4585

MUC4

mucin 4, cell surface associated;MUC4;mucin 4, cell surface associated

ascites sialoglycoprotein|mucin 4, tracheobronchial|mucin-4|pancreatic adenocarcinoma mucin|testis mucin|tracheobronchial mucin

3q29

protein-coding

OBJECTIVE: The aim of our study was to determine the diagnostic value of MUC1 and MUC4 for distinguishing between metastatic adenocarcinoma cells (MAC) and reactive mesothelial cells (RMC) in effusion fluids. STUDY DESIGN: A total of 237 cell block specimens from pleural and peritoneal effusions, including 196 malignant effusions with MAC and 41 benign effusions with RMC, were stained with antibodies against MUC1 and MUC4. Membranous staining with or without cytoplasmic staining was considered to be positive. RESULTS: MUC1 immunoreactivity was observed in 194 (99.0%) of 196 cases of MAC and in 20 (48.8%) of 41 cases of RMC. MUC4 immunoreactivity was observed in 174 (88.8%) of 196 cases of MAC and in 4 (9.8%) of 41 cases of RMC. For distinguishing MAC from RMC, the MUC1 reactivity was found to be 99.0% sensitive and 51.2% specific with a positive predictive value of 90.7% and a negative predictive value of 91.3%. The sensitivity of MUC4 for MAC was 88.8%, the specificity was 90.2%, the negative predictive value was 62.7%, and the positive predictive value was 97.8%. CONCLUSION: Our data suggest that MUC4 appears to be a sensitive and specific marker for differentiating between MAC and RMC.

The MUC4 mucin is a high molecular weight membrane-bound glycoprotein. It is aberrantly expressed in pancreatic tumors and tumor cell lines with no detectable expression in the normal pancreas. A progressive increase of MUC4 expression has also been observed in pancreatic intraepithelial neoplasia, suggesting its association with disease development. Here, we investigated the consequences of silencing MUC4 expression in an aggressive and highly metastatic pancreatic tumor cell line CD18/HPAF that expresses high levels of MUC4. The expression of MUC4 was down-regulated by the stable integration of a plasmid-construct expressing antisense-MUC4 RNA. A decrease in MUC4 expression, confirmed by Western blot and immunofluorescence analyses, resulted in diminished growth and clonogenic ability of antisense-MUC4-transfected (EIAS19) cells compared with parental, empty vector (ZEO) and sense transfected (ES6) control cells. In addition, EIAS19 cells displayed a significant decrease in tumor growth and metastatic properties when transplanted orthotopically into the immunodeficient mice. In vitro biological assays for motility, adhesion, and aggregation demonstrated a 3-fold decrease in motility of EIAS19 cells compared with control cells, whereas these cells adhered more and showed an increase in cellular aggregation. Interestingly, MUC4 down-regulation also correlated with the reduced expression of its putative interacting partner, HER2/neu, in antisense-MUC4-transfected cells. In conclusion, the present work demonstrates, for the first time, a direct association of the MUC4 mucin with the metastatic pancreatic cancer phenotype and provides experimental evidence for a functional role of MUC4 in altered growth and behavioral properties of the tumor cell.

MUC4 is a large transmembrane type I glycoprotein that is overexpressed in pancreatic cancer (PC) and has been shown to be associated with its progression and metastasis. However, the exact cellular and molecular mechanism(s) through which MUC4 promotes metastasis of PC cells has been sparsely studied. Here we showed that the nidogen-like (NIDO) domain of MUC4, which is similar to the G1-domain present in the nidogen or entactin (an extracellular matrix protein), contributes to the protein-protein interaction property of MUC4. By this interaction, MUC4 promotes breaching of basement membrane (BM) integrity, and spreading of cancer cells. These observations are corroborated with the data from our study using an engineered MUC4 protein without the NIDO domain, which was ectopically expressed in the MiaPaCa PC cells, lacking endogenous MUC4 and nidogen protein. The in vitro studies demonstrated an enhanced invasiveness of MiaPaCa cells expressing MUC4 (MiaPaCa-MUC4) compared with vector-transfected cells (MiaPaCa-Vec; P=0.003) or cells expressing MUC4 without the NIDO domain (MiaPaCa-MUC4-NIDO(Delta); P=0.03). However, the absence of NIDO-domain has no significant role on cell growth and motility (P=0.93). In the in vivo studies, all the mice orthotopically implanted with MiPaCa-MUC4 cells developed metastasis to the liver as compared with MiaPaCa-Vec or the MiaPaCa-MUC4-NIDO(Delta) group, hence, supporting our in vitro observations. Additionally, a reduced binding (P=0.0004) of MiaPaCa-MUC4-NIDO(Delta) cells to the fibulin-2 coated plates compared with MiaPaCa-MUC4 cells indicated a possible interaction between the MUC4-NIDO domain and fibulin-2, a nidogen-interacting protein. Furthermore, in PC tissue samples, MUC4 colocalized with the fibulin-2 present in the BM. Altogether, our findings demonstrate that the MUC4-NIDO domain significantly contributes to the MUC4-mediated metastasis of PC cells. This may be partly due to the interaction between the MUC4-NIDO domain and fibulin-2.

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared with the normal pancreas, and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette smoke extract and nicotine, which is the major component of CS, significantly upregulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via the alpha7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the alpha7nAChR antagonists, alpha-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. In addition, we demonstrated that nicotine-mediated MUC4 upregulation promotes the PC cell migration through the activation of the downstream effectors, such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in vivo studies showed a marked increase in the mean pancreatic tumor weight (low dose (100 mg/m(3) total suspended particulate (TSP)), P=0.014; high dose (247 mg/m(3) TSP), P=0.02) and significant tumor metastasis to various distant organs in the CS-exposed mice, orthotopically implanted with luciferase-transfected PC cells, as compared with the sham controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine (low dose, 155.88+/-35.96 ng/ml; high dose, 216.25+/-29.95 ng/ml) and increased MUC4, alpha7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS upregulates the MUC4 mucin in PC via the alpha7nAChR/JAK2/STAT3 downstream signaling cascade, thereby promoting metastasis of PC.

