Abstract 3012: The CaSm oncogene promotes proliferation, invasion, and chemoresistance of pancreatic cancer cells.

Abstract

Abstract Introduction: The Cancer associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and its expression is necessary for in vivo tumor formation. Evidence supports that CaSm modulates mRNA degradation; however, CaSm's target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. We hypothesize that CaSm overexpression promotes neoplastic development by altering several hallmarks of cancer - including proliferation, metastasis, and chemoresistance. Methods and Results: Human CaSm was overexpressed in Panc1 cells (tet-on CaSm Panc1) using the Clontech Retro-X doxycycline inducible system with tet-on driver Panc1 cells used as controls in all experiments. Trypan blue cell counts presented a 2-fold increase in cellular proliferation following CaSm overexpression compared with driver and noninduced controls. Using SRB assays to evaluate cytotoxicity, CaSm induction increased resistance to gemcitabine at all drug concentrations (0.1-100uM) and CaSm overexpression also significantly decreased apoptosis (sub-G1 DNA content by cell cycle analysis) after treatment with 200nM gemcitabine. Real-time PCR-based microarray analysis was used to identify altered gene expression upon CaSm induction, showing decreased Bad and E2F1 expression and increased Bcl-xL mRNA levels; results were confirmed using qPCR and western blot analysis. These genes have been implicated in gemcitabine resistance in multiple cancers and provide a possible mechanism behind CaSm-mediated chemoresistance. To analyze migration and invasion, tet-on CaSm and tet-on driver Panc1 cells were plated onto fibronectin- or matrigel-coated Boyden chambers for 4 and 48 hours respectively. CaSm overexpression significantly increased transwell migration by more than 2-fold and invasion by nearly 4-fold. Microarrays identified increased MMP1 and uPAR mRNA upon CaSm induction, congruent with increased metastasis in vivo. Furthermore, western blot demonstrated that CaSm overexpression decreased E-cadherin and increased N-cadherin, characteristic of epithelial-to-mesenchymal transition (EMT) that is also known to promote both metastasis and chemoresistance in vivo. Investigation of EMT transcription factors by qPCR revealed that CaSm overexpression increases mRNA levels of Slug demonstrating a possible mechanism behind CaSm-induced EMT. Conclusions: Induced CaSm expression results in increased proliferation, decreased chemotherapeutic sensitivity, and enhanced migration/invasion in pancreatic cancer cells. CaSm upregulation alters the gene expression of critical mediators of apoptosis, metastasis, and EMT, which complements CaSm's proposed role in mRNA regulation and provides a mechanism for CaSm-mediated neoplastic progression. Citation Format: Elizabeth C. Little, E. Ramsay Camp, Cindy Wang, Patricia M. Watson, Dennis K. Watson, David J. Cole. The CaSm oncogene promotes proliferation, invasion, and chemoresistance of 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 3012. doi:10.1158/1538-7445.AM2013-3012