Abstract
The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood1. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel–Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.
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Acknowledgements
We thank O. Wessely, G. Del Sal, C. Oakman and A. Di Leo for comments; and W. Kaelin, C. Borner, S. Libutti, Y. Maeda and Y. Kato for gifts of reagents. M.M. is a recipient of a FIRC (Federazione Italiana Ricerca Cancro) fellowship. We are in debt to E. Tagliafico and the Modena Affimetrix platform team for help with microarrays. E.E. is a recipient of a Cariparo PhD fellowship. M.M. was a recipient of an AIRC (Italian Association for Cancer Research) fellowship. This work is supported by a Young Italian Researchers grant of the Italian Welfare Ministry and an AIRC (Associazione Italiana per la Ricerca sul Cancro) MFAG grant to M.C.; a Fondazione Città della Speranza Grant, and MIUR (Ministero dell'Istruzione dell'Università e della Ricerca Italia) and PRIN grants to G.B; and an AIRC Principal Investigator grant, an AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ grant, an HSFP grant, a University of Padua Strategic grant, an IIT Excellence grant, a CNR–Miur Epigenetics Flagship project, and a Comitato Promotore Telethon grant to S.P.
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M.M., M.C. and S.P. designed research, and M.M., E.E, M.F., M.C., E.R., G.B., G.L. and F.Z. performed experiments. M.C., S.B. and M.F. performed bioinformatics analysis, and A.R. and A.P. helped with assays in mice and tumour pathology. M.M., M.C. and S.P. wrote the manuscript.
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Supplementary information
Supplementary Information 1
This file contains Supplementary Figures 1-29, Supplementary Methods, Supplementary Tables 1-2, 4, and 6-11 (see separate files for Supplementary Tables 3 and 5) and Supplementary references. (PDF 15593 kb)
Supplementary Table 3
This file contains a table with the Characteristics of the 250 TNBC samples. (XLS 92 kb)
Supplementary Table 5
This file contains a table with the Genes identified by SAM as up-regulated in Sharp1/CyclinG2 Low TNBCs compared to Sharp1/CyclinG2 High TNBCs. (XLS 550 kb)
Supplementary Information 2
This file contains the Sweave document in .pdf format for the analysis of gene expression data from MDA-MB-231 cells depleted of HIF-1α and HIF-2α (MDA-shHIF) and MDA-MB-231 cells overexpressing Sharp1 (MDA-Sharp1). (PDF 272 kb)
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Montagner, M., Enzo, E., Forcato, M. et al. SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors. Nature 487, 380–384 (2012). https://doi.org/10.1038/nature11207
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DOI: https://doi.org/10.1038/nature11207
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