Melanoma is a common form of skin cancer and, despite years of research, metastatic disease is often fatal1
. Although some targeted treatment has been effective2
, the results of these treatments are not long lasting, showing a need for new clinically relevant targets. GPCRs, which are activated by ligands, are integral in regulating various signaling pathways3,4
. The importance these molecules play in human diseases is evident by the fact that 50–60% of the US Food and Drug Administration–approved therapeutics target GPCRs5
. As GPCRs regulate pathways that affect cell growth, their genetic analysis in cancer is warranted.
To identify somatic mutations in GPCRs, we performed a GPCR-targeted mutational analysis in tumor DNA derived from 11 melanoma samples. For each sample, we performed DNA capture using molecular inversion probes of 7,059 regions corresponding to 2,400 GPCR exons encoding 734 GPCR genes6
. We then analyzed DNA samples enriched for GPCR exons by massively parallel sequencing using the Illumina GAII platform. We identified 755 potential non-synonymous mutations when comparing the molecular inversion probe results to the known HapMap sequence. To determine which alterations were somatic, we examined the genomic DNA derived from matched normal tissue. From these alterations, we confirmed 106 to be somatic in 94 different genes (Supplementary Table 1
). Eleven of these genes harbored at least two somatic mutations (Supplementary Table 2
). The coding sequence of these 11 genes was analyzed for somatic mutations in a total of 80 melanoma samples, as described previously7
. Supplementary Figure 1
depicts the stages described above.
From the ~3.4 Mb of sequencing information obtained, we identified 115 non-synonymous, somatic mutations in 42 of the 80 tumors (). The number of C>T mutations was significantly greater than the number of other substitutions, resulting in a high prevalence of C:G>T:A transitions (P
< 1 × 10−6
) (Supplementary Fig. 2
), consistent with melanoma mutation signatures8
. We found a total of seven nonsense mutations and two splice site alterations, potentially resulting in aberrant or truncated proteins for five of the genes. We identified recurring alterations in CHRM3, RXFP1, OR8B8 and OR1J2, which harbored p.Pro421Leu/p.Pro421Ser, p.Ser269Phe, p.Ser74Phe and p.Asp109Asn, respectively, in two individuals. Using SIFT (sorting intolerant from tolerant)9
, we determined a computational estimation of the effects of the mutations (Supplementary Table 3
). A description of these genes and the likely nature of their mutations is given in the Supplementary Note
Somatic mutations identified in recurrently mutated GPCRs
were the most frequently mutated genes in our screen. GRM3
had a 16.3% mutation rate, with 18 non-synonymous mutations in 13 of 80 tumors. GPR98
had a total of 42 non-synonymous mutations in 22 of 80 tumors (27.5%). As GRM3
belongs to the metabotropic family and was previously shown to be linked to tumorigenesis10
, it was evaluated genetically in an additional panel consisting of 57 melanoma specimens11
. In this screen, we discovered 11 non-synonymous alterations in nine tumors affecting 15.7% of the individuals analyzed and yielding a non-synonymous to synonymous ratio of 29:7, which is significantly higher than the non-synonymous to synonymous ratio of 2:1 predicted for nonselected passenger mutations (P
< 0.05). This investigation allowed the identification of a mutational hotspot in GRM3; we found p.Glu870Lys in four different individuals with melanoma (one individual from the original panel and three from the second panel) (Supplementary Table 4
and Supplementary Fig. 3
). The likelihood for the occurrence of four identical mutations is approximately 1.8 × 10−12
, suggesting that the GRM3
hotspot mutation is functionally important. Clinical information for all tumors harboring GPCR mutations is given in Supplementary Table 5
We focused on GRM3 (the group II metabotropic glutamate receptor-3 gene, or mGluR3), as two genetic observations suggested that the mutations in this gene may be functionally important for melanoma tumori-genesis: (i) GRM3 was one of the most highly mutated genes in the screen, and (ii) it contained a mini-hotspot (p.Glu870Lys).
