Nuclear Factor-kappaB in Ovarian Cancer

核因子-kB途径促进癌细胞存活。我对卵巢癌的研究始于确定核因子-kB在这种疾病中的激活状态和生物学相关性。核转录因子-kB家族广泛表达。核因子-kB信号与卵巢癌有关，但核因子-kB信号在卵巢癌中的意义和作用机制尚不清楚。有先例表明，核因子-kB在癌症中是一种关键的信号机制。我最初的假设是，在有更多攻击性行为的卵巢癌中，核因子-kB途径被过度激活。在体外，核因子-kB途径参与了卵巢癌细胞的增殖和细胞因子的分泌，并参与了卵巢癌细胞的化疗耐药。因此，我试图确定核因子-kB途径蛋白在原发卵巢癌组织中的表达模式和预后相关性。我证明了核因子-kB亚单位p50在诊断时的过度表达在这些患者中传达了不良的结局。核因子-kB在卵巢癌中的生物学相关性是在我的实验室建立的。在证明了核因子-kB机制在卵巢癌中的协同存在后，我试图调节它的活动。核因子-kB的抑制物(IkB)被标记为通过蛋白酶体被IKB激酶(IKK)特异地诱导磷酸化而降解。因此，靶向抑制IKKS可以分离出作为卵巢癌发病机制的核因子-kB。抑制IKKB对卵巢癌细胞生长、黏附、侵袭和细胞因子分泌的影响。我利用IKKB的药理学和遗传操作，开发了卵巢癌中IKKB信号的基因表达特征。这一信号使我深入了解了基于卵巢癌特定靶基因已知功能的核因子-kB在卵巢癌中的结果，并使我能够探索已建立的卵巢癌数据库，以评估核因子-kB信号对卵巢癌女性患者生存的相对影响。更高的核因子-kB活性意味着更糟糕的结果，这表明IKKB的调节可能有利于肿瘤显示出靶基因表达升高的患者。这项工作的一个关键发现是核因子-kB信号的组织特异性。卵巢癌实验中定义的9个基因特征与我之前在多发性骨髓瘤中确定的11个基因完全不同。该项目的总体目标是剖析卵巢癌中核因子-kB信号的分子结构，目的是开发依赖于核因子-kB的生物标志物，以及新的治疗干预点。我们完成了两个shRNA文库筛选，一个与IKKbeta的抑制剂相结合，另一个与针对IKKepsilon的shRNA相结合。这些研究发现了卵巢癌中核因子-kB途径之间的新的相互作用。结合IKKbeta，我们发现caspase 8在保护细胞免受坏死性下垂方面具有协同作用。这项工作继续设计了一种翻译策略，以改善患有caspase 8缺陷癌症的女性的临床结果。我们计划在治疗上针对坏死性下垂途径，绕过介导对标准化疗方案耐药的有缺陷的细胞凋亡。在一个相关的途径上，我们继续研究卵巢癌肿瘤启动细胞中的核因子-kB信号。我们通过免疫荧光观察到，根据细胞核中核因子-kB p65的存在，在任何给定的时间，经典的核因子-kB似乎只在培养的亚群中活跃。在前列腺癌、乳腺癌和卵巢肿瘤的肿瘤起始细胞(TIC)中已经观察到经典的核因子-kB信号的升高，但对替代的核因子-kB信号的研究有限。经典和替代信号级联都是维持和促进乳腺癌抽搐所必需的。经典和替代的核因子-kB通路可以相互调节，并与其他信号通路整合，以微调功能输出。因此，核因子-kB的不同和复杂的作用表明该转录因子家族以一种上下文依赖的方式调节细胞功能，并可能是维持异质性的关键因素。我们目前正在测试这一假说，即经典和替代的核因子-kB通路支持卵巢癌启动肿瘤的不同亚群，这些亚群协作形成化疗后的继发性肿瘤。我们最近的研究结果表明，核因子-kB支持TIC表型，并且核因子-kB的功能输出与细胞的增殖、化疗耐药、分化和多能性有关。更好地了解TIC和非TIC肿瘤细胞中潜在的NF-kB信号的生物学将指导设计更有效的治疗方法来克服化疗耐药，防止复发，并提高卵巢癌女性患者的生存率。从治疗的角度来看，我们之前在复发和难治性卵巢癌患者中使用Smac模拟物biinapant完成了一项2期临床试验-它可以通过抑制IAP蛋白来靶向NF-kB信号转导。没有临床反应。因此，通过NCI的重大机遇，我们完成了一次基质药物筛选，测试了2000种化合物与比利奈特的联合作用，以确定协同作用的组合走向临床。我们确定了符合协同作用标准的药物类别，并正在用小鼠模型进行测试。正在进行的工作是在体外和小鼠模型中测试Smac模拟物与HDAC抑制剂或紫杉烷化疗的组合。临床试验将在这项临床前工作的基础上进行。

The NF-kB pathway promotes survival of cancer cells. My research in ovarian cancer began with characterizing the activation state and biological relevance of NF-kB in this disease. The NF-kB family of transcription factors is ubiquitously expressed. NF-kB signaling has been implicated in ovarian cancer, but the significance and mechanism of NF-kB signaling in ovarian cancer is unknown. There is precedent to propose that NF-kB is a critical signaling mechanism in cancer. I initially hypothesized that the NF-kB pathway is over-activated in ovarian cancers with more aggressive behavior. The NF-kB pathway was implicated in ovarian cancer proliferation and cytokine secretion in vitro, and contributed to chemoresistance of ovarian cancer cell lines. I therefore sought to determine the expression patterns and prognostic associations of NF-kB pathway proteins in primary ovarian cancer tissues. I demonstrated that overexpression of the NF-kB subunit p50 at diagnosis conveyed poor outcome in these patients. The biological relevance of NF-kB in ovarian cancer was established in my laboratory. Having demonstrated the coordinate presence of NF-kB machinery in ovarian cancers, I sought to modulate its activity. Inhibitors of NF-kB (IkBs) are tagged for degradation through the proteasome upon specific inducible phosphorylation by IkB kinases (IKKs). Therefore, targeted inhibition of IKKs could isolate NF-kB as a mechanism for ovarian cancer pathogenesis. A subset of ovarian cancer cell lines was affected by inhibition of IKKb in properties of growth, adhesion, invasion and cytokine secretion. I developed a gene expression signature of IKKb signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKb. This signature gave insight into the results of NF-kB in ovarian cancer, based on known functions of the ovarian cancer-specific target genes, and allowed me to probe established ovarian cancer databases in order to estimate the relative