Date of Award

5-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Carlos A. Molina

Committee Member

Alexis Khursigara

Committee Member

Reginald Halaby

Abstract

The Inducible cAMP Early Repressor (ICER) comprises a group of isoforms that function as transcription factors within the cyclic AMP (cAMP) signaling pathway. These isoforms are encoded by the cAMP Response Element Modulator (CREM) gene. The cAMP signaling pathway and its associated transcriptional regulators have been implicated in the development of resistance to BRAF/MEK-targeted therapies in melanoma. Recent findings from our laboratory demonstrated that transgenic ICER expression in a zebrafish melanoma model promotes the formation of aggressive melanomas. Cell lines were established from tumors expressing ICER, as well as from control tumors expressing Green Fluorescent Protein (GFP). To begin characterizing the malignancy of these cell lines, we investigated whether ICER expression affects promoter activity of PRKACAA, a gene encoding the catalytic subunit of protein kinase A. We conducted a series of expression assays comparing two melanoma cell lines: 447A, which expresses NHA-wtICER and displays a more aggressive phenotype, and 446A, which expresses EGFP and is less aggressive. Western blot analyses were used to quantify HA-tagged ICER expression in total, nuclear, and cytosolic fractions of 447A cells. Immunocytochemistry further confirmed that ICER predominantly localizes to the nucleus. To assess transcriptional repression, we performed a dual-luciferase reporter assay targeting the PRKACAA promoter, comparing activity in both cell lines with and without forskolin (FSK) treatment, a known activator of the cAMP pathway. Our results showed that PRKACAA promoter activity was significantly lower in 447A cells compared to 446A, and that chronic FSK treatment further reduced promoter activity. To further explore these observations, we examined CREB phosphorylation levels and analyzed cell morphology in both lines before and after chronic FSK treatment. While morphological differences were evident between the two cell lines, no significant differences in CREB phosphorylation were detected. Electrophoretic mobility shift assays (EMSA) revealed distinct differences in protein-DNA binding at putative cAMP response elements (CREs) within the PRKACAA promoter, suggesting altered transcriptional regulation linked to ICER expression.

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