The Role of CtBP1 in UV-mediated Melanoma Carcinogenesis

描述(由申请人提供)： 摘要在美国，黑色素瘤的发病率正在迅速增加，包括退伍军人中的黑色素瘤发病率，在美国服兵役期间暴露在阳光下与黑色素瘤发病率的增加有关。尽管黑色素瘤不是最常见的皮肤癌，但它是最致命的形式。美国国家癌症研究所预测，2013年有76,690例黑色素瘤确诊病例和9,480例死亡病例。确定与黑色素瘤有关的分子和途径对于合理开发新的预防和治疗策略至关重要。CtBP1(羧基末端结合蛋白1)是一种依赖于NADH的转录调节因子，在体外可以抑制多种肿瘤抑制因子的转录。这项建议侧重于了解CtBP1在紫外线(UV)介导的黑色素瘤癌变过程中的高表达作用。我们的长期目标是开发基于CtBP1的黑色素瘤新的预防/治疗方法。我们的初步数据表明，CtBP1在人类黑色素瘤中过表达，这抑制了参与核苷酸切除修复(NER)途径的关键成员的转录，从而抑制了紫外线照射后的DNA损伤修复。此外，角质形成细胞中CtBP1的过度表达会导致紫外线致癌过程中常见的几种细胞因子的产生。根据我们的初步数据，我们假设：CtBP1的过表达抑制了黑素细胞修复紫外线介导的DNA损伤的关键基因，并激活了角质形成细胞中参与免疫抑制、炎症和血管生成的紫外线信号细胞因子，从而促进了紫外线致癌。为了验证我们的假设，目标1将评估CtBP1在体内紫外线致癌中的作用。Tyr-H-rasG12V/Ink4a基因缺失的小鼠出现自发性皮肤黑色素瘤，与紫外线诱导的恶性黑色素瘤非常相似。我们将把Tyr-H-rasG12V/Ink4a缺失的小鼠与CtBP1基因敲除的小鼠进行杂交，以评估降低CtBP1是否抑制紫外线致癌作用，以及与CtBP1过度表达相关的任何病理变化是否有助于黑色素瘤的发展。此外，我们还建立了K5.CtBP1转基因小鼠，在角质形成细胞中过表达CtBP1，并表现出皮下炎症和血管生成。我们将把Tyr-H-rasG12V/Ink4a缺失的小鼠与我们的K5.CtBP1小鼠杂交，研究CtBP1诱导的免疫抑制、炎症和血管生成细胞因子对紫外线介导的黑色素瘤癌变的影响。我们的初步研究表明，紫外线照射皮肤会增加CtBP1和NADH的水平。我们首次发现CtBP1的激活依赖于NADH，并且最近发现NADH阻断剂Tempol1抑制了CtBP1的功能。因此，我们将确定紫外光介导的黑色素瘤癌变是否能被坦普尔抑制。这些分析将为通过靶向CtBP1来开发紫外线介导的黑色素瘤的预防或治疗方法铺平道路。目的2研究CtBP1抑制NER途径关键介质的分子机制。我们将评估CtBP1介导的NER基因转录抑制在黑色素瘤发病机制中的作用，明确CtBP1转录调控NER基因的分子机制，并确定NER基因的CtBP1转录辅助因子。我们还将测试在紫外线致癌过程中，NER基因的CtBP1调节是否发生变化。这些分析将确定CtBP1功能亢进如何抑制NER基因，并抑制紫外线诱导的黑素细胞DNA损伤修复。目的3鉴定角质形成细胞产生的紫外线信号细胞因子，这些细胞因子是影响黑色素瘤微环境的CtBP1转录靶点。我们的初步数据显示，CtBP1可以在角质形成细胞中直接反式激活几种参与免疫抑制、炎症和间质血管生成的紫外线特征细胞因子。在AIM 1中产生的样本将用于识别这些CtBP1转录靶标。体内敲除候选CtBP1靶标将用于验证它们在紫外线致癌中的作用。

DESCRIPTION (provided by applicant): Summary The incidence of melanoma is rapidly increasing in the United States including in VA populations, and sun exposure during US military service has been linked to increased melanoma incidence. Even though melanoma is not the most common skin cancer, it is the most deadly form. The National Cancer Institute predicted 76,690 melanoma diagnoses and 9,480 deaths for 2013. Identification of the molecules and pathways responsible for melanoma is critical to the rational development of novel preventive and therapeutic strategies. CtBP1 (Carboxyl-terminal Binding Protein 1) is an NADH-dependent transcriptional regulator shown to repress transcription of multiple tumor suppressors in vitro. This proposal focuses on understanding the role of CtBP1 over expression in ultraviolet (UV)-mediated melanoma carcinogenesis. Our long-term goal is to develop CtBP1-based novel preventative/therapeutic approaches for melanoma. Our preliminary data show that CtBP1 is over expressed in human melanomas, which represses transcription of key players involved in the nucleotide excision repair (NER) pathway, thus inhibiting DNA damage repair following UV irradiation. Furthermore, CtBP1 over expression in keratinocytes causes production of several cytokines commonly seen during