The two genes with the highest somatic mutation rate were GNG10 and GNAZ (Suppl. Fig. 1
); these genes were each found to have non-synonymous somatic mutations in 4/80 (5%) tumors. The four novel mutations in GNAZ were all found to be in the G protein alpha subunit domain; they affect amino acids residues that are highly conserved across species (Suppl. Fig. 2
). The alpha subunit is an important component of G protein interaction with receptors and effectors, and contains the guanine nucleotide binding site and regulates the kinetics of signaling through an intrinsic GTPase activity. Therefore, mutations in the G protein alpha subunit domain in GNAZ may affect critical functions of this gene.
GNAZ is distinct from other G-alpha genes; its relatively low sequence homology (60%) with the other members of the Gi
protein subfamily is reflected in its unique biochemical properties within the subfamily.9
G-alpha z is subjected to a variety of covalent modifications, and hydrolyzes GTP with slower kinetics than the other G-alpha subunits.10
Although the relationship between G-alpha z and the MAPK signaling pathway is not yet fully understood, there is evidence that G-alpha z might contribute to the regulation of specific subfamilies of MAPKs, including extracellular signal-regulated kinases and Jun N-terminal kinases.11,12
Previous biochemical studies on GNAZ elucidating these relationships have not been conducted in cancer cells; however, as these signaling pathways are known to be important in cancer progression, future studies examining the impact of mutations in GNAZ in tumor cells are warranted.
Of the four somatic mutations in GNG10, only two fall into the G protein gamma subunit-like domain. These two missense mutations found in GNG10 do not display high levels of evolutionary conservation; however, both p.P8L and p.E52A result in a reverse in charge of the amino acid. The change from hydrophilic to hydrophobic may have particular functional importance for p.E52A as the G protein gamma subunit is known to have hydrophobic interactions with the G protein beta subunit at p.D51 and p.L53.13
We detected one NS somatic mutation in GNA12. Several previous studies have begun to elucidate the importance and functional roles of GNA12 in cancer development and progression. Members of the G12 family, which includes G-alpha 12 and 13, were found to promote the growth and oncogenic transformation of murine fibroblasts.14,15
Kelley et al. have shown increased expression of G-alpha 12 in breast cancer, and that signaling through the G12 family of G proteins promotes breast cancer cell invasion, and that inhibition of G12 signaling reduces breast cancer metastases in vivo.16
A follow-up study by the same group showed that G12 family proteins were also regulators of prostate cancer invasion, and blocking the G12 signaling pathways reduced cell invasion in vitro.17
GNA12 has also previously been shown to be differentially expressed between oral squamous cell carcinoma and normal oral mucosa, suggesting that it may play a role in oral squamous cell carcinoma carcinogenesis.18
We identified two novel changes in GNB3. GNB3 and its pathways have also been shown to be functionally important in cancer progression. Previous studies have shown that a C825T polymorphism results in a splice variant with a deletion of 41 amino acids and increased G protein activation. This polymorphism has been associated with oncogenesis and increased metastases in a variety of cancers, including breast, colorectal, head and neck squamous cell, and thyroid cancers.19–23
The results of these studies, combined with our data showing a mutation in malignant melanoma in GNB3, suggest that further examination of pathway is warranted.
GNA11 has previously been sequenced in melanoma by two different groups. One study reported a mutation rate of 29% (2/7) in malignant melanoma (www.sanger.ac.uk/genetics/CGP/Census
); the second study sequenced only exon 5 of GNA11, but found no mutations in the 24 melanomas examined.7
Although we only found one somatic mutation in GNA11, this gene is of great interest phenotypically as it has previously been shown to have reduced expression in breast cancer.24
mRNA expression was found to be decreased in 62.5% (10/16) of human breast cancers by RT-PCR, and the immunoreactivity of the G-alpha 11 protein reduced in 14 of 16 cancers.24
This study by Asada et al. suggests that reduced G-alpha 11 levels are advantageous for the growth of breast cancer cells; functional work on GNA11 could help elucidate the mechanisms behind this phenotype.
GNA14 and GNA15 are both members of the Gq
class of G-alpha proteins, along with GNA11, GNA16 and GNAQ. Although few functional studies have been conducted on these two genes, it is of note that we found mutations in 3 of the 5 members of this class: GNA11, GNA14 and GNA15. This class of G proteins is largely involved in inositol lipid signaling through the activation of beta-isoforms of phospholipase C.25
Receptors coupled to the Gq
class of G-alpha proteins mediate a wide range of cellular responses, including cell growth and proliferation, glucose secretion, and leukocyte activation.25
The unique distribution patterns of the members of this class of proteins suggests unique signaling functions, particularly for G-alpha 14 and 15, which have a more limited distribution pattern than the ubiquitously expressed G-alpha 11.25
Combined, this data suggests that each of these genes is worth studying individually to determine its role in melanoma development and progression.
Interestingly, our results showed that there were no GNAQ mutations in any of the tumors. This result is consistent with previously published studies showing that GNAQ has a high mutation rate in blue naevi and uveal melanoma, but not other types of melanoma.6,7
In melanoma subtypes with no H-, N-, K-RAS or BRAF mutations, GNAQ has been shown to act as an oncogene. The lack of mutations in GNAQ found in this study supports the conclusion that GNAQ is a critical cancer gene in blue naevi and uveal melanoma, but not metastatic melanoma.
In conclusion, we have completed the first systematic genetic analysis of the heterotrimeric G protein gene family in melanoma. This lead to the identification of 18 non-synonymous somatic mutations in 7 different genes in melanoma; 5 of which have not previously been reported to be mutated in cancer. The NS:S mutation ratio, which was significantly higher than the expected non selected passenger mutation ratio, suggest that mutated G proteins could play a functional role in melanoma progression.