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Even though tamoxifen has significantly improved the survival of estrogen receptor positive (ER+) mammary carcinoma (MC) patients, the development of drug resistance with consequent disease recurrence has limited its therapeutic efficacy. Trefoil factor-3 (TFF3) has been previously reported to mediate anti-estrogen resistance in ER+MC. Herein, the efficacy of a small molecule inhibitor of TFF3 (AMPC) in enhancing sensitivity and mitigating acquired resistance to tamoxifen in ER+MC cells was investigated. AMPC induced apoptosis of tamoxifen-sensitive and resistant ER+MC cells and significantly reduced cell survival in 2D and 3D culture in vitro. In addition, AMPC reduced cancer stem cell (CSC)-like behavior in ER+MC cells in a BCL2-dependent manner. Synergistic effects of AMPC and tamoxifen were demonstrated in ER+MC cells and AMPC was observed to improve tamoxifen efficacy in tamoxifen-sensitive cells and to re-sensitize cells to tamoxifen in tamoxifen-resistant ER+MC in vitro and in vivo. Additionally, tamoxifen-resistant ER+MC cells were concomitantly resistant to anthracycline, platinum and fluoropyrimidine drugs, but not to Taxanes. Taxane treatment of tamoxifen-sensitive and resistant ER+MC cells increased TFF3 expression indicating a combination vulnerability for tamoxifen-resistant ER+MC cells. Taxanes increased CSC-like behavior of tamoxifen-sensitive and resistant ER+MC cells which was reduced by AMPC treatment. Taxanes synergized with AMPC to promote apoptosis and reduce CSC-like behavior in vitro and in vivo. Hence, AMPC restored the sensitivity of tamoxifen and enhanced the efficacy of Taxanes in tamoxifen-resistant ER+MC. In conclusion, pharmacological inhibition of TFF3 may serve as an effective combinatorial therapeutic strategy for the treatment of tamoxifen-resistant ER+MC., Competing Interests: Declaration of competing interest The authors declare the following competing interests: P.E.L. and T.Z. 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., (Copyright © 2023. Published by Elsevier B.V.)

Abstract: It is now apparent that growth hormone (GH), an anterior pituitary hormone predominantly regulating postnatal somatic growth and metabolism, is also expressed in extrapituitary tissues. An extrapituitary synthetic site of GH that has garnered interest is the de novo or enhanced expression of GH in carcinoma or other cancers. In a number of cancers, including carcinoma of the mammary gland, endometrium, liver, prostate, and colon, the expression of GH is independently associated with more advanced clinicopathologic parameters of the cancer. In some of these cancers, tumor human growth hormone (hGH) expression portends worse survival outcomes for patients. Functionally, tumor-derived hGH exerts both autocrine and paracrine functions on carcinoma cells and cancer-associated stroma. Expression of autocrine/paracrine hGH in cancer drives tumor growth, angiogenesis, metastasis, and resistance to therapy by promotion of cancer cell proliferation, survival, epithelial-to-mesenchymal transition, motility, invasion, cancer stem cell-like behavior, and metastasis. Autocrine/paracrine hGH activates oncogenic signaling pathways and specific transcriptome signatures and enhances the expression of an oncogenic secretome to promote these functions. Hence, extrapituitary expression of GH in cancer promotes cancer progression independent of endocrine hGH, and may be considered as a validated target in oncology.

While tremendous improvement has been made for the treatment of breast cancers, the treatment of triple negative breast cancer (TNBC) still remains a challenge due to its aggressive characteristics and limited treatment options. Most of the studies on TNBC were conducted in Western population and TNBC is reported to be more frequent in the African women. This review encapsulates the studies conducted on TNBC patients in Asian population and elucidates the similarities and differences between these two regions. The current treatment of TNBC includes surgery, radiotherapy and chemotherapy. In addition to the current chemotherapies, which mainly include cytotoxic agents, such as taxanes and anthracyclines, many clinical trials are investigating the potential use of other chemotherapy drugs, targeted therapeutics and combinational therapies to treat TNBC. Moreover, this review also integrates the studies involving novel markers, which will help us to dissect the pathologic process of TNBC and in turn facilitate the development of better treatment strategies to combat TNBC., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC., Competing Interests: The authors have no conflicts of interest to declare.

