Targeting the NF-kappaB Pathway in Melanoma

描述（由申请方提供）：恶性黑色素瘤发展中的一个关键事件是NF-κ B的失调，其导致炎性介质和趋化因子的组成性产生并逃避细胞凋亡。NF-kB受抑制蛋白IkB调节，IkB将NF-kB p65/p50复合物隔离在细胞质中，防止其易位到细胞核中。当这种IkappaB蛋白被IkappaB激酶（IKK）磷酸化时，它随后被26 S蛋白酶体泛素化和降解。我们已经证明，人黑色素瘤细胞系表现出IKK的组成性激活，增强的磷酸化和IkappaB的降解，以及细胞核中激活的NF-κ B p65/p50复合物的水平升高。这导致增强的NF-κ B介导的基因转录，导致趋化性细胞因子（趋化因子）、VEGF、IL-6和增强肿瘤细胞生长、促进肿瘤血管生成和转移的其它调节因子的组成型表达。我们的假设是，在肿瘤进展过程中，黑素细胞和/或基质细胞中NF-κ B的失调导致炎症介质的产生增加和黑素细胞肿瘤进展。此外，我们提出，阻断NF-κ B将干扰肿瘤生长和/或转移。我们的初步数据表明，在裸鼠上生长的人黑素瘤细胞中阻断NF-κ B靶向细胞凋亡并抑制肿瘤生长。为了进一步阐明在免疫活性环境中抑制NF-κ B用于黑素瘤治疗的潜力，我们提出了以下具体目标：la）确定敲低IKKa和IKKb对体外和体内黑素瘤肿瘤生长和转移的影响;（b）为了确定黑素细胞中表达组成型活性IKKb对黑色素瘤发病率的影响，以及黑素细胞特异性表达IKKb对黑色素瘤发病率的影响，2）确定IKKb特异性抑制剂BMS-345541单独和与DNA烷化剂替莫唑胺组合对免疫活性小鼠中黑色素瘤肿瘤的生长和转移以及对肿瘤的免疫应答的影响; 3）探讨抑制IKKb诱导黑色素瘤细胞凋亡的机制。重要性：黑色素瘤是美国增长最快的肿瘤类型之一，由于阳光和化学品暴露，老年人受到的影响越来越大。在肿瘤侵袭之前进行手术干预是降低这种癌症死亡率的关键因素。对于转移性疾病，免疫治疗，通常与化疗联合，是最有前途的方法之一。我们的数据表明，靶向NF-κ B可以靶向肿瘤细胞凋亡，抑制肿瘤血管生成，肿瘤生长和转移。然而，我们需要确定阻断NF-κ B对宿主对肿瘤的反应和黑色素瘤形成的影响。本文设计的研究将有助于设计更好的使用万珂或IKK抑制剂等抑制剂联合化疗的临床试验。

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.