HN1 Is Enriched in the S-Phase, Phosphorylated in Mitosis, and Contributes to Cyclin B1 Degradation in Prostate Cancer Cells

Simple Summary Hematological and Neurological Expressed 1 (HN1) has previously been explored in Prostate cancer, where it was highly expressed as compared to normal Prostate cells. The HN1 co-expression network in Prostate cancer displayed two distinct nodes of G1/S transition-related genes and mitotic protein encoding genes. Interestingly, HN1 expression was found as inversely correlated with Cyclin B1. The mechanistic data for the involvement of HN1 in the cell cycle dynamics and pathways are not available. Here, we employed cell cycle synchronizations coupled with kinase inhibitors and overexpression experiments. HN1 levels fluctuated in the cell cycle with enriched levels in the S-phase, with a distinct phospho-HN1 form appearing in mitosis, which disappeared upon treatment with inhibitors of GSK3 beta and Cdk1 kinases. Mechanistically, HN1 interacted with Cdh1 (a co-factor of APC/C) for its stabilization and degradation of Cyclin B1, via increased ubiquitination. HN1 levels are tightly regulated throughout the cell cycle, as before Nocodazole blocks HN1 overexpression, it led to increased accumulation of cells in the S-phase and after Nocodazole arrest, it led to early mitotic exit. Therefore, HN1 is a novel cell cycle-regulated protein with potentially dual roles, involved in the modulation of cell cycle progression at the S-phase and mitotic exit. HN1 has previously been shown as overexpressed in various cancers. In Prostate cancer, it regulates AR signaling and centrosome-related functions. Previously, in two different studies, HN1 expression has been observed as inversely correlated with Cyclin B1. However, HN1 interacting partners and the role of HN1 interactions in cell cycle pathways have not been completely elucidated. Therefore, we used Prostate cancer cell lines again and utilized both transient and stable inducible overexpression systems to delineate the role of HN1 in the cell cycle. HN1 characterization was performed using treatments of kinase inhibitors, western blotting, flow cytometry, immunofluorescence, cellular fractionation, and immunoprecipitation approaches. Our findings suggest that HN1 overexpression before mitosis (post-G2), using both transient and stable expression systems, leads to S-phase accumulation and causes early mitotic exit after post-G2 overexpression. Mechanistically, HN1 interacted with Cyclin B1 and increased its degradation via ubiquitination through stabilized Cdh1, which is a co-factor of the APC/C complex. Stably HN1-expressing cells exhibited a reduced Cdt1 loading onto chromatin, demonstrating an exit from a G1 to S phenotype. We found HN1 and Cdh1 interaction as a new regulator of the Cyclin B1/CDK1 axis in mitotic regulation which can be explored further to dissect the roles of HN1 in the cell cycle.

Automated summary

HN1 is highly expressed in prostate cancer compared to normal prostate cells. It is involved in the regulation of cell cycle progression at the S-phase and mitotic exit. HN1 interacts with Cdh1 to stabilize and degrade Cyclin B1 through increased ubiquitination.