Abstract
We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27Kip1. To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27Kip1. However, in contrast to previous findings in fibroblasts, the stabilization of p27Kip1 upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27Kip1 and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27Kip1 levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.
Highlights
Eukaryotic cell cycle progression is dependent on the precisely timed activation and inactivation of a series of cyclin-dependent kinases (CDKs) [1,2]
This was tested by the stable overexpression in DU145 cells of rat USP19 that is resistant to targeting by human USP19 small interfering RNA (siRNA)
Our strategy resulted in a cell line in which USP19 levels did not fall and in which growth was no longer inhibited upon USP19 depletion (Fig. 1B), thereby confirming the ability of rat USP19 to reverse the effect of human USP19 siRNA and specificity of action of the siRNA oligonucleotide
Summary
Eukaryotic cell cycle progression is dependent on the precisely timed activation and inactivation of a series of cyclin-dependent kinases (CDKs) [1,2]. Upon binding to the cyclin E-CDK2 complex, p27Kip inhibits CDK2 activity, thereby inhibiting cell cycle progression and cell proliferation. Silencing of p27Kip results in enhanced cell proliferation [4]. Consistent with this growth inhibitory role, p27Kip knockout mice are larger in size than wild-type littermates. They develop multi-organ hyperplasia and exhibit increased susceptibility to cancer [5,6,7]
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