Abstract B15: NuSAP is regulated by RB1 and modulates prostate cancer progression
Authors: Catherine A. Gordon, Xue Gong, Zulfiqar G. Gulzar, James D. Brooks
Journal: Cancer Research (2013)

Abstract We have recently shown that nucleolar and spindle-associated protein (NUSAP1; encoding NuSAP) is overexpressed in recurrent prostate cancer tumors. Correlation between NUSAP1 transcript levels and prognosis has been seen in melanomas, glioblastomas, and breast cancers, indicating that NuSAP plays an important role in cancer aggressiveness. Although NuSAP is known to be essential for cell cycle progression, faithfully binding to and stabilizing microtubules during mitosis, little is known about its role in cancer progression. Hence, in this study we performed an extensive analysis to understand the role of NuSAP in aggressive cancer, focusing on prostate cancer. We first aimed to identify a mechanism leading to NuSAP upregulation in prostate cancer. In our previous study we found that E2F transcription factor-1 (E2F1) directly binds to the promoter of NUSAP1, enhancing NUSAP1 expression. Since E2F1 is known to be negatively regulated by the retinoblastoma protein (RB1), and RB1 is frequently lost in aggressive prostate cancer, we hypothesized that the expression of NuSAP is regulated, at least in part, by the RB1/E2F1 axis. We confirmed that NUSAP1 transcripts are anti-correlated with RB1 transcripts and positively correlated with E2F1 transcripts in prostate cancer microarray datasets, and then used lentiviral-based technology to knockdown RB1 in human prostate cancer cell lines (PC3 and LNCaP). RT-qPCR and western blot revealed that NuSAP expression increased upon knockdown of RB1, and was reduced upon further knockdown of E2F1, supporting the notion that NuSAP is regulated by the RB1/E2F1 axis. We next aimed to determine the function of NuSAP in prostate cancer progression. Using lentiviral-based technology we knocked-down and overexpressed NuSAP in prostate cancer cell lines, and examined proliferation, invasion, apoptosis, cell cycle stages, migration, and global gene expression. Proliferation, invasion, and scratch assays revealed that knockdown of NuSAP consistently reduced proliferation, invasion, and migration, respectively, while overexpression of NuSAP significantly increased invasion. Flow cytometry analyses revealed that knockdown of NuSAP led to a significant increase in the number of apoptotic cells and number of cells in the G2/M phase of the cell cycle. RNA-Seq analysis further revealed genes significantly differentially expressed upon knockdown or overexpression of NuSAP. Of note, overexpression led to an activation of genes involved in invasion, cellular movement, angiogenesis, and metastasis. Examination of microarray datasets additionally revealed increased NUSAP1 transcripts in metastatic prostate tumors. Taken together, our work provides a mechanism accounting for NuSAP overexpression in prostate cancer, and identifies novel functions of NuSAP in aggressive cancers. Understanding the role of NuSAP in prostate cancer progression will provide insights into the inner workings of aggressive cancer cells, and may ultimately pave the way for new routes to prognosticate and treat prostate and other cancers. Citation Format: Catherine A. Gordon, Xue Gong, Zulfiqar G. Gulzar, James D. Brooks. NuSAP is regulated by RB1 and modulates prostate cancer progression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B15.