SHARP1 expression is inversely correlated with risk of metastasis of triple negative breast cancer (TNBC): Tumors with lower SHARP1 expression are more likely to metastasize. Using gene set enrichment analyses of TNBC samples, Montagner et al. identified gene sets associated with activity of the hypoxia-inducible factor (HIF) pathway as correlated with low SHARP1 expression compared to those tumors with high SHARP1 expression. Tumors with low SHARP1 expression were also enriched in the expression of HIF-target genes. HIF-1α coimmunoprecipitated with SHARP1 in extracts from three independent TNBC cell lines, one of which was nonmetastatic. In TNBC cell lines exposed to hypoxic conditions, the induction of HIF target genes, as measured by quantitative PCR, was inhibited by overexpression of SHARP1. The authors verified the inverse correlation between HIF activity and SHARP1 expression in primary human TNBC by detecting HIF target proteins in tumors by immunohistochemical analysis. In a lung colonization assay of metastasis, where TNBC cell lines were injected into the tail veins of mice, either overexpression of SHARP1 or inhibition of HIF-1α or HIF-2α expression by short hairpin RNA (shRNA) opposed lung colonization. Inhibition of SHARP1 expression by shRNA increased trans-well migration of a nonmetastatic TNBC cell line, and this effect was rescued by shRNA-mediated inhibition of HIF-1α expression. Western blots showed reduced HIF-1α abundance in extracts from TNBC cell lines overexpressing SHARP1 compared with controls, and immunohistochemical analysis of tumors in mice injected with TNBC cell lines overexpressing SHARP1 showed that the tumors also had less HIF-1α than did tumors produced from control TNBC cell lines. The decrease of HIF-1α abundance occurred in TNBC cells under both hypoxic and normoxic conditions. Knockdown of SHARP1 led to increased abundance of HIF-1α. Treatment of TNBC cell lines with proteasome inhibitors resulted in accumulation of HIF-1α. Decrease of HIF-1α abundance due to overexpression of SHARP1 in cell lines with a temperature-sensitive E1 ubiquitin-activating enzyme occurred at both restrictive and permissive temperatures, suggesting that the ability of SHARP1 to destabilize HIF-1α was independent of ubiquitination. Both SHARP1 and HIF-1α coimmunoprecipitated with the 20S proteasomal subunit; however, HIF-1α did not coimmunoprecipate with the 20S proteasomal subunit in lysates of SHARP1-depleted cells. Coimmunoprecipitation with truncation mutants of SHARP1 showed that the N-terminal basic helix-loop-helix domain was sufficient to trigger degradation of HIF-1α, and a construct lacking this domain stabilized HIF-1α, presumably by preventing it from binding to the proteasome. In TNBC, SHARP1 enables the ubiquitin-independent association of HIF proteins with the proteasome, thus promoting their degradation and preventing the induction of HIF target genes that contribute to cancer pathogenesis and metastasis.
M. Montagner, E. Enzo, M. Forcato, F. Zanconato, A. Parenti, E. Rampazzo, G. Basso, G. Leo, A. Rosato, S. Bicciato, M. Cordenonsi, S. Piccolo, SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors. Nature 487, 380–384 (2012). [PubMed]