Targeting the NF-kappaB Pathway in Melanoma

描述(申请人提供)：恶性黑色素瘤发展中的一个关键事件是核因子-kappa B的失调，导致炎症介质和趋化因子的结构性产生并逃脱细胞凋亡。核因子-kB受一种抑制蛋白-IKB的调节，它将核因子-kappaBp65/p50复合体隔离在细胞质中，防止其移位到细胞核中。当这个IkappaB蛋白被IkappaB激酶(IKK)磷酸化时，它随后变得泛素化，并被26S蛋白酶体降解。我们已经证明，人黑色素瘤细胞系表现出IKK的结构性激活，IkappaB的磷酸化和降解增强，以及核内NF-kappaB p65/p50复合体的激活水平升高。这导致核因子-kappaB介导的基因转录增强，导致趋化细胞因子(趋化因子)、血管内皮生长因子(VEGF)、IL-6和其他调节因子的结构性表达，促进肿瘤细胞的生长，促进肿瘤血管生成和转移。我们的假设是，在肿瘤进展过程中，黑素细胞和/或间质细胞中核因子-kappaB的失调导致炎性介质的产生增加，黑素细胞肿瘤进展。此外，我们认为阻断核因子-kappaB将干扰肿瘤的生长和/或转移。我们的初步数据表明，阻断生长在裸鼠身上的人黑色素瘤细胞中的核因子-kappaB可以靶向细胞凋亡并抑制肿瘤生长。为了进一步阐明在免疫激活的环境下抑制NF-kB治疗黑色素瘤的可能性，我们提出了以下具体目标：a)确定敲除IKKA和IKKB对黑色素瘤生长和转移的影响：1B)确定在黑素细胞中表达具有组成活性的IKKB对黑色素瘤发病率的影响，以及黑素细胞特异性表达超级抑制型IkappaB对肿瘤诱导的抵抗的影响；2)检测IKKb特异性抑制剂BMS-345541单独及与DNA烷化剂替莫唑胺联用对黑色素瘤生长、转移及免疫活性小鼠免疫应答的影响；3)探讨抑制IKK4诱导黑色素瘤细胞凋亡的机制。意义：黑色素瘤是美国增长最快的肿瘤类型之一，由于阳光和化学物质的暴露，老年人受到的影响越来越大。肿瘤侵袭前的手术干预是降低这种癌症病死率的关键因素。对于转移性疾病，免疫疗法通常与化疗相结合，是最有前途的方法之一。我们的研究结果表明，靶向核因子-kappaB可以靶向肿瘤细胞的凋亡，抑制肿瘤血管的生成，抑制肿瘤的生长和转移。然而，我们需要确定阻断NF-kappaB对宿主对肿瘤的反应以及对黑色素瘤形成的影响。这里设计的研究将有助于设计更好的临床试验，将VELCADE或IKK抑制剂等抑制剂与化疗结合使用。

DESCRIPTION (provided by applicant): A key event in the development of malignant melanoma is the disregulation of NF-kappa B which results in constitutive production of inflammatory mediators and chemokines and escape from apoptosis. NF-kB is regulated by an inhibitory protein-IkB which sequesters the NF-kappaBp65/p50 complex in the cytoplasm, preventing its translocation into the nucleus. When this IkappaB protein is phosphorylated by the IkappaB kinase (IKK), it subsequently becomes ubiquitinated and degraded by the 26S proteasome. We have demonstrated that human melanoma cell lines exhibit constitutive activation of IKK, enhanced phosphorylation and degradation of IkappaB, and elevated levels of activated the NF-kappaB p65/p50 complex in the nucleus. This results in enhanced NF-kappaB mediated gene transcription, resulting in constitutive expression of chemotactic cytokines (chemokines), VEGF, IL-6, and other regulatory factors that augment the growth of the tumor cells, promote tumor angiogenesis and metastasis. Our hypothesis is that during tumor progression the disregulation of NF-kappaB in melanocytes and/or stromal cells leads to enhanced production of inflammatory mediators and melanocyte tumor progression. Moreover, we propose that blocking NF-kappaB will interfere with tumor growth and/or metastasis. Our preliminary data suggest that blocking NF-kappaB in human melanoma cells growing on nude mice targets the cells for apoptosis and inhibits tumor growth. To further clarify the potential of inhibition of NF-kB for melanoma therapy in an immunocompetent setting, we propose the following specific objectives: la) To determine the effects of knocking down IKKa and IKKb on the growth and metastasis of melanoma tumors in vitro and in vivo; Ib) To determine the effect of expressing a constitutively active IKKb in melanocytes on incidence of melanoma and the effect of melanocyte specific expression of a super repressor form of IkappaB on resistance to tumor induction by oncogenic Ras; 2) To determine the effects of the IKKb specific inhibitor, BMS-345541, alone and in combination with the DNA alkylating agent, temozolamide, on the growth and metastasis of melanoma tumors and the immune response to the tumor in immunocompetent mice; 3) To determine the mechanism for the induction of apoptosis in melanoma tumor cells by inhibiting IKKb. Significance: Melanoma is one of the fastest growing tumor types in the US and aging persons are increasingly affected due to sun and chemical exposure. Surgical intervention prior to the time of tumor invasion is the key factor in reduction in fatality for this form of cancer. For metastatic disease, immunotherapy, often combined with chemotherapy, is one of the most promising approaches. Our data indicate that targeting NF-kappaB may target tumor cells for apoptosis, inhibit tumor angiogenesis, tumor growth and metastasis. However, we need to determine the effects of blocking NF-kappaB on the host response to the tumor and on the incidence of melanoma tumor formation. The studies designed here will help design better clinical trials using inhibitors like VELCADE or IKK inhibitors in combination with chemotherapy.