Identification of the Hypoxia-inducible Factor 2α Nuclear Interactome in Melanoma Cells Reveals Master Proteins Involved in Melanoma Development

Abstract

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that play a key role in cellular adaptation to hypoxia. HIF proteins are composed of an ␣ subunit regulated by oxygen pressure (essentially HIF1␣ or HIF2␣) and a constitutively expressed ␤ subunit. These proteins are often overexpressed in cancer cells, and HIF overexpression frequently correlates with poor prognosis, making HIF proteins promising therapeutic targets. HIF proteins are involved in melanoma initiation and progression; however, the specific function of HIF2 in melanoma has not yet been studied comprehensively. Identifying protein complexes is a valuable way to uncover protein function, and affinity purification coupled with mass spectrometry and label-free quantification is a reliable method for this approach. We therefore applied quantitative interaction proteomics to identify exhaustively the nuclear complexes containing HIF2␣ in a human melanoma cell line, 501mel. We report, for the first time, a high-throughput analysis of the interactome of an HIF subunit. Seventy proteins were identified that interact with HIF2␣, including some well-known HIF partners and some new interactors. The new HIF2␣ partners microphthalmia-associated transcription factor, SOX10, and AP2␣, which are master actors of melanoma development, were confirmed via coimmunoprecipitation experiments. Their ability to bind to HIF1␣ was also tested: microphthalmia-associated transcription factor and SOX10 were confirmed as HIF1␣ partners, but the transcription factor AP2␣ was not. AP2␣ expression correlates with low invasive capacities. Interestingly, we demonstrated that when HIF2␣ was overexpressed, only cells expressing large amounts of AP2␣ exhibited decreased invasive capacities in hypoxia relative to normoxia. Hypoxia-inducible transcription factors (HIFs)1 are central mediators of cellular adaptation to hypoxia that control the expression of genes involved in anaerobic metabolism, intracellular pH, angiogenesis, and cell growth and survival (1). HIF proteins are heterodimers consisting of an oxygen-regulated ␣ subunit and a constitutively expressed ␤ subunit (also referred to as the arylhydrocarbon receptor nuclear translocator, or ARNT). Three isoforms have been described for each subunit; most HIF function, however, has been attributed to HIF1␣, HIF2␣, and HIF1␤ (2). HIF1␣ is detected in almost all tissues, whereas HIF2␣ (also known as EPAS1, for endothelial PAS protein 1) is restricted to certain tissues. HIF2 is expressed notably in hypoxic endothelium but also in distinct cell populations in the brain, liver, kidney, lung, and intestine (3). HIF␣ proteins are regulated post-translationally as a function of the partial pressure of oxygen. In normoxic conditions, the protein is rapidly hydroxylated by prolyl hydroxylases and then recognized by the von Hippel-Lindau protein, a component of an E3-ubiquitin ligase complex that targets the ␣ subunit for degradation by the proteasome. When the partial pressure of oxygen is low, prolyl hydroxylase activi...

Hypoxia promotes production of neural crest cells in the embryonic head

“…Whereas both Sox9 and Sox10 have been identified as HIF target genes (Amarilio et al, 2007;Steunou et al, 2013;Zhang et al, 2011) and are sufficient to produce neural crest cells at least in the trunk neural tube (Cheung and Briscoe, 2003;McKeown et al, 2005), these genes have different expression profiles and functions. Sox9 is transiently expressed in the dorsal neural tube at the neural crest-emigration stages but not required for the emigration per se (Mori-Akiyama et al, 2003).…”

“…The genes that are directly regulated by HIF and expressed in neural crest cells in amniote embryos include SOX9 (Amarilio et al, 2007), SOX10 (Steunou et al, 2013), SNAIL1 (Luo et al, 2011) and CXCR4 (Staller et al, 2003). Although SNAIL2 has not been shown as a direct target of HIF so far, a strong correlation of its expression with hypoxia-induced metastatic phenotypes was shown (Wang et al, 2014;Zhang et al, 2013) [note that chick embryo neural crest cells express SNAIL2, not SNAIL1, whereas in mouse, Snail1 is the gene expressed in the equivalent structures (Locascio et al, 2002)].…”

“…Twist1 is also important for EMT in some contexts (Lamouille et al, 2014); however, in mouse embryos, Twist1 is expressed in migrating neural crest cells as they colonise the branchial arches (Füchtbauer, 1995) whereas it is not expressed in chick neural crest cells (Bothe and Dietrich, 2006). Among the neural crest specifier genes, Snail1, Sox9, Sox10 and Twist1 are known to be regulated directly by HIF in cancer and/or chondrocytes (Amarilio et al, 2007;Gort et al, 2008;Luo et al, 2011;Steunou et al, 2013;Yang et al, 2008). The chemokine receptor Cxcr4 is also a HIF-1α-regulated gene involved in chemoattraction of neural crest cells to their destination (Barriga et al, 2013;Escot et al, 2013;Rezzoug et al, 2011;Staller et al, 2003).…”

VHL Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma

“…Previous pulldown assays have reported that both HIF2α and HIF1β can interact with histone acetyltransferase p300 (68)(69)(70)(71). Indeed, p300 frequently marks enhancers (43) and is thought to be recruited by tissue-specific transcription factors (72).…”

Protein-protein interaction networks: probing disease mechanisms using model systems

“…Likewise, affinity purification of tagged EGFR (a cell surface receptor that is overexpressed in a number of cancers) led to the identification and quantification (by isobaric tags for relative and absolute quantification (iTRAQ)-based stable isotope labeling) of differential binding partners in lung tumor cell lines [75]. Several proteins with potentially crucial roles in the development of melanoma were elucidated by AP-MS analysis of hypoxia induced factor 2 (HIF2, a transcription factor commonly overexpressed in aggressive cancers) in human melanoma cell lines [76]. Likewise, novel interactors of the adenomatous polyposis coli (APC) oncoprotein were identified by AP-MS in HEK293 cells [77].…”