Sritama Nath

MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition

Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic ductal adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. EMT was significantly reduced when MUC1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT–PCR and western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared with cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus, thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer.

MUC1: a multifaceted oncoprotein with a key role in cancer progression

The transmembrane glycoprotein Mucin 1 (MUC1) is aberrantly glycosylated and overexpressed in a variety of epithelial cancers, and plays a crucial role in progression of the disease. Tumor-associated MUC1 differs from the MUC1 expressed in normal cells with regard to its biochemical features, cellular distribution, and function. In cancer cells, MUC1 participates in intracellular signal transduction pathways and regulates the expression of its target genes at both the transcriptional and post-transcriptional levels. This review highlights the structural and functional differences that exist between normal and tumor-associated MUC1. We also discuss the recent advances made in the use of MUC1 as a biomarker and therapeutic target for cancer.

MUC1 induces drug resistance in pancreatic cancer cells via upregulation of multidrug resistance genes

MUC1 (CD227), a membrane tethered mucin glycoprotein, is overexpressed in >60% of human pancreatic cancers (PCs), and is associated with poor prognosis, enhanced metastasis and chemoresistance. The objective of this study was to delineate the mechanism by which MUC1 induces drug resistance in human (BxPC3 and Capan-1) and mouse (KCKO, KCM) PC cells. We report that PC cells that express high levels of MUC1 exhibit increased resistance to chemotherapeutic drugs (gemcitabine and etoposide) in comparison with cells that express low levels of MUC1. This chemo resistance was attributed to the enhanced expression of multidrug resistance (MDR) genes including ABCC1, ABCC3, ABCC5 and ABCB1. In particular, levels of MRP1 protein encoded by the ABCC1 gene were significantly higher in the MUC1-high PC cells. In BxPC3 and Capan-1 cells MUC1 upregulates MRP1 via an Akt-dependent pathway, whereas in KCM cells MUC1-mediated MRP1 upregulation is via an Akt-independent mechanism. In KCM, BxPC3 and Capan-1 cells, the cytoplasmic tail motif of MUC1 associates directly with the promoter region of the Abcc1/ABCC1 gene, indicating a possible role of MUC1 acting as a transcriptional regulator of this gene. This is the first report to show that MUC1 can directly regulate the expression of MDR genes in PC cells, and thus confer drug resistance.

MUC1 regulates PDGFA expression during pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDA) has one of the worst prognoses of all cancers. Mucin 1 (MUC1), a transmembrane mucin glycoprotein, is a key modulator of several signaling pathways that affect oncogenesis, motility and metastasis. Its expression is known to be associated with poor prognosis in patients. However, the precise mechanism remains elusive. We report a novel association of MUC1 with platelet-derived growth factor-A (PDGFA). PDGFA is one of the many drivers of tumor growth, angiogenesis and metastasis in PDA. Using mouse PDA models as well as human samples, we show clear evidence that MUC1 regulates the expression and secretion of PDGFA. This, in turn, influences proliferation and invasion of pancreatic cancer cells leading to higher tumor burden in vivo. In addition, we reveal that MUC1 overexpressing cells are heavily dependent on PDGFA both for proliferation and invasion, whereas MUC1-null cells are not. Moreover, PDGFA and MUC1 are critical for translocation of β catenin to the nucleus for oncogenesis to ensue. Finally, we elucidate the underlying mechanism by which MUC1 regulates PDGFA expression and secretion in pancreatic cancer cells. We show that MUC1 associates with Hif1-α, a known transcription factor involved in controlling PDGFA expression. Furthermore, MUC1 facilitates Hif1-α translocation to the nucleus. In summary, we have demonstrated that MUC1-induced invasion and proliferation occurs via increased exogenous production of PDGFA. Thus, impeding MUC1 regulation of PDGFA signaling may be therapeutically beneficial for patients with PDA.

