Nuclear Factor-kappaB in Ovarian Cancer

NF-kB通路促进癌细胞的存活。我对卵巢癌的研究开始于描述这种疾病中NF-kB的激活状态和生物学相关性。转录因子的NF-κ B家族广泛表达。NF-kB信号通路与卵巢癌有关，但NF-kB信号通路在卵巢癌中的意义和机制尚不清楚。有先例表明NF-kB是癌症中的关键信号传导机制。我最初假设NF-kB通路在卵巢癌中过度激活，具有更强的侵袭性。NF-κ B通路参与卵巢癌细胞的体外增殖和细胞因子分泌，并导致卵巢癌细胞株的化疗耐药性。因此，我试图确定NF-κ B通路蛋白在原发性卵巢癌组织中的表达模式和预后相关性。我证明了诊断时NF-κ B亚单位p50的过度表达传达了这些患者的不良结局。NF-kB在卵巢癌中的生物学相关性是在我的实验室建立的。在证明了卵巢癌中NF-κ B机制的协调存在后，我试图调节其活性。标记NF-κ B抑制剂（IkB），以便在IkB激酶（IKK）特异性诱导磷酸化后通过蛋白酶体降解。因此，靶向抑制IKK可以分离NF-kB作为卵巢癌发病机制。抑制IKKb可影响卵巢癌细胞系的生长、粘附、侵袭和细胞因子分泌。我开发了一个IKKb信号在卵巢癌中的基因表达签名，使用IKKb的药理学和遗传操作。基于卵巢癌特异性靶基因的已知功能，该特征使我能够深入了解NF-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，以估计NF-kB信号传导对卵巢癌患者生存的相对影响。较高的NF-kB活性传达了更差的结果，表明IKKb的调节可能使肿瘤显示靶基因表达升高的患者受益。这项工作的一个关键发现是NF-κ B信号的组织特异性。在卵巢癌中实验定义的9个基因签名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NF-kB信号传导的分子结构，旨在开发NF-kB依赖性的生物标志物和治疗干预的新点。我们完成了两个shRNA文库筛选，一个与IKK β抑制剂组合，另一个与针对IKK β的shRNA组合。这些研究确定了卵巢癌中NF-κ B通路之间的新相互作用。与IKK β结合，我们发现caspase 8在保护细胞免于坏死性凋亡方面是合作的。这项工作继续设计一种翻译策略，以改善caspase 8缺陷型癌症妇女的临床结局。我们计划针对坏死性凋亡途径进行治疗，以绕过介导对标准化疗方案耐药的缺陷性细胞凋亡。在相关的途径中，我们继续研究卵巢癌肿瘤起始细胞中的NF-κ B信号传导。我们通过免疫荧光观察到，基于核中NF-kB p65的存在，经典NF-kB似乎在任何给定时间仅在培养物的亚群中是活性的。在前列腺、乳腺和卵巢肿瘤的肿瘤起始细胞（TIC）中观察到经典NF-kB信号传导升高，但对替代NF-kB信号传导的研究有限。经典和替代信号级联都是维持和促进乳腺癌TIC所必需的。经典和替代性NF-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，NF-κ B的多样性和复杂的作用表明，这个转录因子家族调节细胞功能的上下文依赖性的方式，并可能是一个关键因素，在维持异质性。我们目前正在测试的假设，经典和替代NF-κ B途径支持不同的亚群卵巢癌肿瘤启动球体，合作，以填充化疗后继发性肿瘤。我们最近的研究结果表明，NF-κ B支持TIC表型和NF-κ B的功能输出负责增殖，化疗耐药性，分化，多能性。更好地了解TIC和非TIC肿瘤细胞群体中NF-κ B信号传导的生物学基础，将指导设计更有效的治疗方法，以克服化疗耐药性，预防复发，提高卵巢癌患者的生存率。从治疗的角度来看，我们之前完成了一项使用SMAC模拟物birinapant的2期临床试验-可以通过抑制IAP蛋白靶向NF-κ B信号传导-在复发性和难治性卵巢癌患者中进行。无临床反应。因此，通过NCI重大机会，我们完成了一项矩阵药物筛选测试，测试了2000种化合物与birinapant的组合，以确定协同组合，以进入临床。我们确定了符合协同作用标准的药物类别，并正在小鼠模型中进行测试。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。临床试验将从临床前工作中发展出来。

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.