Abstract A13: Chemoprevention agent SHetA2 induces G1 arrest through modulation of a biological system driven by Cyclin D1

A13 Flexible heteroarotinoid (Flex-Het) compounds induce apoptosis through direct targeting of mitochondria and Bcl-2 proteins, inhibit angiogenesis through regulation of cytokine expression and induce differentiation through increased E-Cadherin expression. In addition, the lead Flex-Het, SHetA2, induces G1 cell cycle arrest through induction of Cyclin D1 protein phosphorylation, ubiquitination and degradation. Overexpression of Cyclin D1 abrogated the G1 arrest. SHetA2 counteraction of carcinogen-induced Cyclin D1 expression was associated with SHetA2 chemoprevention of transformation in an organotypic model. Cyclin D1 is a tightly controlled protein that drives G1 cell cycle progression. Overexpression of Cyclin D1 is frequently observed in ovarian cancer and is believed to be an early event in ovarian tumorigenesis. The hypothesis of this study was that SHetA2 induces G1 arrest through modulation of a biological system driven by Cyclin D1. Cyclin D1 degradation was associated with several expected down-stream events in 2 ovarian cancer cell lines. Co-IP assays demonstrated that Cyclin D1 loss was associated with relocation of p21 from the Cyclin D1 complex to the Cyclin E2 complex. Consistent with the stimulatory effect of p21 on the CyclinD1-Cdk4/6 complex and the inhibitory effect on the CyclinE2-Cdk2 kinase activity, Western blot analysis demonstrated that phosphorylation of Rb on serine 780, known to be induced by CyclinD1-Cdk4/6-p21, was decreased. This was followed by decreased Rb phosphorylation on serine 612, which is known to be induced by CyclinE2-Cdk2. SHetA2 inhibition of Cyclin A protein expression, confirmed that the repression of Rb phosphorylation prevented the release of E2F from Rb, thus preventing expression of S Phase genes. An rtPCR array demonstrated that 7 genes involved in cell cycle regulation were significantly altered above background in both ovarian cancer cell lines. Ingenuity analysis demonstrated that several of these genes are involved in an interacting biological system driving the cell cycle. A Cdk4 inhibitor, p16, and the down-stream gene Bax were up-regulated, while several Cyclin D1 and E2F interacting genes were also regulated. Study of upstream events driving Cyclin D1 degradation demonstrated that the GSK3β kinase, known to induce Cyclin D1 threonine 286 phosphorylation, was phosphorylated/inhibited by SHetA2 and not involved in the cell cycle arrest. This phosphorylation event could be due to the SHetA2 induced phosphorylation of Akt serine 473, which has been shown to lead to increased GSK3β phosphorylation. Experiments with libraries of chemical and siRNA kinase inhibitors demonstrated that inhibition of kinases, such as EGF-R, mTOR, PKC, DNA-PK, contributed to SHetA2 growth inhibition, depending on their expression and activity patterns in the specific cell lines evaluated. In conclusion, SHetA2 inhibition of Cyclin D1 induced a number of changes in down-stream and associated targets consistent with G1 arrest. Experiments are ongoing to identify the specific kinase responsible for SHetA2 induction of Cyclin D1 phosphorylation. Knowledge of SHetA2 modulation of the biological system regulating cell cycle progression provides important information, such as response biomarkers, that can be used in translational research associated with planned chemoprevention trials. Supported by CA106713 Citation Information: Cancer Prev Res 2008;1(7 Suppl):A13.