MicroRNA miR-308 regulates dMyc through a negative feedback loop in Drosophila

Summary The abundance of Myc protein must be exquisitely controlled to avoid growth abnormalities caused by too much or too little Myc. An intriguing mode of regulation exists in which Myc protein itself leads to reduction in its abundance. We show here that dMyc binds to the miR-308 locus and increases its expression. Using our gain-of-function approach, we show that an increase in miR-308 causes a destabilization of dMyc mRNA and reduced dMyc protein levels. In vivo knockdown of miR-308 confirmed the regulation of dMyc levels in embryos. This regulatory loop is crucial for maintaining appropriate dMyc levels and normal development. Perturbation of the loop, either by elevated miR-308 or elevated dMyc, caused lethality. Combining elevated levels of both, therefore restoring balance between miR-308 and dMyc levels, resulted in lower apoptotic activity and suppression of lethality. These results reveal a sensitive feedback mechanism that is crucial to prevent the pathologies caused by abnormal levels of dMyc.

Mucin 1 Regulates Cox-2 Gene in Pancreatic Cancer

Objective Eighty percent of pancreatic ductal adenocarcinomas (PDAs) overexpress mucin 1 (MUC1), a transmembrane mucin glycoprotein. MUC1high PDA patients also express high levels of cyclooxygenase 2 (COX-2) and show poor prognosis. The cytoplasmic tail of MUC1 (MUC1-CT) partakes in oncogenic signaling, resulting in accelerated cancer progression. Our aim was to understand the regulation of Cox-2 expression by MUC1. Methods Levels of COX-2 and MUC1 were determined in MUC1−/−, MUC1low, and MUC1high PDA cells and tumors using reverse transcriptase–polymerase chain reaction, Western blot, and immunohistochemistry. Proliferative and invasive potential was assessed using MTT and Boyden chamber assays. Chromatin immunoprecipitation was performed to evaluate binding of MUC1-CT to the promoter of COX-2 gene. Results Significantly higher levels of COX-2 mRNA and protein were detected in MUC1high versus MUC1low/null cells, which were recapitulated in vivo. In addition, deletion of MUC1 gene and transient knockdown of MUC1 led to decreased COX-2 level. Also, MUC1-CT associated with the COX-2 promoter at ∼1000 base pairs upstream of the transcription start site, the same gene locus where nuclear factor &kgr;B p65 associates with the COX-2 promoter. Conclusions Data supports a novel regulation of COX-2 gene by MUC1 in PDA, the intervention of which may lead to a better therapeutic targeting in PDA patients.

Pancreatic Cancer Cells Isolated from Muc1-Null Tumors Favor the Generation of a Mature Less Suppressive MDSC Population

Mucin 1 (MUC1) is a transmembrane mucin glycoprotein that is over-expressed and aberrantly glycosylated in >80% of human pancreatic ductal adenocarcinoma (PDA) and is associated with poor prognosis. To understand the role of MUC1 in PDA, we have recently developed two mouse models of spontaneous PDA, one that expresses full-length human MUC1 transgene (KCM mice) and one that is null for MUC1 (KCKO mice). We have previously reported that KCM mice express high levels of myeloid derived suppressor cells (MDSCs) in their tumors and develop highly aggressive PDA. To further understand the underlying mechanism for high MDSC levels in KCM-tumors, we generated primary cell lines from KCM and KCKO-tumors. In this study, we report that MDSCs derived using KCM cells express significantly higher levels of arginase 1 and inducible nitric oxide synthase (markers associated with immune suppression) and lower levels of CD115 (a marker associated with maturation of myeloid cells) as compared to KCKO-derived MDSCs. Functionally, KCM-derived MDSCs secrete significantly higher levels of urea and nitric oxide (NO) when co-cultured with normal splenic cells as compared to KCKO-derived MDSCs. Data indicates that KCM-derived MDSCs remain immature and are more suppressive as compared to KCKO-derived MDSCs. This was further corroborated in vivo where MDSCs isolated from KCM-tumor-bearing mice retained their immature state and were highly suppressive as compared to MDSCs derived from KCKO-tumor-bearing mice. Finally, we show that KCM cells secrete significantly higher levels of prostaglandin E2 (PGE2), a COX-2 metabolite and a known driver of suppressive MDSCs as compared to KCKO cells. Thus, inhibiting PGE2 with a specific COX-2 inhibitor reverses the immunosuppressive and immature phenotype of KCM-derived MDSCs. This is the first report that clearly suggests a functional role of pancreatic tumor-associated MUC1 in the development of functional MDSCs.

