previous study has reported that the forced expression of TFF3 enhanced various RTK activities in ER+HER2+ MC, including c-MET, suggesting a potential regulatory relationship between TFF3 and c-MET[18]. To delineate this potential relationship, the phosphorylation of c-MET was assessed in MDA-MB-361 and BT474 cells. Western blot analysis revealed that the phosphorylation levels of c-MET at Tyrosine 1234/1235 were significantly elevated in MDA-MB-361 cells with forced expression of TFF3 compared to the vector transfected control (Fig. 8A). Conversely, the c-MET phosphorylation levels were markedly decreased in MDA-MB-361 cells with TFF3 depletion compared to control vector cells (Fig. 8A). Moreover, pharmacological inhibition of TFF3 by AMPC induced a dose-dependent decrease in c-MET phosphorylation at Tyrosine 1234/1235 in MDA-MB-361 and BT474 cells (Fig. 8B).\u003c/p\u003e\n\u003cp\u003eGiven the intricate interplay previously reported between HER2 and c-MET[59, 60], alongside their shared attributes as tyrosine receptor kinases and the findings from combination experiments herein demonstrating the suppressive effect of c-METis on TFF3 expression both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Fig. 6D, 7E); whether there is a regulatory association between TFF3 and c-MET was therefore further investigated. It was observed that the expression of TFF3 was markedly suppressed in MDA-MB-361 and BT474 cells following c-MET depletion using two independent siRNAs targeting c-MET (siMET#1 and siMET2#2), when compared to the respective scrambled siRNA transfected cells (Fig. 8C and Supplementary information 10A). Furthermore, the expression of TFF3 exhibited a dose-dependent reduction upon treatment with c-METis in both cell lines (Fig. 8D). Since c-MET also appears to be functionally downstream of TFF3 activated pathways, Co-IP assays were conducted to investigate whether TFF3 itself may be an alternate ligand for c-MET. However, no association of TFF3 to c-MET was observed in MDA-MB-361 cells, whereas the reported c-MET ligand HGF demonstrated interaction between c-MET and HFG as a positive control (Fig. 8E and Supplementary information 10B). Therefore, the phosphorylation of c-MET enhanced by TFF3 may occur indirectly, as for other RTKs[26, 61, 62].\u003c/p\u003e\n\u003cp\u003eThe synergistic inhibition of TFF3 by AMPC combined with c-METis effectively decreased CSC-like phenotype in ER+HER2+ MC cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Fig. 6C and Supplementary information 7G). To further explore the effect of the TFF3-c-MET pathway on CSC-like behavior, ER+HER2+ MC cells with forced expression of TFF3 and with c-MET depletion were examined by western blot and ALDEFLUOR assays. The elevated population of ALDH1-positive cells observed in cells with forced expression of TFF3 was significantly reduced following c-MET depletion compared to the scrambled siRNA transfected cells (Fig. 8F and Supplementary information 10C). Western blot analyses demonstrated that the increased expression of CSC markers BMI1, SOX2, and ALDH1A1 in both MDA-MB-361 and BT474 cells with forced expression of TFF3 was mitigated upon c-MET depletion (Fig. 8G). Collectively, it is apparent that c-MET positively regulates its own signaling through TFF3 with consequent enhancement of CSC-like phenotype in ER+HER2+ MC cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant advances in treatments for both HER2+ and ER+ MC, and the resultant survival benefits for affected patients, ER+HER2+ MC remains an underrepresented subgroup lacking sufficient tailored therapeutic options due to its distinct characteristics from either HER2+ or ER+ MC[63, 64]. Over the past decade, TFF3 has emerged as a promising therapeutic target due to its promotory role in cancer progression, including colorectal, hepatocellular, lung, pancreatic, prostate, cervical, endometrial, and ER+ mammary carcinomas[9, 10, 12-21]. Furthermore, inhibition of TFF3 has been shown to enhance the efficacy of ionizing radiation, Gemicitabine, Taxanes and MEK1/2 inhibitors, and to overcome resistance to 5-FU, anti-estrogen and HER2-targeted therapy[9, 12, 18]. Previous studies have reported that the pharmacological inhibition of TFF3 modulates the PI3K/AKT, MAPK, WNT and JAK/STAT3 signaling pathways in carcinoma cells[9, 12, 13, 20, 30]. Consistently, this study revealed that TFF3 possesses an oncogenic role in ER+HER2+ MC and demonstrated that TFF3 inhibition enhances the effectiveness of inhibition of c-MET. These findings suggest TFF3 as a novel promising target for combination therapeutic strategies in ER+HER2+ MC.