NUSAP1 expression is upregulated by loss of RB1 in prostate cancer cells

Abstract

Overexpression of NUSAP1 is associated with poor prognosis in prostate cancer, but little is known about what leads to its overexpression. Based on previous observations that NUSAP1 expression is enhanced by E2F1, we hypothesized that NUSAP1 expression is regulated, at least in part, by loss of RB1 via the RB1/E2F1 axis. Using Significance Analysis of Microarrays, we examined RB1, E2F1, and NUSAP1 transcript levels in prostate cancer gene expression datasets. We compared NUSAP1 expression levels in DU145, LNCaP, and PC-3 prostate cancer cell lines via use of cDNA microarray data, RT-qPCR, and Western blots. In addition, we used lentiviral expression constructs to knockdown RB1 in prostate cancer cell lines and transient transfections to knockdown E2F1, and investigated RB1, E2F1, and NUSAP1 expression levels with RT-qPCR and Western blots. Finally, in DU145 cells or PC-3 cells that stably underexpress RB1, we used proliferation and invasion assays to assess whether NUSAP1 knockdown affects proliferation or invasion. NUSAP1 transcript levels are positively correlated with E2F1 and negatively correlated with RB1 transcript levels in prostate cancer microarray datasets. NUSAP1 expression is elevated in the RB1-null DU145 prostate cancer cell line, as opposed to LNCaP and PC-3 cell lines. Furthermore, NUSAP1 expression increases upon knockdown of RB1 in prostate cancer cell lines (LNCaP and PC-3) and decreases after knockdown of E2F1. Lastly, knockdown of NUSAP1 in DU145 cells or PC-3 cells with stable knockdown of RB1 decreases proliferation and invasion of these cells. Our studies support the notion that NUSAP1 expression is upregulated by loss of RB1 via the RB1/E2F1 axis in prostate cancer cells. Such upregulation may promote prostate cancer progression by increasing proliferation and invasion of prostate cancer cells. NUSAP1 may thus represent a novel therapeutic target.