We functionally characterized four somatic mutations (resulting in p.Gly561Glu, p.Ser610Leu, p.Glu767Lys and p.Glu870Lys) discovered in GRM3
based on sequence conservation and their location within particular functional domains. To examine the biological effects of GRM3
mutations, we established stable pooled clones expressing either a vector control or wild-type or mutant (p.Gly561Glu, p.Ser610Leu, p.Glu767Lys or p.Glu870Lys) GRM3. We selected the melanoma cell lines A375 or Mel-STR, as they express wild-type GRM3. We saw similar levels of expression of GRM3 protein in both the A375 and Mel-STR stable clone cell lines, except for in the p.Glu767Lys and p.Glu870Lys mutants, which had reduced protein expression (Supplementary Fig. 4a,b
To examine the effects of GRM3
mutations on cell growth, we investigated growth rate on plastic (). In the presence of media containing 10% serum, all clones grew similarly (Supplementary Fig. 5a,b
). However, if we reduced the serum concentration, wild-type clones grew at a lower rate than mutant clones, except for the clone expressing the p.Glu870Lys alteration ( and Supplementary Fig. 5c
). We also observed this difference in cell growth when we assessed the cells for anchorage independence, where cells expressing mutant GRM3 formed a significantly higher number of colonies compared to wild type or empty vector (; P
< 0.05 t
Figure 1 Effects of GRM3 alterations on cell growth and MEK phosphorylation. (a) Somatic alterations in GRM3 cause increased proliferation in reduced serum. We seeded A375 pooled GRM3 clones expressing wild-type, p.Gly561Glu, p.Ser610Leu, p.Glu767Lys, p.Glu870Lys (more ...)
The C-terminal region of GPCR proteins is important for binding signaling molecules involved in pathways such as the RAS-RAF-MEK pathway3,4
. Mutations in or near this region of GRM3 may therefore affect signal transduction leading to increased cell proliferation, thus providing the biochemical basis for the growth differences described above. As group 2 metabotropic glutamate receptors can be activated by agonists such as DCG-IV12–14
, we tested the biochemical effects of GRM3 alterations in the presence and absence of DCG–IV. We saw striking differences in MEK1/2 phosphorylation. When we stimulated Mel-STR clones with DCG-IV, there was a sevenfold to tenfold increased phosphorylation of MEK1/2 compared to wild-type-GRM3–expressing cells (). We observed similar results in the A375 clones (). Notably, Mel-STR cells harbor mutant RAS and A375 cells harbor mutant BRAF, both of which are known to activate MEK15
, and so mutant GRM3 thus allowed further activation of the MEK pathway.
Previous studies reported that activation of the MEK pathway increases cell migration16,17
. As GRM3 variants activate the MEK pathway, we determined whether these variants also affect migration. To test this, we seeded A375 or Mel-STR pooled clones in serum-free medium in the presence or absence of DCG-IV and looked for migration (). Mutant GRM3 expression increased migration compared to wild-type GRM3 or an empty vector containing cells in the absence of agonist (; P
< 0.05 t
-test). Upon stimulation with DCG-IV, vector and wild-type–expressing cells migrated similarly to mutant-GRM3–expressing cells in the absence of stimuli (). Our results suggest that expression of mutant GRM3 increases migration in the absence of growth factors or receptor agonists.
GRM3 mutations increase migration in vitro and in vivo
To determine whether these phenotypes occur in vivo
, A375 pooled clones expressing vector, wild-type or mutant GRM3 were administered to NOD/SCID mice by tail vein injection. Nine weeks after injection, macroscopic assessment of lung colonization showed that the groups injected with cells expressing vector, wild-type or mutant p.Gly561Glu had two to three mice with gross lung tumors. In contrast, most of the mice injected with cells expressing the p.Ser610Leu, p.Glu767Lys or p.Glu870Lys GRM3 alterations had pulmonary macrometastases (). Microscopic examination allowed for the detection of micrometastases, with no significant difference being seen in their number or size (Supplementary Fig. 6a,b
). Thus, expression of mutant forms of GRM3 in melanoma cells affects growth in vivo
once the lung is colonized.
To assess if melanoma cells with endogenous GRM3
mutations are dependent on GRM3 signaling for proliferation and migration, we used short hairpin RNA (shRNA) to stably knock down GRM3 protein levels in melanoma cells that harbor either wild-type GRM3
or endogenous mutant GRM3
. We confirmed specific targeting of GRM3
by transient transfection in HEK293T cells and immunoblotting, as well as by quantitative RT-PCR analysis (). The shRNA had little effect on cells harboring wild-type GRM3 but significantly reduced the growth of cells harboring mutant forms of GRM3 ( and Supplementary Fig. 7a,b
< 0.005, P
< 0.008 and P
< 0.004 for , respectively). Depletion of GRM3 by shRNA in mutant-GRM3–expressing cells reduced their ability to migrate significantly compared to wild-type GRM3 cells targeted with GRM3 shRNA ( and Supplementary Fig. 7c,d
). To determine whether similar results occur in vivo
, melanoma cells harboring either wild-type or mutant GRM3 targeted with GRM3 or control shRNA were administered into Nu/Nu mice by subcutaneous injection. Nineteen days after injection, depletion of GRM3 had little effect on in vivo
growth of cells harboring wild-type GRM3. In contrast, GRM3 knockdown significantly reduced the tumor growth induced by cells harboring mutant GRM3 (; P
< 0.0005 and P
< 0.02 for , respectively). As the shRNA-mediated phenotypes could be caused by specific or nonspecific effects, we engineered an exogenous, non-targetable wild-type GRM3 construct that harbors silent mutations in the region of GRM3 targeted by shRNA #3 to rescue the effects of knockdown of endogenous GRM3. Melanoma cells harboring the p.Glu573Lys alterations stably expressing either control or GRM3 shRNA #3 construct were transduced either with the lentiviral non-targetable GRM3 construct or with the empty vector as a control. To show that the non-targetable GRM3 is not knocked down in the presence of GRM3 shRNA #3, we transiently transfected HEK293T cells and immunoblotted for FLAG-GRM3 and GAPDH as a loading control (Supplementary Fig. 8a
). Importantly, non-targetable GRM3-reconstituted cells showed significantly more migration than cells infected with the control vector (Supplementary Fig. 8b
). These results suggest that certain GRM3 mutations are essential for cellular proliferation as well as for cell migration in melanoma cells.
