The integrated stress response and the microenvironment in melanoma progression

摘要 尽管出现了新的治疗方式，包括BRAF抑制剂和免疫疗法，但 预计每年有7100人死于恶性黑色素瘤，主要死于转移性转移。 以及治疗抵抗。拟议的研究充分利用了这两个联合PIs在肿瘤方面的专业知识。 微环境、综合应激反应(ISR)和黑色素瘤进展测试总体 假设作为对肿瘤微环境(TME)应激适应性反应的一部分，双向 黑色素瘤细胞与成纤维细胞、内皮细胞和脂肪细胞之间的相互作用促进生存， 转移性播散和治疗抵抗。两项私人投资指数的初步结果及发表的报告 表明ISR在人类黑色素瘤中被激活，而基因或药物干扰 ISR严重损害多种实验肿瘤的原发肿瘤生长和转移，包括 黑色素瘤。这些研究将描述转录因子ATF4的显著作用，ATF4是一种主要的转录因子 整合应激反应的效应器，在一个促进生存和促进转移的计划中，由非 典型的肿瘤抑制因子Brn2，多种黑色素瘤相关信号的转录因子效应因子 小路。为了检验中心假设，我们将追求三个具体目标：在目标1中，我们将使用一部小说， 条件性全局ATF4基因敲除小鼠模型，以及成纤维细胞(FB)特异性和内皮(EC)- 特异的ATF4基因敲除小鼠和细胞，以确定ATF4在每个TME组分上的表达作用 黑色素瘤的进展和转移。ATF4结果的全局或FB特定删除的初步结果 在肿瘤生长严重不足的侧翼黑色素瘤肿瘤。在目标2中，我们将研究SRC如何促进 黑色素瘤的进展通过ATF4-Brn2的协同作用。Brn2和ATF4均能抑制失巢凋亡， 在初步研究中，我们发现Brn2与ATF4相互作用。因此，我们将检验这一假设 细胞外基质(ECM)驱动的整合素信号转导和单不饱和脂肪酸(MUFA)摄取 脂肪细胞和淋巴聚集在SRC上，施加协同的Brn2-ATF4抗凋亡和促进 转移基因表达程序，部分由河马信号驱动。最后，在目标3下，我们将确定 多不饱和脂肪酸如何决定黑色素瘤的表型。在这里，我们将剖析支撑MUFA的一种新机制- 介导-连环蛋白的核定位，并检验由此产生的SRC驱动的核CAV1- -连环蛋白复合体通过Brn2-ATF4促进肿瘤转移相关基因的表达 ATF4和Brn2对黑色素瘤表型异质性和肿瘤免疫浸润的影响。通过描述如何 ISR使用ATF4来协调TME的输出来塑造黑色素瘤的进展，我们将确定 抗黑色素瘤方法的治疗可利用途径。

SUMMARY Despite the emergence novel therapeutic modalities, including BRAF inhibitors and immunotherapies, over 7,100 people are expected to die each year from malignant melanoma, primarily from metastatic dissemination and therapy resistance. The proposed studies leverage the expertise of the two co-PIs in tumor microenvironment, the Integrated Stress Response (ISR) and melanoma progression to test the overall hypothesis that as part of the adaptive response to tumor microenvironmental (TME) stress, bi-directional interactions between melanoma cells and fibroblasts, endothelial cells and adipocytes promote survival, metastatic dissemination, and therapy resistance. Preliminary results and published reports from the two PIs indicate that the ISR is activated in human melanomas and that genetic or pharmacological disruption of the ISR severely impairs primary tumor growth and metastasis in multiple experimental tumors, including melanomas. The studies will delineate the salient roles of the transcription factor ATF4, a major transcriptional effector of the Integrated Stress Response, in a pro-survival and pro-metastatic program mediated by the non- canonical tumor suppressor BRN2, a transcription factor effector of multiple melanoma-associated signaling pathways. To test the central hypothesis, we will pursue three specific aims: In Aim 1 we will use a novel, conditional global ATF4 knockout mouse model, as well as Fibroblast (FB)-specific and Endothelial (EC)- specific ATF4 knockout mice and cells to determine the role of ATF4 expression in each TME component on melanoma progression and metastasis. Preliminary results with global or FB-specific deletion of ATF4 results in a severe deficiency in tumor growth of flank melanoma tumors. In Aim 2 we will study how SRC promotes melanoma progression via ATF4-BRN2 cooperativity. Both BRN2 and ATF4 can repress anoikis/apoptosis, and in preliminary studies we reveal that BRN2 interacts with ATF4. Therefore, we will test the hypothesis that Extracellular Matrix (ECM)-driven integrin signaling, and monounsaturated fatty acid (MUFA) uptake from adipocytes and lymph converge on SRC to impose a cooperative BRN2-ATF4 anti-apoptotic and pro- metastasis gene expression program, driven in part by hippo signaling. Finally, under Aim 3, we will determine how MUFAs dictate melanoma phenotype. Here we will dissect a novel mechanism underpinning MUFA- mediated nuclear localization of -catenin and test the hypothesis that the resulting SRC-driven nuclear CAV1- -catenin complex promotes a pro-metastasis gene expression program via BRN2-ATF4, and the contribution of ATF4 and BRN2 to melanoma phenotypic heterogeneity and tumor immune infiltration. By delineating how the ISR uses ATF4 to coordinate the output of the TME to shape melanoma progression, we will identify therapeutically exploitable pathways for anti-melanoma approaches.