The Protein Kinase C Inhibitor Enzastaurin Exhibits Antitumor Activity against Uveal Melanoma

Abstract

GNAQ mutations at codon 209 have been recently identified in approximately 50% of uveal melanomas (UM) and are reported to be oncogenic through activating the MAPK/Erk1/2 pathway. Protein kinase C (PKC) is a component of signaling from GNAQ to Erk1/2. Inhibition of PKC might regulate GNAQ mutation-induced Erk1/2 activation, resulting in growth inhibition of UM cells carrying GNAQ mutations. UM cells carrying wild type or mutant GNAQ were treated with the PKC inhibitor enzastaurin. Effects on proliferation, apoptosis, and signaling events were evaluated. Enzastaurin downregulated the expression of several PKC isoforms including PKCβII PKCθ, PKCε and/or their phosphorylation in GNAQ mutated cells. Downregulation of these PKC isoforms in GNAQ mutated cells by shRNA resulted in reduced viability. Enzastaurin exhibited greater antiproliferative effect on GNAQ mutant cells than wild type cells through induction of G1 arrest and apoptosis. Enzastaurin-induced G1 arrest was associated with inhibition of Erk1/2 phosphorylation, downregulation of cyclin D1, and accumulation of cyclin dependent kinase inhibitor p27Kip1. Furthermore, enzastaurin reduced the expression of antiapoptotic Bcl-2 and survivin in GNAQ mutant cells. Inhibition of Erk1/2 phosphorylation with a MEK specific inhibitor enhanced the sensitivity of GNAQ wild type cells to enzastaurin, accompanied by p27Kip1 accumulation and/or inhibition of enzastaurin-induced survivin and Bcl-2 upregulation. PKC inhibitors such as enzastaurin have activity against UM cells carrying GNAQ mutations through inhibition of the PKC/Erk1/2 pathway and induction of G1 arrest and apoptosis. Inhibition of the PKC pathway provides a basis for clinical investigation in patients with UM.

A Role for Proteomics in Identifying Targets for Radiosensitizing Strategies in Melanoma: The FKBP51 Paradigm

“…It has been shown to be effective especially in combination with genotoxic drugs, including melphalan and doxorubicin [33,34]. Previous studies support the efficacy of this agent in malignant melanoma [14,35]. Consistent with previous findings [5,36,37], ionizing radiation produces only a slight effect on melanoma cell death.…”

Specifying protein kinase C functions in melanoma

“…Enzastaurin inhibited the growth of primary melanoma cultures and had a preferential growth inhibitory effect on uveal melanoma cell lines with GNAQ mutations (Hanauske et al, 2007;Wu et al, 2012). GNAQ is a phospholipase C-coupled Ga q subunit commonly mutated in uveal melanomas where it functions as a dominant oncogene (Van Raamsdonk et al, 2009.…”

“…GNAQ is a phospholipase C-coupled Ga q subunit commonly mutated in uveal melanomas where it functions as a dominant oncogene (Van Raamsdonk et al, 2009. Enzastaurin was able to reduce the protein levels and phosphorylation of multiple PKC enzymes (PKCb, PKCe, PKCh) important for cell viability in uveal melanoma cells (Wu et al, 2012). Enzastaurin was also able to reduce viability of cell such as Mel285 lacking detectible expression of PKCb, indicating that it has targets in addition to PKCb (Wu et al, 2012).…”

Immune consequences of kinase inhibitors in development, undergoing clinical trials and in current use in melanoma treatment

“…To date, the strongest support for the use of PKCi in melanoma comes from uveal melanoma, which commonly has mutations in gene-encoding subunits of GTP-binding proteins resulting in constitutive activation of the PKC pathway [196,197]. Blockade of PKC leads to reduced uveal melanoma cell proliferation, invasion and radiosensitivity in vitro and in animal models [198][199][200] and a Phase I clinical trial [201] aims to evaluate this using the PKCi sotrastaurin (AEB071) [202]. Sotrastaurin reduced clinical psoriasis severity and proliferation of lymphocytes following ex vivo stimulation [203] and effectively prevented the conversion of Tregs to IL-17 secreting, Foxp3-negative T cells, thereby enhancing Treg responses while inhibiting effector T-cell responses [204].…”