Background: Artemin (ARTN) has been implicated in promoting oncogenicity, tumor growth and invasiveness in diverse human malignancies. However, the clinical and prognostic significance of upstream ligand binding components, potentially mediating ARTN oncogenicity, largely remain to be determined., Methods: We determined the mRNA and protein expression of three proteins demonstrated to bind ARTN, namely GFRα1, GFRα3 and syndecan-3 (SDC3), in benign breast disease and mammary carcinoma by in situ hybridization and immunohistochemistry, respectively. Their prognostic significance combined with ARTN expression was also investigated in mammary carcinoma., Results: The expression of GFRα1 and GFRα3, but not SDC3, was significantly increased in mammary carcinoma and positively associated with tumor lymph node metastases, higher clinical stage and HER-2 positivity. Moreover, both GFRα1 and GFRα3 expression were significantly associated with survival outcome of patients with mammary carcinoma by univariate and multivariate analyses, whereas expression of SDC3 was not. Co-expression of ARTN with either GFRα1 or GFRα3, but not SDC3, produced synergistic increases in the odds ratio for both relapse-free and overall survival in patients with mammary carcinoma. Furthermore, significant association of GFRα1 and GFRα3 expression with survival outcome observed herein were restricted to ER negative or HER-2 negative mammary carcinoma., Conclusions: The expression of GFRα1 and/or GFRα3, especially when combined with ARTN expression, may be useful predictors of disease progression and outcome in specific subtypes of mammary carcinoma.

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Context: Empirical evidence suggests that autocrine human GH (hGH) may possess a proliferative and oncogenic role in human mammary carcinoma. However, this concept is largely derived from studies using cultured human mammary carcinoma cell (HMCC) lines., Objective: We investigated the expression and functionality of hGH and the hGH receptor in isolated cultures of primary HMCC., Design: Epithelial cell adhesion molecule-positive primary HMCC were isolated from surgical biopsies of patients with mammary carcinoma and cultured in vitro. Expression of hGH and hGH receptor was determined by RT-PCR, immunofluorescence microscopy, and ELISA. The proliferative response of the cultured primary HMCC to hGH stimulation or hGH inhibition with a hGH antagonist was determined., Results: One hundred percent of cultured primary HMCC expressed the hGH receptor, and 52% expressed hGH at the mRNA level. hGH-positive primary HMCC produced hGH protein within the cell and secreted hGH to the media. Both hGH-negative and hGH-positive HMCC responded to hGH stimulation with large increases in cell number. hGH-positive HMCC responded to inhibition of hGH by a hGH antagonist with a decrease in cell number, whereas hGH-negative HMCC did not., Conclusion: Primary HMCC proliferate in response to hGH, and the proliferation of hGH-positive HMCC is inhibited by hGH antagonism. Inhibition of hGH in patients with mammary carcinoma may therefore limit tumor growth.

Deregulated PAX5 expression has been associated with metastatic mammary carcinoma, although the precise role of PAX5 in cancer progression is unclear. Stable forced expression of PAX5alpha in the mammary carcinoma cell lines MCF-7 and MDA-MB-231 reduced cell cycle progression, cell survival, and anchorage-independent cell growth. In xenograft studies, forced expression of PAX5alpha was associated with a significant reduction in tumor volume. Furthermore, forced expression of PAX5alpha in mammary carcinoma cells resulted in altered cell morphology with resultant enhancement of epithelial cell characteristics. Morphologic changes were associated with localization of beta-CATENIN at cell-cell junctions and with altered mRNA expression of mesenchymal markers in mammary carcinoma cells. In addition, forced expression of PAX5alpha in MCF-7 and MDA-MB-231 cells significantly reduced cell migration and invasion. Concomitantly, small interfering RNA-mediated depletion of PAX5alpha increased MCF-7 total cell number, cell motility, migration, and invasion. These studies show that PAX5alpha enhances the epithelial characteristics of mammary carcinoma cells, reminiscent of mesenchymal to epithelial transition.