\u003c/p\u003e\n\u003cp\u003eCSCs are a critical subpopulation of tumor-initiating cells implicated in cancer relapse, metastasis, and resistance to radiotherapy and chemotherapy[65]. Breast cancer stem cells (BCSCs) were initially recognized based on the relative expression of CD44 and CD24[66]. CD44, CD24, and aldehyde dehydrogenase-1 (ALDH1) are now widely used as biomarkers for identifying BCSC characteristics[67]. One of the defining characteristics of ER+HER2+ MC is the presence of ALDH1+ epithelial BCSCs, which is associated with poor clinical prognosis[3]. Moreover, BCSCs have been implicated in the failure of endocrine therapy, chemotherapy, radiotherapy, and immunotherapy in MC treatment, ultimately promoting relapse[68]. Recently, several studies have reported that TFF3 promotes CSC-like phenotype in pancreatic, colorectal, hepatocellular, lung, cervical, and ER+ mammary carcinoma[10, 13, 15, 18-20, 69]. In ER+ MC patients, expression and activation of c-MET is significantly higher in metastatic sites than in primary sites[70]. Furthermore, c-MET expression has been strongly correlated with CSC markers, ALDH1A3 and CD133 in MC[71]. High c-MET expression and its activation are also suggested to be involved in the promotion of \u003cem\u003eALDH1A3\u003c/em\u003e gene expression in the basal-like type of MC[71]. This investigation delineated the role of TFF3 in enhancing CSC-like phenotype in ER+HER2+ MC cells, as evidenced by heightened ALDH1 activity and increased spheroid formation capacity (Fig. 1G, 1H). Additionally, this study revealed that inhibiting TFF3 reduced CSC-like phenotype, and dual inhibition of TFF3 and c-MET led to a further reduction, suggesting a potential mechanism for the decreased metastatic burden observed with combinatorial therapy of AMPC and c-METis in ER+HER2+ MC.\u003c/p\u003e\n\u003cp\u003eIntriguingly, the high-throughput anti-cancer compound screening assays demonstrated that TFF3 inhibition by AMPC synergized most effectively with compounds targeting four distinct RTKs, EGFR, VEGFR, c-KIT, and c-MET in MDA-MB-361 and BT474 cells. These RTKs have all been implicated in oncogenic progression and are potential targets for cancer therapy. Inhibiting EGFR, VEGFR, and c-MET, whether by single drugs or drug combinations, has proven beneficial by halting cell growth, proliferation and metastasis[72, 73] although resistance ultimately develops. Mechanistically, it has been previously shown that TFF3 can competitively bind with LINGO2 to disrupt EGFR-LINGO2 complexes, leading to the release of EGFR activity[74]. Additionally, targeting TFF3 with AMPC resulted in decreased EGFR activity in ER+MC cells[30]. The capacity of TFF3 to enhance the activation of the EGFR further indicates the importance of TFF3 as a therapeutic target in that TFF3 may modulate other RTKs through its involvement in multiple signaling pathways, including p44/42 MAPK[9], PI3K/AKT[28, 29] and STAT3[15, 16]. Given the reported functions of TFF3 in modulating RTK-mediated cellular functions[18, 30], including the data herein, it may be thus reasoned that RTK inhibition in cancer will be rendered more efficacious by TFF3 depletion or inhibition.\u003c/p\u003e\n\u003cp\u003eTo date, no FDA-approved c-MET inhibitor exists for MC, however clinical investigations are currently underway to assess the effectiveness of c-MET-targeted therapies in MC patients. Cabozantinib is a multi-kinase inhibitor targeting c-MET, VEGFR1-3, RET, AXL, FLT3, and c-KIT[72]. In a single-arm Phase II study recruited patients with ER+ MC and bone metastases treated with daily Cabozantinib (NCT01441947) demonstrated efficacy of Cabozantinib[57]. The clinical benefits of Cabozantinib were also explored in ER+ MC and HER2+ MC patients with brain metastases (NCT02260531) in a Phase II trial[75]. Clinical trials involving c-MET-targeted medications in MC have exhibited varied outcomes suggesting that a combination strategy with c-MET inhibition in ER+HER2+ MC may be more useful.\u003c/p\u003e\n\u003cp\u003eIn ER+HER2+ MC, increased expression of TFF3 has been implicated in trastuzumab-resistance, activating both the HER family of tyrosine kinases and crosstalk partners, including c-MET[18]. Moreover, crosstalk of c-MET signaling pathways with ER and HER2 signaling pathways has been reported[76, 77]. Stephen \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003ereported the increased expression of c-MET along with a marked increase in the migratory and invasive capacity of Fulvestrant-resistant MC cells; and observed that increased expression of c-MET in endocrine therapy-resistant epithelial MC cells promoted cancer progression[76]. Furthermore, David \u003cem\u003eet al.