Somatic mutation of GNAQ gene is rare in common solid cancers and leukemias

To the Editor

Guanine nucleotide-binding proteins are a family of proteins that couple cell surface receptors to intracellular signaling pathways. Receptor activation catalyzes the exchange of GTP for GDP bound to the inactive G protein alpha subunit. The G protein subunits are capable of regulating various cellular functions [1], [2]. G-alpha-q (GNAQ) is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of protein kinase C signaling. GNAQ contributes to heart development, platelet functions, and skin color formation [1–3].

Recently, Van Raamsdonk et al. [3] analyzed a broad spectrum of benign and malignant melanocytic neoplasia for the detection of GNAQ somatic mutations. They found a recurrent somatic point mutation of GNAQ (p.Q209L) in malignant blue nevus (50%), blue nevus (83%), Ota nevus (6%), melanoma on skin with chronic sun-induced damage (4%), and ocular malignant melanoma (46%). Functionally, the GNAQ p.Q209L mutation enhanced anchorage-independent cell growth in vitro and tumorigenicity in vivo[3]. The p.Q209L mutation activated protein kinase C signaling, which subsequently activated MAP kinase pathway [3]. Because MAP kinase signaling is constitutively activated not only in malignant melanomas but also in many human cancers [4], it could be hypothesized that the GNAQ p.Q209L mutation could be responsible to the MAP kinase signaling activation in other cancers besides malignant melanomas as well. To explore this issue, in the present study, we analyzed the GNAQ p.Q209L mutation in common solid cancers and leukemias.

For this, we analyzed 667 cancer tissues from Korean patients (breast carcinomas (n = 47), colon carcinomas (n = 79), lung carcinomas (n = 47), stomach carcinomas (n = 47), esophageal squamous cell carcinomas (n = 68), hepatocellular carcinomas (n = 76), hepatoblastomas (n = 56), prostate adenocarcinomas (n = 47), urothelial carcinomas (n = 28), ovarian carcinomas (n = 80), squamous cell carcinomas of uterine cervix (n = 17), skin squamous cell carcinomas (n = 6) and renal cell carcinomas (n = 16), and malignant mesotheliomas (n = 6) and acute leukemias (n = 47)) by a polymerase chain reaction (PCR)- single strand conformation polymorphism (SSCP) assay. The colon carcinomas originated from cecum (n = 2), ascending colon (n = 17), transverse colon (n = 4), descending colon (n = 4), sigmoid colon (n = 20) and rectum (n = 32). The gastric carcinomas consisted of 18 diffuse-type, 15 intestinal-type and 14 mixed-type gastric adenocarcinomas by Lauren's classification, and seven early and 40 advanced gastric carcinomas according to the depth of invasion. The breast carcinomas consisted of seven ductal carcinomas in situ and 40 invasive ductal carcinomas. The ovarian carcinomas consisted of 45 serous and 35 mucinous adenocarcinomas. The NSCLC samples consisted of 25 adenocarcinomas and 22 squamous cell carcinomas. We analyzed the primary tumors, but not the metastatic lesions. In solid tumors, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator, as described previously [5]. The content of the tumor cells in the microdissection was 85 – 97%. DNA extraction from the microdissected tissues was performed by a modified single-step DNA extraction method by proteinase K treatment [5]. Genomic DNA each from tumor cells and normal cells from the same patients were amplified by PCR with a primer pair covering the mutation (p.Q209L) sequence in exon 5 of human GNAQ gene. The primer sequences of GNAS were as follows (forward and reverse, respectively): 5′-attttccctaagtttgtaagtag-3′ and 5′-cagtgtatccattttcttctc-3′. Radioisotope ([³²P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. Other procedures of PCR and SSCP analysis were performed as described previously [5].

On the SSCP autoradiograms, all of the PCR products from the cancers were clearly seen. However, the SSCPs from them did not reveal any aberrantly migrating band compared to wild-type bands from the normal tissues, indicating there was no evidence of the somatic mutation of GNAQ in the cancers. To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure the specificity of the results, and found that the data were consistent. We analyzed the GNAQ mutation in 100 cancers (30 breast, 30 colon, 20 gastric and 20 leukemias) used in this study by direct sequencing as well as by SSCP, and no additional mutation was detected by the direct sequencing.

One of the main concerns in cancer genetics is as to whether any mutation found in a cancer is common in other cancer types. For example, EGFR mutation is specific to few cancer types [6], whereas K-RAS mutation is common to many cancer types [7]. As a possible mechanism of MAP kinase signaling activation in common human cancers, we analyzed GNAQ mutation in a variety of human cancers. However, we detected no GNAQ p.Q209L mutation in the cancers. Compared to the high incidences of the GNAQ mutation in the melanomas and nevi [7], the incidence in the cancers analyzed in this study was significantly low (Fisher's exact test, p < 0.05). Our data indicate that the MAP kinase signaling activation frequently observed in the cancer types may not be a result of the GNAQ mutation. The discovery of the GNAQ p.Q209L mutation as an oncogene offered an opportunity for developing therapeutic as well as diagnostic tools for human cancers. From our observation, however, such approaches may be restricted to melanocytic neoplasia.

Acknowledgements

This study was supported by a grant of the National Cancer Control R&D Program, Republic of Korea (0820080). The prostate cancer tissues were supplied from the Prostate Bank in Korea supported by Korea Science and Engineering Foundation.