Elucidating the role of circPMS1 in melanoma metastasis.

Project Summary/Abstract Melanoma is a highly aggressive cancer from melanocytes, accounting for 1% of skin cancer cases yet causing most skin cancer deaths. The 5-year survival for metastatic melanoma patients is below 30%. The mechanisms driving melanoma metastasis are poorly understood, necessitating thorough investigations to enhance patient survival rates. While targeted therapies like BRAF and MEK inhibitors can lead to initial regression, relapses occur quickly. Immunotherapy offers lower response rates (30-50%) and substantial toxicity, highlighting the need for novel therapeutic targets. The human genome consists of 98% non-coding regions, many of which produce non-coding RNAs essential for cancer development, including melanoma. Circular RNAs (circRNAs), known for their closed structure and degradation resistance, are attracting attention for their potential roles in cancer. However, their specific functions and mechanisms in melanoma metastasis remain largely unexplored. By analyzing RNA-sequencing data, I identified circPMS1 as the most upregulated circRNA in melanoma cells compared to melanocytes. Using previously developed genetic tools for stable circRNA overexpression, I assessed the oncogenic potential of circPMS1. Overexpression of circPMS1 enhanced both melanoma and melanocyte migration and invasion in vitro. Conversely, CasRX-mediated silencing of circPMS1 resulted in decreased migration and invasion. Furthermore, circPMS1-overexpressing cells formed more metastases in lungs and liver when injected into NSG mice, while silencing circPMS1 significantly reduced metastasis formation. Despite the presence of a predicted ORF and IRES, I demonstrated that circPMS1 does not translate into a protein. This leads to my hypothesis that circPMS1 interacts with proteins that influence the metastatic potential of melanoma cells. To investigate this, I pulled down tagged circPMS1 and identified six potential interacting proteins via mass spectrometry. Four of these proteins (ABLIM, CAPZA2, PDLIM7, LMO7) contain LIM domains that may bind nucleic acids and are involved in cytoskeleton rearrangement and metastasis in other cancers. However, the effects of RNA binding on these LIM domain proteins’ biology remain unstudied. In the light of these findings, I hypothesized that circPMS1 has pro-metastatic functions in melanoma by altering the expression, activity, and/or localization of its protein binding partners. In the F99 phase, I will characterize these partners and elucidate the molecular mechanism underlying circPMS1's metastatic phenotype using in vitro assays and genetically engineered melanoma mouse models. In the K00 phase, I will examine the role of circPMS1 in melanoma brain metastasis through in vitro and transplant models, developing therapeutic strategies to target circPMS1 and inhibit its pro-metastatic functions. Additionally, I will identify and characterize circRNAs associated with melanoma brain metastasis through RNA-seq, CRISPR screenings, in vitro assays, and transplant models. The F99/K00 award will provide essential training to become an independent investigator, enabling me to identify circRNAs as potential therapeutic targets for melanoma metastasis.