Abstract A07: Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis

Abstract

Abstract Purpose of the study: The purpose of the study is to identify new effectors of K-Ras mediated and KSR1 dependent colon tumorigenesis. Background: Gene targeting and RNA interference identified Kinase suppressor of Ras1 (KSR1) as an effector of oncogenic Ras-induced tumor formation in mouse embryonic fibroblast (MEF) and human colon tumor cells but dispensable for normal development. Therefore, KSR1 and its downstream signaling molecules are potential targets for therapy in colon tumorigenesis. Previous analyses identified nuclear co-activator Peroxisome proliferator-activated receptor gamma co-activator 1(PGC1) and transcription factor Estrogen Related Receptor α (ERRα) as effectors of Ras-induced and KSR1-dependent cell transformation in MEFs. To identify molecules that are essential for Ras-driven and KSR1-dependent colon tumorigenesis, we performed high-throughput screens with the human colon tumor cell line HCT116, using genome-wide siRNA and natural product libraries. The siRNA screen identified AMP-activated protein kinase subunit γ1 (AMPKγ1) as an effector of KSR1-dependent signaling. Results: Compared to non-targeting siRNA, RNAi of AMPKγ1 in HCT116 cells decreased anchorage-independent growth in polyHEMA-coated plates by 6-fold in 24 h (p<10-3) and by 11-fold in 48 hours (p<10-3). Moreover, RNAi of AMPKγ1 differentially induced apoptosis of HCT116 cells (21%) compared to immortalized normal human colon epithelial cells (HCEC) (0.7%; p<10-4). Similarly, RNAi of AMPK catalytic α subunits (AMPKα1/AMPKα2) induced the death of HCT116 cells (21.5%) compared to non-targeting control siRNA (1.3%). RNAi of K-Ras, KSR1 and AMPKγ1 decreased protein expression of PGC1β and ERRα in HCT116 cells. Protein and mRNA expression of PGC1β and ERRα were significantly up-regulated in colon cancer cell lines compared to HCEC, indicating these are important effectors of K-Ras mediated KSR1 and AMPK-driven cell transformation. Furthermore, stable knockdown of PGC1β and ERRα decreased colony formation by 7-fold (p<10-3) and 3.5-fold (p<10-3) in soft agar and delayed tumor formation in nude mice (p<10-4). In-situ hybridization in human tumor samples revealed PGC1β mRNA to be significantly up-regulated in primary tumor stage I, stage II, and stage IV metastatic tumor samples (p<0.05) compared to normal colonic epithelium. ERRα mRNA was up-regulated in primary tumor stage I, II, III and stage IV metastatic tumors (p<0.05). Assay of natural product library for molecules that phenocopy RNAi of KSR1 and AMPK inhibitor Compound C identified 4-OH-Staurosporine. 4-OH-Staurosporine is a competitive inhibitor ATP binding to recombinant AMPK (α1β1γ1) (Ki=23.5 nM). 4-OH-Staurosporine treatment decreased phosphorylation of AMPK substrates acetyl-CoA carboxylase (ACC) at Ser89 and Raptor at Ser792 and expression of PGC1β and ERRα in a panel of human colon tumor cell lines (HCT116, HCT15, SW480, CACO2, GEO). Treatment with 3.75μM 4-OH-Staurosporine also decreased viability of colon tumor cells (HCT116, 52%; HCT15, 15%; SW480, 12%, CACO2, 58%; DLD1, 16%; GEO, 15%) compared with HCEC (p<10-3). Conclusion: These results demonstrate the importance of AMPK and its downstream signaling effectors PGC1β and ERRα in maintaining colon tumor cell survival driven by oncogenic Ras and demonstrate the value of using KSR1 as a reference standard to detect genes essential to colon tumor survival. Citation Format: Binita Das, Kurt Fisher, Hyunseok Kim, Deanna J. Volle, Deandra R. Smith, Robert S. Livergood, John MacMillan, Michael White, Robert Lewis. Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A07. doi: 10.1158/1557-3125.RASONC14-A07