Nuclear Factor-kappaB in Ovarian Cancer

特异性目的1：表征NF-kappaB在卵巢癌细胞系中的激活状态及其生物学相关性。NF-kappaB通路对多种细胞类型的生长和存活至关重要，但受到IkappaBs抑制剂分子的严格调控。NF-kappaB转录因子通过IkappaB激酶（IKKs）的诱导激活而从这种抑制中释放出来(2)。特异性药物抑制IKKs将分离NF-kappaB作为卵巢癌细胞生长和存活的机制。我们证明了MLN120b对IKKbeta的抑制使我们能够定义nf - kappab依赖性多发性骨髓瘤细胞系的一个亚群(1)。在过去的一年里，我们研究了IKKbeta抑制剂在一系列11种浆液性或未分化性卵巢癌细胞系中的毒性，这些细胞系代表了临床上最普遍和预后最差的卵巢癌组织学亚型。用两倍连续稀释的ikkβ抑制剂孵育3天和7天后，用MTT或XTT染色细胞以量化代谢活性。在6种卵巢癌细胞系中，IKKbeta抑制使增殖降低了50%以上，从而为NF-kappaB信号在该亚群中的必要性提供了强有力的证据。当细胞在软琼脂中接种时，发现了与ikkβ抑制剂相似的结果，这表明NF-kappaB信号在11种卵巢癌细胞系中锚定独立生长的重要性。我们的研究结果表明NF-kappaB信号在一些卵巢癌细胞系中也很活跃。对卵巢癌中涉及的单个NF-kappaB成分的详细了解将确定可能受益于NF-kappaB阻断的患者，并将为未来治疗剂或组合的开发提供具体指导。我们用Western blot对该通路进行生化评估，以评估IKKbeta药物抑制后激活状态的变化。我们发现IKK蛋白本身在卵巢癌细胞系中有差异表达，IKKalpha和IKKepsilon的水平受到IKKbeta抑制的影响。我们目前正在确定这三种IKK酶（α， β和epsilon）在卵巢癌中对NF-kappaB信号传导的具体贡献。为此，我们开发了6个稳定表达四环素阻遏因子的细胞系，以诱导方式表达针对每个IKK的短发夹RNA干扰分子。这些实验正在进行中。该技术还将允许详细研究NF-kappaB信号级联中每个IKK的特定作用，并允许从L Staudt （CCR， NCI代谢分支）设计和构建的文库中高通量筛选NF-kappaB通路相关shrna。特异性目标2：定义NF-kappaB在卵巢癌中的激活特征，并测量其在原发肿瘤中的频率。NF-kappaB激活的生存和增殖效应部分是由NF-kappaB转录因子家族转录调节的基因产物介导的。先前的研究表明NF-kappaB靶点与肿瘤血管生成、侵袭和细胞凋亡抵抗有关。NF-kappaB靶基因已在许多组织中被发现，包括癌性和非恶性组织。一些靶基因是所有组织共有的，而另一些则是细胞类型特异性的。通过监测2个敏感细胞系的基因表达谱，在NF-kappaB途径抑制后0和48小时提取至少6次重复的RNA，鉴定卵巢癌特异性NF-kappaB靶基因。根据IKKbeta抑制0、12、24、48和72小时的初始时间序列结果，我们选择了这个时间框架来监测基因表达变化。NF-kappaB亚基结合DNA的变化预计在IKK抑制后30分钟内发生。NF-kappaB靶基因的信使RNA水平在12小时后开始下降；72小时后，间接目标开始改变。我们将比较和对比2种不同细胞系中IKKbeta抑制后基因表达的变化。通过与M. Birrer （NCI细胞与癌症生物学分部）的合作，利用NF-kappaB药理学抑制定义的基因标记探测了来自25个卵巢癌细胞系和185个患者样本的Affymetrix U133微阵列数据。我们通过细胞系分析中开发的基因表达特征来表征卵巢癌患者样本。这项分析目前正在进行中。该签名将通过计算签名中每个基因与其他基因的相关性来验证和改进。基因集富集分析将用于探索可能在NF-kappaB亚群中协调调节的主要途径。这种基因表达特征不仅能让我们深入了解卵巢癌的发病机制，而且还可以确定一些患者将受益于靶向阻断这一途径。

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.