Researchers discovered an enzyme, originally known to regulate the TGF-β, has a previously unreported role in HIF-1α regulation.
A small regulatory protein can be found not only in all humans, animals, plants and fungi, but in each and every eukaryotic cell. Following the discovery of these ubiquitous proteins in 1974, researchers aptly named them ubiquitin. Enzymes and/or ligases (enzymes that catalyze the formation of chemical bonds), allow ubiquitins to bind to substrate proteins. This process, known as ubiquitination, can initiate or prevent protein interactions, denote them for degradation and alter their activity and cellular location.
Aberrant ubiquitination may play an important role in tumorigenesis. At the core of solid tumors, hypoxia-inducible factor 1α (HIF-1α) is induced (by hypoxia) and allows cancer cells to adapt to the lack of oxygen. The accumulation of HIF-1α can also transcribe a number of genes well-known to be involved in cancer. Therefore, HIF-1α is an attractive, albeit difficult-to-pinpoint, therapeutic target in cancer. Researchers—from Brown University and Lifespan Cancer Institute—previously observed the stabilization of HIF-1α by the protein coding gene cyclin-dependent kinases CDK1. They further proposed that CDK4 may also be an HIF-1α stabilizer. However, the mechanism of HIF-1α’s regulation by CDK4 has yet to be fully elucidated.
“Till now, development of therapies targeting HIF-1α remains hindered. Therefore, it is imperative to explore the mechanism of HIF-1α regulation in cancer cells and investigate new possibilities to therapeutically target HIF-1 signaling.”
In 2021, the same team conducted a new study further investigating the molecular mechanisms of HIF-1α destabilization by CDK1 or CDK4/6 inhibitors in colorectal cancer. Their priority research paper was published as the cover of Oncotarget’s Volume 12, Issue 20, and entitled, “Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase.”
“We hypothesized that there may be a E3 ubiquitin ligase, instead of VHL, that targets HIF-1α for ubiquitination upon CDK4/6 inhibition.”
The Study
In this study, the researchers conducted proteomic analysis of human colon cancer cell lines HCT116 and SW480 and renal cell carcinoma cell line RCC4. They used the CDK1 inhibitor Ro3306, CDK4/6 inhibitor palbociclib, a proteasome inhibitor, HIF-1α and Ran antibodies, plasmids and siRNAs. Their methods included cell transfection, western blot, immunoprecipitation, proteomic mass spectrometry and analysis, E3 ubiquitin ligase prediction, ubiquitination assay, and correlation analysis.
“To improve our understanding of HIF-1α stabilization by CDKs and gain novel insights into the mechanisms of HIF-1α regulation in cancer, we performed a proteomic analysis on immunoprecipitated HIF-1α from colorectal cancer cells treated in hypoxia.”
The team identified E3 ubiquitin protein ligase 2 (Smurf2) as potentially involved in HIF-1α destabilization. Originally known to regulate the TGF-β signaling pathway, Smurf2 overexpression inhibited HIF-1α expression levels in SW480 and RCC4 cell lines. This HIF-1α degrading role for Smurf2 was previously unidentified.
“Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1α destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1α regulation.”
Conclusion
“In summary, we propose a non-canonical mechanism involving Smurf2 in HIF-1α degradation upon CDK4/6 inhibitor treatment, which provides novel insights in HIF-1α regulation. It sheds light on the HIF-1α stabilization in cancer as well as suggests new possibilities of therapeutic angiogenesis.”
Click here to read the full priority research paper, published by Oncotarget.
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