Here, we provide evidence for a functional role of artemin (ARTN) in progression of endometrial carcinoma (EC). Increased ARTN protein expression was observed in EC compared with normal endometrial tissue, and ARTN protein expression in EC was significantly associated with higher tumor grade and invasiveness. Forced expression of ARTN in EC cells significantly increased total cell number as a result of enhanced cell cycle progression and cell survival. In addition, forced expression of ARTN significantly enhanced anchorage-independent growth and invasiveness of EC cells. Moreover, forced expression of ARTN increased tumor size in xenograft models and produced highly proliferative, poorly differentiated, and invasive tumors. The ARTN-stimulated increases in oncogenicity and invasion were mediated by increased expression and activity of AKT1. Small interfering RNA-mediated depletion or antibody inhibition of ARTN significantly reduced oncogenicity and invasion of EC cells. Thus, inhibition of ARTN may be considered as a potential therapeutic strategy to retard progression of EC.

Increased activation of signal transducer and activator of transcription (STAT)-5 has been reported in various malignancies including mammary carcinoma. However, it is only recently that potentially distinct roles of STAT5A and STAT5B in neoplasia have begun to emerge. Herein we systematically delineate the functions of STAT5A and STAT5B in human mammary carcinoma cell lines MCF-7 and T47D. Forced expression of constitutively active (CA) STAT5A enhanced both survival and anchorage-independent growth of human mammary carcinoma cells but concordantly suppressed cell motility as revealed in colony scattering, cell migration, and invasion assays. In contrast, forced expression of CA STAT5B exhibited lower potency than CA STAT5A in enhancing survival and anchorage-independent growth of mammary carcinoma cells and exerted no effects on cell motility. Differential expression of genes that regulate cellular survival and motility was concomitantly observed on forced expression of CA STAT5A or CA STAT5B. Small interfering RNA-mediated depletion of STAT5A significantly impaired anchorage-independent growth of human mammary carcinoma cells, whereas a smaller reduction was observed upon small interfering RNA-mediated depletion of STAT5B. Depletion of endogenous STAT5A also significantly enhanced cell motility, whereas depletion of endogenous STAT5B exhibited no effect. Xenograft studies provided data concordant with the in vitro effects of the two STAT5 isoforms. We therefore demonstrate that STAT5A and STAT5B differentially regulate behavior of human mammary carcinoma cells.

The functional role of autocrine trefoil factor-1 (TFF1) in mammary carcinoma has not been previously elucidated. Herein, we demonstrate that forced expression of TFF1 in mammary carcinoma cells resulted in increased total cell number as a consequence of increased cell proliferation and survival. Forced expression of TFF1 enhanced anchorage-independent growth and promoted scattered cell morphology with increased cell migration and invasion. Moreover, forced expression of TFF1 increased tumor size in xenograft models. Conversely, RNA interference-mediated depletion of TFF1 in mammary carcinoma cells significantly reduced anchorage-independent growth and migration. Furthermore, neutralization of secreted TFF1 protein by polyclonal antibody decreased mammary carcinoma cell viability in vitro and resulted in regression of mammary carcinoma xenografts. We have therefore demonstrated that TFF1 possesses oncogenic functions in mammary carcinoma cells. Functional antagonism of TFF1 can therefore be considered as a novel therapeutic strategy for mammary carcinoma.

