ASPP2 Is Phosphorylated by CDK1 during Mitosis and Required for Pancreatic Cancer Cell Proliferation

SIMPLE SUMMARY: Pancreatic cancer is one of the leading causes of cancer death. Identifying and elucidating important molecular mechanisms underlying pancreatic cancer cell proliferation will provide opportunities for therapeutic interventions. This study aims to examine the role of apoptosis-stimulating protein of p53-2 (ASPP2) in pancreatic cancer. We found that ASPP2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) during mitosis. Results further indicate that depletion of ASPP2 attenuates pancreatic cancer cell growth in vitro and in vivo. Finally, our results show that ASPP2 markedly affects the transcriptome landscape of yes-associated protein (YAP)-related genes. These findings, for the first time, reveal the CDK1-mediated phosphorylation sites of ASPP2 during mitosis. These findings also demonstrate the essential role of ASPP2 in pancreatic cancer cell growth and identify the key targets for ASPP2-loss-induced growth remission. ABSTRACT: (1) Background: pancreatic cancer is highly lethal. The role of apoptosis-stimulating protein of p53-2 (ASPP2) in this lethal disease remains unclear. This protein belongs to the ASPP family of p53 interacting proteins. Previous studies in this lab used phosphate-binding tag (Phos-tag) sodium dodecyl sulfate (SDS) polyacrylamide gels and identified a motility upshift of the ASPP family of proteins during mitosis. (2) Purpose: this study expands on previous findings to identify the detailed phosphorylation regulation of ASPP2 during mitosis, as well as the function of ASPP2 in pancreatic cancer. (3) Methods: the Phos-tag technique was used to investigate the phosphorylation mechanism of ASPP2 during mitosis. Phospho-specific antibodies were generated to validate the phosphorylation of ASPP2, and ASPP2-inducible expression cell lines were established to determine the role of ASPP2 in pancreatic cancer. RNA sequencing (RNA-Seq) was used to uncover the downstream targets of ASPP2. (4) Results: results demonstrate that ASPP2 is phosphorylated during mitosis by cyclin-dependent kinase 1 (CDK1) at sites S562 and S704. In vitro and in vivo results show that ASPP2 is required for pancreatic cancer growth. Furthermore, the expressions of yes-associated protein (YAP)-related genes are found to be dramatically altered by ASPP2 depletion. Together, these findings reveal the phosphorylation mechanism of ASPP2 during mitosis. Collectively, results strongly indicate that ASPP2 is a potential target for abating tumor cell growth in pancreatic cancer.