Proteasome Inhibition and ER Stress

项目总结 目前正在对基于蛋白酶体抑制剂(PI)的联合化疗进行评估，以治疗 胰腺癌和其他实体恶性肿瘤。我们的假设是PI引起内质网(ER) 压力作用于癌细胞，而这种压力会导致细胞死亡。此外，我们已经获得了初步证据 PI对细胞死亡的影响是高度不同的，并且与它们是否诱导 EIF2？的磷酸化，这是未折叠蛋白反应(UPR)的一个组成部分，介导抑制 全球蛋白质合成。具体地说，PI促进eIF2的强烈磷酸化。 对PI诱导的细胞凋亡有相对抵抗力的细胞系，但它们未能做到这一点(或衰减 翻译)在最敏感的细胞系中。确定这种异质性的生化基础 能够对最有可能对基于PI的组合产生反应的肿瘤进行前瞻性识别 化疗，并应产生新的治疗干预的目标。我们还希望更好地定义 一种最有希望的PI类化合物诱导细胞凋亡的分子机制 联合方案，即硼替佐米+组蛋白脱乙酰酶(HDAC)抑制剂。 我们已经获得了良好的初步证据，表明HDAC抑制剂促进蛋白酶体抑制物介导的 通过破坏被称为侵袭体的细胞保护结构进行细胞凋亡，侵袭体似乎具有缓解ER的功能 压力。基因沉默研究表明，导致侵袭性破坏的HDAC是 HDAC6，并且更具选择性的HDAC6抑制剂有可能产生与之相当或更好的肿瘤细胞杀伤 比PAN HDAC抑制剂(如SAHA)毒性更小。为了直接测试我们的假设，我们提出了 遵循特定的目标。(1)明确控制硼替佐米诱导的分子机制 EIF2的磷酸化。我们将测试PI通过诱导PERK激活抑制PERK激活的假设 分子查帕酮(HSP70？)的表达阻止药物敏感细胞中的PERK同种聚集；(2) 确定内质网应激在PI诱导的细胞凋亡中的作用。在这里，我们将评估ROS、Ca2+、 JNK、Noxa、Caspase-4或PI诱导的细胞凋亡；(3)：测定JNK、Noxa和Caspase-4的毒性和抗肿瘤效果 异种移植中PIs和HDAC抑制剂的联合治疗。我们将比较以下几种方法的效果 Pis联合SAHA(一种PAN HDAC抑制剂)、Tubacin(HDAC6选择性)或SNDX-275联合治疗 (I型HDAC特异性)在体外和来自敏感和耐药细胞系的原位肿瘤中。我们会 还研究了药物-靶点相互作用和生物反应的药效学标志物 可以在这些动物的外周血液中测量，并应用这些方法来测量 Bortezomib联合SAHA治疗胰腺癌的II期临床试验 癌症。

PROJECT SUMMARY Proteasome inhibitor (PI)-based combination chemotherapy is currently being evaluated for the treatment of pancreatic cancer and other solid malignancies. Our hypothesis is that PIs cause endoplasmic reticular (ER) stress in cancer cells and this stress mediates cell killing. Furthermore, we have obtained preliminary evidence that the effects of PIs on cell death are highly heterogeneous and are linked to whether or not they induce phosphorylation of eIF2¿, a component of the unfolded protein response (UPR) that mediates suppression of global protein synthesis. Specifically, PIs promote strong phosphorylation of eIF2¿ phosphorylation in the cell lines that are relatively resistant to PI-induced apoptosis, but they fail to do so (or attenuate translation) in the cell lines that are most sensitive. Identifying the biochemical basis for this heterogeneity could enable the prospective identification of tumors that are most likely to respond to PI-based combination chemotherapy and should yield new targets for therapeutic intervention. We also wish to better define the molecular mechanisms involved in the apoptosis that is induced by one of the most promising PI-based combination regimens, namely, the combination of bortezomib plus histone deacetylase (HDAC) inhibitors. We have obtained good preliminary evidence that HDAC inhibitors promote proteasome inhibitor-mediated apoptosis by disrupting cytoprotective structures known as aggresomes that appear to function to alleviate ER stress. Gene silencing studies have demonstrated that the HDAC responsible for aggresome disruption is HDAC6, and it is possible that more selective HDAC6 inhibitors will yield comparable or better tumor cell killing than pan HDAC inhibitors (like SAHA) with less toxicity. To directly test our hypotheses we propose the following Specific Aims. (1) Define the molecular mechanisms that control bortezomib-induced phosphorylation of eIF2¿. We will test the hypothesis that PIs inhibit PERK activation by inducing the expression of a molecular chaparone (HSP70?) that blocks PERK homoaggregation in drug-sensitive cells; (2) Determine role of ER stress in PI-induced apoptosis. Here we will assess the contributions of ROS, Ca2+, JNK, Noxa, and caspase-4 ot PI-induced apoptosis; (3): Determine the toxicity and anti-tumor efficacy of combination therapy with PIs and HDAC inhibitors in xenografts. We will compare the effects of combination therapy with PIs plus SAHA (a pan HDAC inhibitor), tubacin (HDAC6-selective), or SNDX-275 (type I HDAC-specific) in vitro and in orthotopic tumors derived from sensitive and resistant cell lines. We will also investigate whether or not pharmacodynamic markers of drug-target interaction and biological response can be measured in the peripheral blood of these animals and apply these methods to measure the effects of therapy with bortezomib plus SAHA within the context of a Phase II clinical trial in patients with pancreatic cancer.