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黑色素瘤发生和维持的新机制，最终目标是找到更合理的治疗方法。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（nevi），在MAPK通路中表达促癌突变，通常在BRAF或NRAS基因中。尽管如此，这些突变的痣细胞可以在几十年内保持有丝分裂不活跃。阻止痣恶性转化为黑色素瘤的分子基础尚不明确。我们之前报道过，细胞衰老可能是体外和体内黑色素细胞沉默表达致癌BRAF的一个有效机制。然而，痣的大小和组织学特征是出了名的不同。因此，单一的抑制反应不太可能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蛋白被两种不同的动力学、形态学和机制的抗增殖反应所反对。BRAFV600E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。相反，NRASQ61R，特别是HRASG12V，参与了与细胞结构的大规模破坏和内质网（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阻滞。在本提案中，我们将验证内质网是癌基因和药物诱导的应激信号的关键变阻器的假设，因此它在黑色素瘤的发生、进展和化疗耐药中起着关键作用。为了验证这一假设，我们将探讨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，通过基因修饰选择性靶向er调节剂，并在三维模型中确定其对黑素细胞瘤的影响（目的2）。同时，荧光和核磁共振成像的结合将用于解剖UPR在动物模型中对黑色素瘤维持和化疗耐药的功能贡献（目标3）。识别和表征新的肿瘤抑制机制(s)在黑色素瘤可能具有重要的临床意义。此外，由于应激反应的改变是多种肿瘤类型的普遍特征，因此拟议的研究应该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制在6-10个月，自20世纪70年代以来一直没有改善。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。本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侵袭行为及其极端化疗耐药性的贡献。我们认为这一建议最具创新性的方面之一是具有特定遗传缺陷的黑素细胞病变的工程设计，以及随后在标准组织培养系统之外的体外和体内功能研究。此外，与国内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。我们期望我们的研究将为黑色素瘤进展的分子基础提供新的知识，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.