MUC4 is a type-1 transmembrane mucin differentially expressed in multiple cancers and has previously been shown to potentiate progression and metastasis of pancreatic cancer. In this study, we investigated the molecular mechanisms associated with the MUC4-induced invasion and metastasis in pancreatic cancer. Stable silencing of MUC4 in multiple pancreatic cancer cells resulted in the downregulation of N-cadherin and its interacting partner fibroblast growth factor receptor 1 (FGFR1) through downregulation of partly by pFAK, pMKK7, pJNK and pc-Jun pathway and partly through PI-3K/Akt pathway. The downregulation of FGFR1 in turn led to downregulation of pAkt, pERK1/2, pNF-kappaB, pIkBalpha, uPA, MMP-9, vimentin, N-cadherin, Twist, Slug and Zeb1 and upregulation of E-cadherin, Occludin, Cytokeratin-18 and Caspase-9 in MUC4 knockdown BXPC3 and Capan1 cells compared with scramble vector transfected cells. Further, downregulation of FGFR1 was associated with a significant change in morphology and reorganization of the actin-cytoskeleton, leading to a significant decrease in motility (P < 0.00001) and invasion (P < 0.0001) in vitro and decreased tumorigenicity and incidence of metastasis in vivo upon orthotopic implantation in the athymic mice. Taken together, the results of the present study suggest that MUC4 promotes invasion and metastasis by FGFR1 stabilization through the N-cadherin upregulation.

OBJECTIVE. Mucin 4 (MUC4) is a transmembrane glycoprotein that is expressed in pancreatic ductal adenocarcinoma (PDAC), but not in normal pancreatic tissue. MUC4 has a proposed role in pancreatic tumor progression and metastasis. The purpose of this pilot study was to investigate MUC4 expression during PDAC metastasis by comparing the expression in the primary tumor and paired lymph node metastases from the same patient. MATERIAL AND METHODS. Surgical specimens from 17 cases of primary PDAC and paired lymph node metastases were immunohistochemically analyzed for MUC4 expression. The modified histochemical score (H-score) was used for staining assessment. RESULTS. Positive staining for MUC4 was detected in most primary and metastatic PDAC tumors (15/17 vs. 14/17). The concordance for MUC4 expression in primary tumors and corresponding lymph node metastases was 82%. In two cases, the primary tumor was MUC4-positive and the lymph node metastases were negative, while in one patient with a MUC4-negative primary tumor, the lymph node metastasis was positive. The distribution of H-score for expression of MUC4 significantly correlated (r = 0.615; p = 0.009) between primary tumors and paired metastatic lesions. CONCLUSIONS: MUC4 was observed in both primary and matched metastatic tumors with a high level of concordance, suggesting that MUC4 expression is retained following PDAC metastasis.

The membrane mucin MUC4 is aberrantly expressed in multiple cancers and is of clinical significance to diagnosis and prognosis in pancreatic cancer. However, the role of MUC4 in angiogenesis and the potential association among these malignant capabilities have not been explored. In this study, we investigated the collective signaling mechanisms associated with MUC4-induced growth, metastasis and angiogenesis in pancreatic cancer. Knockdown of MUC4 in two pancreatic cancer cell lines led to downregulation of lysosomal degradation of E-cadherin by Src kinase through downregulation of pFAK and pSrc pathway. The downregulation of lysosomal degradation of E-cadherin in turn induced the formation of E-cadherin/beta-catenin complex and membrane translocation of beta-catenin, resulting in the downregulation of Wnt/beta-catenin signaling pathway. Thus, the Wnt/beta-catenin target genes c-Myc, Cyclin D1, CD44 and VEGF were down-regulated and their malignant functions proliferation, metastasis and angiogenesis were reduced. Taken together, MUC4-induced nuclear translocation of beta-catenin is a novel mechanism for growth, metastasis and angiogenesis of pancreatic cancer.

Molecular changes are vital for the development of prognostic markers and therapeutic modalities of prostate cancer (CaP). There is growing interest in mucins as treatment targets in human malignancies, including CaP. The role of their expression in the progression of CaP is however unclear. We examined the expressions MUC1, MUC2, MUC4, MUC5AC and MUC6 in CaP tissues using tissue microarrays (TMAs) to look for tumor-associated antigens (TAAs) for targeted therapy. In this study, 120 paraffin-embedded specimens were selected from patients who underwent radical retro-pubic prostatectomy (RRP) or trans-urethral-resection of the prostate (TURP) for primary, untreated CaP and 10 matched lymph node metastases. A series of MUC monoclonal antibodies (mAbs) was used on TMAs by standard immunohistochemistry. Our results indicate that the over-expression of MUC1 was detected in 58% of primary CaP tissues and 90% of lymph node metastases but not in normal prostate or benign tissues, while the expression of MUC2, MUC4, MUC5AC and MUC6 was found to be negative in both normal and cancer tissues. Of the MUC1 positive tumors 86% were Gleason grade 7 or higher. Over-expression of MUC1 was found in late stage CaP while MUC2, 4, 5AC and 6 were negative in CaP. MUC1 is a TAA that is highly related to tumor progression in CaP patients. This antigen is ideal for targeted therapy to control micrometastases and hormone refractory disease but additional studies are necessary to assess its usefulness in patient biopsies and CaP bone metastases before clinical trial.