Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Abstract

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor-mediated or mitochondriamediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucoseregulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stressinduced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway.

Treatment with Panobinostat Induces Glucose-Regulated Protein 78 Acetylation and Endoplasmic Reticulum Stress in Breast Cancer Cells

“…Our findings are consistent with this report and add GRP78 to the list of molecular chaperones that are acetylated following inhibition of HDAC6, i.e., HDAC6 "acetylome", which includes a growing list of proteins (36). In transformed cells, activation of the RAS-RAF-MEK-ERK pathway and a basal state of proteotoxic stress induces GRP78 and HDAC6 levels (33,(37)(38)(39). Increased levels of GRP78 also result from an adaptive response to proteotoxic stress and UPR and confer resistance to apoptosis (2,(4)(5)(6)(7)20).…”

The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family

“…Human caspase-4 is at least partially localized to the ER, and, like murine caspase-12, appears to be selectively activated in response to ER stress (Hitomi et al, 2004b). A functional role for caspase-4 has been shown in amyloid b-mediated cytotoxicity (Hitomi et al, 2004b) as well as in ER stress-associated death in response to a variety of stimuli, including TG and TN, as well as proteasome inhibition and kinase inhibition (Hitomi et al, 2004b;Nawrocki et al, 2005;LopezAnton et al, 2006;Jiang et al, 2007;Rahmani et al, 2007).…”

“…Although an association between IRE-1 and caspase-12 has not been shown, it is possible that IRE-1 recruitment of the TRAF2/caspase-12 complex provides a scaffold for caspase-12 processing during ER stress (Yoneda et al, 2001). Less is known about caspase-4 than about caspase-12, but it requires both dimerization and interdomain processing for activation (Karki et al, 2007), and, like caspase-12, can be activated by calpain and is likely inhibited by interaction with GRP78 (Jiang et al, 2007;Oda et al, 2008).…”

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

“…4,5 Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression, 6 loss of PTEN function, 7 dysregulation of Bcl-2 expression, 8 upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression 9,10 and the ER chaperone GRP78. [11][12][13] Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression. 14 Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF V600E representing more than 90% of BRAF mutations, 15,16 the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.…”