Abstract LB-192: DNA damage checkpoints control spindle assembly checkpoint by regulating Mad2
Authors: Changxian Shen, Peter Houghton
Journal: Cancer Research (2011)

Abstract DNA damage checkpoints coordinate with the spindle assembly checkpoint (SAC) to maintain genome integrity. In yeast Saccharomyces cerevisiae, ATM/ATR-Chk2/Chk1 checkpoint enhances SAC through phosphorylation and stabilization of securin by Chk1 and inhibition of APCCDC20 by Chk2 in response to DNA damage. The control of SAC by DNA damage checkpoints in mammalian cells is unclear. Here we report that DNA damage checkpoint signals to SAC by regulating Mad2 in human rhabdomyosarcoma (RMS) cells. In vitro, treatment of RMS Rh30 cells with DNA strand crosslinkers melphalan, cisplatin or mitomycin C for 5 hours resulted in elevation of Mad2 protein, which was companied with ATM/ATR checkpoint activation as shown by increase of ATM-S1981, Chk2-T68 and Chk1-S345. It has been shown that Mad2 is controlled by the Rb-E2F1 pathway and overexpression of Mad2 leads to genome instability and tumorigenesis. Consistently, treatment of Rh30 cells with inhibitors of either mTOR, Akt or CDK1/2 lead to dephosphorylation of Rb and decrease of Mad2. However, we found that DNA damage-induced increase of Mad2 is independent on Rb-E2F1 pathway as treatment of Rh30 cells with DNA damage agents resulted in dephosphorylation of Rb, suggesting inhibition of the E2F1 pathway. Moreover, we found that Mad2 is controlled by ubiquitin-proteasome signaling as MG132 treatment increased Mad2 protein levels. In vivo, we determined the Mad2 levels in 50 pediatric xenograft tumor models (PPTP) and found that Mad2 is overexpressed in most RMS (7/8). Elevated Mad2 is associated with elevated spontaneous DNA damage response in RMS as demonstrated by relatively high levels of Chk2-T68 and Chk1-S345 phosphorylation. The deregulation of the Rb-E2F1 pathway seems not to be the cause of Mad2 overexpression in RMS as Rb-S780 phosphorylation is comparable to other PPTP tumors without Mad2 overexpression. Further, gene expression and copy number analysis found that chromosome 2p24, in which MYCN is located, is amplified in most alveolar RMS xenografts (58.3%). Consequently, both MYCN mRNA and protein are overexpressed in these 2p24 amplified RMS. It has been well documented that oncogene activation leads to DNA replication stress and consequently DNA damage response in most cancers especially late-stage and relapse cancers. Alveolar RMS is genomically unstable as shown by increased tetraploid cells. We thus hypothesize that chromosome 2p24 amplification results in overexpression of MYCN oncogene, which leads to replication stress and hence DNA damage, and this oncogene induced DNA damage may lead to overexpression of Mad2 and consequently chromosome instability in RMS. Taken together, we speculate that DNA damage response stabilizes Mad2 protein independently of the Rb-E2F1 pathway in vitro. In vivo, oncogene amplification leading to an enhanced DNA damage response may stabilize Mad2 overexpression, potentially leading to chromosome instability. Supported by CA77776. 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 LB-192. doi:10.1158/1538-7445.AM2011-LB-192