Abstract 368: Shoc2 scaffold protein role in Ras-MAPK signaling and cell survival in pancreatic adenocarcinoma cells
Authors: Camilla R. Borges, Pamela V. Andrade, Mirella Baroni, Vanessa S. Silveira
Journal: Cancer Research (2017)

Abstract Background: The highly malignant potential and aggressiveness of pancreatic adenocarcinoma cells is mainly promoted by oncogenic KRAS activation, which triggers innumerous signaling pathways and controls key processes for tumor progression such as cell proliferation, cell survival and metabolic reprograming. Interestingly, recent studies suggest that the major adjustments in metabolic reprogramming, mediated by KRAS activity, are regulated by MAP kinase activities. As SHOC2 scaffold protein plays an important role linking RAS to downstream signal transducers in the RAS/ERK MAP kinase signaling cascade, this study aimed to evaluate the mechanisms underlying RAS/ERK activation mediated by SHOC2 and the outcome in pancreatic adenocarcinoma cells proliferation and survival. Experimental design: RNAseq data from Pancreatic Adenocarcinoma (data generated by the TCGA Research Network: http://cancergenome.nih.gov/) was analyzed by using the cBioPortal (Sci. Signal. 6, pl1 (2013)). Gene expression analysis and protein expression was performed by quantitative RT-PCR and western blot respectively in pancreatic cell lines and normal pancreatic tissue. To address the role of SHOC2 in human pancreatic carcinoma cell lines (AsPC-1, PANC-1 and MIA PaCa-2) shRNA lentiviral constructs containing shRNA for SHOC2 or empty vector were used. Puromycin selection was performed to generate stable transformed lineages. Gene knockdown was confirmed by qRT-PCR and western blot. Doubling time and cell proliferation assay was performed in a 4-day MTT assay. Colony-forming assay (CFA) was tested in triplicate and t-test was used to analyze the data (alpha value was set at 5%). Results: Gene expression analysis by RNA-seq revealed that SHOC2 is highly expressed (a 10-fold log scale upregulation) in pancreatic adenocarcinoma samples. SHOC2 was also upregulated in pancreatic carcinoma cell lines in comparison to normal pancreatic tissue in both mRNA and protein analysis. After SHOC2 gene knockdown cell lines showed proliferation rates reduction and clearly increased their doubling time. In survival analysis both PANC-1 and MIA PaCa-2 cell lines showed a significant decrease in self-renewal capability (p = 0.003 and p = 0.002 respectively) in shRNA#SHOC2 transduced cells compared to controls (empty vector) suggesting that SHOC2 may play a role in pancreatic carcinoma cells survival. To further address if this outcome was ERK-mediated, we investigate ERK activation after SHOC2 knockdown. Unexpectedly, we did not observe any modulation on ERK phosphorylation, suggesting that SHOC2 function in pancreatic carcinoma cell survival may occur through other molecules rather than only ERK-mediated. SHOC2 knockdown had no major effects in ERK activation and cell growth in AsPC-1 cells. Conclusion: In pancreatic carcinoma cells we suggest that SHOC2 can play an important role in oncogenic signaling and lead to tumor cell survival. Citation Format: Camilla R. Borges, Pamela V. Andrade, Mirella Baroni, Vanessa S. Silveira. Shoc2 scaffold protein role in Ras-MAPK signaling and cell survival in pancreatic adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 368. doi:10.1158/1538-7445.AM2017-368