Abstract 3119: MUC1 transcriptionally regulates COX-2 gene in pancreatic cancer cells.

Introduction: Pancreatic Cancer (PC) is the fourth leading cause of cancer-related deaths in the United States. Over 80% of human pancreatic ductal adenocarcinomas (PDA) overexpress MUC1, a transmembrane mucin glycoprotein. MUC1high tumors are aggressive and patients with such tumors have poor prognosis. Interestingly, MUC1high PDA tumors overexpress cyclooxygenase-2 (COX-2), an inducible pro-inflammatory enzyme known to promote tumor progression and metastasis. Since the cytoplasmic domain of MUC1 (MUC1-C) is critically involved in the signal transduction cascade that promotes tumor progression and metastasis, we hypothesize that MUC1 induces its effect via regulating COX-2 expression and function. Thus, we aim to establish the relationship between MUC1 and COX-2 expression and determine the mechanism by which MUC1 regulates COX-2 expression. Cell lines: Human BxPC3 and mouse Panc02 PC cell lines that express low endogenous MUC1 were stably transfected with full length MUC1 (MUC1), empty vector (Neo), or full length MUC1 with all 7 tyrosine residues of MUC1 CT mutated to Phenylalanine (Y0). Other cell lines: human HPAFII and Capan-1 and mouse KCM cells that express high endogenous MUC1; and mouse KCKO cells that genetically lack Muc1. Method: We measured the level of COX-2 mRNA and protein by RT-PCR and Western blot respectively. MUC1 was transiently knocked down using a smart pool of MUC1 specific siRNA in MUC1high cells. Immunohistochemistry (IHC) was performed on tumor sections to determine the in situ expression of COX-2 and MUC1. Chromatin immunoprecipitation (ChIP) assay was conducted 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 PC cells expressing high MUC1 compared to cells with low MUC1. This was recapitulated in vivo with higher COX-2 expression in MUC1high versus MUC1low tumors. A subsequent decrease in COX-2 protein was observed upon MUC1 knock down in the MUC1high PC cells. Thus, a direct correlation exists between MUC1 and COX-2 expression in PC cells. Importantly, this correlation was lost in the Y0 cells in which COX-2 expression was significantly decreased even though there was high MUC1 expression. Data clearly suggests that signaling through the tyrosine residues of MUC1 CT is essential for MUC1 induced COX-2 gene expression. Further, in the MUC1high cells, MUC1 CT associates with the COX-2 promoter around 1000 bp upstream of the transcription start site. This association does not occur in the Y0 cells suggesting that an intact CT is necessary for this interaction. Interestingly, MUC1 CT interacts with the COX-2 promoter around the same gene locus where NF-kB associates with the COX-2 promoter. This raises the possibility that MUC1 CT associates with NF-kB via its tyrosine residues and the MUC1/NFkB complex acts as a transcriptional regulator of the COX-2 gene. Citation Format: Sritama Nath, Lopamudra Das Roy, Shanti Rao, Priyanka Grover, Pinku Mukherjee. MUC1 transcriptionally regulates COX-2 gene in pancreatic cancer cells. [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 3119. doi:10.1158/1538-7445.AM2013-3119