Elucidating the Regulation of Delayed-Early Gene Targets of Sustained MAP Kinase Pathway Activation

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PROJECT ABSTRACT RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer including melanoma. BRAFV600E is the most common mutation in cutaneous melanoma. This constitutively activating mutation induces sustained signaling of the Mitogen-Activated Protein Kinase (MAPK) pathway through MEKERK to regulate key cancer cell hallmarks such as progress through the cell division cycle, reduced programmed cell death and enhanced cell motility. Amongst the list of RAF-regulated genes are those encoding integrins, alpha-beta heterodimeric transmembrane proteins that regulate cell adhesion to extracellular matrix. Altered integrin expression has been linked to the acquisition of more aggressive behavior by melanoma, lung and breast cancer cells leading to diminished survival of cancer patients. This study aims to elucidate the regulation of integrin beta3 (ITGB3) and determine the biological role it has in the progression of melanoma. We have previously documented the ability of the RAF->MEK->ERK MAP kinase pathway to induce the expression of ITGB3 in several different cell types. RAF-mediated induction of ITGB3 mRNA requires sustained, high-level activation of ERK signaling mediated by oncogene activation and is classified as “delayed- early”, in that it is sensitive to the protein synthesis inhibitor cycloheximide. However, to date, the regulatory mechanisms that allow for induced ITGB3 downstream of sustained, high-level activation of RAF->MEK->ERK signaling remain obscure. To date, we have identified a number of genes using RNA-sequencing, including those expressing additional cell surface proteins, that display similar regulatory characteristics as ITGB3. We aim to relate altered expression of genes to RAF-induced changes in chromatin structure to determine if there is an underlying regulatory logic to the observed effects of activated RAF on delayed-early genes by ATAC-seq. Given the large numbers of human cancers with elevated RAF->MEK->ERK signaling, the mechanisms identified to regulate these genes by this strategy may have potential for use as either biomarkers of metastasis potential or as novel therapeutic targets. Due to the correlative evidence between increased ITGB3 expression and metastatic phenotypes, it is important to find the link between the two. With the use of congenic mouse melanoma cell lines that can be transplanted into C57BL/6 mouse hosts, ITGB3 will be genetically ablated by CRISPR/Cas9 strategies to evaluate the necessity and sufficiency of this gene in the progression of this disease. Results accumulated from the study will lead to further findings that in turn will improve the health of melanoma patients, including expanding the average life span after diagnosis. The applicant, Kali Dale, will trained in a supportive environment for her to become an independent research scientist from formal and informal mentors to be proficient in transcription focused research.