Preventing the unfolded protein response via aberrant activation of 4E‐binding protein 1 by versipelostatin

Abstract

We recently isolated a macrocyclic compound, versipelostatin (VST), that exerts in vivo antitumor activity. VST shows unique, selective cytotoxicity to glucose-deprived tumor cells by preventing the unfolded protein response (UPR). Here we show that eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of eukaryotic initiation factor 4E-mediated protein translation, plays a role in the UPR-inhibitory action of VST. Indeed, 4E-BP1 is aberrantly activated by VST. This activation occurs specifically during glucose deprivation and results in profound translation repression and prevents induction of the typical UPR markers glucose-regulated protein (GRP) 78 and activating transcription factor (ATF) 4. Our overexpression and knockdown experiments showed that 4E-BP1 can regulate GRP78 and ATF4 expression. These mechanisms appear to be specific for VST. By contrast, rapamycin, which activates 4E-BP1 regardless of cellular glucose availability, has only marginal effects on the expression of GRP78 and ATF4. Our present findings demonstrate that aberrant 4E-BP1 activation can contribute to UPR preventing by VST, possibly through a mechanism that does not operate in rapamycin-treated cells. (Cancer Sci 2009; 100: 327-333) S olid tumors have regions of low glucose and low oxygen (hypoxia) that arise from immature and irregular distribution of microvasculature.(1,2) In this stressful microenvironment, tumor cells are thought to survive by activating adaptive response pathways.(3) An important response for tumor development is the unfolded protein response (UPR), which can be activated in tumor cells during glucose deprivation as well as hypoxia.(4,5) The UPR has also been associated with lowered chemosensitivity in breast cancer and gliomas. (6,7) The UPR is a regulatory network that allows the cells to cope with stress that leads to the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER).(8) The main signaling pathways are sensed and initiated by the ERlocalized transmembrane proteins Double-standard RNA-dependent Protein Kinase (PKR)-like ER kinase (PERK), inositol-requiring 1 (IRE1), and activating transcription factor (ATF) 6.(8) These signaling pathways reduce global translation and produce several different active transcription factors to induce divergent UPR target genes, such as the ER-resident molecular chaperones glucose-regulated protein (GRP) 78 and GRP94. (5,8,9) Thus, during the UPR, both translational and transcriptional control mechanisms operate to relieve ER stress to allow for cell survival. (9)(10)(11)(12) In the case of intolerable levels of ER stress, however, the sensor proteins can contribute to apoptosis. (9)(10)(11)(12)(13) In the UPR translational control, PERK plays a major role by phosphorylating eukaryotic initiation factor (eIF) 2α at Ser51. (12,14) Phosphorylation of eIF2α reduces global translation and, paradoxically, directs preferential translation of ATF4, a UPR transcription activator.(15) The signaling pathway is further regulated by...

Heteroclitic XBP1 peptides evoke tumor-specific memory cytotoxic T lymphocytes against breast cancer, colon cancer, and pancreatic cancer cells

“…[41][42][43][44][45][46][47] A few examples of UPR-targeted cancer drugs in development and their mechanisms include Bortezomib (proteasome inhibitor), Tanespimycin (HSP90 inhibitor), Retaspimycin (HSP90 inhibitor), Versipelostatin (GRP78 inhibitor), and Irestatins (IRE1a inhibitor). [48][49][50] The purpose of these studies was to evaluate an immunotherapeutic approach for targeting the XBP1, an important transcription activator of UPR, in a variety of solid tumor. Previously, we identified highly immunogenic heteroclitic XBP1 US [184][185][186][187][188][189][190][191][192] (YISPWILAV) and XBP1 SP 367-375 (YLFPQLISV) peptides with higher HLA-A2 binding affinities and stabilities than their native counterparts.…”

Hyperactivation of 4E-Binding Protein 1 as a Mediator of Biguanide-Induced Cytotoxicity during Glucose Deprivation

“…Cell lysates were prepared as previously described (22). The following antibodies were used for immunoblotting: anti-eIF2a (Abcam), anti-phospho eIF2a, anti-4EBP1, anti-phospho-4EBP1 (Ser65; 174A9), anti-phospho-4EBP1 (Thr70), anti-eIF4E, anti-eIF4G, anti-AMPK, anti-phospho-AMPK (Cell Signaling Technology), anti-KDEL (for GRP78 detection; Stressgen), anti-ATF4 (Santa Cruz Biotechnologies), and anti-b-actin (Sigma).…”

“…Cell extracts (500 mg) were incubated with 7mGTP-conjugated Sepharose beads (GE Healthcare UK Ltd.) in the lysis buffer at 4 C for 2 hours (22). After 3 washings in the lysis buffer, the beads were boiled with 2ÂSDS sample loading buffer at 100 C. Each sample was analyzed by immunoblot.…”

“…MTT (Sigma) was then added to the culture medium. After 3 hours, the absorbance of each well was determined, as described previously (22). Relative cell survival was calculated by setting each control absorbance from untreated cells as 100%.…”

Functional Proteomic Analysis of Experimental Autoimmune Myocarditis-Induced Chronic Heart Failure in the Rat

“…Moreover, GRP78-related pathways are inhibited by versipelostatin, and this inhibition causes cancer cell death. 20) Using this agent, the cause and effect of GRP78 might be proved. HSP70 and 72 are from the same family as GRP78, and it was reported that levels of these proteins were increased during heart failure including in humans.…”