Gr(i)p the ER to Stress Out Melanoma
Authors: Wanping Xu, Len Neckers
Journal: Cancer Cell (2016)

Because of their elevated steady-state stress level, cancer cells are particularly sensitive to perturbation of mechanisms regulating protein homeostasis. In this issue, Cerezo and colleagues show that pharmacologic modulation of GRP78, master regulator of the unfolded protein response in the endoplasmic reticulum, can be exploited for cancer treatment. Because of their elevated steady-state stress level, cancer cells are particularly sensitive to perturbation of mechanisms regulating protein homeostasis. In this issue, Cerezo and colleagues show that pharmacologic modulation of GRP78, master regulator of the unfolded protein response in the endoplasmic reticulum, can be exploited for cancer treatment. As the most aggressive form of skin cancer, melanoma accounts for 80% of skin cancer-related deaths, and its incidence is doubling every 10 years. Gain-of-function mutation in BRAF kinase is a common cause of melanoma, and inhibitors targeting BRAF cause non-durable regression of melanoma carrying BRAF mutations in the majority of patients (Flaherty et al., 2012Flaherty K.T. Infante J.R. Daud A. Gonzalez R. Kefford R.F. Sosman J. Hamid O. Schuchter L. Cebon J. Ibrahim N. et al.N. Engl. J. Med. 2012; 367: 1694-1703Crossref PubMed Scopus (2140) Google Scholar). Although long-lasting responses have been achieved by targeting CTLA4 or PD1, response rates are only 15%–35% (Eggermont et al., 2014Eggermont A.M. Spatz A. Robert C. Lancet. 2014; 383: 816-827Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Thus, drugs targeting a common vulnerability of melanoma remain an urgent need. Cerezo and colleagues now report development of the small molecule HA15, which is cytotoxic for melanoma cell lines and cultured melanoma cells freshly isolated from patient tumors, independent of the mutation status of BRAF, TP53, or NRAS (Cerezo et al., 2016Cerezo M. Lehraiki A. Millet A. Rouaud F. Plaisant M. Jaune E. Botton T. Ronco C. Abbe P. Amdouni H. et al.Cancer Cell. 2016; 29 (this issue): 805-819Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). To identify molecular target(s) of HA15, the authors performed proteomic analysis of biotinylated HA15 affinity pull downs, identifying 20 associating proteins. Among these was GRP78, a chaperone residing in the endoplasmic reticulum (ER). Considering the dramatically increased expression of genes involved in ER stress in HA15-treated melanoma cells and the pivotal role played by GRP78 in the ER stress response (unfolded protein response, UPR), the authors directed their focus on this chaperone. In vitro binding assay with recombinant proteins showed that HA15 interacts directly with GRP78, but not with other proteins involved in ER stress. The drug-protein interaction was confirmed using several approaches. Further, HA15 inhibited the intrinsic ATPase activity of GRP78. Together, these results suggest that HA15 may interfere with GRP78 function. Indeed, HA15 treatment reduced the association of GRP78 with the ER transmembrane UPR effectors PERK, IRE1α, and ATF6, leading to their activation. Under normal conditions, GRP78 binds PERK, IRE1α, and ATF6 in the ER lumen and inhibits their activity (Hassan et al., 2015Hassan M. Selimovic D. Hannig M. Haikel Y. Brodell R.T. Megahed M. World. J. Exp. Med. 2015; 5: 206-217Crossref PubMed Google Scholar). Elevated misfolded/unfolded protein load in the ER causes GRP78 to dissociate from these effectors, leading to their activation and initiation of the UPR. Freed from GRP78, PERK activates its intrinsic kinase activity, causing phosphorylation of the translation initiation factor eIF2α and resulting in suppression of global translation, thus reducing the folding demand placed on the ER. Like PERK, IRE1α is also activated upon dissociation of GRP78. Activated IRE1α removes 26 nucleotides from XBP1 mRNA, generating an isoform encoding a functional transcription factor (XBP1s). The UPR effector, ATF6, when dissociated from GRP78, translocates to the Golgi, where it undergoes proteolytic cleavage, releasing its cytosolic domain. This domain and XBP1s translocate to the nucleus and activate transcription of genes encoding ER chaperones and proteins involved in ER-associated degradation (ERAD). ERAD is a vital mechanism by which misfolded proteins in the ER are retro-translocated to the cytosol, ubiquitinated, and degraded in the proteasome (see Figure 1). By decreasing the influx of proteins into the ER and increasing the removal/refolding of unfolded proteins, transient activation of the UPR restores protein homeostasis (proteostasis). When these measures are unable to restore proteostasis in the face of severe or sustained ER stress, their prolonged activity leads to initiation of autophagy and apoptosis. Autophagy is a catabolic process that degrades and recycles subcellular components. A survival mechanism under adverse conditions, autophagy leads to cell death when it is activated for an extended time (Morselli et al., 2009Morselli E. Galluzzi L. Kepp O. Vicencio J.M. Criollo A. Maiuri M.C. Kroemer G. Biochim. Biophys. Acta. 2009; 1793: 1524-1532Crossref PubMed Scopus (320) Google Scholar). IRE1α activates ASK1 through the adaptor protein TRAF2. Activated ASK1 phosphorylates and inactivates the inhibitory partners of Beclin-1, leading to activation of autophagy (Sykes et al., 2016Sykes E.K. Mactier S. Christopherson R.I. Cancers (Basel). 