p53 in Melanomagenesis

 描述(由申请人提供)：侵袭性和致命性转移性黑色素瘤与过度暴露于紫外线辐射(UVR)有关。UVR诱导癌基因如BRAF的激活突变，从而启动色素病变(Nevi)和黑色素瘤的发展；然而，人们也认为UVR促进黑色素瘤的发生不依赖于其诱变作用。最近出现了一种UVR促肿瘤的分子模型，其中黑素细胞增殖似乎受到角质形成细胞来源的黑素细胞生长因子的调控，转录因子和关键的肿瘤抑制因子P53对UVR的反应。此外，在携带BRAF(BRAFV600E)或CDK4(CDK4R24C/R24C)突变的黑色素瘤易感小鼠中，黑素细胞生长因子的表达增加加速了色素病变的形成，并促进了黑色素瘤的形成。与此一致的是，我们独特的角质形成细胞特异性P53高表达的小鼠模型在遵循致癌方案时显示出发生痣和黑色素瘤的倾向。这些发现导致我们假设角质形成细胞P53释放旁分泌黑素细胞生长因子，这些生长因子与黑素细胞中的致癌突变(如BRAFV600E)协同作用，促进启动的黑素细胞的增殖，从而促进黑色素瘤。这一新颖而重要的概念首次提供了一种解释UVR促进肿瘤的分子途径，并将P53的作用扩展到其典型的肿瘤抑制之外，包括在黑色素瘤发生中的组织特异性功能。为了验证这一假设，我们将使用复杂的人类黑素细胞和角质形成细胞培养系统(目标1)和3D人类皮肤等效异种移植小鼠模型(目标2)。在目标1中，原代人类角质形成细胞将受到紫外线照射或用特定的P53激活药物Nutlin-3a处理，以产生角质形成细胞条件黑素细胞生长介质。转导对照GFP和GFP标记的BRAFV600E慢病毒的原代人黑素细胞将在有或没有UVR或Nutlin 3a条件的培养液中生长。我们将监测UVR和P53条件培养液对转导的黑素细胞行为(增殖、凋亡、衰老、迁移和色素沉着)的影响。我们预计，条件培养液中的旁分泌因子将增加BRAF突变的黑素细胞的增殖和迁移能力，并减少其癌基因诱导的培养衰老。利用慢病毒shRNA，我们将确定对黑素细胞增殖和转化最关键的生长因子。在目标2中，将产生含有BRAFV600E-GFP和GFP对照黑素细胞的人体3D皮肤等效异种移植。这些移植物将接受紫外线照射或用Nutlin-3a治疗，以检查目标1中探索的依赖于p53的生长因子对黑素细胞行为的体内影响。在长期研究中，将检查移植物的组织学黑素细胞变化、黑素细胞损伤或黑色素瘤。总之，这些目标将探索UVR、BRAF突变和黑色素瘤发生之间的联系，这对于我们理解这种致命的肿瘤是必不可少的。我们的发现将证明旁分泌P53信号参与黑色素瘤的发展，并可能导致新的黑色素瘤预防和治疗策略。

 DESCRIPTION (provided by applicant): Invasive and deadly metastatic melanoma is associated with excessive exposure to ultraviolet radiation (UVR). UVR induces activating mutations in oncogenes such as BRAF which initiates the development of pigmented lesions (nevi) and melanoma; however it is also believed that UVR promotes melanomagenesis independently of its mutagenic action. Recently, a molecular model of UVR tumor promotion has emerged where melanocyte proliferation seems to be up-regulated by keratinocyte-derived melanocyte growth factors under the control of the transcription factor and key tumor suppressor p53 in response to UVR. Moreover, in melanoma prone mice that carry mutations in Braf (BrafV600E) or CDK4 (CDK4R24C/R24C), increased expression of melanocyte growth factors accelerated the formation of pigmented lesions and promoted melanoma. In line with this, our unique mouse model with constitutively high expression of keratinocyte-specific p53 displayed a propensity to develop nevi and melanoma when subjected to a carcinogenesis protocol. These findings lead us to the hypothesis that keratinocyte p53 releases paracrine melanocyte growth factors that cooperate with oncogenic mutations such as BRAFV600E in melanocytes to promote proliferation of initiated melanocytes and thus melanoma. This novel and important concept provides for the first time a molecular pathway explaining UVR tumor promotion, and extends the role of p53 beyond its canonical tumor suppression to include a tissue- specific function in melanomagenesis. To test this hypothesis we will use intricate human melanocyte and keratinocyte culture systems (Aim 1) and a 3D human skin equivalent xenografted mouse model (Aim 2). In Aim 1, primary human keratinocytes will be UV-irradiated or treated with a specific p53 activating drug, Nutlin- 3a, to generate keratinocyte-conditioned melanocyte growth media. Primary human melanocytes transduced with a control GFP and a GFP-tagged BRAFV600E lentivirus will be grown in the presence or absence of the UVR- or Nutlin 3a-conditioned media. We will monitor the impact of UVR- and p53- conditioned media on the behavior of transduced melanocytes (proliferation, apoptosis, senescence, migration and pigmentation). We expect that paracrine factors in the conditioned media will increase the proliferation and migration potential of BRAF-mutated melanocytes, and decrease their oncogene-induced senescence in culture. Using lentiviral shRNAs, we will identify the most crucial growth factors for melanocytic proliferation and transformation. In Aim 2, human 3D skin equivalent xenografts will be generated that contain the BRAFV600E-GFP and GFP control melanocytes. These grafts will be UV-irradiated or treated with Nutlin-3a to examine the in vivo impact of p53-dependent growth factors on melanocyte behavior as explored in Aim 1. In longer term studies, grafts will be examined for histological melanocytic changes, melanocytic lesions or melanoma. Together, these aims will explore a link that is still not well defined between UVR, BRAF mutations and melanomagenesis which is imperative for our understanding of this deadly tumor. Our findings will demonstrate the involvement of a paracrine p53 signaling in melanoma development and may lead to new strategies for melanoma prevention and treatment.