MUC1 is an oncogenic mucin overexpressed in several epithelial cancers, including pancreatic ductal adenocarcinoma, and is considered as a potent target for cancer therapy. To control cancer progression, miRNAs became very recently, major targets and tools to inhibit oncogene expression. Inhibiting MUC1 using miRNAs appears thus as an attractive strategy to reduce cancer progression. However, potent miRNAs and associated mechanisms regulating MUC1 expression remain to be identified. To this aim, we undertook to study MUC1 regulation by miRNAs in pancreatic cancer cells and identify those with tumor suppressive activity. MiRNAs potentially targeting the 3'-UTR, the coding region, or the 5'-UTR of MUC1 were selected using an in silico approach. Our in vitro and in vivo experiments indicate that miR-29a and miR-330-5p are strong inhibitors of MUC1 expression in pancreatic cancer cells through direct binding to MUC1 3'-UTR. MUC1 regulation by the other selected miRNAs (miR-183, miR-200a, miR-876-3p and miR-939) was found to be indirect. MiR-29a and miR-330-5p are also deregulated in human pancreatic cancer cell lines and tissues and in pancreatic tissues of Kras(G12D) mice. In vitro, miR-29a and miR-330-5p inhibit cell proliferation, cell migration, cell invasion and sensitize pancreatic cancer cells to gemcitabine. In vivo intra-tumoral injection of these two miRNAs in xenografted pancreatic tumors led to reduced tumor growth. Altogether, we have identified miR-29a and miR-330-5p as two new tumor suppressive miRNAs that inhibit the expression of MUC1 oncogenic mucin in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers in the world with one of the worst outcome. The oncogenic mucin MUC4 has been identified as an actor of pancreatic carcinogenesis as it is involved in many processes regulating pancreatic cancer cell biology. MUC4 is not expressed in healthy pancreas whereas it is expressed very early in pancreatic carcinogenesis. Targeting MUC4 in these early steps may thus appear as a promising strategy to slow-down pancreatic tumorigenesis. miRNA negative regulation of MUC4 could be one mechanism to efficiently downregulate MUC4 gene expression in early pancreatic neoplastic lesions. Using in silico studies, we found two putative binding sites for miR-219-1-3p in the 3'-UTR of MUC4 and showed that miR-219-1-3p expression is downregulated both in PDAC-derived cell lines and human PDAC tissues compared with their normal counterparts. We then showed that miR-219-1-3p negatively regulates MUC4 mucin expression via its direct binding to MUC4 3'-UTR. MiR-219-1-3p overexpression (transient and stable) in pancreatic cancer cell lines induced a decrease of cell proliferation associated with a decrease of cyclin D1 and a decrease of Akt and Erk pathway activation. MiR-219-1-3p overexpression also decreased cell migration. Furthermore, miR-219-1-3p expression was found to be conversely correlated with Muc4 expression in early pancreatic intraepithelial neoplasia lesions of Pdx1-Cre;LSL-Kras(G12D) mice. Most interestingly, in vivo studies showed that miR-219-1-3p injection in xenografted pancreatic tumors in mice decreased both tumor growth and MUC4 mucin expression. Altogether, these results identify miR-219-1-3p as a new negative regulator of MUC4 oncomucin that possesses tumor-suppressor activity in PDAC.

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

Growing body of evidence suggests that epithelial‐mesenchymal transition (EMT) is a critical process in tumor progression and chemoresistance in pancreatic cancer (PC). The aim of this study was to analyze the role of EMT‐like changes in acquisition of resistance to gemcitabine in pancreatic cells of the mesenchymal or epithelial phenotype. Therefore, chemoresistant BxPC‐3, Capan‐2, Panc‐1, and MiaPaca‐2 cells were selected by chronic exposure to increasing concentrations of gemcitabine. We show that gemcitabine‐resistant Panc‐1 and MiaPaca‐2 cells of mesenchymal‐like phenotype undergo further EMT‐like molecular changes mediated by ERK‐ZEB‐1 pathway, and that inhibition of ERK1/2 phosphorylation or ZEB‐1 expression resulted in a decrease in chemoresistance. Conversely, gemcitabine‐resistant BxPC‐3 and Capan‐2 cells of epithelial‐like phenotype did not show such typical EMT‐like molecular changes although the expression of the tight junction marker occludin could be found decreased. In pancreatic cancer patients, high ZEB‐1 expression was associated with tumor invasion and tumor budding. In addition, tumor budding was essentially observed in patients treated with neoadjuvant chemotherapy. These findings support the notion that gemcitabine treatment induces EMT‐like changes that sustain invasion and chemoresistance in PC cells.

Secreted mucins are large O-glycosylated proteins that participate in the protection/defence of underlying mucosae in normal adults. Alteration of their expression is a hallmark of numerous epithelial cancers and has often been correlated to bad prognosis of the tumour. The secreted mucin MUC5B is overexpressed in certain subtypes of gastric and intestinal cancers, but the consequences of this altered expression on the cancer cell behaviour are not known. To investigate the role of MUC5B in carcinogenesis, its expression was knocked-down in the human gastric cancer cell line KATO-III and in the colonic cancer cell line LS174T by using transient and stable approaches. Consequences of MUC5B knocking-down on cancer cells were studied with respect to proliferation, migration and invasion, and on tumour growth using a mouse subcutaneous xenograft model. Western blotting, luciferase assay and qRT-PCR were used to identify proteins and signalling pathways involved. MUC5B down-regulation leads to a decrease in proliferation, migration and invasion properties in both cell lines. Molecular mechanisms involved the alteration of β-catenin expression, localization and activity and decreased expression of several of its target genes. xenografts of MUC5B-deficient cells induced a decrease in tumour growth when compared with MUC5B-expressing Mock cells. Altogether, the present study shows that down-regulation of MUC5B profoundly alters proliferation, migration and invasion of human gastrointestinal cancer cells and that these alterations may be, in part, mediated by the Wnt/β-catenin pathway emphasizing the potential of MUC5B as an actor of gastrointestinal carcinogenesis.

MUC1 is a membrane-bound mucin known to participate in tumor proliferation. It has been shown that MUC1 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplasia to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is gastro-esophageal reflux and MUC1 was previously shown to be up-regulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC1 plays a role in biological properties of human esophageal cancer cells. For that, a stable MUC1-deficient esophageal cancer cell line was established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of MUC1-deficient cells were analyzed. Our results show that esophageal cancer cells lacking MUC1 were less proliferative and had decreased migration and invasion properties. These alterations were accompanied by a decreased activity of NFKB p65, Akt and MAPK (p44/42, JNK and p38) pathways. MCM6 and TSG101 tumor-associated markers were also decreased. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC1. Altogether, the data indicate that MUC1 plays a key role in proliferative, migrating and invasive properties of esophageal cancer cells as well as in tumor growth promotion. MUC1 mucin appears thus as a good therapeutic target to slow down esophageal tumor progression.

The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their “growth factor-like” biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and β-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

No abstract