The Unfolded Protein Response in Melanoma Progression and Chemoresistance

描述（由申请人提供）：本提案的长期目标是确定涉及黑色素瘤启动和维持的新机制，最终目标是更合理的治疗方法。几乎所有白皙皮肤的个体都患有多发性良性黑色素细胞肿瘤（痣），其表达MAPK途径中的原癌基因突变，通常在BRAF或NRAS基因中。尽管如此，这些突变的痣细胞可以保持几十年的有丝分裂不活跃。阻止痣恶性转化为黑色素瘤的分子基础尚不清楚。我们以前曾报道，细胞衰老可能是一种有效的机制，占沉默的黑素细胞表达致癌BRAF，在体外和体内。然而，痣的大小和组织学特征是众所周知的异质性。因此，不太可能单一的抑制反应控制黑素细胞转化的早期阶段。我们最近发现，肿瘤相关的BRAF和RAS蛋白是由两个动力学，形态学和机制不同的抗增殖反应。BRAFV 600 E诱导了一个缓慢的衰老程序，可以通过额外的癌基因来挽救。相比之下，NRASQ 61 R，特别是HRASG 12 V，参与了与细胞结构的大规模解体和内质网（ER）相关应激（特别是未折叠蛋白反应，UPR）的激活相关的快速细胞周期停滞。在这项提案中，我们将测试的假设，ER是癌基因和药物诱导的应激信号的关键变阻器，因此，它在黑色素瘤的发生，发展和耐药性中起着关键作用。为了验证这一假设，我们将解决RAS蛋白诱导黑素细胞衰老的分子基础（目的1）。特别强调将致力于重建人类皮肤，基因改造，选择性地针对ER-调节剂，并确定其对黑素细胞瘤的三维模型的影响（目标2）。同时，将使用基于荧光和核磁共振成像的组合来剖析UPR对动物模型中黑色素瘤维持和化疗耐药性的功能贡献（目的3）。新的肿瘤抑制机制在黑色素瘤中的鉴定和表征可能具有重要的临床意义。此外，由于改变的应激反应是多种肿瘤类型的一般特征，因此拟议的研究应与各种其他侵袭性癌症相关，并可能为药物开发提供一类新的靶点。公共卫生相关性：黑色素瘤是最具侵袭性的皮肤癌。IV期（转移性）黑色素瘤患者的长期生存期通常限于6-10个月，自20世纪70年代以来没有改善130，131。因此，黑色素瘤领域迫切需要新的疾病标志物和改进的抗癌策略。该项目的主要目的是评估依赖于内质网的生存信号对黑色素瘤细胞的侵袭行为及其极端化学抗性的贡献。我们认为，这一建议的最具创新性的方面之一是工程的黑素细胞病变与特定的遗传缺陷，以及随后的功能研究，在体外和体内超出标准的组织培养系统。此外，与该国最大的多学科黑色素瘤诊所之一的密切合作将有助于测试和验证我们假设的生理影响。我们希望我们的研究将提供黑色素瘤进展的分子基础的新知识，并揭示治疗干预的新靶点。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to identify novel mechanisms involved in melanoma initiation and maintenance with the ultimate objective of a more rational approach to treatment. Nearly all individuals of fair skin bear multiple benign melanocytic neoplasms (nevi) expressing pro- oncogenic mutations in the MAPK pathway, usually in the BRAF or NRAS genes. Still, these mutated nevus cells can remain mitotically inactive for decades. The molecular bases blocking the malignant transformation of nevi into melanoma are poorly defined. We have previously reported that cellular senescence may be a potent mechanism accounting for the silent nature of melanocytes expressing oncogenic BRAF, both in vitro and in vivo. However, the size and histological features of nevi are notoriously heterogeneous. Therefore, it is unlikely that a single suppressive response controls early stages of melanocyte transformation. We have recently found that tumor-associated BRAF and RAS proteins are opposed by two kinetically, morphologically and mechanistically different anti-proliferative responses. BRAFV600E induced a slow program of senescence that could be rescued by additional oncogenes. In contrast, NRASQ61R and particularly HRASG12V, engaged a fast cell cycle arrest associated with massive disorganization of the cellular architecture and the activation of endoplasmic reticulum (ER)-associated stress (specifically, the Unfolded Protein Response, UPR). In this proposal we will test the hypothesis that the ER is a key rheostat of oncogene and drug-induced stress signals, and as such it plays a critical role in melanoma initiation, progression and chemoresistance. To test this hypothesis, we will address the molecular bases underlying the induction of melanocyte senescence by RAS proteins (Aim 1). Special emphasis will be dedicated to reconstructed human skin, genetically modified to selectively target ER-modulators and determine their impact on melanocytic neoplasia in a three dimensional model (Aim 2). In parallel, a combination of fluorescence-based and nuclear magnetic resonance imaging will be used to dissect the functional contribution of the UPR to melanoma maintenance and chemoresistance in animal models (Aim 3). The identification and characterization of novel tumor suppressor mechanism(s) in melanoma is likely to have important clinical implications. In addition, since altered stress responses are a general feature of multiple tumor types, the proposed studies should be relevant to a wide variety of other aggressive cancers, and may provide a new class of targets for drug development. PUBLIC HEALTH RELEVANCE: Melanoma is the most aggressive form of skin cancer. The long-term survival for patients with Stage IV (metastatic) melanoma is typically limited to 6-10 months, and has not improved since the 1970s130,131. Therefore, the melanoma field is in urgent need of new markers of the disease and improved anticancer strategies. The primary objective of this project is to evaluate the contribution of survival signals dependent on the endoplasmic reticulum to the aggressive behavior of melanoma cells and their extreme chemoresistance. We consider that one of the most innovative aspects of this proposal is the engineering of melanocytic lesions with specific genetic defects, and subsequent functional studies in vitro and in vivo beyond standard tissue culture systems. In addition, a close collaboration with one of the largest Multidisciplinary Melanoma Clinics of the country will help testing and validating the physiological impact of our hypotheses. We expect that our studies will provide new knowledge of the molecular basis of melanoma progression, and reveal novel targets for therapeutic intervention.