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.
UV irradiation is the major environmental carcinogen for skin cancer including melanoma, posing high threat to human health, especially to the soldiers in the combat zone and other areas with excess UV exposure. Improving treatment strategies requires a thorough understanding of molecular mechanisms underlying the pathophysiology of melanoma. This application will use state-of-the-art approaches to identify molecular mechanisms involved in this deadly disease, thus providing novel targets for future prevention or therapeutic strategies for melanoma.