Increasing Melanoma Cell Death Using Inhibitors of Protein Disulfide Isomerases to Abrogate Survival Responses to Endoplasmic Reticulum Stress

Abstract

Exploiting vulnerabilities in the intracellular signaling pathways of tumor cells is a key strategy for the development of new drugs. The activation of cellular stress responses mediated by the endoplasmic reticulum (ER) allows cancer cells to survive outside their normal environment. Many proteins that protect cells against ER stress are active as protein disulfide isomerases (PDI) and the aim of this study was to test the hypothesis that apoptosis in response to ER stress can be increased by inhibiting PDI activity. We show that the novel chemotherapeutic drugs fenretinide and velcade induce ER stress-mediated apoptosis in melanoma cells. Both stress response and apoptosis were enhanced by the PDI inhibitor bacitracin. Overexpression of the main cellular PDI, procollagen-proline, 2-oxoglutarate-4-dioxygenase B subunit (P4HB), resulted in increased PDI activity and abrogated the apoptosis-enhancing effect of bacitracin. In contrast, overexpression of a mutant P4HB lacking PDI activity did not increase cellular PDI activity or block the effects of bacitracin. These results show that inhibition of PDI activity increases apoptosis in response to agents which induce ER stress and suggest that the development of potent, small-molecule PDI inhibitors has significant potential as a powerful tool for enhancing the efficacy of chemotherapy in melanoma. [Cancer Res 2008;68(13):5363-9]

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

“…45 In cutaneous metastatic melanoma, we have recently demonstrated the clinical potential of ER stress-induced apoptosis, with BRAF wt melanomas showing greater sensitivity, compared with tumours harbouring hyperactivating mutations in the BRAF protein kinase. 32,33 Moreover, we have also shown (and confirmed in the present study) that BRAF mutation is associated with increased levels of basal autophagy. 20 Interestingly, ER stress-induced apoptosis is reduced in melanoma cells harbouring oncogenic BRAF compared with the observed induction in BRAF wt melanoma cells, and in this context and in contrast to observations in BRAF mutant melanoma cells, we have also shown that autophagy inhibition significantly sensitizes BRAF wt melanoma cells to ER stressmediated apoptosis.…”

“…We have previously shown that BRAF-mutated melanoma cells display reduced sensitivity to ER stress activation compared with wt cells, suggesting that an abrogated ER stress response may limit drug-induced apoptosis in these cells 20,32 (Figure 1). We therefore hypothesised that the reduced ER stress response observed in BRAF mutant melanoma cells results from chronic ER stress that desensitizes them to further stress stimuli.…”

“…As shown in Figure 2a, BRAF V600E melanoma cell lines displayed approximately twofold greater expression of mRNA coding for ERdj5, ERp57, ATF4 and Xbp-I 33 compared with BRAF wt cells, indicating that a mild but consistent induction of ER stress is conferred by an activating mutation in the BRAF protein kinase. 28,32,33 To rule out the possibility that differences in ER stress induction were due to effects other than BRAF mutation, we expressed constitutively active BRAF V600E in the BRAF wt SKMel-110 melanoma cell line in order to compare melanoma cells with identical genetic background except for BRAF status. Cells stably expressing BRAF V600E or GFP were selected for more than 1 month in culture, in order to better represent in vivo tumour development and adaptation to BRAF activating mutation.…”

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal–epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

“…15,[35][36][37] In this study, we show that ERp29 is a novel regulator in modulating cell growth arrest and phenotypic changes associated with MET in proliferative/invasive breast cancer cells. Importantly, we show that ERp29 is a potential tumor suppressor in breast cancer progression.…”

The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma

“…With the aim of identifying PDI isoform(s) responsible for CDDP resistance, we transfected pools of siRNAs to selectively and individually inhibit the expression of various PDI isoforms. Four isoforms have been selected because of their previously characterized role in survival to ER stress (PDIA1), 30 in Ca 2 þ exchange (PDIA3) 31 and their overexpression in the present study (PDIA4 and PDIA6). Thus, siRNAs against these four isoforms allowed efficient knockdown in A549-resistant clones with limited off-target effects in 48 h (Supplementary Figure S3).…”