Role of Notch Signaling in Melanoma Transdifferentiation and Tumor Progression

描述(由申请人提供)： 皮肤黑色素瘤是皮肤癌中最致命的一种，它起源于神经脊来源的黑素细胞。在描述启动导致转移性黑色素瘤的一连串恶变的分子事件方面已经取得了很大进展。这些进展导致了黑色素瘤治疗方面的一些显着改进。然而，臭名昭著的对传统疗法的内在抵抗力和对靶向疗法的治疗抵抗力的发展仍然是令人头疼的问题，突出了探索预防黑色素瘤发展和进展的其他途径的必要性。在此背景下，Notch信号在黑色素瘤中的作用仍有待充分了解和利用。Notch信号是一种重要的细胞信号机制，已知在黑素细胞发育中发挥作用。然而，Notch在黑色素瘤发生发展中的分子机制和具体作用尚不完全清楚。在我的实验室获得的初步数据显示，Notch信号在黑色素瘤肿瘤进展中可能起到双相作用。在皮肤驻留的良性新生黑素细胞和早期原发黑色素瘤中，Notch信号及其效应因子HES1(一种转录抑制因子)的下调允许神经元分化的阐述。这是 MAP2蛋白的表达表明，MAP2通常只存在于缺乏分裂能力的终末分化的神经元中。一项回顾性生存分析显示，MAP2阳性的原发皮肤肿瘤患者无黑色素瘤复发的存活时间明显长于MAP2阴性黑色素瘤的患者。MAP2在转移性黑色素瘤中缺失或较少出现。与其结合和稳定微管的主要功能一致，MAP2改变了微管的动态不稳定性，并诱导了黑色素瘤中有丝分裂的纺锤体缺陷。初步数据还显示，启动黑素细胞恶变的分子参与者也在激活这种神经元分化标记物方面发挥了作用。因此，在黑素细胞恶性转化的早期阶段，神经元特异性基因的激活与体外抑制增殖和体内肿瘤生长和进展相关。基于这些数据，我们假设Notch信号在黑色素瘤中具有双相或双相作用。首先，在恶性转化的早期阶段，Notch信号被下调，持续的致癌信号促进神经元分化，作为对抗黑色素瘤发生的故障安全机制，以及其他遗传事件，如PTEN的丢失，然后导致Notch信号上调，以支持黑色素瘤的进展。为了验证这一假设，我们将使用体外和体内相结合的方法来实现以下目标：在目标1中，我们将使用原代黑素细胞和一组特征良好的黑色素瘤细胞株来研究黑素细胞和患者来源的黑色素瘤的转分化机制，我们将探讨其分子机制。在目标2中，我们将研究Notch信号和神经元分化在转移性黑色素瘤细胞黑色素瘤发生中的作用，这些黑色素瘤细胞具有稳定的HES1基因敲除和/或免疫低下小鼠中Notch信号的抑制剂治疗，以及在黑素细胞中可诱导MPA2表达的转基因小鼠和BrafCa/Pten-/-小鼠之间的遗传杂交。在目标3中，我们将评估Notch在黑色素瘤中的预后意义，应用定量免疫组织化学方法分析由225例具有完整临床病史的I-III期原发黑色素瘤标本组成的内部创建的黑色素瘤组织芯片。关于Notch在转分化中的双重作用的假设，以及结合使用小鼠遗传模型和完全注释的黑色素瘤组织微阵列是拟议研究的创新方面。从这些研究中获得的数据有可能发现更可靠的黑色素瘤侵袭性预后标志物，并影响侵袭性皮肤黑色素瘤的治疗。

DESCRIPTION (provided by applicant): Cutaneous melanoma, the deadliest form of skin cancer, arises from neural crest derived melanocytes. Much progress has been made in delineating molecular events that initiate the cascade of malignant changes leading to metastatic melanoma. These advances have led to some remarkable improvements in treatment of melanoma. However, the notorious intrinsic resistance to conventional therapies and development of treatment resistance to targeted therapies continue to be vexing problems highlighting the need to explore additional avenues for preventing melanoma development and progression. In this context, the role for Notch signaling in melanoma remains to be fully understood and exploited. Notch signaling, an important cell signaling mechanism, is known to play a role in melanocyte development. However, molecular mechanisms and the specific roles Notch plays in melanoma development and progression are not completely understood. Preliminary data obtained in my laboratory showed a possible biphasic role for Notch signaling in melanoma tumor progression. In skin resident benign neo-plastic melanocytes and early primary melanoma, a downregulation of Notch signaling and its effector HES1, a transcriptional repressor, allows elaboration of neuronal differentiation. This is indicated by the expression of a protein, MAP2 that is normally present only in terminally differentiated neurons that lack ability to divide. A retrospective survival analysis showed that patients whose primary skin tumors were MAP2-positive survived significantly longer without melanoma recurrence than those diagnosed with MAP2-negative melanoma. MAP2 is absent or found less frequently in metastatic melanoma. In agreement with its primary function of binding to and stabilizing microtubules, MAP2 alters the dynamic instability of microtubules and induces mitotic spindle defects in melanoma. Preliminary data also showed that molecular players that initiate the malignant changes in melanocytes also play a role in activation of this neuronal differentiation marker. Thus, activation of a neuron-specific gene in melanocytes at early stages of malignant transformation correlates with inhibition of proliferation in vitro and tumor growth and progression in vivo. Based on these data, we hypothesized that Notch signaling, has a dual or biphasic role in melanoma. First, Notch signaling is downregulated during early steps in malignant transformation by sustained oncogenic signaling to promote neuronal differentiation as a fail-safe mechanism against melanoma tumorigenesis, and additional genetic events such as loss of PTEN then lead to upregulation of Notch signaling to support melanoma tumor progression. To test this hypothesis, we will use a combination of in vitro and in vivo approaches to accomplish the following Aims: In Aim 1, we will study mechanisms of transdifferentiation in melanocytes and patient-derived melanoma using primary melanocytes and a panel of well-characterized melanoma cell lines, we will investigate the molecular mechanisms. In Aim 2, we will investigate the role of Notch signaling and neuronal differentiation in melanomagenesis using metastatic melanoma cells with stable knockdown of HES1and/or treated with inhibitors of Notch signaling in immunocompromised mice, and a genetic cross between transgenic mice with inducible MPA2 expression in melanocytes and BrafCa/Pten-/- mice. In Aim 3, we will evaluate the prognostic significance of Notch in melanoma employing quantitative immunohistochemical method to analyze an in- house created melanoma tissue microarray consisting of 225 stage I-III primary melanoma specimens with complete clinical history. The hypothesis on the dual role of Notch in transdifferentiation and use of combination of mouse genetic model and a fully annotated melanoma tissue microarray are the innovative aspects of the proposed research. Data obtained from these studies have the potential to uncover more reliable prognostic markers for melanoma aggressiveness and impact the management of aggressive cutaneous melanoma.