UV carcinogenesis. Based on our preliminary data, we hypothesize: CtBP1 over expression suppresses genes critical for repairing UV-mediated DNA damage in melanocytes and activates UV signature cytokines in keratinocytes involved in immune suppression, inflammation, and angiogenesis, thus contributing to UV carcinogenesis. To test our hypotheses, Aim 1 will assess the role of CtBP1 in UV carcinogenesis in vivo. The Tyr-H-rasG12V/Ink4a-null mice develop spontaneous cutaneous melanomas, highly resemble the UV-induced malignant melanoma. We will cross the Tyr-H-rasG12V/Ink4a-null mice with CtBP1 knockout mice to assess whether decreasing CtBP1 inhibits UV carcinogenesis and if any pathological alterations associated with CtBP1 over expression contributes to melanoma development. In addition, we have generated the K5.CtBP1 transgenic mice, which over express CtBP1 in keratinocytes and display subcutaneous inflammation and angiogenesis. We will cross the Tyr-H-rasG12V/Ink4a-null mice with our K5.CtBP1 mice to study the impact of the CtBP1-induced immune suppressive, inflammatory, and angiogenic cytokines on UV-mediated melanoma carcinogenesis. Our preliminary study shows that UV irradiation of the skin increases CtBP1 and NADH levels. We are the first to identify the NADH-dependent activation of CtBP1 and have recently shown the inhibition of CtBP1 function by the NADH-blocker Tempol. Therefore, we will determine if UV-mediated melanoma carcinogenesis can be inhibited by Tempol. These analyses will pave the road for developing the preventative or therapeutic approaches for UV-mediated melanoma by targeting CtBP1. Aim 2 will examine the molecular mechanisms by which CtBP1 represses key mediators of the NER pathway. We will assess the impact of CtBP1-mediated repression of NER genes in the pathogenesis of melanoma, define the molecular mechanism of CtBP1 transcriptional regulation of NER genes, and identify CtBP1 transcriptional co-factors of NER genes. We will also test if CtBP1 regulation of NER genes is altered during UV carcinogenesis. These analyses will determine how CtBP1 hyperfunction suppresses the NER genes and inhibits UV-induced DNA damage repair in melanocytes. Aim 3 will identify UV signature cytokines produced in keratinocytes, which are CtBP1 transcriptional targets that affect melanoma microenvironment. Our preliminary data revealed that several UV signature cytokines involved in immune suppression, inflammation, and angiogenesis in the stroma could be directly transactivated by CtBP1 in keratinocytes. Samples generated in Aim 1 will be used to identify these CtBP1 transcriptional targets. In vivo knockdown of candidate CtBP1 targets will be used to validate their roles in UV carcinogenesis.