Abstract 3113: Hyperactivation of FOXM1 drives ovarian cancer growth and metastasis independent of the G2-M cell cycle checkpoint.

Abstract

Abstract Forkhead box M1 (FOXM1) is a TP53- and phosphorylation-dependent transcription factor that plays a critical role in cell cycle progression. Recent analyses by the Cancer Genome Atlas (TCGA) consortium indicate that FOXM1 overexpression may be a key, early event driving the growth of epithelial ovarian cancer (EOC). However, this hypothesis has not been critically tested. Using RT-qPCR, we examined the expression of each of the known FOXM1 splice variants. We found that levels of FOXM1c but not FOXM1b or the translationally inactive FOXM1a are significantly higher in specimens of EOC (n=5) than normal ovary (n=3) or fallopian tube (n=3). By western blot, we found that FOXM1 is highly phosphorylated in EOC, suggesting that its hyperactivation is a consistent feature of ovarian cancer. When FOXM1 expression was targeted in TP53-wt HEYA8, TP53-mutated OVCAR8, and TP53-deficient SkOViP31 ovarian cancer cells by siRNAs, we found that ovarian cancer cell lines with reduced FOXM1 expression grow much more slowly than control cultures transfected with a non-targeting siRNA control and that a loss of FOXM1 results in G2-M cell cycle arrest that can be detected by flow cytometry using propidium iodide stained cells. Reduced FOXM1 expression also inhibited migration and invasion when OVCAR8 and HeyA8 cells were evaluated using standard Boyden chamber assays. Using qPCR and Western blot, we examined the ability of FOXM1 to directly regulate >19 gene products that are highly expressed in EOC. Despite the fact that FOXM1 regulates many of these genes (Cyclin B1, Plk1, and CENPF) in other cells and tissues, levels of their expression were not observed when FOXM1 was knocked down SKOV3ip1, HEY8 and OVCAR8 cells. Therefore, we analyzed patterns of gene expression from 581 TCGA ovarian cancer specimens using serial linear regression with L1 normalization (Lasso analysis) to test for correlations with FOXM1 expression. These analyses identified multiple gene products whose expression is potentially regulated by FOXM1 in ovarian cancer. Altered levels of expression for multiple gene products identified by these analyses have been examined by qPCR and Western blot in ovarian cancer cell lines transfected with shRNA targeting FOXM1 expression. These included multiple gene products, including VEGF-B and -C known to regulate angiogenesis. To explore the in vivo impact of targeting FOXM1 expression, a xenograft model was established by inoculating Fox1Nu mice with OVCAR8 cells stably transfected with either shRNA targeting FOXM1 and a non-targeting control. Collectively, these results indicate that hyperactivation of FOX1-mediated transcriptional activity is a consistent feature of EOC and plays a critical role in regulating ovarian cancer metastasis in addition to proliferation. FOXM1 may be a valuable therapeutic target for overcoming the genetic heterogeneity that limits current ovarian cancer treatments. Citation Format: Triparna Ghosh-Choudhury, Holli A. Loomans, Ying-Wooi Wan, Zhangdon Liu, Shannon M. Hawkins, Matthew L. Anderson. Hyperactivation of FOXM1 drives ovarian cancer growth and metastasis independent of the G2-M cell cycle checkpoint. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3113. doi:10.1158/1538-7445.AM2013-3113