Abstract 2115: CASZ1 suppresses neuroblastoma cell growth by restoring Rb activity in G1 and regulating kinases involved in G2/M progression

Abstract

Abstract A hallmark of cancer is the disruption of cell cycle regulation. Frequently this occurs in the Rb signal transduction pathway and causes tumor cells to become insensitive to antigrowth signals. In neuroblastoma (NB), poor prognosis tumors have been found to over-express multiple cell cycle regulatory genes, including those that participate in Rb regulation. Specifically, while amplification of Cyclin D1 and Cdk4 and mutations in Cdk6 occur at low frequency in NB, more than 75% of tumors contain elevated cyclin D1 expression. Recently, we identified CASZ1 to be a chromosome 1p36 tumor suppressor gene that suppresses NB tumor growth. To determine how loss of the CASZ1 tumor suppressor might contribute to cell cycle deregulation in NB tumors, we investigated how CASZ1 reconstitution affects proteins required for transition of NB cells through the G1/S and G2/M phases of the cell cycle by western blot analysis. CASZ1 restoration causes a 2-fold increase in the time required for NB cells to progress through the cell cycle with no major changes in any particular phase of the cell cycle. CASZ1 decreases DNA synthesis by ∼30% using a Click-iT EdU assay. Within 24 hr of CASZ1 induction there is a 2-fold increase in the level of the Cdk2/4 inhibitor p21 and a 1.5-fold increase in p27 levels at 72 hr. Steady-state levels of p16 levels do not change. There is also a 50% decrease in the steady-state levels of Cdk2 and Cdk6 at 24 hr. Consistent with the increase of Cdk inhibitors and decrease in Cdks levels, there is a 60% decrease in phosphorylation of their target Rb at p-RbS780 and p-RbS807/S811. Thus, increased levels of CASZ1 restore Rb activity, which regulates the G1/S transition. CASZ1 causes a near 40% decrease of cyclin E at 24 hr, and Cyclin E:Cdk2 is required for G1/S transition in addition to Rb. Cyclin A decreases about 40% at 24 hr after CASZ1 induction and cyclin A:Cdk2 is required for the cell to progress through the S phase. Cyclin B:Cdk1 is required for G2/M progression, CASZ1 induces 70% decrease of cyclin B1 and 40% decrease of its up-stream activator phosphates Cdc25c although Cdk1 level does not change. CASZ1 causes a 70% decrease in the steady-state levels of Aurora A kinase, which a key regulatory kinase in mitosis. These decreases occur at 48 hr suggesting that they may be related to changes occurring in cells progressing through G1. Taken together, this study indicates that restoration of CASZ1 activates Rb in G1 and inhibits the G2/M regulators cyclin B1, Cdc25c and aurora kinase. The decreases in the key regulators controlling G1/S and G2/M transition leads to a lengthening of NB cell cycle progression and a subsequent decreased growth in vitro and in vivo. 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 2115. doi:10.1158/1538-7445.AM2011-2115