Endoplasmic Reticulum Stress-Activated Transcription Factor ATF6α Requires the Disulfide Isomerase PDIA5 To Modulate Chemoresistance

Abstract

e ATF6␣, a membrane-anchored transcription factor from the endoplasmic reticulum (ER) that modulates the cellular response to stress as an effector of the unfolded-protein response (UPR), is a key player in the development of tumors of different origin. ATF6␣ activation has been linked to oncogenic transformation and tumor maintenance; however, the mechanism(s) underlying this phenomenon remains elusive. Here, using a phenotypic small interfering RNA (siRNA) screening, we identified a novel role for ATF6␣ in chemoresistance and defined the protein disulfide isomerase A5 (PDIA5) as necessary for ATF6␣ activation upon ER stress. PDIA5 contributed to disulfide bond rearrangement in ATF6␣ under stress conditions, thereby leading to ATF6␣ export from the ER and activation of its target genes. Further analysis of the mechanism demonstrated that PDIA5 promotes ATF6␣ packaging into coat protein complex II (COPII) vesicles and that the PDIA5/ATF6␣ activation loop is essential to confer chemoresistance on cancer cells. Genetic and pharmacological inhibition of the PDIA5/ATF6␣ axis restored sensitivity to the drug treatment. This work defines the mechanisms underlying the role of ATF6␣ activation in carcinogenesis and chemoresistance; furthermore, it identifies PDIA5 as a key regulator ATF6␣-mediated cellular functions in cancer. P rotein folding in the endoplasmic reticulum (ER) can be particularly affected by the presence of mutations in secretory proteins or by dynamic changes in the cellular microenvironment, events which are often encountered in cancers. In the ER, these events are sensed by specific sensors, which in turn trigger select signaling pathways, collectively named the unfolded-protein response (UPR) (1). The UPR is an adaptive response that allows the cells to either overcome the stress or promote cell death in the case of overwhelming burden (1). Three ER-resident proteins, namely, the protein kinase PKR-like ER kinase (PERK), the inositol-requiring protein 1 alpha (IRE1␣), and the activating transcription factor 6 alpha (ATF6␣), have been identified as the major transducers of the UPR in mammals. They display an ER luminal domain that senses misfolded proteins and are activated by a common mechanism involving the dissociation of the ER chaperone BiP/GRP78. PERK is responsible for translational attenuation through the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2␣) (2). IRE1␣ mediates the unconventional splicing of X-box binding protein 1 (Xbp1) mRNA (3) as well as mRNA expression levels through regulated IRE1␣-dependent mRNA decay (RIDD) (4) and controls the activation of the c-jun N-terminal kinase (JNK) pathway. The third arm of the UPR is controlled by ATF6␣. This membrane-anchored transcription factor is a type II transmembrane protein regulated by intramembrane proteolysis by the Golgi apparatuslocalized site 1 and site 2 proteases (S1P and S2P) upon ER stress (5). Indeed, upon ER stress, BiP dissociates from the luminal domain of ATF6␣, thereby unmaski...

Redox controls UPR to control redox

“…1B). Not surprisingly, this occurs more rapidly in cells that are treated with DTT compared with cells that are treated with other pharmacological UPR triggers (DuRose et al, 2006;Higa et al, 2014;Nadanaka et al, 2007). In cells that have been depleted of PDIA5 (also known as PDIR), but not of other PDIs, oligomer dissociation, ER-to-Golgi migration and induction of ATF6 target genes are all inhibited, strongly suggesting that PDIA5 cleaves the disulfide bonds in ATF6 (Higa et al, 2014).…”

“…Not surprisingly, this occurs more rapidly in cells that are treated with DTT compared with cells that are treated with other pharmacological UPR triggers (DuRose et al, 2006;Higa et al, 2014;Nadanaka et al, 2007). In cells that have been depleted of PDIA5 (also known as PDIR), but not of other PDIs, oligomer dissociation, ER-to-Golgi migration and induction of ATF6 target genes are all inhibited, strongly suggesting that PDIA5 cleaves the disulfide bonds in ATF6 (Higa et al, 2014). ER-stress-induced dissociation of BiP from ATF6, however, occurs independently of PDIA5 (Higa et al, 2014) and is still a prerequisite for ER-to-Golgi transport of monomeric ATF6 in which cysteine residues 467 and 618 are mutated (Nadanaka et al, 2007).…”

“…In cells that have been depleted of PDIA5 (also known as PDIR), but not of other PDIs, oligomer dissociation, ER-to-Golgi migration and induction of ATF6 target genes are all inhibited, strongly suggesting that PDIA5 cleaves the disulfide bonds in ATF6 (Higa et al, 2014). ER-stress-induced dissociation of BiP from ATF6, however, occurs independently of PDIA5 (Higa et al, 2014) and is still a prerequisite for ER-to-Golgi transport of monomeric ATF6 in which cysteine residues 467 and 618 are mutated (Nadanaka et al, 2007). Thus, PDIA5-mediated reduction of ATF6 is necessary but not sufficient for Golgi targeting, which in addition requires the dissociation of BiP.…”

“…Upregulation of both PDIA5 and PDIA6 is essential for chemoresistant cancer cell growth. Specifically, resistance of leukemic cells to the tyrosine kinase inhibitor Imatinib depends on PDIA5 and pro-survival ATF6 signaling (Higa et al, 2014), and non-small lung cancer cells require robust overexpression of PDIA6 (and PDIA4, also known as ERp72) to withstand treatment with cytotoxic Cisplatin (Tufo et al, 2014). It is attractive to speculate that these pro-survival roles of PDIA5 and PDIA6 in cancer are mainly due to redox regulation of UPR sensors, i.e.…”

“…BiP, which binds to all three sensors in their inactive state, is titrated off when the unfolded protein load rises in the ER, and UPR sensors can also directly sense unfolded proteins, leading to their activation (Walter and Ron, 2011). Two studies have recently described redox regulation of the lumenal domains of the ER sensors that is mediated by dedicated PDIs (Eletto et al, 2014;Higa et al, 2014). This regulation impacts the onset, amplitude and duration of signaling by each of the sensors, thus providing a mechanistically novel concept of sensor-specific UPR activation.…”