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.

Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2␣ at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2␣ was able to attenuate the phosphorylation of eIF2␣ and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2␣-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.Mammalian cells have the ability to alter their gene expression in order to adapt to a variety of environmental stresses, including nutrient depletion, oxidative stress, UV irradiation, reductive stress, exposure to toxins, and hypoxia, although the exact molecular events controlling the stress response have not been fully elucidated.Hypoxic stress plays a pivotal role in normal human development and physiology, including embryogenesis and wound repair, and has been well studied for its importance in the pathogenesis of several human diseases, namely, heart disease, stroke, diabetes, and cancer (for a detailed review, see reference 52). Hypoxia results when oxygen availability does not meet the demand of the surrounding tissue, resulting in decreased oxygen tension. In cancer this is initiated by the rapid proliferation of tumor cells, which gives rise to abnormal and chaotic vasculature, leading to the development of occlusions, blind ends, and vesicular shunts (4). The presence of hypoxic cells in solid tumors is well documented, and both clinical and experimental evidence suggests that the hypoxic microenvironment of a tumor helps to produce a more aggressive phenotype (62) by functioning as a selective pressure for the clonal expansion of apoptotically insensitive cells (16). The presence of hypoxic regions in solid tumors also correlates with a poor prognosis and has been shown to limit the efficacy of standard anticancer treatments, such as radiotherapy and chemotherapy (5). A key element to cellular survival and adaptation during hypo...

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cel-

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

No abstract

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.

We have previously cloned and identified a novel esophageal cancer related gene 2 (ECRG2; GenBank Accession Number AF268198), which is down-regulated in esophageal squamous cell carcinoma (ESCC) and involved in the induction of the apoptosis in esophageal cancer cell lines. In the present study, we have found a short tandem repeat (STR) polymorphism in the noncoding region of the exon 4 of the ECRG2 gene by using PCR-denaturing high-performance liquid chromatography (DHPLC Esophageal cancer, with a 5-year survival rate Ͻ10%, is one of the most common fetal cancers worldwide and occurs at very high frequencies in certain areas of China, Iran, South Africa, Uruguay, France and Italy. 1 Fifty percent of esophageal cancer cases in the world occur in China. Among high-risk areas of esophageal squamous cell carcinoma (ESCC), Linxian County in Henan Province has the highest age-adjusted mortality rate of this cancer. 2 Epidemiological studies have revealed that the incidence of ESCC is associated with several factors, such as N-nitrosamines, which have been shown to be involved in the etiology of ESCC in Linxian. 3 However, studies in high-risk areas have demonstrated a strong tendency towards familial aggregation or clustering of cases within families, suggesting that genetic susceptibility factors may also play an important role in the etiology of ESCC.Previous studies have shown that several genetic abnormalities including amplification of C-myc, Int-2 and Hst, 4,5 mutation and/or deletion of p53 and Rb 2 and allelic deletion 6 have frequently occurred in human ESCC and esophageal cancer cell lines. However, the genetic events that lead to the development of esophageal cancer are not clear yet. In recent years, many studies of esophageal cancer are focused on the cloning and identification of novel esophageal cancer-related genes, which might play an important role in the carcinogenesis and development of the cancer. [7][8][9] Recently, we have cloned and identified a novel ESCC-related gene, ECRG2 (Genbank Accession Number AF 268198), from human normal esophageal epithelia. Firstly, we obtained an EST fragment by mRNA differential display techniques from 3 normal esophageal epithelia and 2 primary ESCC tissues from high incidence families in Linxian County and named it ECRG2. The RT-PCR detection results demonstrated that the expression of ECRG2 was down-regulated in ESCC as well as colon and brain tumors. 10 Secondly, we obtained the 569 bp full-length cDNA of the ECRG2 gene by SMARTTM RACE technique. We found that the gene contains 4 exons, spans about 3,540 bp on chromosome 5q32-33 and has a 258 bp open reading frame encoding an 85-amino acids polypeptide. 11 Analysis by bioinformatics has shown that 97% of the amino acid sequences of ECRG2 are homologous to a tumor associated KAZAL-type serine protease inhibitor that may play a central role in the protection of esophageal mucosa. 12 Thirdly, we studied the function of the ECRG2 gene by yeast 2-hybrid system and identified that the ECRG2 gene interacts directly ...