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MUCIN-4 (MUC4) is a novel tumor antigen in pancreatic cancer immunotherapy.

ABSTRACT: Pancreatic cancer (PC) is a highly lethal malignancy with a dismal five-year survival rate. This is due to its asymptomatic nature, lack of reliable biomarkers, poor resectability, early metastasis, and high recurrence rate. Limited efficacies of current treatment modalities treatment-associated toxicity underscore the need for the development of immunotherapy-based approaches. For non-resectable, locally advanced metastatic PC, immunotherapy-based approaches including vaccines, antibody-targeted, immune checkpoint inhibition, CAR-T-cells, and adoptive T-cell transfer could be valuable additions to existing treatment modalities. Thus far, the vaccine candidates in PC have demonstrated modest immunological responses in different treatment modalities. The identification of tumor-associated antigens (TAA) and their successful implication in PC treatment is still a challenge. MUC4, a high molecular weight glycoprotein that functionally contributes to PC pathogenesis, is an attractive TAA. It is not detected in the normal pancreas; however, it is overexpressed in mouse and human pancreatic tumors. The recombinant MUC4 domain, as well as predicted immunogenic T-cell epitopes, elicited cellular and humoral anti-MUC4 response, suggesting its ulility as a vaccine candidate for PC therapy. Existence of PC-associated MUC4 splice variants, autoantibodies against overexpressed and aberrantly glycosylated MUC4 and presence of T-cell clones against the mutations present in MUC4 further reinforce its significance as a tumor antigen for vaccine development. Herein, we review the significance of MUC4 as a tumor antigen in PC immunotherapy and discuss both, the development and challenges associated with MUC4 based immunotherapy. Lastly, we will present our perspective on MUC4 antigenicity for the future development of MUC4-based PC immunotherapy.

SUBMITTER: Gautam SK

PROVIDER: S-EPMC7160012 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Project description:The MUC4 mucin is a high molecular weight, membrane-bound, and highly glycosylated protein. It is a multi-domain protein that is putatively cleaved into a large mucin-like subunit (MUC4?) and a C-terminal growth-factor like subunit (MUC4?). MUC4 plays critical roles in physiological and pathological conditions and is aberrantly overexpressed in several cancers, including those of the pancreas, cervix, breast and lung. It is also a potential biomarker for the diagnosis, prognosis and progression of several malignancies. Further, MUC4 plays diverse functional roles in cancer initiation and progression as evident from its involvement in oncogenic transformation, proliferation, inhibition of apoptosis, motility and invasion, and resistance to chemotherapy in human cancer cells. We have previously generated a monoclonal antibody 8G7, which is directed against the TR region of MUC4, and has been extensively used to study the expression of MUC4 in several malignancies. Here, we describe the generation of anti-MUC4 antibodies directed against the non-TR regions of MUC4. Recombinant glutathione-S-transferase (GST)-fused MUC4? fragments, both upstream (MUC4?-N-Ter) and downstream (MUC4?-C-Ter) of the TR domain, were used as immunogens to immunize BALB/c mice. Following cell fusion, hybridomas were screened using the aforementioned recombinant proteins ad lysates from human pancreatic cell lines. Three anti MUC4?-N-Ter and one anti-MUC4?-C-Ter antibodies were characterized by several inmmunoassays including enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescene, flow cytometry and immunoprecipitation using MUC4 expressing human pancreatic cancer cell lines. The antibodies also reacted with the MUC4 in human pancreatic tumor sections in immunohistochemical analysis. The new domain-specific anti-MUC4 antibodies will serve as important reagents to study the structure-function relationship of MUC4 domains and for the development of MUC4-based diagnostics and therapeutics.

Project description:The fluorinated analog of deoxycytidine, Gemcitabine (Gemzar), is the main chemotherapeutic drug in pancreatic cancer, but survival remains weak mainly because of the high resistance of tumors to the drug. Recent works have shown that the mucin MUC4 may confer an advantage to pancreatic tumor cells by modifying their susceptibility to drugs. However, the cellular mechanism(s) responsible for this MUC4-mediated resistance is unknown. The aim of this work was to identify the cellular mechanisms responsible for gemcitabine resistance linked to MUC4 expression. CAPAN-2 and CAPAN-1 adenocarcinomatous pancreatic cancer (PC) cell lines were used to establish stable MUC4-deficient clones (MUC4-KD) by shRNA interference. Measurement of the IC50 index using tetrazolium salt test indicated that MUC4-deficient cells were more sensitive to gemcitabine. This was correlated with increased Bax/BclXL ratio and apoptotic cell number. Expression of Equilibrative/Concentrative Nucleoside Transporter (hENT1, hCNT1/3), deoxycytidine kinase (dCK), ribonucleotide reductase (RRM1/2) and Multidrug-Resistance Protein (MRP3/4/5) was evaluated by quantitative RT-PCR (qRT-PCR) and western blotting. Alteration of MRP3, MRP4, hCNT1 and hCNT3 expression was observed in MUC4-KD cells, but only hCNT1 alteration was correlated to MUC4 expression and sensitivity to gemcitabine. Decreased activation of MAPK, JNK and NF-?B pathways was observed in MUC4-deficient cells, in which the NF-?B pathway was found to have an important role in both sensitivity to gemcitabine and hCNT1 regulation. Finally, and in accordance with our in vitro data, we found that MUC4 expression was conversely correlated to that of hCNT1 in tissues from patients with pancreatic adenocarcinoma. This work describes a new mechanism of PC cell resistance to gemcitabine, in which the MUC4 mucin negatively regulates the hCNT1 transporter expression via the NF-?B pathway. Altogether, these data point out to MUC4 and hCNT1 as potential targets to ameliorate the response of pancreatic tumors to gemcitabine treatment.

Project description: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(?); 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(?) group, hence, supporting our in vitro observations. Additionally, a reduced binding (P=0.0004) of MiaPaCa-MUC4-NIDO(?) 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.

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MUCIN-4 (MUC4) is a novel tumor antigen in pancreatic cancer immunotherapy.

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