To unravel the mechanisms underlying the impacts of HADHA on ovarian cancer development, we conducted an Identified Peptide Mass Spectrometry (IPMS) analysis to identify proteins that interact with HADHA. Next, we examined the status of these proteins in ovarian cancer and selected the top 5 proteins for further investigation. We assessed the expression levels of these proteins in HADHA knockdown cells, and the results revealed a significant reduction in CDK1 expression, as depicted in Fig.
3A. We also conducted an assessment of the expression levels of HADHA and CDK1 using multicenter high-throughput datasets from the GEO database. The results were presented in Figure
S2, which revealed that HADHA exhibited significant upregulation in ovarian cancer samples compared to normal samples within the GSE66957 dataset. Conversely, CDK1 displayed pronounced upregulation in ovarian cancer samples relative to normal samples across multiple datasets, including GSE40595, GSE36668, GSE69428, GSE54388, and GSE26712. Through a comprehensive analysis of these multiple datasets, we identified a positive correlation between the expression levels of HADHA and CDK1 in ovarian cancer samples specifically within the GSE26712 dataset (
P < 0.05).These led us to hypothesize that CDK1 might be a downstream target of HADHA involved in the regulation of ovarian cancer. To delve deeper into the mechanistic aspects of how the loss of HADHA impacts CDK1 expression, we made an incidental discovery that endogenously expressed HADHA specifically interacts with CDK1, indicating a protein-protein interaction between HADHA and CDK1 (Fig.
3B). To further study the mechanism by which HADHA depletion mediates the reduction in CDK1 protein expression, we treated HADHA-depleted HO8910 and SK-OV-3 cells with cycloheximide (CHX) to assess the half-life of CDK1. Western blotting analysis showed that the inhibition of HADHA accelerated the degradation of CDK1 protein (Fig.
3C). This effect was partially rescued by the addition of the proteasome inhibitor MG132 (Fig.
3D). These findings imply that HADHA may regulate CDK1 through the ubiquitin-proteasome system (UPS). Given that protein ubiquitination is commonly associated with proteasome-mediated degradation [
13,
14], we further evaluated the ubiquitination levels of CDK1. As anticipated, HADHA depletion augmented the ubiquitination of CDK1 (Fig.
3E). Thus, we proposed that HADHA could stabilize CDK1 by affecting CDK1 ubiquitination.