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Ric‐8A deletion as tumor suppressor of oncogenic G‐protein alpha subunit alleles (842.8)
Author(s) -
Patel Bharti,
Tall Gregory
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.842.8
Subject(s) - microbiology and biotechnology , cre recombinase , gene knockout , mutant , melanoma , protein subunit , knockout mouse , biology , cancer research , cytotoxic t cell , chemistry , gene , transgene , genetics , genetically modified mouse , in vitro
Constitutively‐active Q209L (QL) mutations in Gαq and Gα11 were found in 83% of human ocular melanomas. Ectopically expressed Gα11‐QL drove metastatic melanoma from xenografted melan‐a cells. No therapeutics target the melanoma‐driving mutant G proteins. Ric‐8A and Ric‐8B are molecular chaperones required for Gα subunit biosynthesis and maintenance of cellular Gαq/11 levels. Blocking Ric‐8A function may represent a viable means to attenuate the abundance of mutant, oncogenic G proteins. To test this, we created a Ric‐8A conditional knockout mouse. Homofloxed Ric‐8A mouse embryonic fibroblasts (MEFs) were isolated and transduced by lentivirus to express Cre‐recombinase and induce a Ric‐8A knockout (KO). Dramatic decreases in endogenous Gαi/o and Gαq/11 subunit levels were observed in Ric‐8A KO MEFs. Ric‐8A KO and control MEFs were examined for known cytotoxicity elicited by Gα11‐QL overexpression. Cell growth rates were measured and correlated to cytotoxic cell death measured by lactate dehydrogenase activity in culture media. Ric‐8A induced KO in MEFs reduced expressed Gα11‐QL levels and rescued cytotoxicity. To study the effect of Ric‐8A gene ablation on suppression of GNA11‐QL‐driven mouse melanoma, a Ric‐8A homofloxed mouse with melanocyte‐specific, tamoxifen‐inducible Cre‐recombinase was created. Inducible Ric‐8A KO melanocytes were immortalized and stably transduced with GNA11‐QL. Melanocytes will be xenografted in immunodeficient mice to induce Gα11‐QL driven melanoma. The efficacy of tamoxifen‐induced Ric‐8A deletion in injected melanocytes (or derived tumors) to inhibit the Gα11‐QL‐dependent melanoma will be examined. Tumor growth rates, metastases, and Kaplan‐Meier survival analyses will be presented. We anticipate that Ric‐8A KO‐dependent Gα abundance decrease will attenuate oncogenic Gα11‐QL driven melanoma and increase survival. Our work will establish the tenability of Ric‐8A as a potential drug target for disease caused by mutant G proteins. Grant Funding Source : Supported by NIH grant RGM088242A