Michaël G. Magagnin

Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control

Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2α and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1–2 h and is due to phosphorylation of eIF2α. Continued hypoxic exposure activates a second, eIF2α‐independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E‐BP1 and transporter 4E‐T. Quantitative RT–PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2α. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.

The mTOR target 4E-BP1 contributes to differential protein expression during normoxia and hypoxia through changes in mRNA translation efficiency.

Hypoxia causes a rapid and sustained inhibition in mRNA translation that is characterized by both a transient phosphorylation of eukaryotic initiation factor 2‐alpha (eIF2α) and by inhibition of the mRNA cap binding protein eIF4E via activation of two distinct inhibitory proteins, the mammalian target of rapamycin (mTOR) target 4E‐BP1 and the eIF4E transporter 4E‐T. Although the importance of eIF2α phosphorylation during hypoxia has been clearly demonstrated, there is little information on the potential relevance of eIF4E regulation. We generated HeLa cells stably expressing a short hairpin interfering RNA (shRNA) against 4E‐BP1 and found that despite efficient knockdown, no significant changes occurred in the overall inhibition of mRNA translation during hypoxia. However, using a proteomics approach we identified seven proteins that were exclusively expressed in the 4E‐BP1 knockdown cells during both normoxic and hypoxic conditions. Further investigation of the transcriptional and translational regulation of these genes by quantitative RT‐PCR indicated that the loss of 4E‐BP1 causes a significant increase in the rate of protein synthesis of S100 calcium‐binding protein A4 (S100A4) and transgelin 2. These 4E‐BP1 regulated proteins have previously been associated with tumor cell motility, invasion and metastasis and may thus contribute to an adverse tumor phenotype.

The prognostic value of temporal in vitro and in vivo derived hypoxia gene-expression signatures in breast cancer

BACKGROUND AND PURPOSE Recent data suggest that in vitro and in vivo derived hypoxia gene-expression signatures have prognostic power in breast and possibly other cancers. However, both tumour hypoxia and the biological adaptation to this stress are highly dynamic. Assessment of time-dependent gene-expression changes in response to hypoxia may thus provide additional biological insights and assist in predicting the impact of hypoxia on patient prognosis. MATERIALS AND METHODS Transcriptome profiling was performed for three cell lines derived from diverse tumour-types after hypoxic exposure at eight time-points, which include a normoxic time-point. Time-dependent sets of co-regulated genes were identified from these data. Subsequently, gene ontology (GO) and pathway analyses were performed. The prognostic power of these novel signatures was assessed in parallel with previous in vitro and in vivo derived hypoxia signatures in a large breast cancer microarray meta-dataset (n=2312). RESULTS We identified seven recurrent temporal and two general hypoxia signatures. GO and pathway analyses revealed regulation of both common and unique underlying biological processes within these signatures. None of the new or previously published in vitro signatures consisting of hypoxia-induced genes were prognostic in the large breast cancer dataset. In contrast, signatures of repressed genes, as well as the in vivo derived signatures of hypoxia-induced genes showed clear prognostic power. CONCLUSIONS Only a subset of hypoxia-induced genes in vitro demonstrates prognostic value when evaluated in a large clinical dataset. Despite clear evidence of temporal patterns of gene-expression in vitro, the subset of prognostic hypoxia regulated genes cannot be identified based on temporal pattern alone. In vivo derived signatures appear to identify the prognostic hypoxia induced genes. The prognostic value of hypoxia-repressed genes is likely a surrogate for the known importance of proliferation in breast cancer outcome.

Inhibition of 4E-BP1 Sensitizes U87 Glioblastoma Xenograft Tumors to Irradiation by Decreasing Hypoxia Tolerance

PURPOSE Eukaryotic initiation factor 4E (eIF4E) is an essential rate-limiting factor for cap-dependent translation in eukaryotic cells. Elevated eIF4E activity is common in many human tumors and is associated with disease progression. The growth-promoting effects of eIF4E are in turn negatively regulated by 4E-BP1. However, although 4E-BP1 harbors anti-growth activity, its expression is paradoxically elevated in some tumors. The aim of this study was to investigate the functional role of 4E-BP1 in the context of solid tumors. METHODS AND MATERIALS In vitro and in vivo growth properties, hypoxia tolerance, and response to radiation were assessed for HeLa and U87 cells, after stable expression of shRNA specific for 4E-BP1. RESULTS We found that loss of 4E-BP1 expression did not significantly alter in vitro growth but did accelerate the growth of U87 tumor xenografts, consistent with the growth-promoting function of deregulated eIF4E. However, cells lacking 4E-BP1 were significantly more sensitive to hypoxia-induced cell death in vitro. Furthermore, 4E-BP1 knockdown cells produced tumors more sensitive to radiation because of a reduction in the viable fraction of radioresistant hypoxic cells. Decreased hypoxia tolerance in the 4E-BP1 knockdown tumors was evident by increased cleaved caspase-3 levels and was associated with a reduction in adenosine triphosphate (ATP). CONCLUSIONS Our results suggest that although tumors often demonstrate increases in cap-dependent translation, regulation of this activity is required to facilitate energy conservation, hypoxia tolerance, and tumor radioresistance. Furthermore, we suggest that targeting translational control may be an effective way to target hypoxic cells and radioresistance in metabolically hyperactive tumors.

Translational control is a major contributor to hypoxia induced gene expression

BACKGROUND AND PURPOSE Hypoxia is a common feature of solid tumors that is associated with an aggressive phenotype, resistance to therapy and poor prognosis. Major contributors to these adverse effects are the transcriptional program activated by the HIF family of transcription factors as well as the translational response mediated by PERK-dependent phosphorylation of eIF2α and inhibition of mTORC1 activity. In this study we determined the relative contribution of both transcriptional and translational responses to changes in hypoxia induced gene expression. MATERIAL AND METHODS Total and efficiently translated (polysomal) mRNA was isolated from DU145 prostate carcinoma cells that were exposed for up to 24 h of hypoxia (<0.02% O(2)). Changes in transcription and translation were assessed using affymetrix microarray technology. RESULTS Our data reveal an unexpectedly large contribution of translation control on both induced and repressed gene expression at all hypoxic time points, particularly during acute hypoxia (2-4 h). Gene ontology analysis revealed that gene classes like transcription and signal transduction are stimulated by translational control whereas expression of genes involved in cell growth and protein metabolism are repressed during hypoxic conditions by translational control. CONCLUSIONS Our data indicate that translation influences gene expression during hypoxia on a scale comparable to that of transcription.