\u003c/em\u003e reported that c-MET is frequently co-expressed with HER2 in HER2+ MC and contributes to trastuzumab resistance of HER2+ MC cells through sustained AKT activation; whereas the loss of c-MET function, either through RNA interference–mediated depletion or small molecule–mediated inhibition, significantly improves the response to trastuzumab[77]. c-MET enhances the activation of PI3K/AKT and p44/42 MAPK signaling, two downstream signaling pathways also enhanced by TFF3[7], and which display heightened activity in ER+HER2+ MC and in lymph node metastases of this MC subtype[7, 72, 78]. This may explain the lack of a complete response observed for xenograft growth and lung metastasis in xenograft models with the combination treatment of AMPC and Cabozantinib (Fig. 7A and Supplementary information 9H). However, given the observed efficacy, it may be postulated that prolonging the treatment duration or optimizing the dosage/dose intensity may markedly enhance the therapeutic response. Additionally, supplementing with a third drug, such as selective estrogen receptor modulators (SERMs) or HER2 targeting agents, might provide a more efficacious approach to treating ER+HER2+ MC and improving patient outcomes.\u003c/p\u003e\n\u003cp\u003eConsistent with a previous study for HER2 [18], it is herein hypothesized that c-MET regulates its own signaling; although through a positive feedback loop by TFF3 in ER+HER2+ MC cell lines. However, co-immunoprecipitation assays failed to demonstrate direct binding of TFF3 and c-MET (Fig. 8E). This is consistent with other RTKs, as TFF3-stimulated EGFR activation was achieved without direct binding or colocalization of TFF3 and EGFR[61, 62]. A possible mechanism through which TFF3 activates c-MET in ER+HER2+ MC is activation via crosstalk pathways including HER2, as TFF3 has been previously shown to activate HER2, a previously demonstrated heterodimeric partner of c-MET[18, 79, 80]. There may also exist a mechanism analogous to TFF3-LINGO2-EGFR[26] in which TFF3 sequesters proteins interacting with c-MET that inhibit its activation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe investigations herein highlight the pivotal role of TFF3 in the oncogenicity of ER+HER2+ MC cells and disease progression. This study therefore enhances the understanding of HER+ER+ MC progression by delineating the bidirectional control mechanisms involving c-MET and TFF3 in ER+HER2+ MC cells. TFF3 enhanced phosphorylation of c-MET and c-MET signaling was abrogated by TFF3 inhibition or depletion. Furthermore, TFF3 expression was increased by c-MET activation, and decreased by inhibition of c-MET. Hence, the complex molecular landscape of this subtype has been further clarified by the delineation of the interactions of TFF3 and c-MET signaling pathways, offering insight into the potential amelioration of targeted therapy for ER+HER2+ MC. The collective evidence suggests that targeting TFF3 and c-MET represents a promising and potentially efficacious treatment approach for addressing the unique challenges posed by ER+HER2+ MC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (82172618 and 82102768), China; Guangdong Basic and Applied Basic Research Foundation (2020A1515111064), China; the Shenzhen Key Laboratory of Innovative Oncotherapeutics (ZDSYS20200820165400003) (Shenzhen Science and Technology Innovation Commission), China; Universities Stable Funding Key Projects (WDZC20200821150704001), China; China Postdoctoral Science Foundation (2022M721894), China; Overseas Research Cooperation Project (HW2020008) (Tsinghua Shenzhen International Graduate School), China and The Shenzhen Bay Laboratory, Oncotherapeutics (21310031), China.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare the following competing interests: P.E.L. have previously consulted for Perseis Therapeutics Ltd. P.E.L. are named on PCT application numbers WO 2006/69253 and WO 2008/042435 and US provisional application number 61/059558 and derivatives thereof. V.P., B.B., and P.E.L. are named as inventors on PCT application WO/2018/226155 (PCT/SG2018/050277), Compounds, As Inhibitors of TFF3 Dimerization, Methods and Applications Thereof (and derivatives thereof including US Patent 11,141,402). P.E.L. is an equity holder in Sinotar Pharmaceuticals Ltd which currently holds PCT/SG2018/050277 and derivatives thereof including issued US Patent no. 11,141,402. All other authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used in this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003ePeter E. Lobie, Vijay Pandey and Chuyu He designed the study; Basappa synthesized AMPC. Chuyu He, Xuejuan Wang, Yi-Shiou Chiou performed the experiments; Chuyu He, Xuejuan Wang, Yi-Shiou Chiou, Tao Zhu, Vijay Pandey and Peter E. Lobie analyzed the data; Chuyu He, Vijay Pandey and Peter E. Lobie wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e\n\u003cp\u003eAll animal care and experimental protocols for \u003cem\u003ein vivo\u003c/em\u003e studies was approved by the Laboratory Animal Ethics Committee (Certificate number: YW) at Peking University Shenzhen, and ethical approval was obtained from Tsinghua Shenzhen International Graduate School (Number: 9, Year 2020). 