Heterotrimeric G‐protein α Subunit Chaperone Ric‐8A as a Novel Target for Cancer Therapeutics

Constitutively‐active Q209L (QL) mutations in Gαq and Gα11 were found in 83% of human ocular melanomas. Ectopically expressed Gαq‐QL drove metastatic melanoma from xenografted melanocytes. Melanocyte specific expression of the GNAQ‐ QL oncogene in mouse model drove metastatic melanoma and recapitulated the characteristics of human uveal melanoma. There are no therapeutics that target cancer‐causing mutated G proteins. Ric‐8A and Ric‐8B are molecular chaperones required for Gα subunit biosynthesis and maintenance of cellular Gαq/11 levels. Targeting Ric‐8A function may represent a viable means to attenuate the abundance of mutant, oncogenic G proteins. To study the effect of Ric‐8A gene ablation on suppression of GNAQ ‐QL‐driven melanocyte transformation and mouse melanoma, a Ric‐8A homofloxed mouse with tamoxifen‐inducible Cre‐recombinase was created. Inducible Ric‐8A knockout (KO) mouse melanocytes were isolated, cultured and immortalized by serial passage. In these melanocytes, deletion of Ric‐8A moderately enhanced the growth rate and decreased the levels of Gαi, Gαq and Gα13 proteins. Stable transduction of Inducible Ric‐8A KO melanocytes with GNAQ ‐QL overcomes the requirement of growth factor 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA) for continuous proliferation in culture. Stable expression of GNAQ ‐QL in xenografted inducible Ric‐8A KO melanocytes was sufficient to drive melanoma tumor formation in immunodeficient mice. GNAQ ‐QL driven melanoma tumor cells were isolated, cultured and examined for effect of Ric‐8A KO on TPA independent growth and melanoma tumorigenesis. Deletion of Ric‐8A ameliorates the TPA independent growth of GNAQ ‐QL stable melanocytes and tumor explant cultures by decreasing the abundances of Gαq‐QL oncoprotein. Ric‐8A deletion substantially attenuated GNAQ ‐QL stable melanocytes and tumor explant cultures driven melanoma tumor progression. Forced expression of Ric‐8A transgene restores the defect in abundance of expressed Gαq‐QL protein and rescues the inhibition of Gαq‐QL driven tumor growth mediated by induced genetic ablation of Ric‐8A . Deletion of Ric‐8A in‐vivo in GNAQ ‐QL stable melanocytes driven tumors result in tumor regression and decrease in lung metastases. Our work demonstrated the tenability of Ric‐8A as a potential target for design of therapeutics that can reduce the levels of disease‐causing Gα subunits in G proteins driven diseases. Support or Funding Information NIH grant R01 GM088242 to G.G.T. Conditional Ric‐8A Flox alleleA . Ric‐8A knockout strategy. B. Genotyping PCR analysis of mice with Ric‐8A Flox and RosaCreER alleles. C. Schematic of RT‐PCR products obtained from Ric‐8A Flox and Ric‐8A KO allele transcripts. D. Characterization of Ric‐8A KO and Ric‐8A Flox allele transcripts by size of RT‐PCR products. E. DNA sequencing chromatogram of RT‐PCR product from Ric‐8A Flox and Ric‐8A KO allele transcript. G. Western blot analysis of control and Cre‐expressing Ric‐8A Flox/Flox MEFs.Ric‐8A deletion confers growth advantage to melanocytesA. Bright field images of untreated or 4‐hydroxy tamoxifen (4OHT)‐treated Ric‐8A Flox/Flox osaCreER +/− melanocytes grown in presence of CTX, or TPA or both. B. Growth rates of control, Cre or EGFP expressing Ric‐8A Flox/Flox Rosa‐CreER +/− melanocytes. B. Western blot analyses of Ric‐8A and Gα subunits in Cre‐lentivirus‐or 4OHT‐treated melanocyte cell lysates. α‐tubulin or GAPDH levels were measured as loading controls.Ric‐8A knockout suppresses the Gαq‐Q209L driven TPA‐independent growth of cultured melanocytesRic‐8A Flox/Flox RosaCreER +/− melanocytes stably transduced with GFP, GNAQ‐ WT or GNAQ ‐Q209L were treated with solvent or 4OHT to induce Ric‐8A Knockout (KO) and cultured in the absence or presence of TPA. A. Western blot analyses of Ric‐8A, Gαq/11 and Gαq‐Q209L in Ric‐8A Flox/Flox or Ric‐8A KO/KO melanocytes. B–C DAPI nuclear stained cells were counted using Image J and fold cell growth was quantified.Ric‐8A deletion inhibits Gαq‐Q209L driven tumor progressionA. Western blot analyses of Ric‐8A and Gαq levels in GFP and GNAQ ‐Q209L(QL) stable Ric‐8 Flox/Flox RosaCreER +/− melanocyte. B. Rate of tumor growth by Ric‐8A Flox or Ric‐8A KO Gαq‐QL transformed melanocytes. C–D. Gαq‐QL driven tumors weight. E. Western blot analyses of Ric‐8A and Gα levels in Gαq‐QL driven tumor explant cultures. F. Tumor explant cultures driven tumor progression. G. Average weight of tumor grown by tumor explant cultures.