Abstract 2478: Silencing Aurora Kinase A radio-sensitizes glioma cells

Abstract

Abstract Purpose: It has been reported that Aurora Kinase A (AurKA) is overexpressed in many cancer types, including breast, ovarian, pancreatic, head and neck, esophageal, renal, and lung. Upregulation of AurKA can lead to oncogenic transformation and aneuploidy. AurKA is a member of the serine/threonine family of protein kinases that is localized to the mitotic spindle during cell division. AurKA is involved in spindle assembly and regulated by phosphorylation-dependent proteosomal degradation. Cells that overexpress AurKA surpass the G2/M checkpoint prematurely, resulting in chromosomal abnormalities. Silencing AurKA will prevent premature progression through the G2/M checkpoint, allowing for cells to be targeted in the radiosensitive G2/M phase. Materials and Methods: We first looked at the expression of AurKA in established (ATCC) and primary glioblastoma (GBM) cell lines. Stable silencing of AurKA was done via a lentiviral transfection with shRNA in U87, LN18, and LN229 cell lines. We performed clonogenic survival and MTS assays to measure the cell death after radiation of these AurKA knockdown (AurKA KD) cells versus cells transfected with non-targeting (NT) shRNA. Western blot was performed using these same cells post-radiation treatment to investigate the expression of proteins in both apoptotic and pro-survival pathways. To investigate the effect of AurKA KD on the formation of polyploidy cells and cell cycle regulation. Cell cycle analysis on AurKA KD and NT cells was conducted using flow cytometry. AurKA is also known to phosphorylate tumor suppressor protein p53 at Ser315, which leads to degradation of p53 via ubiquitination by Mdm2. We carried out experiments where AurKA KD cells, with either intact or mutant p53, were treated with a p53 inhibitor Pifithrin α and subsequently measured apoptosis using Annexin V and Western blot. Initial sequencing results of AurKA in our GBM cell lines did not lead to any conclusive results. Furthermore, we assessed the copy number variation of AurKA to correlate with radiosensitivity of our GBM cell lines. Results and Conclusions: Radiation treatment increases AurKA expression in our established and primary GBM cell lines. Next we silenced AurKA using lentiviral mediated shRNA transduction. AurKA KD cells exhibit greater radiosensitivity and reduced proliferation when compared to control cells, as shown by the clonogenic survival and MTS assays, respectively. Increased expression of apoptotic proteins, such as cleaved PARP and cleaved caspase-3, is seen in AurKA KD cells after treatment with radiation when compared to controls. Moreover, silencing AurKA prevented the accumulation of polyploid cells after radiation treatment. Our data suggest that AurKA could be a promising therapeutic target for radiosensitizing of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2011-2478