Cancer Letters

Cancer Letters

Volume 346, Issue 2, 1 May 2014, Pages 225-236
Cancer Letters

MUC1 drives epithelial–mesenchymal transition in renal carcinoma through Wnt/β-catenin pathway and interaction with SNAIL promoter

https://doi.org/10.1016/j.canlet.2013.12.029Get rights and content

Abstract

MUC1 is overexpressed in human carcinomas. The transcription factor SNAIL can activate epithelial–mesenchymal transition (EMT) in cancer cells. In this study, in renal carcinoma, we demonstrate that (i) MUC1 and SNAIL were overexpressed in human sarcomatoid carcinomas, (ii) SNAIL increased indirectly MUC1 expression, (iii) MUC1 overexpression induced EMT, (iv) MUC1 C-terminal domain (MUC1-C) and β-catenin increased SNAIL transcriptional activity by interaction with its promoter and (v) blocking MUC1-C nuclear localization decreased Wnt/β-catenin signaling pathway activation and SNAIL expression. Altogether, our findings demonstrate that MUC1 is an actor in EMT and appears as a new therapeutic target.

Introduction

Mucin 1 (MUC1) is a large O-glycoprotein type I translated as a single polypeptide that undergoes autocleavage into N-terminal (MUC1-N) and C-terminal (MUC1-C) subunits allowing the formation of a heterodimer through a stable non-covalent association [1]. MUC1-N is an extracellular domain containing extensively O-glycosylated tandem repeat (TR) 20 amino acid (AA) sequence and protudes far away from the apical side of the cell (200–500 nm). The MUC1-C includes a 58-AA extracellular domain, a 28-AA transmembrane domain and a 72-AA cytoplasmic tail (CT). In adult, MUC1 expression is cell- and tissue-specific and is altered during carcinogenesis. MUC1 has been shown to induce cell growth and tumor progression through activation of various signaling pathway [2]. In normal kidney, MUC1 localizes to the apical membrane of distal convoluted tubule and collecting ducts. Clear renal cell carcinoma (cRCC) is the main histological subtype of renal cell carcinoma. Previous studies have shown that MUC1 is diffusely overexpressed in cRCC with correlation to a worse outcome and metastatic disease [3], [4].

Epithelial–mesenchymal transition (EMT) is defined as a dynamic process characterized by changes in cell phenotype between epithelial and mesenchymal states. EMT involvement in embryonic development and cancer dissemination has been largely recognized [5]. The term EMT refers to a complex molecular and cellular program by which epithelial cells loose apico-basal polarity, reorganize cytoskeletal elements and acquire the ability to invade and move into the extracellular matrix. EMT has been found to contribute to invasion, metastatic dissemination and acquisition of therapeutic resistance [6]. One example of EMT-associated cancer is represented by sarcomatoid carcinoma, characterized by spindle carcinomatous cells conferring a worse prognosis [7], [8]. Various signaling pathways have been demonstrated to regulate EMT and the reverse program termed mesenchymal–epithelial transition (MET). Canonical Wnt/β-catenin pathway is one of the most involved pathways in EMT process [9]. Nuclear accumulation of β-catenin associated with activation of Wnt/β-catenin pathway resulted in activation of target genes, notably those encoding for mesenchymal markers, such as vimentin [10] and EMT partners [11]. SNAIL, an EMT-associated transcription factor, is the most widely recognized EMT effector and suppressor of E-cadherin expression. SNAIL expression has been described in several human aggressive dedifferentiated carcinomas such as renal carcinomas [12].

MUC1 is classically considered as an epithelial marker [5] and one previous study has shown MUC1 transcriptional repression trigged by SNAIL activation in colonic HT29 cells [13]. Nevertheless, more recent studies reported a direct role of MUC1 and also another membrane bound mucin MUC4 in initiating EMT during pancreatic, mammary and ovarian carcinomas [14], [15], [16].

In this study, in renal cancer, we show that (i) MUC1 cytoplasmic and SNAIL nuclear expressions were associated to the human sarcomatoid component of cRCC which is related to an EMT model, suggesting a coregulation of both proteins; (ii) SNAIL induced MUC1 indirectly; (iii) also, MUC1 overexpression was sufficient to induce EMT features and SNAIL activation and (iv) a direct interaction between MUC1-C in cooperation with β-catenin on the −120/−12 region of the SNAIL promoter was associated with SNAIL upregulation.

Section snippets

Tissue microarray (TMA)

Twenty-two formalin-fixed and paraffin-embedded primary cRCC samples with sarcomatoid component were retrieved from the archives of the Department of Pathology of the University hospital of Lille. A consent form was obtained from each patient. Three core tissue biopsies, 0.6 mm in diameter, were taken from selected morphologically representative regions of each cRCC (conventional and sarcomatoid components), distant to necrotic areas, and precisely arrayed using a tissue arrayer (Tissue Arrayer

MUC1 and SNAIL are overexpressed in renal sarcomatoid carcinomas

Sarcomatoid carcinoma in cRCC depicts a complete EMT pattern [7], [8]. Using a TMA sampling 22 cRCC with a sarcomatoid component, we showed by immunohistochemistry, that MUC1 was significantly overexpressed in sarcomatoid compared with conventional carcinomatous component (mean, 95% versus 31.8%; p < 0.001). In conventional cRCC, MUC1 staining was commonly restricted to the cytoplasmic membrane (Fig. 1A), whereas in sarcomatoid cRCC, MUC1 staining was diffusely cytoplasmic associated or not with

MUC1 is overexpressed in renal cancer-associated EMT

Originally described as markers of epithelial differentiation, mucins, especially MUC1, were recognized to be repressed during EMT [5], [28]. Thus, as E-cadherin, MUC1 was showed to be transcriptionally repressed by SNAIL in HT29 colon cancer cell line [13]. Furthermore, phosphorylation of MUC1-CT by Met was associated with inhibition of two EMT properties, i.e. migration and invasion, in pancreatic cancer cells via p53 signaling [29]. However, several recent studies in cellular models have

Financial support

This work is supported in part by the following grants: Agence Nationale de la Recherche (ANR-09-JCJC-0002; MP), Fondation pour la Recherche Médicale (MP), Comité du Nord de la Ligue Nationale contre le Cancer (SA) and the Conseil Régional du Nord Pas de Calais (CC, NP). AB is a recipient of a doctoral fellowship from “Région Nord-Pas-de-Calais” and INSERM. KG is a recipient of a doctoral fellowship from “Région Nord-Pas-de-Calais” and CHRU de Lille.

Conflict of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors gratefully acknowledge Marc Samyn (Institute of Pathology, CHRU Lille) and Rose-Marie Siminski & Marie-Hélène Gevaert (Department of Histology, Faculté de Médecine, Lille) for their technical help. MUC1 expressing vectors (MUC1FL, MUC1ΔTR, MUC1ΔCT) and empty vector were a gift from S.J. Gendler (Mayo Clinic, Scottsdale, AZ, USA). The human SNAIL promoter construct was a gift from L. Larue (CNRS UMR3347, INSERM U1021, Institut Curie, 91405 Orsay, France). The p-cDNA3 SNAIL-tagged HA

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