Apoptosis of skin melanoma by the new Hsp H11

描述（由申请人提供）：黑色素瘤是一种主要的医学问题，发病率迅速增加，终生风险不断增加。治疗选择、治愈率和生存率随着疾病进展而急剧下降。尽管协同努力，化疗方案迄今为止产生了令人失望的结果，与内在耐药性有关。基于凋亡的化学基因治疗（定义为药物诱导的靶基因上调/激活）正在成为传统化疗的一种可行且有前途的替代方案。然而，选择合适的靶点是一个重大的临床挑战。我们最近克隆了一种新的热休克蛋白，H11，它不同于其他已知的家族成员，因为它具有细胞类型和刺激特异性的促凋亡活性。在黑色素瘤中，H11被异常的DNA甲基化沉默。通过药物诱导的去甲基化（即用Aza-C）强制表达H11引发生长停滞/凋亡，这被H11特异性寡核苷酸（ODNs）抑制，表明H11是化学基因治疗的有希望的靶点。提出了三个具体目标来检验这一假设。目的应用真实的实时甲基化特异性PCR和定量RT-PCR检测H11基因在黑色素瘤和痣组织、正常黑素细胞培养物和早期传代新鲜分离的黑色素瘤细胞培养物中的沉默频率。目的II将使用去甲基化（Aza-C，有或没有H11反义ODN和对照）和Dox处理稳定转染有四环素调节的H11的黑素瘤细胞，以扩展目前关于TAK 1在Nil诱导的生长停滞/凋亡中的功能的发现。研究将证实H11/TAK 1结合，鉴定结合位点，确定H11诱导的TAK 1活化的机制，并验证TAK 1介导的β-连环蛋白抑制的作用。将研究额外的早期传代黑色素瘤培养物（5-6/年）。对正常黑素细胞和角质形成细胞的研究将证实Aza-C未能引起H11过载和生长停滞/凋亡，验证H11通过Hsp 27结合的隔离以及H11增强Hsp 27介导的AKT活化/细胞存活的能力。目的III将证明H11过载杀死黑素瘤异种移植物，并检查H11过载在培养和体内使黑素瘤细胞对细胞毒性药物敏感的能力。拟议的研究将阐明热休克蛋白在癌细胞命运决定中的功能的新范式，开发一种急需的新的黑色素瘤化学基因治疗，并确定未来基于H11的治疗靶点。

DESCRIPTION (provided by applicant): Melanoma is a major medical problem with a rapidly increasing incidence and a growing lifetime risk. Treatment options, cure rates and survival decrease dramatically with disease progression. Despite concerted efforts, chemotherapy regimens have so far yielded disappointing results, related to intrinsic resistance. Apoptosis-based chemogene therapy (defined as drug-induced target gene upregulation/ activation) is emerging as a viable and promising alternative to conventional chemotherapy. However, the selection of the appropriate target is a major clinical challenge. We have recently cloned a novel heat shock protein, H11, that differs from other known family members in that it has cell type and stimulus specific pro- apoptotic activity upon overload. In melanoma, H11 is silenced by aberrant DNA methylation. Its forced expression by drug-induced de-methylation (viz. with Aza-C) triggers growth arrest/apoptosis, which are inhibited by H11 specific oligonucleotides (ODNs), suggesting that H11 is a promising target for chemogene therapy. Three specific Aims are proposed to test this hypothesis. Aim I will use real time methylation specific PCR and quantitative RT-PCR to examine the frequency of H11 silencing in melanoma and nevi tissues, normal melanocyte cultures and early passage freshly isolated melanoma cultures. Aim II will use de-methylation (Aza-C, with or without H11 antisense ODN and controls) and Dox treatment of melanoma cells stably transfected with Tetracycline regulated H11, to extend present findings for TAK1 function in Nil-induced growth arrest/apoptosis. Studies will confirm H11/TAK1 binding, identify binding sites, define the mechanism of H11-induced TAK1 activation, and verify the role of TAK1-mediated inhibition of beta- catenin. Additional early passage melanoma cultures (5-6/year) will be studied. Studies of normal melanocytes and keratinocytes will confirm the failure of Aza-C to cause H11 overload and growth arrest/ apoptosis, verify H11 sequestration through Hsp27 binding and the ability of H11 to potentiate Hsp27- mediated AKT activation/cell survival. Aim III will document that H11 overload kills melanoma xenografts and examine the ability of H11 overload to sensitize melanoma cells to cytotoxic drugs in culture and in vivo. The proposed studies will elucidate a novel paradigm for Hsp function in cancer cell fate determination, develop a much needed novel chemogene therapy for melanoma and identify targets for future H11-based therapies.