Elucidating neural crest-like reprogramming in melanoma

描述（由申请人提供）：黑色素瘤是最致命的皮肤癌类型，晚期癌症的五年生存率仅为15%。这种肿瘤预后差的主要原因是其高转移率和对治疗的抵抗。迄今为止，我们对黑色素瘤中转移性表型的获得和治疗耐药性的分子途径知之甚少。越来越多的证据表明，癌细胞利用发育程序进行肿瘤的传播、繁殖和对现有治疗方法的抵抗。我们的总体假设是黑素瘤细胞劫持了神经嵴的发育程序。我们最近发现，当Msh同源盒1 （MSX1）过表达时，在黑素细胞中实现神经嵴样重编程。表达msx1的黑素细胞表现出与人类皮肤神经嵴样前体细胞相似的形态和表型。在胚胎中，MSX1是神经嵴发育所必需的转录抑制因子。我们的数据表明MSX1结合并抑制小眼相关转录因子（MITF）的近端启动子，MITF是黑素细胞分化的主要转录调节因子。MSX1的表达与黑色素瘤的进展相关，我们的数据显示，转移细胞中MSX1的敲除显著损害体外运动，并减少体内肝脏定植。微环境中的炎症因子上调MSX1的表达，已被证明在黑色素瘤的细胞可塑性和免疫治疗抵抗中发挥作用。在本提案中，我们利用我们的肿瘤生物学和肿瘤免疫学知识，建议研究癌症胚胎表型与对癌细胞的免疫反应之间的新联系。我们的目标是描述MSX1如何在黑色素瘤细胞中指示神经嵴样塑性表型的机制，这有助于它们逃避免疫监视，并最终促进其转移。在特异性目标1中，我们将定义神经嵴样重编程在免疫治疗耐药性中的作用。我们将采用功能获得（cDNA过表达）和功能丧失（RNAi）方法，利用慢病毒载体，利用细胞毒性T淋巴细胞测定和免疫能力、同基因、同种异体移植小鼠黑色素瘤系统，评估msx1诱导的人类黑色素瘤细胞神经嵴样去分化的生物学意义。我们期望通过靶向神经嵴样重编程，将黑色素瘤细胞的表型冻结在更分化和更免疫原性的状态，从而使黑色素瘤细胞对目前可用的免疫疗法敏感。小分子抑制转录因子的设计是一个挑战；因此，我们将探索MSX1的上游诱导剂和下游靶点，以确定特异性靶2中MSX1驱动的去分化的黑色素瘤特异性药物靶点。这项研究的成功完成将确定黑色素瘤特异性去分化机制，并提供靶向黑色素瘤细胞可塑性的原理证明，特别是与免疫治疗相结合。

DESCRIPTION (provided by applicant): Melanoma is the most deadly type of skin cancer with a five-year survival rate for late stage cancer of only 15%. Key reasons for the poor prognosis of this tumor type are its high rate of metastasis and resistance to therapy. To date, very little is known about the molecular pathways governing the acquisition of the metastatic phenotype and therapy resistance in melanoma. Emerging evidence suggests that cancer cells use developmental programs for tumor dissemination, propagation, and resistance to currently available therapies. Our overall hypothesis is that melanoma cells hijack the neural crest developmental program for their progression. We recently discovered that neural crest-like reprogramming is achieved in melanocytes when Msh homeobox 1 (MSX1) is overexpressed. MSX1-expressing melanocytes exhibit a similar morphology and phenotype to neural crest-like precursor cells in human skin. In the embryo, MSX1 is a transcriptional repressor essential for neural crest development. Our data demonstrate that MSX1 binds and suppresses the proximal promoter of microphthalmia-associated transcription factor (MITF), the master transcriptional regulator of melanocyte differentiation. Expression of MSX1 correlates with melanoma progression, and our data show that knockdown of MSX1 in metastatic cells significantly impairs motility in vitro and reduces liver colonization in vivo. MSX1 expression is upregulated by inflammatory factors in the microenvironment, which has been shown to play a role in cellular plasticity and immunotherapy resistance in melanoma. In this proposal, using our knowledge of tumor biology and tumor immunology, we propose to study a novel link between the embryonic phenotype of cancer and immune response to cancer cells. Our goal is to delineate the mechanisms for how MSX1 dictates a neural crest-like plastic phenotype in melanoma cells, which helps them escape immune surveillance, and ultimately promotes their metastasis. In Specific Aim 1, we will define the role of neural crest-like reprogramming in resistance to immunotherapy. We will take gain-of-function (cDNA over-expression) and loss-of-function (RNAi) approaches using lentiviral vectors to assess the biological significance of MSX1-induced neural crest-like dedifferentiation in human melanoma cells using the cytotoxic T lymphocyte assay and an immune competent, syngeneic, allograft mouse melanoma system. We expect that by targeting neural crest-like reprogramming, this will freeze the phenotype of melanoma cells in more differentiated and more immunogenic state, thereby sensitizing melanoma cells to currently available immunotherapies. The design of small molecules inhibiting transcription factors is a challenge; therefore we will explore upstream inducers and downstream targets of MSX1 to identify melanoma-specific druggable targets for MSX1-driven dedifferentiation in Specific Aim 2. Successful completion of this study will identify melanoma-specific dedifferentiation mechanisms and provide a proof-of-principle to target cellular plasticit in melanoma, especially in combination with immunotherapy.