Targeting Endoplasmic Reticulum Stress Response for Cancer Therapy

描述(由申请人提供)：这项提案的总体目标是验证IERSR可以作为癌症治疗的药理靶点的假设。实体瘤的血运很差，因此不能获得足够的氧气和营养，特别是在血运最少的区域。这会导致内质网(ER)中未折叠蛋白的积累，称为内质网应激。整合内质网应激反应(IERSR)的激活对肿瘤的生长和存活起着至关重要的作用。IERSR包括抑制翻译起始以减少对内质网折叠能力的需求，并激活转录程序以扩大内质网的大小和折叠能力。翻译起始是通过激活蛋白激酶R(PKR)样的ER驻留激酶PERK和真核细胞翻译起始因子2APLHA(EIF2a)的磷酸化来抑制的。增加内质网大小和折叠能力的转录程序是通过激活关键转录因子如X盒结合蛋白-1 XBP-1来完成的，这些转录因子控制内质网伴侣、内质网生物发生和内质网相关逆行蛋白运输和降解基因的表达。然而，IERSR必须在空间和时间上进行调控，因为无论是IERSR的激活失败还是IERSR的持续激活都会降低应激细胞的存活率。我们假设肿瘤细胞以一种空间和时间调节的方式利用IERSR来生存内质网应激，或避免延长IERSR的细胞抑制和细胞毒效应。我们进一步假设，限制肿瘤激活IERSR的能力或导致持续和夸大的IERSR将导致肿瘤选择性死亡。我们已经开发了IERSR的化学调节剂，并通过基因工程获得了对这些药物具有抗药性的人类癌细胞株。如果这项提议获得资助，我们将利用我们的转基因细胞系和IERSR的化学调节剂来检验我们的假设，并最终确定IERSR是否可以作为癌症治疗的药理靶点。1)我们将验证N，N‘-二芳基脲诱导的持续eIF2a磷酸化将抑制肿瘤生长的假设。我们将研究精选/优化的N，N‘-二芳基脲的药代动力学和急性毒性。我们将通过治疗一侧表达eIF2a-WT的双侧肿瘤和另一侧携带非磷酸化突变体eIF2a-S51a的小鼠(特定目标1)来确定它们的有效性和机制特异性。2)我们将通过研究精选/优化的二芳基氧化吲哚的药代动力学和急性毒性来验证二芳基氧化吲哚抑制XBP-1剪接抑制肿瘤生长的假说。我们将通过治疗一侧仅表达内源性XBP-1而另一侧已剪接XBP-1的小鼠来确定它们的有效性和机制特异性(特异性目标2)，以及3)我们将验证抑制XBP-1剪接和诱导eIF2a磷酸化将协同抑制肿瘤生长和转移的假设(特异性目标3)。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to test the hypothesis that IERSR can be pharmacologically targeted for cancer therapy. Solid tumors are poorly vascularized and therefore cannot receive sufficient oxygen and nutrients particularly in the least vascularized regions. This causes accumulation of unfolded proteins in endoplasmic reticulum (ER), termed ER-stress. Activation of integrated ER-stress response (IERSR) critically contributes to tumor growth and survival. The IERSR involves inhibiting translation initiation to reduce the demand on the folding capacity of the ER and activating a transcription program to enlarge the size and the folding capacity of the ER. Translation initiation is inhibited through activation of protein kinase R (PKR)-like ER resident kinase PERK and phosphorylation of eukaryotic translation initiation factor 2 aplha (eIF2a). The transcription program to increase the size and the folding capacity of the ER is accomplished by activating key transcription factors such as X box binding protein-1 Xbp-1 that control expression of ER-chaperons, ER biogenesis and ER-associated retrograde protein transport and degradation genes. However, IERSR must be regulated in a spatial and temporal manner because either the failure to activate IERSR or sustained activation of IERSR will reduce survival of stressed cells. We hypothesize that tumor cells utilize IERSR in a spatially and temporally regulated manner to survive ER-stress or avoid the cytostatic and cytotoxic effects of prolonged IERSR. We further hypothesize that limiting the ability of tumors to activate IERSR or causing sustained and exaggerated IERSR will cause selective demise of tumors. We have developed chemical modulators of the IERSR and genetically engineered human cancer cells lines resistant to these agents. If this proposal is funded, we will utilize our transgenic cell lines and chemical modulators of IERSR to test our hypothesis and to determine conclusively if the IERSR can be pharmacologically targeted for cancer therapy. 1) We will test the hypothesis that N,N'-diarylurea induced sustained eIF2a phosphorylation will inhibit tumor growth. We will study the pharmacokinetic profile and acute toxicity of selected/optimized N,N'-diarylureas. We will determine their efficacy and mechanism specificity by treating mice carrying bilateral tumors expressing eIF2a-WT on one side and non-phosphorylatable mutant, eIF2a-S51A on the other side (Specific Aim 1). 2) We will test the hypothesis that inhibition of Xbp-1 splicing by diaryl-oxindoles inhibit tumor growth by studying the pharmacokinetic profile and acute toxicity of selected/optimized diaryl-oxindole. We will determine their efficacy and mechanism specificity by treating mice carrying bilateral tumors expressing only endogenous Xbp-1 on one side and already spliced Xbp-1 on the other side (Specific Aim 2), and 3) We will test the hypothesis that inhibition of Xbp-1 splicing and induction of eIF2a phosphorylation will synergistically inhibit tumor growth and metastasis (Specific Aim 3).