Nuclear Factor-kappaB in Ovarian Cancer

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SPECIFIC AIM #1: To characterize NF-kappaB activation state and biological relevance in ovarian cancer cell lines. The NF-kappaB pathway is essential to the growth and survival of diverse cell types but is under tight regulation by inhibitor molecules, the IkappaBs. NF-kappaB transcription factors are released from this inhibition by inducible activation of IkappaB kinases (IKKs) (2). Specific pharmacologic inhibition of IKKs will isolate NF-kappaB as a mechanism for the growth and survival of ovarian cancer cells. We demonstrated that inhibition of IKKbeta with MLN120b allowed us to define a subset of NF-kappaB-dependent multiple myeloma cell lines (1). During the past year, we have examined the toxicity of IKKbeta inhibitors in a series of 11 ovarian cancer cell lines of serous or undifferentiated origin, which represent the most clinically prevalent and poorest prognosis histological subtypes of ovarian cancer. Cells were stained with MTT or XTT to quantify metabolic activity after 3 and 7 days of incubation with two-fold serial dilutions of the IKKbeta inhibitor. Proliferation decreased by greater than 50% with IKKbeta inhibition in six ovarian cancer cell lines, thus providing strong evidence for the necessity of NF-kappaB signaling in this subset. Similar results with the IKKbeta inhibitors were seen when cells were plated in soft agar suggesting the importance of NF-kappaB signaling in anchorage independent growth of each of the 11 ovarian cancer cell lines. Our results suggest that NF-kappaB signaling is also active in some ovarian cancer cell lines. A detailed understanding of the individual NF-kappaB components involved in ovarian cancer would identify patients who may benefit from NF-kappaB blockade, and would provide specific guidance to the development of future therapeutic agents or combinations. We assessed the pathway biochemically with Western blot to assess the changes in activation state after pharmacologic IKKbeta inhibition. We found that the IKK proteins themselves were differentially expressed among ovarian cancer cell lines, and that the levels of IKKalpha and IKKepsilon were affected by IKKbeta inhibition. We are currently in the process of identifying the specific contribution of each of these three IKK enzymens (alpha, beta and epsilon) to NF-kappaB signaling in ovarian cancer. For this goal, we have developed 6 cell lines stably expressing the Tetracycline Repressor, in order to express short-hairpin RNA interference molecules targeted to each IKK in an inducible fashion. These experiments are ongoing. This technology also will allow detailed investigation of the specific effect of each IKK within the NF-kappaB signaling cascade, and will permit high-throughput screen of NF-kappaB pathway-related shRNAs from a library designed and constructed by L Staudt (Metabolism Branch, CCR, NCI). SPECIFIC AIM #2: To define a signature of NF-kappaB activation in ovarian cancer and measure its frequency in primary tumors. The survival and proliferative effects of NF-kappaB activation are mediated in part by the products of genes transcriptionally regulated by the NF-kappaB family of transcription factors. Previous studies have implicated NF-kappaB targets in tumor angiogenesis, invasion, and resistance to apoptosis. NF-kappaB target genes have been identified in many tissues, both cancerous and non-malignant. Some target genes are common to all tissues, while others are cell-type specific. Ovarian cancer-specific NF-kappaB target genes were identified by monitoring gene expression profiles of 2 sensitive cell lines, in at least 6 replicates of RNA taken at 0 and 48 hours after NF-kappaB pathway inhibition. We selected this time frame for monitoring gene expression changes based on results from our initial timecourses ranging from 0, 12, 24, 48 and 72 hours of IKKbeta inhibition. Changes in DNA binding by NF-kappaB subunits are expected to occur within 30 minutes of IKK inhibition. Messenger RNA levels of the NF-kappaB target genes begin to decrease by 12 hours; after 72 hours, indirect targets begin to change. We will compare and contrast gene expression changes after IKKbeta inhibition, in the 2 different cell lines. The gene signature(s) defined by pharmacologic inhibition of NF-kappaB were used to probe Affymetrix U133 microarray data from 25 ovarian cancer cell lines and 185 patient samples, available through collaboration with M. Birrer (Cell and Cancer Biology Branch, NCI). We characterized ovarian cancer patient samples by the gene expression signature developed in cell line analysis. This analysis is currently ongoing. The signature will be validated and refined by calculating the correlation of each gene with the others in the signature. Gene set enrichment analysis will be used to explore for major pathways that might be coordinately regulated within the NF-kappaB subset of cases. The gene expression signature will not only give insight in to the mechanisms of ovarian cancer pathogenesis, but may also define a subset of patients who would benefit from targeted blockade of this pathway.