Steunou AL, Ducoux-Petit M, Lazar I, Monsarrat B, Erard M, Muller C, Clottes E, Schiltz O, Nieto L

Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that play a key role in cellular adaptation to hypoxia. HIF proteins are composed of an α subunit regulated by oxygen pressure (essentially HIF1α or HIF2α) and a constitutively expressed β subunit. These proteins are often overexpressed in cancer cells and HIF overexpression ...

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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 to mass spectrometry and label-free quantification provides a reliable method for this approach. We therefore applied quantitative interaction proteomics to identify exhaustively the nuclear complexes containing HIF2α in a human melanoma cell line, 501mel. We report, for the first time, a high throughput analysis of the interactome of a HIF subunit. Seventy proteins were identified that interact with HIF2α, including some well-known HIF partners as well as some new interactors. The new HIF2α partners MITF, SOX10 and AP2α which are master actors of melanoma development, were confirmed by coimmunoprecipitation experiments. Their ability to bind to HIF1α was also tested: MITF and SOX10 were confirmed as HIF1α partners, but the transcription factor AP2α was not. AP2α expression correlates with low invasive capacities. Interestingly, we demonstrated that, when HIF2α was overexpressed, only cells expressing large amounts of AP2α exhibited decreased invasive capacities in hypoxia compared to normoxia. The simultaneous presence of both transcription factors, therefore, reduces cells invasive properties. Knowledge of the HIF2α interactome thus provides a useful resource to investigate the general mechanisms of HIF function and regulation, and here has revealed unexpected distinct roles for the HIF1 and HIF2 isoforms in melanoma progression.

Date: Dec. 28, 2012

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