Abstract 475: Combinational treatment with MUC1 vaccine and Indomethacin reduces breast tumor burden via a COX-independent pathway.

Introduction: While much advancement has been made in breast cancer treatment, metastatic breast cancer remains an incurable disease. The glycoprotein MUC1 is over-expressed and underglycosylated in over 90% of human breast tumors and 100% of metastatic lesions, and thus was ranked the second most targetable tumor antigen (TA) by NCI. Vaccines against TAs have several benefits, including the chance to eliminate metastatic lesions that express the TA. To this end, we have proposed vaccinating with peptides from the MUC1 protein core, which is only visible to the immune system on the tumor-associated form of the protein. We have previously shown that vaccination does elicit a MUC1-specific immune response that can only be functional if the immunosuppressive tumor microenvironment is blocked to allow efficient tumor cell killing. We investigated the effectiveness of MUC1 vaccination in combination with drugs known to inhibit immunosuppression. Methods: Mice that are transgenic for human MUC1 were orthotopically injected with syngenic breast cancer cells expressing human MUC1. Mice were vaccinated after palpable tumor formation three times in 10 day intervals. Previous work has shown that blocking the cyclooxygenase pathway (COX) resulted in an inhibition of immunosuppression. Thus we treated daily with following drugs in combination with the MUC1-vaccine therapy: Indomethacine (COX1/2 inhibitor), Celecoxib (COX2 inhibitor), 1-methyl tryptophan (IDO inhibitor), and AH6809 (EP2 receptor antagonist). Tumor volume was monitored every day by caliper measurements. Immune activation was measured via an IFN-γ ELISPOT assay using T cells from tumor-draining lymph nodes as responders and dendritic cells pulsed with the vaccinating peptides as simulators. Results: MUC1 vaccine therapy alone causes a slight reduction in tumor burden, although not significant, but does induce IFN-γ production from T cells, indicating immune cell activation. Only the combinational therapy of Vaccine + Indomethacin results in a significant reduction in tumor size. All drugs cause significantly higher levels of IFN-γ production from T cells compared to vaccine alone but no reduction in tumor burden. The combinational treatments of Vaccine + Indomethacine and Vaccine + Celecoxib reduce intra-tumoral PGE2 levels compared to vaccine alone. In a repeat experiment, we found that vaccine alone and indomethacin alone caused a slight reduction in tumor burden, but tumor burden was only significantly reduced when the therapy was given in combination. Conclusion: In this model, MUC1 vaccine efficacy can be enhanced when given in combination with Indomethacin. Since Indomethacin but not Celecoxib reduced tumor burden when given in combination with the MUC1 vaccine, we suggest that Indomethacin enhanced vaccine efficacy via a COX-independent pathway. Future experiments will explore this mechanism. Citation Format: Jennifer M. Curry, Dahlia M. Besmer, Lopamudra Das Roy, Priyanka Grover, Sritama Nath, Shanti Rao, Pinku Mukherjee. Combinational treatment with MUC1 vaccine and Indomethacin reduces breast tumor burden via a COX-independent pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 475. doi:10.1158/1538-7445.AM2013-475

SMAD4-independent activation of TGF-β signaling by MUC1 in a human pancreatic cancer cell line