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A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer. Sci Transl Med. 2019;11(476).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ER+ HER2 + mammary carcinoma, TFF3, AMPC, c-MET inhibitors, combination therapy","lastPublishedDoi":"10.21203/rs.3.rs-4982898/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4982898/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe interaction between HER2 and ERα signaling pathways contributes to resistance to anti-estrogen and HER2-targeted therapies, presenting substantial treatment challenges in ER-positive (ER+) HER2-positive (HER2+) mammary carcinoma (MC). Trefoil Factor-3 (TFF3) has been reported to mediate resistance to both anti-estrogen and anti-HER2 targeted therapies in ER+ and ER+HER2+ MC, respectively. Herein, the function and mechanism of TFF3 in ER+HER2+ MC was delineated; and novel combinatorial therapeutic strategies were identified. Elevated expression of TFF3 promoted the oncogenicity of ER+HER2+ MC cells, including enhanced cell proliferation, survival, anchorage-independent growth, \u003cem\u003e3D\u003c/em\u003e growth, cancer stem cell-like (CSC-like) phenotype, invasion, migration, and xenograft growth. Targeting TFF3 with an interfering RNA plasmid or a small-molecule inhibitor (AMPC) inhibited these oncogenic characteristics, highlighting the therapeutic potential of targeting TFF3 in ER+HER2+ MC. Furthermore, a high-throughput combinatorial anti-cancer compound library screening revealed that AMPC preferentially synergized with receptor tyrosine kinase c-MET inhibitors (c-METis) to reduce cell survival and the CSC-like phenotype. The combination of AMPC and c-METis also synergistically suppressed \u003cem\u003ein vivo\u003c/em\u003e growth of ER+HER2+ MC cell-derived xenografts and abrogated lung metastasis. Mechanistically, TFF3 was observed to activate c-MET signaling through a positive-feedback loop to enhance the CSC-like phenotype of ER+HER2+ MC. Therefore, proof of concept is provided herein that antagonizing of TFF3 is a promising therapeutic strategy in combination with c-MET inhibition for the treatment of ER+HER2+ MC.\u003c/p\u003e","manuscriptTitle":"Inhibition of TFF3 Synergizes with c-MET Inhibitors to Decrease the CSC-like Phenotype and Metastatic Burden in ER+HER2+ Mammary Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-10 12:26:30","doi":"10.21203/rs.3.rs-4982898/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-10-08T14:46:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-10-04T17:47:00+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-29T01:10:02+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-17T15:31:44+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-16T16:45:51+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-09-08T15:39:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-28T10:11:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-08-27T08:39:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-27T08:39:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08b9aa65-469b-42b4-b42d-1c2742cb733e","owner":[],"postedDate":"October 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":37281599,"name":"Biological sciences/Cancer/Breast cancer"},{"id":37281600,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"}],"tags":[],"updatedAt":"2025-02-07T08:10:33+00:00","versionOfRecord":{"articleIdentity":"rs-4982898","link":"https://doi.org/10.1038/s41419-025-07387-5","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-02-07 05:00:00","publishedOnDateReadable":"February 7th, 2025"},"versionCreatedAt":"2024-10-10 12:26:30","video":"","vorDoi":"10.1038/s41419-025-07387-5","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07387-5","workflowStages":[]},"version":"v1","identity":"rs-4982898","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4982898","identity":"rs-4982898","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}