Abrogating the oncogenic signaling associated with tumor MUC1 in Pancreatic Ductal Adenocarcinoma
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human cancers. The incidence rate of PDA nearly matches its mortality rate and the best treatment till date is surgical resection for which only 25\% are eligible. Tumor recurrence and metastasis are the main causes of cancer-related mortality. MUC1 is a transmembrane glycoprotein expressed on most epithelial cells. It is overexpressed and aberrantly glycosylated in cancer and is known as tumor-associated MUC1 (tMUC1). More than 80\% of PDAs express tMUC1. tMUC1 expression is found in the early stages of PDA development with subsequent increase in later stages.Transforming Growth Factor $\beta$ (TGF-$\beta$) is a cytokine that switches from a tumor-suppressor at early stages to a tumor promoter in the late stages of tumor development, by yet unknown mechanisms. Analysis of human PDA samples from TCGA database showed significant differences in gene expression and survival profiles between low and high MUC1 samples. Further, high MUC1 expression was found to positively correlate to TGF-$\beta$RII expression and negatively correlate to TGF-$\beta$RI expression in PDA cell lines. We hypothesized that MUC1 overexpression induces TGF-$\beta$ mediated non-canonical signaling pathway which is known to be associated with poor prognosis. In this study, we report that MUC1 overexpression in PDA cells directly activates the JNK pathway in response to TGF-$\beta$, and leads to increased cell viability via up-regulation and stabilization of c-Myc. Conversely, in low MUC1 expressing PDA cells, TGF-$\beta$ preserves its tumor-suppressive function and inhibits phosphorylation of JNK and stabilization of c-Myc. Knockdown of MUC1 in PDA cells also results in decreased phosphorylation of JNK and c-Myc in response to TGF-$\beta$ treatment. Taken together, the results indicate that overexpression of MUC1 plays a significant role in switching the function of TGF-$\beta$ from a tumor-suppressor to a tumor promoter by directly activating JNK. Lastly, we report that high-MUC1 PDA tumors respond to TGF-$\beta$ neutralizing antibody in vivo showing significantly reduced tumor growth while low-MUC1 tumors do not respond to TGF-$\beta$ neutralizing antibody further confirming our hypothesis. STAT3 is a transcription factor known to regulate proliferation, stemness, migration, invasion and apoptosis in cancer cells in a contextual manner. MUC1 and STAT3 have been reported to be involved in an auto-inductive loop and regulate each other's expressions in cancer cells. Phosphorylation of STAT3 at Y705 residue is associated with its activation and at S727 is related to its degradation, however, the role of differential phosphorylation of STAT3 in regulating cell fate and the factors regulating it have not been fully elucidated. Here we report, that MUC1 expression levels regulate the differential phosphorylation status of STAT3 in PDA cell lines. We report that STAT3-MUC1 pathway is constitively activated in high-MUC1 cancer cells, and therefore these cells are more sensitive to STAT3-inhibitor Napabucasin. A monoclonal antibody called TAB004, has been developed specifically against human tMUC1 extracellular domain. We report that treatment with TAB004 significantly reduced the colony forming potential and survival of multiple PDA cell lines while sparing normal pancreatic epithelial cell line. Binding of TAB004 to tMUC1 induced cytoskeleton remodeling, ER stress and anoikis in PDA cells. The mechanisms underlying the anti-tumor effects of TAB004 were found to be reduced activation of the EGFR-PI3K signaling pathway, and degradation of tMUC1, thereby reducing its binding to the desmosomal proteins desmoplakin and junction plakoglobin ($\gamma$-catenin) and $\beta$-catenin and other oncogenic partners, thus compromising the ability of the cells to form colonies. Furthermore, TAB004 treatment reduced expression of the transcriptional targets of MUC1, for example, c-SRC and c-MYC. These reduction in oncogenic signaling triggered anoikis as measured by reduced expression of anti-apoptotic proteins, PTRH2 and BCL2. TAB004 treatment slowed the growth of PDA xenograft compared to IgG control and enhanced survival of mice when combined with 5-FU. Since TAB004 significantly reduced colony forming potential and triggered anoikis in the PDA cells, we suggest that it could be used as a potential prophylactic agent to curb tumor relapse after surgery, prevent metastasis and help increase the efficacy of chemotherapeutic agents.