NHGRI-led researchers suggest glutamate signaling pathway could represent new target for skin cancer therapeutics.

A whole-exome sequencing project has generated what researchers claim is the most comprehensive map of genetic alterations in melanoma assembled to date. The project, carried out by a National Human Genome Research Institute (NHGRI)-led team, found that the majority of melanoma tumors analyzed had alterations in glutamate signaling pathway genes, suggesting that this pathway could represent an important target for the development of therapeutics.

Most significantly, the study found that some 33% of melanoma samples had mutations in a newly identified glutamate receptor subunit gene, GRIN2A. In addition, 4% of tumor samples analyzed sported an identical mutation in the TRRAP gene. The results are published in Nature Genetics in a paper titled “Exome sequencing identifies GRIN2A as frequently mutated in melanoma.”

 Approximately 69,000 individuals were diagnosed with invasive melanoma in the U.S. in 2010, and there were about 8,700 deaths from the disease the same year, report senior author Yardena Samuels, Ph.D., at the Cancer Genetics Branch of the NHGRI’s intramural research division, and colleagues. Moreover, while whole-genome analysis of a single melanoma has been completed and other studies have linked the disease with mutations in genes such as BRAF, cKit, and ERBB4, no compre­hensive analysis of this tumor type has yet been performed.

To further mine the genetic basis of melanoma and home in on true disease-related genes, the researchers carried out whole-exome sequencing of 14 metastatic melamona tumor DNAs from untreated individuals and matched normal controls. The resequencing project was carried out at the NIH Intramural Sequencing Center.

The analysis involved a number of steps to eliminate germline mutations and try to ensure only potentially tumor-specific somatic mutations were included in their final count. One step involved eliminating inherited alterations already annotated in dbSNP and the 1,000 Genomes Project.

A number of techniques were then applied to identify true mutations from all the sequence alterations identified. The researchers claim these refinements removed about 18% of the genetic alterations and provided a 97.9% coverage rate, a 2.4% false-negative rate, and a sensitivity of 81%. In further analytical steps the researchers pinpointed which of the somatic mutations were likely to be true ‘driver’ mutations that played a role in melanoma neoplasia and which were functionally inert ‘passenger’ mutations.

Of 86 genetic changes that appeared to be somatically mutated at higher frequencies in melanomas, 16 genes, including BRAF, were characterized as melanoma drivers. Significantly, a gene known as GRIN2A, which is located on chromosome 16p13.2, was found to be mutated in 33% of the original tumor sample set and in 25% of a larger set of melanoma samples. GRIN2A encodes a glutamate (N-methyl-(D)-aspartic acid (NMDA)) receptor subunit, and there is some indication that this gene acts as a tumor suppressor, Dr. Samuels notes.

“The mechanism by which mutations in GRIN2A have a tumorigenic effect is unclear; however, their frequency strongly suggests that they play a major role in melanoma and are worthy of future investigation,” the authors write.

In addition to BRAF, the researchers identified nine more genes that harbored a recurrent, or hot-spot mutation. Screening hot-spot mutations that occurred in different tumors within the initial sample set against an additional 153 melanomas found that an identical mutation in the gene for transformation/transcription domain-associated protein (TRRAP) was present in six separate melanomas. “This was one of the most important discoveries in the study, since we never expected to identify novel not-spot mutations,” Dr. Samuels admits.

“The likelihood for the occurrence of six identical mutations is approximately 5 × 10−20, suggesting that the TRRAP hotspot mutation is important,” the authors write. They suggest the nature, pattern, and functional evaluation of the TRRAP recurrent mutation suggest that the gene does function as a true oncogene. “The positions of the mutations in TRRAP imply that they are likely to be oncogenic, as we observed no truncating mutations, and 100% of the alterations occurred in one location. Subsequent sequence analysis of all coding exons of TRRAP in 25 additional melanomas revealed no additional nonsynonymous alter­ations.”

Interestingly, knocking out TRRAP in cell lines had minimal effect on the survival of cells expressing the wild-type gene but substantially increased the apoptosis rates of melanoma lines carrying mutant TRRAP gene. This confirms the putative role of mutant TRRAP cancer cell survival. “Thus, mutant TRRAP is essential for melanoma cell survival, which is consistent with previous results showing that TRRAP knockout mice are embryonic lethal,” the researchers note.

The approach to genetic analysis used for the study also provided the researchers with an opportunity to look for new gene pathways involved in melanoma These results highlighted the glutamate signaling pathway as highly significant. Interestingly, in addition to GRIN2A, another highly mutated gene identified by the genetic analysis was PLCB4, which encodes a downstream protein involved in metabotropic glutamate receptor-related signal transduction.

“Some of our findings have potential therapeutic implications, as gluta­mate pathway modification has previously been shown to limit tumor growth,” the authors conclude. “Further investigation of this pathway in melanoma as well as development of such inhibitors is warranted.”

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