Accumulating literature implicates pathological angiogenesis and lymphangiogenesis as playing key roles in tumor progression. Autocrine human growth hormone (hGH) is a wild-type orthotopically expressed oncogene for the human mammary epithelial cell. Herein we demonstrate that autocrine hGH expression in the human mammary carcinoma cell line MCF-7 stimulated the survival, proliferation, migration, and invasion of a human microvascular endothelial cell line (HMEC-1). Autocrine/paracrine hGH secreted from mammary carcinoma cells also promoted HMEC-1 in vitro tube formation as a consequence of increased vascular endothelial growth factor-A (VEGF-A) expression. Semiquantitative RT-PCR analysis demonstrated that HMEC-1 cells express both hGH and the hGH receptor (hGHR). Functional antagonism of HMEC-1-derived hGH reduced HMEC-1 survival, proliferation, migration/invasion, and tube formation in vitro. Autocrine/paracrine hGH secreted by mammary carcinoma cells increased tumor blood and lymphatic microvessel density in a xenograft model of human mammary carcinoma. Autocrine hGH is therefore a potential master regulator of tumor neovascularization, coordinating two critical processes in mammary neoplastic progression, angiogenesis and lymphangiogenesis. Consideration of hGH antagonism to inhibit angiogenic processes in mammary carcinoma is therefore warranted.

Recent published data have demonstrated elevated levels of human GH (hGH) in endometriosis and endometrial adenocarcinoma. Herein, we demonstrate that autocrine production of hGH can enhance the in vitro and in vivo oncogenic potential of endometrial carcinoma cells. Forced expression of hGH in endometrial carcinoma cell lines RL95-2 and AN3 resulted in an increased total cell number through enhanced cell cycle progression and decreased apoptotic cell death. In addition, autocrine hGH expression in endometrial carcinoma cells promoted anchorage-independent growth and increased cell migration/invasion in vitro. In a xenograft model of human endometrial carcinoma, autocrine hGH enhanced tumor size and progression. Changes in endometrial carcinoma cell gene expression stimulated by autocrine hGH was consistent with the altered in vitro and in vivo behavior. Functional antagonism of hGH in wild-type RL95-2 cells significantly reduced cell proliferation, cell survival, and anchorage-independent cell growth. These studies demonstrate a functional role for autocrine hGH in the development and progression of endometrial carcinoma and indicate potential therapeutic relevance of hGH antagonism in the treatment of endometrial carcinoma.

It is now apparent that growth hormone (GH), an anterior pituitary hormone predominantly regulating postnatal somatic growth and metabolism, is also expressed in extrapituitary tissues. An extrapituitary synthetic site of GH that has garnered interest is the de novo or enhanced expression of GH in carcinoma or other cancers. In a number of cancers, including carcinoma of the mammary gland, endometrium, liver, prostate, and colon, the expression of GH is independently associated with more advanced clinicopathologic parameters of the cancer. In some of these cancers, tumor human growth hormone (hGH) expression portends worse survival outcomes for patients. Functionally, tumor-derived hGH exerts both autocrine and paracrine functions on carcinoma cells and cancer-associated stroma. Expression of autocrine/paracrine hGH in cancer drives tumor growth, angiogenesis, metastasis, and resistance to therapy by promotion of cancer cell proliferation, survival, epithelial-to-mesenchymal transition, motility, invasion, cancer stem cell-like behavior, and metastasis. Autocrine/paracrine hGH activates oncogenic signaling pathways and specific transcriptome signatures and enhances the expression of an oncogenic secretome to promote these functions. Hence, extrapituitary expression of GH in cancer promotes cancer progression independent of endocrine hGH, and may be considered as a validated target in oncology. [ABSTRACT FROM AUTHOR]

