Combinatorial microRNA signaling in melanoma

描述（由申请人提供）：B-RafV600E突变存在于超过60%的黑色素瘤中，并导致B-Raf/MKK/ERK MAPK信号通路的组成性激活和不受控制的细胞增殖和致癌转化。在临床上，B-Raf抑制在黑色素瘤患者中产生不同的结果，大多数患者只经历了肿瘤生长的暂时减少。有必要进一步研究B-Raf/MKK/ERK的应答，以确定治疗黑色素瘤的更有效的药物靶点。在此之前，我们实验室发现了一组在黑色素瘤中不受调控、直接受B-Raf/MKK/ERK信号通路调控的microRNAs，并建立了单个B-RafV600E microRNAs的重要靶点和功能。然而，这些microrna的调节机制以及它们如何相互作用以控制重要的致癌表型尚未确定。我的建议涉及解决这些问题的三个关键目标。首先，我将确定致癌B-Raf信号协调调节许多microrna的机制。在存在和不存在MKK/Raf抑制剂的情况下，c-Myc的抑制、沉默和过表达将通过使用qRT-PCR测量microRNA的表达，以确定c-Myc对于b - rafv600e依赖性microRNA的表达是否必要和/或充分。其次，我将确定一种不寻常行为背后的机制，在这种行为中，microrna在组合表达时相互干扰。这一目标将通过Argonaute免疫沉淀，随后进行microRNA定量，以及测量microRNA特异性荧光素酶活性报告和3'-UTR荧光素酶报告的转录本来解决。第三，我将测试这些相互作用促进致癌表型的程度，并有助于对B-Raf抑制剂的耐药性。b - raf调控的microRNAs组合将在人类黑色素瘤细胞中过度表达和抑制，以确定它们如何相互作用以调节致癌转录因子LEF1的mRNA和蛋白表达。这个项目将代表我在科罗拉多大学博尔德分校Natalie Ahn博士指导下攻读生物化学博士学位的论文研究。此外，我将完成高级生物化学和细胞信号高级主题的正式课程，并进一步提高我作为临床医生的技能，与科罗拉多州儿童医院的儿科肿瘤学家一起工作。在完成博士学位后，我将回到科罗拉多大学医学院完成我的医学课程和临床实习。

DESCRIPTION (provided by applicant): The B-RafV600E mutation is present in over 60% of melanomas and leads to constitutive activation of the B-Raf/MKK/ERK MAPK signaling pathway and uncontrolled cell proliferation and oncogenic transformation. Clinically, B-Raf inhibition produces varied results among melanoma patients and most patients only experience a temporary decrease in tumor growth. Further investigation of responses to B-Raf/MKK/ERK is necessary to identify more effective drug targets for the treatment of melanoma. Previously, our lab identified a set of microRNAs deregulated in melanoma and directly regulated by the B-Raf/MKK/ERK signaling pathway, and established important targets and functions for individual B-RafV600E microRNAs. However, the mechanism by which these microRNAs are regulated and how they interact combinatorially to control important oncogenic phenotypes has yet to be determined. My proposal addresses three critical goals to address these problems. First, I will identify the mechanisms by which oncogenic B-Raf signaling coordinately regulates many microRNAs. c-Myc inhibition, silencing and overexpression in the presence and absence of MKK/Raf inhibitors will be followed by measurements of microRNA expression using qRT-PCR, in order to determine if c-myc is necessary and/or sufficient for B-RafV600E-dependent microRNA expression. Second, I will determine the mechanisms underlying a unusual behavior, in which microRNAs interfere with each other when expressed combinatorially. This aim will be addressed using Argonaute immunoprecipitation followed by microRNA quantification, and measurement of transcripts from microRNA-specific luciferase activity reporters and 3'-UTR luciferase reporters. Third, I will test the degree to which these interactions promote the oncogenic phenotype and contribute to drug resistance to B-Raf inhibitors. Combinations of B-Raf-regulated microRNAs will be overexpressed and inhibited in human melanoma cells to determine how they interact to regulate mRNA and protein expression of the oncogenic transcription factor LEF1. This project will represent my thesis research for my Ph.D. degree in Biochemistry under Dr. Natalie Ahn at the University of Colorado, Boulder. In addition, I will complete formal coursework in Advanced Biochemistry and Advanced Topics in Cell Signaling, and also further my skills as a clinician working with a pediatric oncologist at Children's Hospita Colorado. Following the completion of my Ph.D. degree, I will return to the University Of Colorado School Of Medicine to complete my medical coursework and clinical rotations. PUBLIC HEALTH RELEVANCE: Metastatic melanoma is the most lethal form of skin cancer and one of the most difficult types of cancer to treat, with poor prognosis in most patients. Learning more about how mutations in the protein kinase, B-Raf, deregulates pigment cell proliferation and promote cancer progression will yield new insight into how metastatic melanoma develops in patients. This will lead to the identification of better drug targets for therapies to improve the treatment of patients with this disease.