Significance of ubiquitin conjugating enzyme 2C in cell cycle exit and re-entry in prostate cancer cells

Abstract

Prostate cancer (PCa) is one of the most commonly diagnosed cancers and the second leading causes of cancer deaths in western countries. Nearly one-third of patients with PCa experience cancer recurrence or progression following primary treatment such as surgery or radiation therapy. The presence of quiescent cancer cells has been documented in many types of cancers, including prostate. Quiescent cells are Ki-67 protein-negative and reversibly arrested at the G0 phase. In PCa, the percentage of Ki-67-positive PCa cells is low in low risk disease, but increases in high risk and advanced PCa, suggesting that an increase in transition from G0 state to a proliferating state may contribute to PCa progression. Hence, how to increase the proportion of cancer cells at G0 over those in a proliferating state and/or to prevent the transition from G0 to a proliferating state is pertinent to the prevention of cancer progression and recurrence. However, our understanding of the signals required for maintaining cancer cells at G0 and/or impeding the transition from quiescent to a proliferating state is limited. In eukaryotes, the ubiquitin proteasome system (UPS) precisely regulates the cell cycle at key checkpoints by targeting cell cycle regulators for proteasome-mediated degradation. The UPS requires the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and ubiquitin ligases (E3) to work in concert to facilitate ubiquitination of target proteins. Ubiquitin conjugating enzyme 2C (UBE2C) is aberrantly overexpressed in a variety of cancers, including PCa. Accumulation of UBE2C stimulates cell proliferation, whereas silencing of UBE2C decreases cell proliferation. The significance of UBE2C in cell cycle exit and re-entry, however, has not been investigated. The aim of this project was to define the role of UBE2C in the proportion of quiescent over proliferative PCa cells and transition to a proliferating state by quiescent PCa cells. Moreover, the concordance between UBE2C protein/mRNA levels in human PCa tissue and progression of PCa were determined. To establish the relationship of UBE2C the proportion of G0 cells, PCa cell lines were transfected with UBE2C siRNA or UBE2C expression plasmid. Silencing of UBE2C decreased cell proliferation, whereas overexpression of UBE2C increased cell proliferation, determined by measuring DNA content. Flow cytometric analysis of both DNA and RNA content were subsequently undertaken to investigate if manipulation of UBE2C changed the proportion of G0. Depletion of UBE2C led to accumulation of quiescent cells, while overexpression of UBE2C decreased the proportion of quiescent PCa cells. To further evaluate the relationship of UBE2C with cell cycle re-entry, we utilised contact inhibition in PC-3 or serum withdrawal in LNCaP and C4-2B cells to induce experimental quiescence. The quiescent state of PCa cells was confirmed by flow analysis. Cell cycle re-entry was rendered by replating the contact-inhibited quiescent cells at low density, or by restoring serum to serum-deprived cells. UBE2C protein levels were decreased during cell cycle exit and increased upon cell cycle re-entry. Overexpression of UBE2C impeded cell cycle exit, and knockdown of UBE2C delayed cell cycle re-entry by quiescent PCa cells. Next, to determine the molecular mechanism underlying UBE2C action on cell cycle exit and re-entry, G0 regulatory proteins were examined by immunoblot. Knockdown of UBE2C expression increased the protein levels of p27 and decreased protein levels of SKP2 and PIRH2; both are components of E3 ligases responsible for p27 degradation. Additionally, the level of c-MYC was also decreased by UBE2C siRNA. Concomitantly, FBXW7, which targets c-MYC for degradation, was increased. However, the silencing of UBE2C did not change protein levels of p27 nuclear exporter CRM1. Lastly, to examine the concordance between UBE2C mRNA levels and progression of PCa, 12 datasets on PCa from ONCOMINE, an online database for genetic variation and mRNA expression in cancer, were analysed. Across these datasets, UBE2C mRNA was highly expressed in the metastasised form of PCa compared with organ-confined primary PCa or benign hyperplastic tissues. At protein levels, the aberrant overexpression of UBE2C was confirmed by immunostaining in PCa tissue specimens (n=70), and was found to be correlated with cancer progression in paired specimens of hormone-sensitive PCa and castration-resistant PCa from the same patient (n=8). In summary, this study has revealed that UBE2C plays a previously unrecognised vital role in the regulation of the proportion and transition of quiescent PCa cells. These findings suggest: (i) UBE2C blocks cell cycle exit and promotes cell cycle re-entry; (ii) UBE2C can downregulate p27 expression, possibly through augmenting SKP2 and PIRH2 expression; (iii) UBE2C can upregulate c-MYC expression, possibly through reducing FBXW7 expression; and (iv) UBE2C mRNA and/or protein levels are aberrantly increased in the advanced form of PCa, and the increased expression of UBE2C is associated with PCa progression.

Date of Award	2016
Original language	English