Mammary carcinoma cells produce pro-angiogenic factors to stimulate angiogenesis and tumor growth. Trefoil factor-3 (TFF3) is an oncogene secreted from mammary carcinoma cells and associated with poor prognosis. Herein, we demonstrate that TFF3 produced in mammary carcinoma cells functions as a promoter of tumor angiogenesis. Forced expression of TFF3 in mammary carcinoma cells promoted proliferation, survival, invasion and in vitro tubule formation of human umbilical vein endothelial cells (HUVEC). MCF7-TFF3 cells with forced expression of TFF3 generated tumors with enhanced microvessel density as compared to tumors formed by vector control cells. Depletion of TFF3 in mammary carcinoma cells by siRNA concordantly decreased the angiogenic behavior of HUVEC. Forced expression of TFF3 in mammary carcinoma cells stimulated IL-8 transcription and subsequently enhanced IL-8 expression in both mammary carcinoma cells and HUVEC. Depletion of IL-8 in mammary carcinoma cells with forced expression of TFF3, or antibody inhibition of IL-8, partially abrogated mammary carcinoma cell TFF3-stimulated HUVEC angiogenic behavior in vitro, as did inhibition of the IL-8 receptor, CXCR2. Depletion of STAT3 by siRNA in MCF-7 cells with forced expression of TFF3 partially diminished the angiogenic capability of TFF3 on stimulation of cellular processes of HUVEC. Exogenous recombinant hTFF3 also directly promoted the angiogenic behavior of HUVEC. Hence, TFF3 is a potent angiogenic factor and functions as a promoter of de novo angiogenesis in mammary carcinoma, which may co-coordinate with the growth promoting and metastatic actions of TFF3 in mammary carcinoma to enhance tumor progression. [ABSTRACT FROM AUTHOR]

Introduction Recurrence or early metastasis remains the predominant cause of mortality in patients with estrogen receptor positive (ER+) mammary carcinoma (MC). However, the molecular mechanisms underlying the initial progression of ER+ MC to metastasis remains poorly understood. Trefoil factor 3 (TFF3) is an estrogen-responsive oncogene in MC. Herein, we provide evidence for a functional role of TFF3 in metastatic progression of ER+ MC. Methods The association of TFF3 expression with clinicopathological parameters and survival outcome in a cohort of MC patients was assessed by immunohistochemistry. The expression of TFF3 in MCF7 and T47D cells was modulated by forced expression or siRNA-mediated depletion of TFF3. mRNA and protein levels were determined using qPCR and western blot. The functional effect of modulation of TFF3 expression in MC cells was determined in vitro and in vivo. Mechanistic analyses were performed using reporter constructs, modulation of signal transducer and activator of transcription 3 (STAT3) expression, and pharmacological inhibitors against c-SRC and STAT3 activity. Results TFF3 protein expression was positively associated with larger tumor size, lymph node metastasis, higher stage, and poor survival outcome. Forced expression of TFF3 in ER+ MC cells stimulated colony scattering, cell adhesion to a Collagen I coated matrix, colony formation on a Collagen I or Matrigel coated matrix, endothelial cell adhesion, and transmigration through an endothelial cell barrier. In vivo, forced expression of TFF3 in MCF7 cells stimulated the formation of metastatic nodules in animal lungs. TFF3 regulation of the mRNA levels of epithelial, mesenchymal, and metastatic-related genes in ER+ MC cells were consistent with the altered cell behaviour. Forced expression of TFF3 in ER+ MC cells stimulated phosphorylation of c-SRC that subsequently increased STAT3 activity, which lead to the downregulation of E-cadherin. siRNA-mediated depletion of TFF3 reduced the invasiveness of ER+ MC cells. Conclusion TFF3 expression predicts metastasis and poor survival outcome of patients with MC and functionally stimulates cellular invasion and metastasis of ER+ MC cells. Adjuvant functional inhibition of TFF3 may therefore be considered to ameliorate outcome of ER+ MC patients. [ABSTRACT FROM AUTHOR]