Figure 3 Expression of mutant GRM3 provides cell proliferation and survival signals in melanoma. (a) Our protein blot analysis shows that expressing GRM3 shRNA decreases endogenous GRM3 levels. We analyzed HEK 293T cells co-transfected with shRNA targeting GRM3 (more ...)
To evaluate whether inhibition of GRM3 signaling would result in a similar phenotype to depletion of endogenous GRM3, we exposed melanoma cells harboring either wild-type GRM3
or mutant GRM3
to AZD-6244 (Selumetinib, ARRY-142886), which is a selective, non-ATP–competitive small molecule inhibitor of MEK1/2 that is being tested in phase 2 clinical trials (see URLs
). Exposure of melanoma cells to AZD-6244 inhibited MEK in mutant as well as in wild-type cells (). However, the relative degree of inhibition was concentration dependent, resulting in greater inhibition of the mutant cells compared to wild-type cells. This suggested that mutant cells would be more sensitive to growth inhibition by AZD-6244 than wild-type cells. Indeed, exposure of most melanoma cells expressing mutant forms of GRM3 were 2–200-fold more sensitive to AZD-6244 compared to wild-type GRM3 cells ( and ). The GRM3 mutations may be dependent on MEK signaling via BRAF, as evidenced by the genotypes of the investigated cells (Supplementary Table 6
). To further investigate the relevance of mutant GRM3 to AZD-6244 sensitivity, we tested whether the sensitivity to MEK inhibition can be altered by modulating GRM3. To do this, we established a stable cell line overexpressing wild-type GRM3 in a mutant GRM3 background (Supplementary Fig. 9a
). Exposure of these stable pools to AZD-6244 resulted in reduced cell proliferation, with a fourfold increased resistance in cells overexpressing wild-type GRM3 compared to control infected cells (Supplementary Fig. 9b,c
). These results further suggest that AZD-6244 preferentially inhibits the signaling of cells expressing mutant GRM3.
Figure 4 Melanoma cell lines expressing GRM3 mutants show increased sensitivity to inhibition of MEK by AZD-6244. (a) Immunoblot analysis of representative melanoma cell lines harboring either wild-type or mutant GRM3. We treated the cells with the indicated concentration (more ...)
The decreased growth of the mutant cells in the presence of AZD-6244 could have arisen either through alteration of the cell cycle or through increased cell death. To distinguish between these two possibilities, we performed FACS analysis and found that cells harboring mutant GRM3 showed a substantial increase in the levels of subG1-population cells (apoptotic cells) compared to wild-type cells (). We observed similar results when we performed the experiment on a larger panel of mutant GRM3 cell lines (; P < 0.05 t-test). We confirmed the apoptotic events by protein blot analysis of the cell lysates analyzed by FACS, which showed increased levels of cleaved PARP in mutant cells compared to wild-type cells (). Thus, melanoma cell lines harboring mutant GRM3 are markedly more sensitive to MEK1/2 inhibition by AZD-6244, leading to increased cell death.
In this study, we used a systematic approach combining exon capture and massively parallel sequencing to genetically characterize the GPCR gene family, allowing for the identification of GRM3 somatic mutations in melanoma. The high frequency of mutations found in GRM3, the finding of a mutational hotspot, as well as the functional assays, suggest GRM3 to be a driver in melanoma.
Taken together, our data highlight a model for melanoma pathogenesis in which activation of MEK by GRM3 alterations promotes the proliferation and migration of melanoma cells. Although further investigation into the mechanism of GRM3 activation of the MEK pathway is required, our study suggests that the presence of GRM3
mutations is expected to indicate subpopulations of individuals whose tumors are dependent on MEK signaling. The prior failure of MEK inhibitors to obtain significant tumor responses in many BRAF
may have resulted at least in part from the absence of additional mutations that activate the MEK pathway, such as those in GRM3
. Therefore, targeting MEK signaling in the presence of GRM3
mutations may have a role in the treatment of melanoma.