An Investigation into GRIN2A Mutations in Human Melanoma

Abstract

Previous exome sequencing studies have reported that the GRIN2A gene is frequently mutated in melanoma. GRIN2A encodes the regulatory GluN2A subunit of the N-methyl-D-aspartate receptor (NMDAR), a glutamate gated calcium ion channel. This thesis investigated the prevalence and significance of GRIN2A mutations in melanoma cell lines developed at the Auckland Cancer Society Research Centre. It also investigated other aspects of NMDAR activity in melanoma proliferation and invasion. Sanger sequencing revealed that amongst 20 cell lines, 20% carried five non-synonymous mutations in GRIN2A. Patients from whom GRIN2A-mutant cell lines were developed showed faster disease spread to lymph nodes and distant organs and shorter overall survival. All nonsynonumous GRIN2A substitutions were computationally predicted to disrupt GluN2A function. The G762E mutation, located in the hinge region of the glutamate-binding domain was modelled into the crystal structure of GluN2A, which predicted that the G762E mutation could alter NMDAR subunit interactions. Transcripts of genes encoding GluN1, the structural subunit of the NMDAR and five of six possible regulatory subunits were present in all melanoma cell lines, with some heterogeneity in expression. Western blotting detected the GluN1 protein in all melanoma cell lines; in contrast, GluN2A expression was detected in only one cell line. NMDAR functionality as a calcium ion channel was suggested from studies that monitored cytosolic calcium fluxes in melanoma cells in response to NMDAR agonists. NMDAR inhibitors exerted antiproliferative and anti-invasive effects on melanoma cells. However, no association was found between the presence of GRIN2A mutations and melanoma cell proliferation or invasion. Lastly, melanoma cells were found to release glutamate when cultured in medium containing less glutamate, and cells carrying GRIN2A mutations expressed the transcriptional repressor Slug. We present a model in which glutamate is required for melanoma progression and is secreted by the cells themselves. We conclude that cells escape glutamate-mediated cytotoxicity by down regulating NMDAR expression or accumulating mutations in GRIN2A, indicating a tumour suppressor role of this subunit. Under low glutamate concentrations encountered in vivo, GRIN2A mutations may confer an oncogenic effect that facilitates melanoma cell survival, proliferation and invasion. Thus, GRIN2A can act as an oncogene or a tumour suppressor, in a context dependent manner dictated by extracellular glutamate levels.