2016; 8: E30Crossref PubMed Scopus (34) Google Scholar). Likewise, besides suppressing global translation, PERK-mediated eIF2α phosphorylation also induces ATF4 expression, which in turn transactivates the transcription factor CHOP, whose expression is also induced by activated ATF6 and XBP1s. CHOP promotes autophagy under high levels of ER stress. Indeed, HA15 increased the level of Beclin-1 and LC3B-II, molecules associated with the autophagosome, in melanoma cells. CHOP also drives the transcription of genes involved in apoptosis, including TNFRSF10B (DR5) and TRIB3 (TRB3). Further, CHOP decreases Bcl-2 levels and causes Bax to translocate to the mitochondria, promoting the release of cytochrome C to initiate apoptosis. ER stress-induced IRE1α activation also results in procaspase-12 cleavage, leading to caspase-3 activation via caspase-9 (see Figure 1). Bcl-2 levels are decreased in HA15-treated melanoma cells, as is the zymogenic form of caspase-3, indicative of its activation. Electron microscopy revealed membrane blebs, which are characteristic of apoptosis, consistent with increased cell labeling with annexin V antibody. The importance of ER stress in determining HA15 activity was verified by silencing CHOP expression, leading to dramatic inhibition of PARP cleavage and conversion of LC3B to LC3B-II, while also abrogating HA15 cytotoxicity. Blocking autophagy with siRNA targeting LC3B partially inhibited cell death, as did blocking apoptosis with QVD, a PARP cleavage inhibitor. Importantly, concomitant administration of both agents yielded complete protection from HA15-induced cell death. HA15 did not affect the viability of normal human melanocytes or fibroblasts. Consistent with the absence of cell death, HA15 did not induce PARP cleavage in these cells. Further, mice carrying melanoma xenografts showed no hepatic toxicity or changes in behavior and body mass upon administration of HA15 at doses that induced tumor ER stress, autophagy, and apoptosis and markedly attenuated xenograft growth. These findings suggest that HA15, by targeting a key component of the proteostasis machinery, may represent a promising chemotherapeutic reagent for melanoma. Importantly, efficacy of HA15 for melanoma was not affected by resistance to the BRAF inhibitor PLX4032. Moreover, HA15 proved cytotoxic to many other cancers, including prostate, breast, colon, and glioma, and its activity was likewise unaffected by resistance to conventional chemotherapies. Due to their high proliferation rates and limited nutrient and oxygen supply, cancer cells must constantly cope with many forms of stress, and the UPR in cancer cells, like the proteostasis network in general, is usually highly activated. Indeed, GRP78 is upreglated in several cancers, correlates with tumor stage and poor prognosis in pancreatic cancer, and, when downregulated, increases chemosensitivity in malignant glioma and breast cancer (Niu et al., 2015Niu Z. Wang M. Zhou L. Yao L. Liao Q. Zhao Y. Sci. Rep. 2015; 5: 16067Crossref PubMed Scopus (69) Google Scholar). Prolonged perturbation of the UPR in cancer cells exacerbates cellular stress and is likely to tip the balance between survival and death (see Figure 1). Indeed, GRP78 knockdown in melanoma cells has previously been shown to result in decreased proliferation (Croft et al., 2014Croft A. Tay K.H. Boyd S.C. Guo S.T. Jiang C.C. Lai F. Tseng H.Y. Jin L. Rizos H. Hersey P. Zhang X.D. J. Invest. Dermatol. 2014; 134: 488-497Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), an observation fully consistent with this report. Elevated GRP78 expression levels have also been associated with metastatic potential, and increased GRP78 levels have been found in cells at the invasive front of melanoma metastases (Papalas et al., 2010Papalas J.A. Vollmer R.T. Gonzalez-Gronow M. Pizzo S.V. Burchette J. Youens K.E. Johnson K.B. Selim M.A. Mod. Pathol. 2010; 23: 134-143Crossref PubMed Scopus (28) Google Scholar). Thus, it would be interesting to test whether HA15 impedes metastasis of tumor cells in general and of melanoma in particular. Prolonged disruption of the UPR can also be achieved by other means, including inhibition of Hsp90, a major chaperone in the cytosol and nucleus, and inhibition of the proteasome, a protein degradation apparatus required for ERAD. Hsp90 and proteasome inhibitors have displayed dramatic anti-cancer activity in preclinical and/or clinical settings (Miyata et al., 2013Miyata Y. Nakamoto H. Neckers L. Curr. Pharm. Des. 2013; 19: 347-365Crossref PubMed Scopus (226) Google Scholar). A combination of HA15 with one of these other proteostasis inhibitors may demonstrate even better anti-cancer activity. The impressive findings reported in this issue by Cerezo and colleagues support further development of HA15 as an anti-cancer agent, and they provide additional support for attacking the proteostasis network as a therapeutic strategy in cancer (Sykes et al., 2016Sykes E.K. Mactier S. Christopherson R.I. Cancers (Basel). 2016; 8: E30Crossref PubMed Scopus (34) Google Scholar). Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor ResistanceCerezo et al.Cancer CellMay 26, 2016In BriefCerezo et al. show that HA15, the lead compound of a series of thiazole benzenesulfonamides that they have developed, kills cancer cells by targeting BiP to increase ER stress. HA15 exhibits strong efficacy in melanoma cells, including those resistant to BRAF inhibitors. Full-Text PDF Open Archive