Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis

Unfolded protein response(UPR) is an adaptive reaction for cells to reduce endoplasmic reticulum(ER) stress. In many types of cancers, such as lung cancer and pancreatic cancer, cancer cells may harness ER stress to facilitate their survival and growth. Prion protein(PrP) is a glycosylated cell surface protein that has been shown to be up-regulated in many cancer cells. Since PrP is a protein prone to misfolding, ER stress can result in under-glycosylated PrP, which in turn may activate ER stress. To assess whether ER stress leads to the production of under-glycosylated PrP and whether underglycosylated PrP may contribute to ER stress thus leading to cancer cell apoptosis, we treated different cancer cells with brefeldin A(BFA), thapsigargin(Thps), and tunicamycin(TM). We found that although BFA, Thps, and TM treatment activated UPR, only ATF4 was consistently activated by these reagents, but not other branches of ER stress. However, the canonical PERK-eIF2α-ATF4 did not account for the observed activation of ATF4 in lung cancer cells. In addition, BFA,but neither Thps nor TM, significantly stimulated the expression of cytosolic PrP. Finally, we found that the levels of PrP contributed to anti-apoptosis activity of BFA-induced cancer cell death. Thus, the pathway of BFA-induced persistent ER stress may be targeted for lung and pancreatic cancer treatment.     

Expression of the hyperphosphorylated tau attenuates ER stress-induced apoptosis with upregulation of unfolded protein response

The neural dysfunction in Alzheimer's disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.     

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.     

Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis

Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA–MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1α). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

Characterization of stanniocalcin 2, a novel target of the mammalian unfolded protein response with cytoprotective properties

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a highly conserved homeostatic response in all eukaryotic cells, termed the unfolded-protein response (UPR). Here we describe the characterization of stanniocalcin 2 (STC2), a mammalian homologue of a calcium- and phosphate-regulating hormone first identified in fish, as a novel target of the UPR. Expression of STC2 gene is rapidly upregulated in cultured cells after exposure to tunicamycin and thapsigargin, by ATF4 after activation of the ER-resident kinase PERK. In addition, STC2 expression is also activated in neuronal cells by oxidative stress and hypoxia but not by several cellular stresses unrelated to the UPR. In contrast, expression of another homologue, STC1, is only upregulated by hypoxia independent of PERK or ATF4 expression. In vivo studies revealed that rat cortical neurons rapidly upregulate STC2 after transient middle cerebral artery occlusion. Finally, siRNA-mediated inhibition of STC2 expression renders N2a neuroblastoma cells and HeLa cells significantly more vulnerable to apoptotic cell death after treatment with thapsigargin, and overexpression of STC2 attenuated thapsigargin-induced cell death. Consequently, induced STC2 expression is an essential feature of survival component of the UPR.     

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.