The Mucin MUC4 and Its Membrane Partner ErbB2 Regulate Biological Properties of Human CAPAN-2 Pancreatic Cancer Cells via Different Signalling Pathways

Abstract

The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.

Mucins in pancreatic cancer and its microenvironment

“…33 Interestingly, transmembrane mucins are characterized by the presence of multiple structural motifs with sequence homology to EGF (referred to as EGF-like), which are thought to mediate heterodimerization of mucins with ERBB receptors (Box 1). 5,34,35 The affinities and functional relevance of these interactions for altered oncogenic signalling are being explored.…”

Micro-RNAs miR-29a and miR-330-5p function as tumor suppressors by targeting the MUC1 mucin in pancreatic cancer cells

“…Total proteins were extracted, electro-transferred, immunostained, and visualized as described before [5,23,26,28,29]. Antibodies used are MUC1 (M8, 1/250, from Pr D. Swallow); β-actin (A5441 AC15, 1/5000) from Sigma-Aldrich; cyclin D1 (sc-718, 1/250)and CDK6 (sc-177, 1/ 250) from Santa Cruz; EGFR (#4267s, 1/500), Bcl-2 (#2872, 1/250), phospho P53 (#9284, 1/500), P53 (#9282, 1/500), phospho p42-44 MAPK (#9101, 1/500), p42-44 MAPK (#9102, 1/500), phospho Stat3 (#9145, 1/250), Stat3 (#9139, 1/500), β-catenin (#8480s, 1/1000), phospho AKT (#4060, 1/500), and AKT (#4691s, 1/500) from Cell Signaling, Ozyme.…”

MUC4 mucin- a therapeutic target for pancreatic ductal adenocarcinoma

“…Carraway et al demonstrated that of the two EGF-like domains present in rat Muc4/Asgp2/Sialomucin complex, interaction with ErbB2 was mediated by EGF1 domain [38]. Human MUC4 contains three EGF domains, and a glutathione S-transferase (GST) fusion construct containing EGF3–EGF1–EGF2 domains of MUC4 forms a stable heterodimer with HER2, while in the HER2 knockdown cells MUC4 can interact with HER3 [39,40]. These interactions subsequently activate various signaling cascades including MAPK, JNK, and STAT-1 that promote cell proliferation and migration in vitro and enhance tumorigenicity and metastasis in vivo (Figure 2) [8,9,12,18,39,40].…”

“…Human MUC4 contains three EGF domains, and a glutathione S-transferase (GST) fusion construct containing EGF3–EGF1–EGF2 domains of MUC4 forms a stable heterodimer with HER2, while in the HER2 knockdown cells MUC4 can interact with HER3 [39,40]. These interactions subsequently activate various signaling cascades including MAPK, JNK, and STAT-1 that promote cell proliferation and migration in vitro and enhance tumorigenicity and metastasis in vivo (Figure 2) [8,9,12,18,39,40]. Additionally, MUC4 can modulate E-cadherin and N-cadherin signaling to promote contact-independent proliferation and drive epithelial-to-mesenchymal transition [9,41].…”

“…These antitumor effects were, in part, attributed to reduced HER2 levels upon MUC4 silencing. MUC4 expression was also silenced transiently, using MUC4-targeted siRNA oligonucleotides [72], or stably, using a retroviral vector encoding MUC4-targeted shRNA [40], to validate the interaction of MUC4 with HER2 and determine its impact on HER2 stability. Retroviral vector-mediated knockdown of MUC4 expression was also used to demonstrate the involvement of MUC4 in gemcitabine resistance [15] and elucidate the metastasis-promoting molecular mechanisms mediated by MUC4 in PDAC cell lines [41].…”