ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities

Replication and Cytopathic Effect of Oncolytic Vesicular Stomatitis Virus in Hypoxic Tumor Cells In Vitro and In Vivo

Tumor hypoxia presents an obstacle to the effectiveness of most antitumor therapies, including treatment with oncolytic viruses. In particular, an oncolytic virus must be resistant to the inhibition of DNA, RNA, and protein synthesis that occurs during hypoxic stress. Here we show that vesicular stomatitis virus (VSV), an oncolytic RNA virus, is capable of replication under hypoxic conditions. In cells undergoing hypoxic stress, VSV infection produced larger amounts of mRNA than under normoxic conditions. However, translation of these mRNAs was reduced at earlier times postinfection in hypoxia-adapted cells than in normoxic cells. At later times postinfection, VSV overcame a hypoxia-associated increase in α subunit of eukaryotic initiation factor 2 (eIF-2α) phosphorylation and initial suppression of viral protein synthesis in hypoxic cells to produce large amounts of viral protein. VSV infection caused the dephosphorylation of the translation initiation factor eIF-4E and inhibited host translation similarly under both normoxic and hypoxic conditions. VSV produced progeny virus to similar levels in hypoxic and normoxic cells and showed the ability to expand from an initial infection of 1% of hypoxic cells to spread through an entire population. In all cases, virus infection induced classical cytopathic effects and apoptotic cell death. When VSV was used to treat tumors established in nude mice, we found VSV replication in hypoxic areas of these tumors. This occurred whether the virus was administered intratumorally or intravenously. These results show for the first time that VSV has an inherent capacity for infecting and killing hypoxic cancer cells. This ability could represent a critical advantage over existing therapies in treating established tumors

Regulation of Protein Synthesis by Hypoxia via Activation of the Endoplasmic Reticulum Kinase PERK and Phosphorylation of the Translation Initiation Factor eIF2α

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2α on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2α, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2α attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2α kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2α. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2α and reduced inhibition of protein synthesis in response to hypoxia. PERK(−/−) mouse embryo fibroblasts failed to phosphorylate eIF2α and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2α and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response

SPARC Inhibits Metabolic Plasticity in Ovarian Cancer

The tropism of ovarian cancer (OvCa) to the peritoneal cavity is implicated in widespread dissemination, suboptimal surgery, and poor prognosis. This tropism is influenced by stromal factors that are not only critical for the oncogenic and metastatic cascades, but also in the modulation of cancer cell metabolic plasticity to fulfill their high energy demands. In this respect, we investigated the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in metabolic plasticity of OvCa. We used a syngeneic model of OvCa in Sparc-deficient and proficient mice to gain comprehensive insight into the paracrine effect of stromal-SPARC in metabolic programming of OvCa in the peritoneal milieu. Metabolomic and transcriptomic profiling of micro-dissected syngeneic peritoneal tumors revealed that the absence of stromal-Sparc led to significant upregulation of the enzymes involved in glycolysis, TCA cycle, and mitochondrial electron transport chain (ETC), and their metabolic intermediates. Absence of stromal-Sparc increased reactive oxygen species and perturbed redox homeostasis. Recombinant SPARC exerted a dose-dependent inhibitory effect on glycolysis, mitochondrial respiration, ATP production and ROS generation. Comparative analysis with human tumors revealed that SPARC-regulated ETC-signature inversely correlated with SPARC transcripts. Targeting mitochondrial ETC by phenformin treatment of tumor-bearing Sparc-deficient and proficient mice mitigated the effect of SPARC-deficiency and significantly reduced tumor burden, ROS, and oxidative tissue damage in syngeneic tumors. In summary, our findings provide novel insights into the role of SPARC in regulating metabolic plasticity and bioenergetics in OvCa, and shines light on its potential therapeutic efficacy

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities