Dhanya Haridas

Targeting the EGFR signaling pathway in cancer therapy

Introduction: Cancer is a devastating disease; however, several therapeutic advances have recently been made, wherein EGFR and its family members have emerged as useful biomarkers and therapeutic targets. EGFR, a transmembrane glycoprotein is a member of the ERBB receptor tyrosine kinase superfamily. EGFR binds to its cognate ligand EGF, which further induces tyrosine phosphorylation and receptor dimerization with other family members leading to enhanced uncontrolled proliferation. Several anti-EGFR therapies such as monoclonal antibodies and tyrosine kinase inhibitors have been developed, which has enabled clinicians to identify and treat specific patient cohorts. Areas covered: This review covers the basic mechanism of EGFR activation and the role of EGFR signaling in cancer progression. Furthermore, current developments made toward targeting the EGFR signaling pathway for the treatment of epithelial cancers and a summary of the various anti-EGFR therapeutic agents that are currently in use are also presented in this review. Expert opinion: EGFR signaling is a part of a complex network that has been the target of effective cancer therapies. However, a further understanding of the system is required to develop an effective anticancer regimen. A combination therapy that comprises an anti-EGFR and a chemotherapeutic/chemopreventive agent will exhibit a multi-pronged approach that can be developed into a highly attractive and specific molecular oriented remedy.

MUC16 induced rapid G2/M transition via interactions with JAK2 for increased proliferation and anti-apoptosis in breast cancer cells

MUC16/CA125 is a tumor marker currently used in clinics for the follow-up of patients with ovarian cancer. However, MUC16 expression is not entirely restricted to ovarian malignancies and has been reported in other cancers including breast cancer. Although it is well established as a biomarker, function of MUC16 in cancer remains to be elucidated. In the present study, we investigated the role of MUC16 in breast cancer and its underlying mechanisms. Interestingly, our results showed that MUC16 is overexpressed in breast cancer tissues whereas not expressed in non-neoplastic ducts. Further, stable knockdown of MUC16 in breast cancer cells (MDA MB 231 and HBL100) resulted in significant decrease in the rate of cell growth, tumorigenicity and increased apoptosis. In search of a mechanism for breast cancer cell proliferation we found that MUC16 interacts with the ezrin/radixin/moesin domain-containing protein of Janus kinase (JAK2) as demonstrated by the reciprocal immunoprecipitation method. These interactions mediate phosphorylation of STAT3 (Tyr705), which might be a potential mechanism for MUC16-induced proliferation of breast cancer cells by a subsequent co-transactivation of transcription factor c-Jun. Furthermore, silencing of MUC16 induced G2/M arrest in breast cancer cells through downregulation of Cyclin B1 and decreased phosphorylation of Aurora kinase A. This in turn led to enhanced apoptosis in the MUC16-knockdown breast cancer cells through Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated extrinsic apoptotic pathway with the help of c-Jun N-terminal kinase signaling. Collectively, our results suggest that MUC16 has a dual role in breast cancer cell proliferation by interacting with JAK2 and by inhibiting the apoptotic process through downregulation of TRAIL.

Pathobiological Implications of MUC16 Expression in Pancreatic Cancer

MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease.

MUC4 potentiates invasion and metastasis of pancreatic cancer cells through stabilization of fibroblast growth factor receptor 1

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-κB, pIkBα, 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.

Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy

BackgroundPancreatic cancer (PC) is a lethal malignancy primarily driven by activated Kras mutations and characterized by the deregulation of several genes including mucins. Previous studies on mucins have identified their significant role in both benign and malignant human diseases including PC progression and metastasis. However, the initiation of MUC expression during PC remains unknown because of lack of early stage tumor tissues from PC patients.MethodsIn the present study, we have evaluated stage specific expression patterns of mucins during mouse PC progression in (KrasG12D;Pdx1-Cre (KC)) murine PC model from pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC) by immunohistochemistry and quantitative real-time PCR.ResultsIn agreement with previous studies on human PC, we observed a progressive increase in the expression of mucins particularly Muc1, Muc4 and Muc5AC in the pancreas of KC (as early as PanIN I) mice with advancement of PanIN lesions and PDAC both at mRNA and protein levels. Additionally, mucin expression correlated with the increased expression of inflammatory cytokines IFN-γ (p < 0.0062), CXCL1 (p < 0.00014) and CXCL2 (p < 0.08) in the pancreas of KC mice, which are known to induce mucin expression. Further, we also observed progressive increase in inflammation in pancreas of KC mice from 10 to 50 weeks of age as indicated by the increase in the macrophage infiltration. Overall, this study corroborates with previous human studies that indicated the aberrant overexpression of MUC1, MUC4 and MUC5AC mucins during the progression of PC.ConclusionsOur study reinforces the potential utility of the KC murine model for determining the functional role of mucins in PC pathogenesis by crossing KC mice with corresponding mucin knockout mice and evaluating mucin based diagnostic and therapeutic approaches for lethal PC.

