The RAS/RAF/MEK/ERK (MAPK) signaling cascade is essential for cell inter- and intra-cellular communication, which regulates fundamental cell functions such as growth, survival, and differentiation. The MAPK pathway also integrates signals from complex intracellular networks in performing cellular functions. Despite the initial discovery of the core elements of the MAPK pathways nearly four decades ago, additional findings continue to make a thorough understanding of the molecular mechanisms involved in the regulation of this pathway challenging. Considerable effort has been focused on the regulation of RAF, especially after the discovery of drug resistance and paradoxical activation upon inhibitor binding to the kinase. RAF activity is regulated by phosphorylation and conformation-dependent regulation, including auto-inhibition and dimerization. In this review, we summarize the recent major findings in the study of the RAS/RAF/MEK/ERK signaling cascade, particularly with respect to the impact on clinical cancer therapy.

A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336–4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G₁ phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G₁ cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy.

Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational C-terminal processing of prenylated proteins, suppresses tumor cell growth and induces cell death. Icmt inhibition by either a small molecule inhibitor termed as cysmethynil or inhibitory RNA induces marked autophagy leading to cell death. HepG2 cells were used to investigate the function of autophagy in tumor cell death. Suppression of autophagy, either pharmacologically or through knockdown of the autophagy essential proteins, Atg5 or Atg1, inhibits not only cysmethynil-induced autophagy, but also apoptosis in HepG2 cells. The dependence of cysmethynil-induced apoptosis on autophagy was further shown using autophagy-deficient mouse embryonic fibroblast (MEF) cells. Atg5^−/− MEF cells were found to be resistant to cysmethynil-induced apoptosis, whereas wild-type MEFs showed high sensitivity to apoptosis induction. These data indicate that inhibition of Icmt can elicit cell death through two linked mechanisms, autophagy and apoptosis, and that autophagy can be an active player upstream of apoptosis in cell types capable of apoptotic cell death, such as HepG2 and MEFs. Further, treatment of mice-bearing HepG2-derived tumors with cysmethynil resulted in marked inhibition of tumor growth; analysis of tumor tissue from these mice revealed markers consistent with autophagy induction and cell growth arrest.