Abstract 1065: Regulation of autophagy and MAPK signaling by glutathione S-transferase-π in KRAS mutated cancer cells

Background: Mutated KRAS (mKRAS) is the most commonly found oncogene which may play a role in carcinogenesis at relatively early stage. Although Raf-1/MEK/ERK pathway has been well characterized as a proliferative signal at downstream of mKRAS, other signals related to senescence or apoptosis were also found at downstream of mKRAS and thus mKRAS function is still needed to be explored at a molecular level. We have hitherto disclosed a close correlation of GST-π expression and mKRAS in cancer tissues, induction of GST-π expression by KRAS transfection (Gastroenterology121:865-874, 2001) and significant reduction of mKRAS positive colon polyps in GST-π KO mice (unpublished observation), suggesting some cooperative function of GST-π for mKRAS. Objective: In the present study, we attempted to clarify the molecular mechanisms of the cooperative function of GST-π for mKRAS activity in tumor cells. Results: We first intended to confirm the correlation between GST-π expression and KRAS mutation in various human cancer cell lines by Western blotting. Those cells with no mKRAS (HepG2, HeLa, MCF7) were negative for GST-π expression while mKRAS-expressing cells (PANC-1, A549, M7609) were positive for GST-π. We then using M7609 cells, examined the effect of GST-π siRNA on their growth and found a significant impairment of cell proliferation. When GST-π KD M7609 cells were examined under electron microscopy, 40% of cells showed intracytoplasmic vesicles. The vesicles were positive for LC3 punctate signal by immunofluorescent staining and showed a characteristic feature of autophagosome, an organelle enclosed by a double lipid bilayer, by electron microscopy. In these cells, phosphorylation of Akt and EGFR was substantially reduced. Since interaction of GST-π with EGFR has been reported, it is plausible that this interaction positively regulates EGFR/class I PI3K/Akt/mTOR signal which in turn inhibits autophagy formation. Another mKRAS related signal: Raf-1/MEK/ERK was also explored in its relation to GST-π function. In M7609 KD cells, both Raf-1 protein and p-Raf-1 (S338), active form of Raf-1, were significantly reduced and phosphorylation of S621 residue which is known to play a crucial role in prevention of ubiquitination of Raf-1 protein was suppressed. In these cells, phosphorylation of downstream kinases of MEK and ERK were also reduced though their total protein levels were unchanged. When the cells were treated with proteasome inhibitor (MG132), recovery of Raf-1 and p-Raf-1 protein was observed. In parental M7609 cells, GST-π was found to be preferentially bound to p-Raf-1 on co-immunoprecipitation analysis, indicating that GST-π forms complex with Raf-1to prevent the Raf-1 from proteasomal degradation. Conclusion: GST-π, on one hand prevents the cancer cells from autophagy formation and on the other hand, stimulates Raf-1/MEK/ERK signal by stabilizing Raf-1 protein in mKRAS cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1065. doi:10.1158/1538-7445.AM2011-1065