MUC16: molecular analysis and its functional implications in benign and malignant conditions

MUC16 is a high‐molecular‐weight glycoprotein that is expressed by the various epithelial cell surfaces of the human body to protect the cell layer from a myriad of insults. It is the largest mucin known to date, with an ~22,152 aa sequence. Structurally, MUC16 is characterized into 3 distinct domains: the amino terminal, the tandem repeat, and the carboxyl terminal domain, with each domain having unique attributes. The extracellular portion of MUC16 is shed into the bloodstream and serves as a biomarker for diagnosing and monitoring patients with cancer; however, its functional role in cancer is yet to be elucidated. Several factors contribute to this challenge, which include the large protein size; the extensive glycosylation that the protein undergoes, which confers functional heterogeneity; lack of specific antibodies that detect the unique domains of MUC16; and the existence of splicing variants. Despite these limitations, MUC16 has been established as a molecule of significant application in cancer. Hence, in this review, we discuss the various aspects of MUC16, which include its discovery, structure, and biological significance both in benign and malignant conditions with an attempt to dissect its functional relevance.— Haridas, D., Ponnusamy, M. P., Chugh, S., Lakshmanan, I., Seshacharyulu, P., and Batra, S. K., MUC16: molecular analysis and its functional implications in benign and malignant conditions. FASEB J. 28, 4183‐4199 (2014). www.fasebj.org

Mucins in lung cancer: diagnostic, prognostic, and therapeutic implications.

Aberrant expression of mucins is associated with cancer development and metastasis. An overexpression of few mucins contributes to oncogenesis by enhancing cancer cell growth and providing constitutive survival signals. This review focuses on the importance of mucins both in the normal bronchial epithelial cells and the malignant tumors of the lung and their contribution in the diagnosis and prognosis of lung cancer patients. During lung cancer progression, mucins either alone or through their interaction with many receptor tyrosine kinases mediate cell signals for growth and survival of cancer cells. Also, stage-specific expression of certain mucins, like MUC1, is associated with poor prognosis from lung cancer. Thus, mucins are emerging as attractive targets for developing novel therapeutic approaches for lung cancer. Several strategies targeting mucin expression and function are currently being investigated to control lung cancer progression.

MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism.

MUC16, a heavily glycosylated type-I transmembrane mucin is overexpressed in several cancers including pancreatic ductal adenocarcinoma (PDAC). Previously, we have shown that MUC16 is significantly overexpressed in human PDAC tissues. However, the functional consequences and its role in PDAC is poorly understood. Here, we show that MUC16 knockdown decreases PDAC cell proliferation, colony formation and migration in vitro. Also, MUC16 knockdown decreases the tumor formation and metastasis in orthotopic xenograft mouse model. Mechanistically, immunoprecipitation and immunofluorescence analyses confirms MUC16 interaction with galectin-3 and mesothelin in PDAC cells. Adhesion assay displayed decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 protein. Further, CRISPR/Cas9-mediated MUC16 knockout cells show decreased tumor-associated carbohydrate antigens (T and Tn) in PDAC cells. Importantly, carbohydrate antigens were decreased in the region that corresponds to MUC16 and suggests for the decreased MUC16-galectin interactions. Co-immunoprecipitation also revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal and increased expression of epithelial markers in MUC16-silenced cells. Additionally, MUC16 loss showed a decreased FAK-mediated Akt and ERK/MAPK activation. Altogether, these findings suggest that MUC16-focal adhesion signaling may play a critical role in facilitating PDAC growth and metastasis.

