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 eIF2a 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.

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 eIF2␣ 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 eIF2␣, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2␣ attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2␣ kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2␣. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2␣ and reduced inhibition of protein synthesis in response to hypoxia. PERK ؊/؊ mouse embryo fibroblasts failed to phosphorylate eIF2␣ 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 eIF2␣ and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.Tumor hypoxia is a well-characterized feature of the solidtumor microenvironment. The development of hypoxia has profound consequences on tumor growth characteristics and on tumor response to radiotherapy and chemotherapy. Hypoxic tumors are more metastatic, are more resistant to radiotherapy and chemotherapy, and have a poorer prognosis than better-oxygenated ones, irrespective of therapy (28,29,72). Delineating the mechanisms by which hypoxia affects tumor physiology at the cellular and molecular levels is crucial for a better understanding of the process of tumor development and metastasis and for the design of better antitumor modalities.At the cellular level, exposure of cells to hypoxia has an immediate and reversible effect on proliferation. In Ehrlich ascites and HeLa cells, replicons stop firing within minutes of exposure to hypoxia, resulting in a G 1 /S-phase arrest. Upon reoxygenation, replicons begin extending, again within minutes (50-52). This hypoxia-induced G 1 /S arrest is independent of functional p53 tumor suppressor protein, since cells with mutant p53 and p53 knockout cells also arrest at the G 1 /S interface under hypoxic conditions (22). These studies point toward a unique mechanism of checkpoint control that differs from those induced by glucose deprivation or ionizing radiation, which require considerably more time for reinitiation of the cel...

1,25-dihydroxyvitamin D(3), (1,25(OH)(2)D(3); calcitriol), the hormonal form of vitamin D, exerts growth-inhibitory, pro-apoptotic and anti-metastatic effects on tumor cells in vitro and in vivo but its clinical use is limited by its calcemic effects. Previous studies have shown that the antiproliferative effects of the less calcemic calcitriol analog 19-nor-1,25-(OH)(2)D(2) (paricalcitol) on prostate tumor cell lines are indistinguishable from those of 1,25(OH)(2)D(3). We therefore investigated the anti-proliferative effects of paricalcitol on the growth of pancreatic tumor cell lines in vitro and in vivo. Both 1,25(OH)(2)D(3) and paricalcitol inhibited the growth of BxPC-3, Hs700T and AsPC-1 lines in a dose-dependent manner. This antiproliferative activity correlated with upregulation of the cell cycle inhibitors p21 (Waf1/CIP1) and p27(Kip1). A fourth pancreatic cell line, Hs766T was unresponsive to both paricalcitol and calcitriol. Hs766T cells also failed to upregulate p21/Waf-1/Cip1 or p27/KiP in response to treatments with these agents. Paricalcitol, given three times per week inhibited the growth of AsPC-1 pancreatic tumor cell xenografts in nude mice at a dose that did not cause hypercalcaemia. Tumor inhibition was accompanied by in vivo upregulation of p21 and p27 expression. Given the few therapeutic options for patients with pancreatic cancer, further exploration of paricalcitol, an FDA-approved medication, is warranted.

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 ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣) 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.

The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (␥-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with controlinfected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.DNA double strand breaks (DSBs) 1 occur naturally during DNA replication and V(D)J recombination but are also produced during the treatment of human malignancies with ionizing radiation (IR) and genotoxic drugs. In mammalian cells, the predominant pathway for repairing DSBs is non-homologous end joining (NHEJ). The DNA-dependent protein kinase (DNA-PK) complex is required for NHEJ. This complex, which includes Ku70, Ku80, and the 450-kDa DNA-PK catalytic subunit (DNA-PKcs), recruits several other repair proteins, including the MRE11/Rad50/NBS1 complex, XRCC4, and ligase IV (1). Despite the large number of proteins and seemingly redundant pathways involved in the processing of DNA damage, targeted gene deletions or mutations of any of the NHEJ proteins in mice results in growth deficiency, immune deficiency from defective V(D)J recombination, hypersensitivity to IR, neuronal apoptosis, and in some cases, tumorigenesis due to increased genomic instability (1, 2). These consequences of defective NHEJ demonstrate the importance of DSBR proteins in maintaining genomic integrity and cellular viability. Conversely, the hypersensitivity of DSBR-deficient cells to IR makes repair proteins an attractive target for radiosensitization of tumor cells with...