The design and development of a small molecule named NPB [3-{(4(2,3-dichlorophenyl)piperazin-1-yl}{2-hydroxyphenyl)methyl}-N-cyclopentylbenzamide], which specifically inhibited the phosphorylation of BAD at Ser99 in human carcinoma cells has been previously reported. Herein, the synthesis, characterization, and effect on cancer cell viability of NPB analogs, and the single-crystal X-ray crystallographic studies of an example compound (4r), which was grown via slow-solvent evaporation technique is reported. Screening for loss of viability in mammary carcinoma cells revealed that compounds such as 2[(4(2,3-dichlorophenyl)piperazin-1-yl][naphthalen-1-yl]methyl)phenol (4e), 5[(4(2,3-dichlorophenyl)piperazin-1-yl][2-hydroxyphenyl)methyl)uran-2-carbaldehyde (4f), 3[(2-hydroxyphenyl][4(p-tolyl)piperazin-1-yl)methyl)benzaldehyde (4i), and NPB inhibited the viability of MCF-7 cells with IC50 values of 5.90, 3.11, 7.68, and 6.5 µM, respectively. The loss of cell viability was enhanced by the NPB analogs synthesized by adding newer rings such as naphthalene and furan-2-carbaldehyde in place of N-cyclopentyl-benzamide of NPB. Furthermore, these compounds decreased Ser99 phosphorylation of hBAD. Additional in silico density functional theory calculations suggested possibilities for other analogs of NPB that may be more suitable for further development. [ABSTRACT FROM AUTHOR]

Herein, the activity of adamantanyl-tethered-biphenyl amines (ATBAs) as oestrogen receptor alpha (ERα) modulating ligands is reported. Using an ERα competitor assay it was demonstrated that ATBA compound 3-(adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline (AMTA) exhibited an inhibitory concentration 50% (IC50) value of 62.84 nM and demonstrated better binding affinity compared to tamoxifen (IC50 = 79.48 nM). Treatment of ERα positive (ER+) mammary carcinoma (MC) cells (Michigan Cancer Foundation-7 (MCF7)) with AMTA significantly decreased cell viability at an IC50 value of 6.4 μM. AMTA treatment of MC cell-generated three-dimensional (3D) spheroids resulted in significantly decreased cell viability. AMTA demonstrated a superior inhibitory effect compared to tamoxifen-treated MC cell spheroids. Subsequently, by use of an oestrogen response element (ERE) luciferase reporter construct, it was demonstrated that AMTA treatment significantly deceased ERE transcriptional activity in MC cells. Concordantly, AMTA treatment of MC cells also significantly decreased protein levels of oestrogen-regulated CCND1 in a dose-dependent manner. In silico molecular docking analysis suggested that AMTA compounds interact with the ligand-binding domain of ERα compared to the co-crystal ligand, 5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7- methylnaphthalen-2-ol. Therefore, an analogue of AMTA may provide a structural basis to develop a newer class of ERα partial agonists. [ABSTRACT FROM AUTHOR]

Increased expression of trefoil factor 3 (TFF3) has been reported in colorectal carcinoma (CRC), being correlated with distant metastasis and poor clinical outcomes. Amongst the CRC subtypes, mesenchymal (CMS4) CRC is associated with the worst survival outcome. Herein, the functional roles of TFF3 and the pharmacological inhibition of TFF3 by a novel specific small molecule TFF3 inhibitor—2-amino-4-(4-(6-fluoro-5-methylpyridin-3-yl)phenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (AMPC) in CMS4 CRC was explored. Forced expression of TFF3 in CMS4 CRC cells promoted cell proliferation, cell survival, foci formation, invasion, migration, cancer stem cell like behaviour and growth in 3D Matrigel. In contrast, siRNA-mediated depletion of TFF3 or AMPC inhibition of TFF3 in CMS4 CRC cells decreased oncogenic behaviour as indicated by the above cell function assays. AMPC also inhibited tumour growth in vivo. The TFF3-stimulated oncogenic behaviour of CMS4 CRC cells was dependent on TFF3 activation of the p44/42 MAPK (ERK1/2) pathway. Furthermore, the forced expression of TFF3 decreased the sensitivity of CMS4 CRC cells to 5-fluorouracil (5-FU); while depleted TFF3 expression enhanced 5-FU sensitivity in CMS4 CRC cells. 5-FU treatment induced TFF3 expression in CMS4 CRC cells. AMPC, when used in combination with 5-FU in CMS4 CRC cells exhibited a synergistic inhibitory effect. In summary, this study provides functional evidence for TFF3 as a therapeutic target in CMS4 CRC. [ABSTRACT FROM AUTHOR]