BACR 7: Role of WSB-1 in the biology of tumour hypoxia in breast cancer
1Hypoxia and Tumour Microenvironment Lab, School of Biological, Biomedical and Environmental Sciences, The University of Hull, Hull, UK,2Proteomics & Analytical Biochemistry Laboratory, Technology Facility, Department of Biology, University of York, York, UK
The tumour microenvironment is a key regulator of cancer progression. Specifically, tumour hypoxia is associated with therapy resistance and increased metastatic potential. As most solid tumours contain hypoxic regions, it is important to better understand the role of hypoxia and hypoxia-responsive factors in tumour progression. WSB-1 is an E3 ubiquitin ligase previously indicated to be hypoxia-induced. Moreover, WSB-1 has been described to be involved in the development of pancreatic cancer and neuroblastoma.
Effect of hypoxia on WSB-1 expression and transcription was assessed by Western blot and qPCR analyses after exposing breast cancer cells to normoxia, 0.5% O2 or 2% O2. Impact of WSB-1 on cell motility was evaluated using migration and invasion assays, and expression and activity of proteases was monitored by Western blotting and zymography. Putative binding partners of WSB-1 were identified by mass spectrometry analyses of HEK293T cells overexpressing a Flag-tagged version WSB-1.
Here, we show that the oxygen-dependent expression of WSB-1 is regulated by HIF-1? specifically. Additionally, our data suggest a crosstalk between WSB-1 and the HIF pathway. WSB-1 knockdown led to a significant decrease in the migration ability of MCF7 cells and alterations in invasiveness of MCF7 and MDA-MB-231 cells at 2% O2. We have indications this is dependent on altered MMPs function. Furthermore, immunoprecipitation of Flag-tagged WSB-1 and subsequent MALDI-MS and LC-MS analyses highlighted significant interactions of WSB-1 with proteins involved notably in mRNA processing and protein folding, relevant for hypoxic biology. Finally, in silico analysis of a cohort of breast cancer patients revealed a correlation between decreased distant metastasis-free survival and high WSB-1 levels.
This work highlights the importance of hypoxia and hypoxia-associated pathways in the modulation of WSB-1 expression; establishes WSB-1 as a regulator of cellular motility and invasiveness in hypoxia; and presents WSB-1 as a promising marker of breast cancer progression.