Immunocytochemistry for MUC4 and MUC16 Is a Useful Adjunct in the Diagnosis of Pancreatic Adenocarcinoma on Fine-Needle Aspiration Cytology

CONTEXT Diagnoses rendered as atypical/suspicious for malignancy on fine-needle aspiration (FNA) of pancreatic mass lesions range from 2% to 29% in various studies. We have identified the expression of 3 genes, MUC4, MUC16, and NGAL that are highly upregulated in pancreatic adenocarcinoma. In this study, we analyzed the expression of these markers in FNA samples to determine whether they could improve sensitivity and specificity. OBJECTIVE To evaluate the utility of MUC4, MUC16, and NGAL in the evaluation of pancreatic FNA specimens. DESIGN Records of pancreatic FNAs performed during 10 consecutive years were reviewed. Unstained sections from corresponding cell blocks were immunostained for MUC4, MUC16, and NGAL (polyclonal). Immunostaining was assessed using the H-score (range, 0-3). Any case with an H-score of >0.5 was considered positive. RESULTS Cases were classified using cytomorphologic criteria as adenocarcinoma (31 of 64; 48.4%), benign (17 of 64; 26.6%), and atypical/suspicious (16 of 64; 25%). On follow-up, all cases (100%; 31 of 31) diagnosed as carcinoma on cytology were confirmed on biopsy/resection samples or by clinical follow-up (such as unresectable disease). Of the cases diagnosed as atypical/suspicious, 69% (11 of 16) were found to be positive for adenocarcinoma and 31% (5 of 16) were benign on subsequent follow-up. Overall sensitivity and specificity, respectively, for the various markers for the detection of pancreatic adenocarcinoma were as follows: MUC4 (74% and 100%), MUC16 (62.9% and 100%), and NGAL (61.3% and 58.8%). In cases that were atypical/suspicious on cytology, expression of MUC4 and MUC16 was 100% specific for carcinoma with sensitivities of 63.6% and 66.7%, respectively. CONCLUSION Immunocytochemistry for MUC4 and MUC16 appears to be a useful adjunct in the classification of pancreatic FNA samples, especially in cases that are equivocal (atypical/suspicious) for adenocarcinoma on cytomorphologic assessment.

Abstract 1629: Novel interaction of MUC16 with FAK activate EMT process and metastasis of pancreatic ductal adenocarcinoma

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: MUC16 is a heavily glycosylated, type I transmembrane mucin, which is over expressed in different cancers. We have previously shown that significant overexpression of MUC16 in human PDAC tissues with disease progression compared to normal pancreas. However, the functional consequences of MUC16 and their role in PDAC is poorly understood. Based on this our hypothesis is that MUC16 can drive pancreatic cancer metastasis through FAK-mediated Akt and ERK/MAPK signaling activation and altering EMT markers. Methods: We have developed MUC16 knockdown Capan1 and Colo-357 PDAC cells to study the functional impacts. Congenic cell survival, soft-agar colony formation, and trans-well chamber assays were performed to determine the in vitro tumorigenicity. Orthotopic implantation was carried out using capan-1 and colo-357 PDAC cells to determine the oncogenic and metastatic potential of MUC16. Binding assay was performed to determine the cell adhesion property of MUC16 in colo-357 cells. The physical interaction between MUC16 and mesothelin, galectin-3 and FAK were evaluated by confocal and immunoprecipitation analysis. Immunoblot analyses were performed to determine the downstream signaling in MUC16 knockdown cells. Results: MUC16 knockdown in capan-1 and colo-357 PDAC cell lines resulted in significantly decreased cell proliferation (P<0.05), colony formation (P<0.01), and migration (P<0.01) in vitro. Further, MUC16 knockdown capan-1 and colo-357 cells significantly decreases the tumor formation (P<0.05) and metastasis (liver P<0.05, spleen P<0.001, intestinal wall P<0.01, diaphragm P<0.01 and peritoneum P<0.001) in orthotopic xenograft mouse model. Adhesion assay displays decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 proteins. Immunoprecipitation and immunofluorescence studies confirmed that MUC16 interaction with mesothelin and galectin-3 in PDAC cells. Co-immunoprecipitation revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal markers (N-cadherin and Zeb1) and increased expression of epithelial markers (E-cadherin and CK18) in MUC16 silenced PDAC cells, correlating with the decrease in metastasis. Moreover, MUC16 knockdown show decreased FAK-mediated Akt and ERK/MAPK activation in PDAC cells. Conclusion: Overall our study concludes that MUC16 interacts with FAK leads to the activation of EMT markers for enhancing pancreatic cancer metastasis. Citation Format: Sakthivel Muniyan, Dhanya Haridas, Satyanarayana Rachagani, Imayavaramban Lakshmanan, Suprit Gupta, Seema Chugh, Parthasarathy Seshacharyulu, Moorthy P. Ponnusamy, Surinder K. Batra. Novel interaction of MUC16 with FAK activate EMT process and metastasis of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1629.