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Ric-8A gene deletion or phorbol ester suppresses tumorigenesis in a mouse model of GNAQ(Q209L)-driven melanoma.

ABSTRACT: The heterotrimeric G protein ? subunit oncogenes GNAQ or GNA11 carry Q209X or R183X activating mutations and are present with ~90% frequency in human uveal melanomas. Forced expression of GNAQ/11(Q209L) in melanocytes is sufficient to drive metastatic melanoma in immune-compromised mice. No known drugs directly target these oncogenic G proteins. Ric-8A is the molecular chaperone that selectively folds G?q/i/13 subunits. Targeting Ric-8A serves as a rational, yet unexplored approach to reduce the functional abundance of oncogenic G?q/11 in order to blunt cancer signaling. Here, using mouse melanocyte cell graft tumorigenesis models, we determined that Ric-8A genetic ablation attenuated the abundance and melanoma-driving potential of G?q-Q209L. A new conditional Ric-8A(Flox/Flox); Rosa-CreER(+/)(-) mouse strain was derived and used as a tissue source to culture an immortalized, tamoxifen-inducible Ric-8A knockout melanocyte cell line that required 12-O-tetradecanoylphorbol-13-acetate (TPA, phorbol ester) for growth. The cell line failed to grow tumors when grafted into immune-compromised mice regardless of Ric-8A expression. Stable expression of human GNAQ(Q209L), but not GNAQ(WT) in the cell line promoted TPA-independent cell proliferation, and upon cell grafting in mice, the initiation and robust growth of darkly-pigmented melanoma tumors. Deletion of Ric-8A in GNAQ(Q209L) cells restored TPA-dependent growth, reduced G?q-Q209L below detectable levels and completely mitigated tumorigenesis from primary or secondary cell line grafts. Interestingly, TPA treatment of cultured GNAQ(Q209L) cells or host animals grafted with GNAQ(Q209L) cells also sharply reduced G?q-Q209L abundance and tumorigenic capacity. Finally, tumorigenesis initiated from GNAQ(Q209L) cell grafts, followed by host mouse systemic tamoxifen treatment to delete Ric-8A in the grafted cells completely abrogated GNAQ(Q209L)-driven tumor progression unless a stable human RIC-8A transgene was used to rescue the floxed Ric-8A alleles. Our work defines two new rational targets that may be developed as potential uveal melanoma therapies through reduction of G?q/11-Q209L oncoprotein abundance: (1) Ric-8A inhibition and (2) phorbol ester treatment.

SUBMITTER: Patel BR

PROVIDER: S-EPMC4945744 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Ric-8A gene deletion or phorbol ester suppresses tumorigenesis in a mouse model of GNAQ(Q209L)-driven melanoma.

Patel B R BR Tall G G GG

Oncogenesis 20160627 6

The heterotrimeric G protein α subunit oncogenes GNAQ or GNA11 carry Q209X or R183X activating mutations and are present with ~90% frequency in human uveal melanomas. Forced expression of GNAQ/11(Q209L) in melanocytes is sufficient to drive metastatic melanoma in immune-compromised mice. No known drugs directly target these oncogenic G proteins. Ric-8A is the molecular chaperone that selectively folds Gαq/i/13 subunits. Targeting Ric-8A serves as a rational, yet unexplored approach to reduce the ...[more]

PMID: 27348266

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Project description:Purpose: Uveal melanoma (UM) is the most frequent metastatic ocular tumor in adults. Therapeutic intervention remains ineffective since none of the novel procedures used to treat this disease increased survival rates. To deal with this limitation, additional studies are required to clarify its pathogenesis. The current study focused on describing how epigenetic modulation by miR-142-3p affects changes in some cellular functions underlying UM pathogenesis. Methods and results: Microarray analysis identified 374 miRNAs which were differentially expressed between UM cells and uveal melanocytes. miR-142-3p was one of the 10 most downregulated miRNAs. Quantitative RT-PCR analysis confirmed that miR-142-3p expression levels were significantly decreased in both UM cell lines and clinical specimens. The results of the MTS, clone formation, scratch wound, transwell assays, and in vivo biofluorescence imaging showed that miR-142-3p overexpression significantly inhibited cell proliferation, migration, and invasiveness. Nevertheless, miR-142-3p did not affect cell apoptotic activity or sensitivity to doxorubicin. Cell cycle and EdU analysis showed that miR-142-3p overexpression induced G1/G2 cell cycle arrest and reduced DNA synthesis in UM cells. Microarray analysis showed that miR-142-3p mainly regulates the TGFβ signaling pathway, and those in which MAPK and PI3K-Akt are constituents. Functional interactions between miR-142-3p and CDC25C, TGFβR1, GNAQ, WASL, and RAC1 target genes were confirmed based on the results of the luciferase reporter assay and Western blot analysis. CDC25C or RAC1 downregulation is in agreement with cell cycle arrest and DNA synthesis disorder induction, while downregulation of TGFβR1, GNAQ, WASL, or RAC1 accounts for declines in cell migration. Conclusion: miR-143-3p is a potential therapeutic target to treat UM since overriding its declines in expression that occur in this disease reversed the pathogenesis of this disease. Such insight reveals novel biomarker for decreasing UM vitality and for improved tracking of tumor progression.

Dataset Information

Ric-8A gene deletion or phorbol ester suppresses tumorigenesis in a mouse model of GNAQ(Q209L)-driven melanoma.

Publications

Ric-8A gene deletion or phorbol ester suppresses tumorigenesis in a mouse model of GNAQ(Q209L)-driven melanoma.

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