Targeting Endoplasmic Reticulum Stress Response for Cancer Therapy

描述（由申请人提供）：本提案的总体目标是检验IERSR可作为癌症治疗靶点的假设。实体瘤的血管化较差，因此不能获得足够的氧气和营养，特别是在血管化最少的区域。这导致内质网（ER）中未折叠蛋白的积累，称为ER应激。整合ER应激反应（IERSR）的激活对肿瘤生长和存活至关重要。IERSR涉及抑制翻译起始以减少对ER折叠能力的需求，并激活转录程序以扩大ER的大小和折叠能力。翻译起始通过蛋白激酶R（PKR）样ER驻留激酶PERK的活化和真核翻译起始因子2aplha（eIF 2a）的磷酸化而被抑制。增加ER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，所述关键转录因子控制ER伴侣、ER生物发生和ER相关的逆行蛋白转运和降解基因的表达。然而，IERSR必须以空间和时间的方式进行调节，因为IERSR的激活失败或持续激活IERSR将降低应激细胞的存活率。我们推测，肿瘤细胞利用IERSR在空间和时间调节的方式生存ER压力或避免细胞生长抑制和细胞毒性作用的延长IERSR。我们进一步假设，限制肿瘤激活IERSR的能力或导致持续和过度的IERSR将导致肿瘤的选择性死亡。我们已经开发了IERSR的化学调节剂和对这些药剂具有抗性的基因工程人类癌细胞系。如果这项提议得到资助，我们将利用我们的转基因细胞系和IERSR的化学调节剂来测试我们的假设，并最终确定IERSR是否可以作为癌症治疗的靶点。1)我们将检验N，N '-二芳基脲诱导的持续eIF 2a磷酸化将抑制肿瘤生长的假设。我们将研究选定/优化的N，N '-二芳基脲类药物的药代动力学特征和急性毒性。我们将通过治疗一侧携带表达eIF 2a-WT而另一侧携带非磷酸化突变体eIF 2a-S51 A的双侧肿瘤的小鼠来确定其疗效和机制特异性（特异性目的1）。2)我们将通过研究选择/优化的二芳基-羟吲哚的药代动力学特征和急性毒性来检验通过二芳基-羟吲哚抑制Xbp-1剪接抑制肿瘤生长的假设。我们将通过治疗携带双侧肿瘤的小鼠来确定它们的功效和机制特异性，所述双侧肿瘤在一侧仅表达内源性Xbp-1并且在另一侧已经剪接的Xbp-1（特异性目标2），和3）我们将测试抑制Xbp-1剪接和诱导eIF 2a磷酸化将协同抑制肿瘤生长和转移的假设（特异性目标3）。 公共卫生相关性：该项目的目的是检验整合的内质应激反应可以通过用单独或组合引起eIF 2a持续磷酸化和/或Xbp-1剪接抑制的药剂治疗荷瘤小鼠来药物靶向癌症治疗的假设。为了验证这一新的假设，我们开发了小分子化学试剂以及乳腺癌和前列腺癌细胞，其中内源性eIF 2a的表达被重组eIF 2a（WT或不可磷酸化的S51 A突变体）或表达已剪接的Xbp-1 mRNA的细胞取代;这些工具使我们能够测试小分子量试剂的疗效和特异性。

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). PUBLIC HEALTH RELEVANCE: The purpose of this project is to test the hypothesis that integrated endoplasmic stress response can be pharmaceutically targeted for cancer therapy by treating tumor bearing mice with agents that cause sustained phosphorylation of eIF2a and or inhibition of Xbp-1 splicing individually or in combination. To test this novel hypothesis we have developed small chemical agents as well as breast and prostate cancer cells in which expression of endogenous eIF2a is replaced by recombinant eIF2a (WT or nonphosphorylatable S51A mutant) or cells that express an already spliced Xbp-1 mRNA; these tools enable us to test the efficacy and specificity of small molecular weight agents.