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.
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.
|Knubel, Kristina H; Pernu, Ben M; Sufit, Alexandra et al. (2014) MerTK inhibition is a novel therapeutic approach for glioblastoma multiforme. Oncotarget 5:1338-51|