USP5 promotes breast cancer proliferation and metastasis by stabilizing HIF2α

Hypoxia drives metastasis

Patients with poorly oxygenated primary tumors have an increased risk of metastasis and mortality. ¹ Response to decreased oxygen availability is regulated by hypoxia inducible factors (HIFs). HIF1 and HIF2 form heterodimers with oxygen-regulated HIF1/HIF2α interacting HIF1ß to drive transcription of hypoxia response genes. Under normoxic conditions, HIF1α and HIF2α are degraded by the proteasome. Under hypoxic conditions, HIF1α and HIF2α can form the heterodimer and bind to hypoxia response elements and drive transcription of HIF target genes.

Hypoxia is a common characteristic of solid tumors and has been implicated in the regulation of each of the steps involved in metastasis. Hypoxia induces epithelial-mesenchymal transition (EMT) by through transcriptional regulators that induce EMT. Increased VEG levels during hypoxia not only promote angiogenesis, but also increase vessel permeabilization, contributing to intravasation and extravasation.³

HIF2α and USP5 in breast cancer

HIF2α plays an important role in sensing and adapting to tumor hypoxia. Multiple types of cancer, including breast cancer, have high HIF2α expression making it a potential diagnostic marker as well as a therapeutic target. While it is well known that Von Hippel-Lindau (VHL) E3 ligase targets HIF2α for proteasomal degradation, which deubiquitinase (DUB) enhances HIF2α stability in breast cancer is unknown. Ubiquitin-specific proteinase 5 (USP5) is a cysteine DUB that is highly expressed in many cancers, including breast cancer. USP5 promotes proliferation and EMT in hepatocellular carcinoma, melanoma, and pancreatic ductal carcinoma by stabilizing EMT transcription regulators. However, its role in breast cancer is unknown.⁴

USP5 deubiquitinates HIF2α to enhance stability and activity

To investigate possible DUBs responsible for stabilizing HIF2α expression, co-immunoprecipitation assays between HIF2α and a DUB library were performed. USP5 was identified as a potential HIF2α interacting partner from the library screen, and these interactions were confirmed with exogenous expression in HEK293T cells and endogenous expression in MDA-MB-231 and MCF7 breast cancer cells. Overexpression of wild type, but not catalytically inactive, USP5 elevated HIF2α levels. USP5 knockdown experiments decreased HIF2α expression, and this decreased expression was restored when USP5 knockdown cells were treated with the proteasome inhibitor MG132. Additionally, ubiquitination assays in HEK293T-HIF2α cells showed elevated levels of HIF2α ubiquitination in the absence of USP5. Taken together, these data suggest that USP5 stabilizes HIF2α via deubiquitination and disruption of proteasomal degradation.

Since HIF1α and HIF2α have redundant functions, HIF1α knockout cells were used to assess whether USP5 affects HIF2α transcriptional activity. USP5 knockdown cells had reduced HIF2α luciferase reporter activity, while overexpression of wild type USP5, but not the catalytic mutant, increased reporter activity. Additionally, RT-qPCR experiments exhibited decreased mRNA levels of known HIF2α target genes when USP5 was decreased. Further support of these observations comes from The Cancer Genome Atlas (TCGA) data showing a positive correlation between USP5 expression and known HIF2α target genes. These data all support the hypothesis that USP5 enhances HIF2α transcriptional activity.

USP5 is a breast cancer oncogene

Since HIF2α plays a critical role in breast cancer growth and metastasis, the researchers investigated whether USP5 does as well. Reducing expression of USP5 in MDA-MB-231 cells by shRNA reduced their proliferation under both normoxic and hypoxic conditions. Consistent with reduced proliferation, decreased levels of USP5 also reduced the number of colonies formed by MDA-MB-231 cells under hypoxic conditions. Additionally, decreased levels of USP5 reduced MDA-MB-231 cell migration and invasion in transwell experiments. Together, these findings support a role for USP5 in breast cancer proliferation and metastasis.

To determine clinical relevance of these observations, the researchers translated their findings to patient samples. Data from TCGA breast tumors (both primary and metastatic) showed higher levels of USP5 compared to adjacent normal tissue. Western blot analysis on seven breast cancer patient samples confirmed increased USP5 protein levels in the tumors compared to adjacent normal tissue. Additionally, HIF2α and USP5 protein levels were positively correlated in six out of seven of the breast cancer samples. Moreover, an online survival analysis tool correlated poor prognosis of breast cancer patients with high USP5 levels.

This research identified USP5 as a novel DUB for HIF2α. USP5 decreases proteasomal degradation of HIF2α, thereby increasing its stability. This increased HIF2α expression increases transcription of metastasis promoting genes. Conversely, decreasing USP5 levels results in decreased breast cancer cell proliferation, migration, and invasion, making USP5 a potential therapeutic target in breast cancer and an additional mechanism for regulating HIF2α in cancer.^​4​

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References

1. Gilkes DM, Semenza GL, Wirtz D. Hypoxia and the extracellular matrix: drivers of tumour metastasis. Nat Rev Cancer. 2014;14(6):430-439. doi:10.1038/nrc3726
2. Gilkes DM, Semenza GL. Role of hypoxia-inducible factors in breast cancer metastasis. Future Oncology. 2013;9(11):1623-1636. doi:10.2217/fon.13.92
3. Lu X, Kang Y. Hypoxia and Hypoxia-Inducible Factors: Master Regulators of Metastasis. Clinical Cancer Research. 2010;16(24):5928-5935. doi:10.1158/1078-0432.CCR-10-1360
4. Huang W, Liu X, Zhang Y, et al. USP5 promotes breast cancer cell proliferation and metastasis by stabilizing HIF2α. J Cell Physiol. 2022;237(4):2211-2219. doi:10.1002/jcp.30686