Heat shock proteins in cancer and signaling

描述(由申请人提供)：热休克蛋白(HSPs)在许多肿瘤中有结构性表达，表明这些蛋白在肿瘤发展中为细胞提供了选择性优势。重组HSP72的表达增强了肿瘤的致瘤性，而从肿瘤细胞(但不是正常细胞)中去除HSP72会降低肿瘤的致瘤性并增加对某些药物的敏感性。这项建议的第一个目标是阐明HSP72在癌细胞中表达升高的作用。我们推测，HSP72可以使肿瘤细胞逃脱癌基因激活诱导的细胞凋亡，并在有毒的肿瘤微环境条件下存活。这一假说将在过表达c-myc癌基因的永生化乳腺上皮细胞中得到验证，并将阐明HSP72介导的抑制细胞凋亡的机制。用在乳腺组织中过表达c-myc和Hsp72的转基因小鼠进行的实验将检验这些发现对癌症发展的意义。某些肿瘤细胞系中HSP72的特异性缺失导致对某些抗癌药物的敏感性增强，生长速度减慢和锚定非依赖性生长，这与核转录因子-kappaB信号通路的下调相一致。我们将验证一种假设，即当HSP72耗尽时，该途径的下调与癌细胞的药物敏感性增强和致瘤性降低有关。第二个目标是确定热休克反应在癌细胞对蛋白酶体和Hsp90抑制剂耐药中的作用。热疗、蛋白酶体抑制剂(如VELCADE)和Hsp90抑制剂(如17-AAG)是三种新的抗癌治疗方法，它们强烈诱导热休克蛋白。我们假设，抑制热休克反应可以增强这些治疗的效力。事实上，MEF细胞中HSFL转录因子的缺失会导致高温、VELCADE和17-AAG后热休克蛋白的诱导受阻，并增强对这些治疗的敏感性。在多发性骨髓瘤、前列腺癌和乳腺肿瘤细胞系中，HSF1将被siRNA取消，这些克隆对VELCADE和17-AAG的敏感性将进行测试。通过异种移植模型，我们将确定HSF1的下调是否增强了肿瘤对VELCADE和17-AAG的敏感性。我们已经从几个化学文库中筛选出35,000个化合物，并鉴定了9个分子，它们(微摩尔和亚微摩尔浓度)有效地抑制热休克、VELCADE和17-AAG诱导的HSPs。我们将分析这两个最有希望的化合物的类似物，以确定其结构中的关键元素，并获得无毒、有效的诱导热休克蛋白的特异性抑制剂。这些抑制剂将在肿瘤细胞培养中测试它们增强VELCADE和17-AAG效力的能力。这些实验将检验一种假设，即热休克反应的抑制剂可以增强蛋白酶体和Hsp90抑制剂的抗癌活性。

DESCRIPTION (provided by applicant): Heat shock proteins (Hsps) are constitutively expressed in many tumors, suggesting that these proteins provide a selective advantage to cells in tumor development. Expression of recombinant Hsp72 enhances tumorigenicity, while abrogation of Hsp72 from tumor cells (but not normal cells) reduces their tumorigenic properties and sensitizes to certain drugs. The first goal of this proposal is to elucidate the role of the elevated expression of Hsp72 in cancer cells. We hypothesize that Hsp72 allows tumor cells to escape apoptosis induced by activated oncogenes and to survive toxic conditions of tumor microenvironments. This hypothesis will be evaluated with immortalized mammary epithelium cells that over-express c-myc oncogene, and the mechanisms of the Hsp72-mediated suppression of apoptosis will be elucidated. Experiments with transgenic mice that overexpress both c-myc and Hsp72 in mammary tissue will test the significance of these findings for cancer development. Specific depletion of Hsp72 in some tumor cell lines leads to enhanced sensitivity to certain anti-cancer agents, and to reduced growth rate and anchorage-independent growth, which coincide with downregulation of the NF-kappaB signaling pathway. We will test a hypothesis that downregulation of this pathway is responsible for enhanced drug sensitivity and reduced tumorigenic properties of cancer cells upon depletion of Hsp72. The second goal is to establish the role of the heat shock response in cancer cell resistance to proteasome and Hsp90 inhibitors. Hyperthermia, proteasome inhibitors (e.g. Velcade) and Hsp90 inhibitors (e.g. 17-AAG) are three novel anti-cancer treatments, which strongly induce heat shock proteins. We hypothesize that inhibition of the heat shock response could enhance the potency of these treatments. In fact, deletion of the Hsfl transcription factor in MEF cells leads to a block of Hsps induction after hyperthermia, Velcade and 17-AAG, and enhances the sensitivity to these treatments. Hsf1 will be abrogated by siRNA in multiple myeloma, prostate and breast tumor cell lines, and sensitivity of these clones to Velcade and 17-AAG will be tested. With xenograft models, we will establish if downregulation of Hsf1 enhances tumor sensitivity to Velcade and 17-AAG. We have screened 35,000 compounds from several chemical libraries and identified 9 molecules, which potently (in micromolar and sub-micromolar concentrations) inhibit induction of Hsps by heat shock, Velcade and 17-AAG. We will analyze analogs of the two most promising compounds to identify critical elements in their structures and obtain non-toxic potent specific inhibitors of induction of Hsps. These inhibitors will be tested for their ability to enhance the potency of Velcade and 17-AAG in tumor cell cultures. These experiments will test a hypothesis that inhibitors of the heat shock response can enhance the anti-cancer activities of proteasome and Hsp90 inhibitors.