SUMMARYProtein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eukaryotic translation initiation factor 4E (eIF4E) binding to the 7-methylguanosine (m7-GpppG) 5′cap of mRNAs1,2. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms3–6. While the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells7,8. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition9. Here, we uncover an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. PAR-CLIP10 analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array mRNAs, including the epidermal growth factor receptor (EGFR). Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5′cap and targets mRNAs to polysomes for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen tension switches the basic translation initiation machinery.

suggests that HIF-2␣ exerts its proliferative effects by endorsing these major pathways. Consistently, silencing these receptors phenocopies the loss of HIF-2␣ oncogenic activity, abrogating the serumindependent growth of human cancer cells in culture. Based on these data, we propose an alternative to the predominant view that cancers exploit independent autonomous growth pathways and reveal HIF-2␣ as a potentially universal culprit in promoting the persistent proliferation of neoplastic cells.epidermal growth factor receptor ͉ growth signaling ͉ hypoxia-inducible factor ͉ insulin-like growth factor receptor ͉ oncogene

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygensensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygensensing pathway and the cellular response to hypoxia.

Inappropriate expression of Ets-1 is observed in a variety of human cancers, and its forced expression in cultured cells results in transformation, autonomous proliferation, and tumor formation. The basis by which Ets-1 confers autonomous growth, one of the primary hallmarks of cancer cells and a critical component of persistent proliferation, has yet to be fully explained. Using a variety of cancer cell lines, we show that inhibition of Ets-1 blocks tumor formation and cell proliferation in vivo and autonomous growth in culture. A screen of multiple diffusible growth factors revealed that inhibition of Ets-1 results in the specific downregulation of transforming growth factor α (TGFα), the proximal promoter region of which contains multiple ETS family DNA binding sites that can be directly bound and regulated by Ets-1. Notably, rescuing TGFα expression in Ets-1-silenced cells was sufficient to restore tumor cell proliferation in vivo and autonomous growth in culture. These results reveal a previously unrecognized mechanism by which Ets-1 oncogenic activity can be explained in human cancer through its ability to regulate the important cellular mitogen TGFα. Cancer Res; 70(2); 730-40. ©2010 AACR.

These results identify a novel nuclear export motif, further highlight the role of nuclear-cytoplasmic shuttling of E3 ligases in degradation of nuclear substrates, and provide evidence that disease-causing mutations can target subcellular trafficking.Ubiquitylation is a multiprotein pathway that destines marked proteins for degradation by the 26S proteasome (22,59). The conjugation of ubiquitin to proteins requires the action of three different enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. The process of ubiquitylation begins with the loading of a ubiquitin molecule onto the E1 ubiquitin-activating enzyme. This is followed by the transfer of ubiquitin from the E1 to the E2 ubiquitin-conjugating enzyme. Finally, transfer of ubiquitin from the E2 to the lysine residue of a target substrate is catalyzed by the E3 ubiquitin ligase. Selectivity of this pathway relies heavily on E3 ubiquitin ligases, which ultimately dictate substrate specificity. E3 ubiquitin ligases can act individually or form a multisubunit complex that may include a member of the Cullin family of proteins to covalently modify a vast array of cellular proteins. In view of the essential role of E3 ubiquitin ligases in regulation of many aspects of cellular functions and biological processes, there is mounting evidence that loss of function or deregulation of E3 ligases contributes to the development of disease.Degradation of nuclear substrates by the ubiquitylation system often requires nuclear-cytoplasmic trafficking of both the E3 ubiquitin ligase and the substrate protein (2, 54). One example is the ubiquitin-mediated degradation of the p53 tumor suppressor protein by the Mdm2 (murine double minute 2) E3 ubiquitin ligase (45, 47). Mdm2 shuttles continuously between the nucleus and the cytoplasm in order to efficiently degrade nuclear p53 (12, 53). Cancer-causing point mutations that disrupt nuclear export of Mdm2 are impaired in mediating proteasomal degradation of p53 (37). Nuclear export of the ROC1-SCF Fbw1a E3 ubiquitin ligase is also required for the proteasomal degradation of the Smad3 transcription factor (13). Another example is the cyclin-dependent kinase inhibitor p27 Kip1 , which requires nuclear export by Jab1 for proteasomemediated degradation. A mutant form of p27Kip1 that fails to assemble with Jab1 cannot be exported from the nucleus and is not degraded by the proteasome (54, 58).The von Hippel-Lindau tumor suppressor protein (VHL) is a vital component of the VBC-Cul2 E3 ubiquitin ligase complex, as it acts as the substrate recognition protein to provide specificity to the degradation process (25,27,30,38,39,50). VHL promotes the recruitment, ubiquitylation, and subsequent proteasomal degradation of the alpha subunit of hypoxia-inducible factor (HIF) in an oxygen-dependent manner (26,41). Under conditions of normal oxygen tension (normoxia), HIF␣ is hydroxylated at key prolyl residues within the oxygen-dependent degradation domain by prolyl hydroxylases (5,8,24,26). This p...