Pancreatic Ductal Adenocarcinoma (PDA) has a mortality rate that nearly matches its incidence rate. Transforming Growth Factor Beta (TGF-β) is a cytokine with a dual role in tumor development switching from a tumor suppressor to a tumor promoter. There is limited knowledge of how TGF-β function switches during tumorigenesis. Mucin 1 (MUC1) is an aberrantly glycosylated, membrane-bound, glycoprotein that is overexpressed in >80% of PDA cases and is associated with poor prognosis. In PDA, MUC1 promotes tumor progression and metastasis via signaling through its cytoplasmic tail (MUC1-CT) and interacting with other oncogenic signaling molecules. We hypothesize that high levels of MUC1 in PDA may be partly responsible for the TGF-β functional switch during oncogenesis. We report that overexpression of MUC1 in BxPC3 human PDA cells (BxPC3.MUC1) enhances the induction of epithelial to mesenchymal transition leading to increased invasiveness in response to exogenous TGF-β1. Simultaneously, these cells resist TGF-β induced apoptosis by downregulating levels of cleaved caspases. We show that mutating the tyrosines in MUC1-CT to phenylalanine reverses the TGF-β induced invasiveness. This suggests that the tyrosine residues in MUC1-CT are required for TGF-β induced invasion. Some of these tyrosines are phosphorylated by the tyrosine kinase c-Src. Thus, treatment of BxPC3.MUC1 cells with a c-Src inhibitor (PP2) significantly reduces TGF-β induced invasiveness. Similar observations were confirmed in the Chinese hamster ovarian (CHO) cell line. Data strongly suggests that MUC1 may regulate TGF-β function in PDA cells and thus have potential clinical relevance in the use of TGF-β inhibitors in clinical trials.

Abstract 2054: In pancreatic cancer, MUC1 regulates function of TGF-β and thus enhances metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background Pancreatic Cancer (PC) is the 4th leading cause of cancer-related deaths in the United States with 94% of PC patients dying within 5 years of diagnosis. Pancreatic Ductal Adenocarcinoma (PDA) characterizes 90% of PC. Over 80% of PDA overexpresses tumor associated Mucin-1 (MUC1), a membrane bound glycoprotein that is hypoglycosylated compared to normally expressed MUC1. Overexpression of MUC1 is associated with increased metastasis and poor prognosis. However the mechanism remains elusive. Transforming growth factor-β (TGF-β) is similarly overexpressed in most PDA. TGF-β is a cytokine with dual functionality. In normal cells, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical Smad pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However, during cancer development, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition, migration, and invasion of tumor cells thus enhancing metastasis. This effect of TGF-β is mediated by activation of the noncanonical Erk1/2 pathway through the engagement of TGF-β Receptor 2 (TGF-βRII). Therefore, we hypothesize that overexpression of MUC1 in PDA transforms the function of TGF-β from a tumor suppressor to a tumor promoter. Further, we postulate that signaling through the MUC1 cytoplasmic tail (CT) is necessary to activate the noncanonical Erk1/2 pathway via phosphorylation of the TGF-βRII, thus leading to enhanced metastasis. Methods We first assessed the ability of TGF-β to induce apoptosis versus invasiveness in PDA cell lines that express variable levels of MUC1 with gain of function (forced expression) and loss of function (knock down using specific MUC1 siRNA) studies. We determined the levels of TGF-βRI, RII, MUC1, and Smad 4 proteins in these PDA cell lines. We evaluated the activation status of the Erk1/2 and Smad pathways. Since c-Src is known to phosphorylate the 6th tyrosine residue of MUC1 CT and is essential for oncogenic signaling, we determined if c-Src is associated with MUC1 and is phosphorylated upon treatment with exogenous TGF-β in PDA cells. Results In MUC1-high expressing PDA cells, TGF-β acts as a tumor promoter while in MUC1-low expressing PDA cells, TGF-β induces apoptosis and acts as a tumor suppressor. We show a clear association between the expression of MUC1 and TGF-βRI and RII activation. In MUC1-high expressing cells, TGF-βRII is over expressed while in MUC1-low expressing cells, TGF-βRI is over expressed and this correlates with the activation status of Erk1/2 and Smad 4. Finally, we show that MUC1 specifically associates with c-Src and without this association, downstream oncogenic signaling is impeded. Conclusion MUC1 expression is directly correlated with TGF-β function and expression of TGF-βRII, while being negatively correlated with TGF-βRI expression. This has high clinical significance for patients with PDA. Citation Format: Priyanka Grover, Sritama Nath, Mohammad Ahmad, Pinku Mukherjee. In pancreatic cancer, MUC1 regulates function of TGF-β and thus enhances metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2015-2054