Control of HIF-1 Expression by eIF2 Phosphorylation-Mediated Translational Repression

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Cancer Research (Impact Factor: 9.33). 03/2009; 69(5):1836-43. DOI: 10.1158/0008-5472.CAN-08-4103
Source: PubMed


Hypoxia inducible factor 1alpha (HIF-1alpha) plays a central role in regulating tumor angiogenesis via its effects on vascular endothelial growth factor (VEGF) transcription, and its expression is regulated through proteasome-mediated degradation. Paradoxically, previous studies have shown that proteasome inhibitors (PI) block tumor angiogensis by reducing VEGF expression, but the mechanisms have not been identified. Here, we report that PIs down-regulated HIF-1alpha protein levels and blocked HIF-1alpha transcriptional activity in human prostate cancer cells. PIs induced phosphorylation of the translation initiation factor 2alpha (eIF2alpha), which caused general translational repression to inhibit HIF-1alpha expression. Furthermore, PIs induced HIF-1alpha accumulation in LNCaP-Pro5 cells depleted of eIF2alpha via siRNA transfection and in MEFs expressing a phosphorylation-deficient mutant form of eIF2alpha. Finally, PIs failed to induce eIF2alpha phosphorylation or translational attenuation in DU145 or 253JB-V cells, and, in these cells, PIs promoted HIF-1alpha accumulation. Our data established that PIs down-regulated HIF-1alpha expression in cells that display activation of the unfolded protein response by stimulating phosphorylation of eIF2alpha and inhibiting HIF-1alpha translation.

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    • "Cap-dependent translation is initiated and regulated by the eukaryote Initiation Factor (eIF) family. Recent work has shed light on the role of some of these family members in regulating HIF-1α translation, including eIF2α [42], [43], eIF3E [44] and eIF4E [45]–[47]. It has been shown that the mammalian Target Of Rapamycin Complex 1 (mTORC1) signalling pathway regulates HIF-1α translation by mediating the phosphorylation of eIF4E binding protein 1 (4E-BP1) [45]–[47], which disrupts its binding to eIF4E thereby stimulating cap-dependent translation [48], [49]. "
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    ABSTRACT: Hypoxia inducible factor 1α (HIF-1α) is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi) block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA) and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor - eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.
    PLoS ONE 08/2014; 9(8):e106224. DOI:10.1371/journal.pone.0106224 · 3.23 Impact Factor
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    • "Recent studies have shown that two distinct pathways regulate HIF-1α protein synthesis. One is the phosphorylation of eIF2α, which is responsible for a rapid inhibition of translation initiation, and the other is a reduction in the phosphorylation of 4E-BP1, a protein that is regulated by mTORC1 [9], [10]. "
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    ABSTRACT: Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Tirapazamine (TPZ), a well-characterized bioreductive anticancer agent, is currently in Phase II and III clinical trials. A major aspect of the anticancer activity of TPZ is its identity as a tumor-specific topoisomerase IIα inhibitor. In the study, for the first time, we found that TPZ acts in a novel manner to inhibit HIF-1α accumulation driven by hypoxia or growth factors in human cancer cells and in HepG2 cell-derived tumors in athymic nude mice. We investigated the mechanism of TPZ on HIF-1α in HeLa human cervical cancer cells by western blot analysis, reverse transcription-PCR assay, luciferase reporter assay and small interfering RNA (siRNA) assay. Mechanistic studies demonstrated that neither HIF-1α mRNA levels nor HIF-1α protein degradation are affected by TPZ. However, TPZ was found to be involved in HIF-1α translational regulation. Further studies revealed that the inhibitory effect of TPZ on HIF-1α protein synthesis is dependent on the phosphorylation of translation initiation factor 2α (eIF2α) rather than the mTOR complex 1/eukaryotic initiation factor 4E-binding protein-1 (mTORC1/4E-BP1) pathway. Immunofluorescence analysis of tumor sections provide the in vivo evidences to support our hypothesis. Additionally, siRNA specifically targeting topoisomerase IIα did not reverse the ability of TPZ to inhibit HIF-1α expression, suggesting that the HIF-1α inhibitory activity of TPZ is independent of its topoisomerase IIα inhibition. In conclusion, our findings suggest that TPZ is a potent regulator of HIF-1α and provide new insight into the potential molecular mechanism whereby TPZ serves to reduce HIF-1α expression.
    PLoS ONE 11/2010; 5(11). DOI:10.1371/journal.pone.0013910 · 3.23 Impact Factor
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    • "For instance, insulin and growth factors such as insulin-like growth factor 1 and 2 and heregulin are known to increase HIF-1α protein concentrations by stimulating its protein synthesis in a 5′untranslated region (5′UTR)-dependent manner via activation of phosphatidylinositol 3-kinase, Akt and mTOR signaling [10], [11]. In addition, HIF-1α protein synthesis has been reported to be regulated by the RNA binding proteins HuR and PTB as well as via stress-induced phosphorylation of eIF2α [12]–[14]. There is also evidence for pathways that control HIF-1α stability in an oxygen-independent manner. "
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    ABSTRACT: Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that is composed of a hypoxia-inducible alpha subunit (HIF-1alpha and HIF-2alpha) and a constitutively expressed beta subunit (HIF-1beta). HIF mediates the adaptation of cells and tissues to low oxygen concentrations. It also plays an important role in tumorigenesis and constitutes an important therapeutic target in anti-tumor therapy. We have screened a number of reported HIF inhibitors for their effects on HIF-transcriptional activity and found that the DNA damage inducing agents camptothecin and mitomycin C produced the most robust effects. Camptothecin is a reported inhibitor of HIF-1alpha translation, while mitomycin C has been reported to induce p53-dependent HIF-1alpha degradation. In this study we demonstrate that the inhibitory effect of mitomycin C on HIF-1alpha protein expression is not dependent on p53 and protein degradation, but also involves HIF-1alpha translational regulation. Initiation of a DNA damage response with the small molecule p53 activator NSC-652287 (RITA) has been reported to inhibit HIF-1alpha protein synthesis by increasing the phosphorylation of eIF2alpha. However, we show here that even when eIF2alpha phosphorylation is prevented, the DNA damage inducing drugs mitomycin C, camptothecin and NSC-652287 still inhibit HIF-1alpha protein synthesis to the same extent. The inhibitory effects of camptothecin on HIF-1alpha expression but not that of mitomycin C and NSC-652287 were dependent on cyclin-dependent kinase activity. In conclusion, specific types of DNA damage can bring about selective inhibition of HIF-1alpha protein synthesis. Further characterization of the involved mechanisms may reveal important novel therapeutic targets.
    PLoS ONE 05/2010; 5(5):e10522. DOI:10.1371/journal.pone.0010522 · 3.23 Impact Factor
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