Therapeutic Discovery PKCd Regulates Death Receptor 5 Expression Induced by PS-341 through ATF4–ATF3/CHOP Axis in Human Lung Cancer Cells

PS-341 (bortezomib), a proteasome inhibitor, has been approved for the treatment ofmultiplemyeloma.Our previous work has shown that PS-341 induces death receptor 5 (DR5)–dependent apoptosis and enhances the TNF-related apoptosis-inducing ligand–induced apoptosis in human non–small cell lung cancer cells. However, the definite mechanism remains undefined. In the present study, we reveal that PKCd and RSK2 mediate PS-341–induced DR5 upregulation, involving coactivation of endoplasmic reticulum (ER) stress. We discovered that PS-341 activated ER stress through elevating the expression of BiP, p-eIF2a, IRE1a, ATF4, ATF3, and CCAAT/enhancer-binding protein homologous protein (CHOP). Further study showed that DR5 upregulation was dependent on ATF4, ATF3, and CHOP expression. Silencing either one of the ATF4, ATF3, and CHOP expression decreased DR5 upregulation and subsequent apoptosis. We determined that ATF4 regulated ATF3 and CHOP expression. Thereafter, ATF3 and CHOP formed a complex and regulated DR5 expression. In addition, we discovered that the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and RSK2 were elevated after PS-341 treatment and inhibition of their phosphorylation using MAP-ERK kinase 1/2 inhibitor decreased the DR5 level, indicating that ERK/RSK2 signaling is involved in DR5 upregulation. Furthermore, we detected the cleavage of PKCd, and the blockage of PKCd expression cut down DR5 upregulation and apoptosis. Importantly, knockdown of PKCd expression decreased the induction of ER stress and the phosphorylation of ERK1/2 and RSK2, suggesting that PKCd regulates DR5 expression through ERK/RSK2 signaling and ATF4–CHOP/ATF3 axis. Collectively, we show that PS-341 induces PKCd-dependent DR5 expression through activation of ERK/RSK2 and ER stress signaling pathway. Mol Cancer Ther; 11(10); 1–9. 2012 AACR. Introduction Death receptor 5 (DR5, also named Apo2), one of the TNF-related apoptosis-inducing ligand (TRAIL) receptors, belongs to the TNF receptor superfamily (1). It is located on the cell surface, and becomes trimerized on binding to its ligand TRAIL and recruits adapter proteins, such as Fas-associated death domain, then forms the death inducing signaling complex, and eventually activates caspase cascades (1). Nowadays, many studies have shown that inducing the expression of DR5 contributes to certain cancer therapeutic agentsinduced apoptosis and enhances TRAIL-induced apoptotic pathway (1). CCAAT/enhancer-binding protein homologous protein (CHOP), a downstream component of endoplasmic reticulum (ER) stress pathways, can be mediated by pancreatic ER kinase-like ER kinase-, activating transcription factor 6 (ATF6)-, and inositol-requiring enzyme 1a (IRE1a)–induced signal transduction pathways (2, 3). Many studies have documented that CHOP promotes apoptosis through upregulation of DR5 expression caused by many agents, such as lonafarnib (4), MG132 (5), CDDO-Me (6) in a variety of cancer cells. Activating transcription factor 4 (ATF4) and ATF3 are stress responsive proteins, which can be induced by ER stress (7, 8). Some earlier studies have found that ATF3 can suppress CHOP gene transcription (9), whereas CHOP inhibits ATF3 protein function (10). However, some data show that ATF3 can promote CHOP expression (11). The protein kinase C (PKC) family is important in regulation of cell proliferation and apoptosis. PKCd is generally considered to be a proapoptotic protein (12, 13). Studies have revealed that PKCd can be cleaved between the regulatory domain and catalytic Authors' Affiliation: Key Laboratory for Experimental Teratology of the Ministry of Education and School of Life Sciences, Shandong University, Jinan, Shandong, China Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). L. Su and L. Xu contributed equally to this work. Corresponding Author: Xiangguo Liu, Shandong University School of Life Sciences, Room 103, South Building, 27 Shanda South Road, Jinan, Shandong 250100, China. Phone: 86-531-88364330; Fax: 86-53188565610; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-12-0602 2012 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org OF1 on June 15, 2017. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst July 30, 2012; DOI: 10.1158/1535-7163.MCT-12-0602