Mechanisms of H‐Ras‐induced autophagic cell death in human glioblastoma

Abstract

Activating mutations in H-Ras (e.g., G12 →V) render the protein oncogenic in many types of cells. However, in glioblastoma cells expression of H-Ras(G12V) triggers caspase-independent cell death that commences with accumulation of vacuoles resembling large autophagosomes and autolysosomes. The cells expressing H-Ras(G12V) lose the ability to form colonies in soft agar and exhibit a marked increase in production of LC3-II, a phosphatidylethanolamine-conjugated protein associated with autophagosome membranes. Therefore, we tested the hypothesis that signals generated by H-Ras in glioblastoma cells may hyper-activate the autophagic machinery that typically responds to nutrient deprivation. The results of immunoblot studies indicate that H-Ras(G12V) does not decrease the activity of the nutrient-sensing kinase, mTOR. Moreover, siRNA gene silencing experiments indicate that accumulation of vacuoles does not require the mammalian autophagy protein, Beclin-1. Studies utilizing Ras-effector domain mutants and an inhibitor of MEK suggest that activation of the Raf-MEK-ERK1/2 signaling cascade is not responsible for induction of non-apoptotic cell death in glioblastoma. Likewise, preliminary studies indicate that vacuolization of the cells probably is not attributable to activation of phosphatidylinositol 3′-kinase (PI3-kinase). Taken together, the results indicate that some of the recently identified H-Ras signaling pathways may trigger autophagic cell death in glioblastoma via a novel mechanism distinct from classical nutrient-dependent macroautophagy. Supported by a grant from the NIH (R01CA34569) to W.A.M.