impact of NF-kB signaling on the survival of women with ovarian cancer. Higher NF-kB activity conveyed a worse outcome, suggesting that modulation of IKKb might benefit patients whose tumors showed elevated target gene expression. A key discovery from this work was the tissue specificity of NF-kB signaling. The 9-gene signature experimentally defined in ovarian cancer was completely different from the 11 genes I previously identified in multiple myeloma. The overall goal of this project is to dissect the molecular structure of NF-kB signaling in ovarian cancer, with the intent to develop biomarkers of dependence on NF-kB, and novel points of therapeutic intervention. We completed two shRNA library screens, one in combination with an inhibitor of IKKbeta, and another in combination with shRNA against IKKepsilon. These studies identified novel interactions between the NF-kB pathway in ovarian cancer. In combination with IKKbeta, we found caspase 8 to be cooperative in protecting the cells from necroptosis. This work continues with designing a translational strategy for improving clinical outcome of women with caspase 8-deficient cancers. We plan to target the necroptosis pathway therapeutically, to bypass the defective apoptosis that mediates resistance to standard chemotherapy regimens. In a related avenue, we continue to study NF-kB signaling in ovarian cancer tumor-initiating cells. We observed by immunofluorescence that classical NF-kB appeared to be active in only a sub-population of the cultures at any given time, based on the presence of NF-kB p65 in the nucleus. Elevated classical NF-kB signaling has been observed in tumor-initiating cells (TICs) of prostate, breast, and ovarian tumors, but there are limited studies examining alternative NF-kB signaling. Both classical and alternative signaling cascades are required for maintenance and promotion of breast cancer TICs. The classical and alternative NF-kB pathways can regulate each other and integrate with other signaling pathways for fine-tuning functional outputs. Thus, the diverse and complex roles of NF-kB suggest this transcription factor family regulates cellular functions in a context dependent manner and may be a key factor in maintaining heterogeneity. We are currently testing the hypothesis that classical and alternative NF-kB pathways support distinct subpopulations of ovarian cancer tumor-initiating spheroids, that collaborate to populate secondary tumors following chemotherapy. Our recent results suggest that NF-kB supports the TIC phenotype and functional outputs of NF-kB are responsible for proliferation, chemoresistance, differentiation, multipotency. A better understanding of the biology underlying NF-kB signaling in TIC and non-TIC populations of tumor cells will guide the design of more effective therapies to overcome chemoresistance, prevent relapse, and improve survival of women with ovarian cancer. From a therapeutic standpoint, we previously completed a phase 2 clinical trial using the SMAC mimetic birinapant - that can target NF-kB signaling via inhibition of IAP proteins - in women with relapsed and refractory ovarian cancer. There were no clinical responses. Therefore, through the NCI Major Opportunities, we completed a matrix drug screen testing 2000 compounds in combination with birinapant, in order to identify synergistic combinations to move to the clinic. We identified drug classes that met the criteria for synergy, and are in the process of testing these in mouse models. Ongoing work is testing the combination of SMAC mimetic with either HDAC inhibitor or Taxane chemotherapy in vitro and in mouse models. Clinical trials will be developed from this preclinical work.