Oncogene database

2768

GNA12

guanine nucleotide binding protein (G protein) alpha 12;GNA12;guanine nucleotide binding protein (G protein) alpha 12

G-protein subunit alpha-12|WUGSC:H_GS165O14.2|g alpha-12|guanine nucleotide-binding protein subunit alpha-12

7p22.2

protein-coding

Galpha(12), the alpha-subunit of G12, which has been referred to as the gep oncogene, stimulates mitogenic pathways in different cell types and readily induces neoplastic transformation of fibroblast cell lines. Recently, we have shown that the oncogenic pathway activated by Galpha(12) involves the receptor tyrosine kinase platelet derived growth factor receptor-alpha (PDGFRalpha) and JAK3. In the present study, we demonstrate that the GTPase-deficient activated mutant of Galpha(12) activates signal transducer and activator of transcription 3 (STAT3) via PDGFRalpha as well as JAK3. Here we show that Galpha(12) stimulates the phosphorylation of STAT3 at both Tyrosine-705 and Serine-727 residues. Studies to delineate the mechanism by which Galpha(12) stimulates STAT3 have indicated that the Tyrosine-705-phosphorylation of STAT3 involves the tyrosine kinases, Janus Kinase-3 as well as Src kinase, whereas the Serine-727 phosphorylation of STAT3 occurs via the receptor tyrosine kinase, PDGFRalpha and phosphatidylinositol 3-OH kinase pathway. Our results also indicate that the coexpression of the dominant negative, DNA binding mutant of STAT3 (STAT3DB) inhibits the foci formation as well as anchorage-independent growth of Galpha(12)QL-transfectants, thereby establishing the critical role of STAT3 in Galpha(12)QL-mediated neoplastic cell growth. The results presented here demonstrate, for the first time, the ability of Galpha(12) to recruit multiple receptor-, nonreceptor-, and Ser/Thr kinases to stimulate STAT3-signaling to promote neoplastic transformation.

Lysophosphatidic acid (LPA) plays a critical role in the pathophysiology of ovarian cancers. Previous studies have shown that LPA stimulates the proliferation of ovarian cancer cells via Galpha12. The present study utilizing Protein/DNA array analyses of LPA-stimulated HeyA8 cells in which the expression of Galpha12 was silenced, demonstrates for the first time that Galpha12-dependent mitogenic signaling by LPA involves the atypical activation cAMP-response element binding protein (CREB). Results indicate that the robust activation of CREB by LPA is an early event that can be monitored by the phosphorylation of SER133 of CREB as early as 3min. The findings that the expression of the constitutively activated mutant of Galpha12 stimulates CREB even in the absence of LPA in multiple ovarian cancer cell lines confirm the direct role of Galpha12 in the activation of CREB. This is further substantiated by the observation that the silencing of Galpha12 drastically attenuates LPA-stimulated phosphorylation of CREB. Our results also establish that LPA-Galpha12-dependent activation of CREB is through a cAMP-independent, but Ras-ERK-dependent mechanism. More significantly, our findings indicate that the expression of the dominant negative S133A mutant of CREB leads to a reduction in LPA-stimulated proliferation of HeyA8 ovarian cancer cells. Thus, results presented here demonstrate for the first time that CREB is a critical signaling node in LPA-LPAR and Galpha12/gep proto-oncogene stimulated oncogenic signaling in ovarian cancer cells.

The high mortality rate of hepatocellular carcinoma (HCC) is associated with its fast-growing malignancy. In tumor microenvironments, certain GPCRs are coupled to Galpha12 for signal transduction. Given the role of forkhead box O1 (FOXO1) in the inhibition of various tumors, this study investigated whether increase of Galpha12 in HCC causes FOXO1 repression, and if so, whether this event occurs through microRNA dysregulation. Overexpression of an active mutant of Galpha12 (Galpha12QL) decreased FOXO1 levels, whereas knockdown of Galpha12 had the opposite effect. Of the microRNAs targeting FOXO1, miR-135b levels were markedly increased by Galpha12 signaling, which led to FOXO1 repression as shown by the experiments using mimic, antisense oligonucleotide or siRNA. Galpha12QL increased the primary form of miR-135b by activating JunB (or c-Jun)/AP-1. Consistently, knockdown of JunB (or c-Jun) decreased miR-135b levels, thereby increasing FOXO1. Moreover, Galpha12QL induced MDM2, the deficiency of which facilitated FOXO1 accumulation. In addition, Galpha12QL repressed miR-194 cluster gene products (194/192/215), which contributed to MDM2-mediated FOXO1 repression. In functional assays, Galpha12QL facilitated tumor cell growth with alterations in cell cycle-associated protein levels, which was antagonized by enforced expression of FOXO1. In human HCCs, FOXO1 levels were decreased as compared with the surrounding liver tissue. Moreover, decrease of FOXO1 or miR-194 was statistically significant between stages T1 and T2, whereas increase of miR-135b discriminated tumor stage T3a versus T1/T2. In conclusion, Galpha12gep oncogene inhibits FOXO1, which may result from the inhibition of FOXO1 de novo synthesis by miR-135b in conjunction with MDM2-mediated destabilization of FOXO1.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Galpha12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Galpha12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Galpha12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Galpha12 (Galpha12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Galpha12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Galpha12. Decreases of miR-200a/b, -192 and -215 by Galpha12 caused ZEB1 induction. The ability of Galpha12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Galpha12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Galpha12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Galpha12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Galpha12 level, a correlation existed in the comparison of relative changes of Galpha12 and ZEB1. In conclusion, Galpha12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Galpha12 upregulation in liver tumor progression, implicating Galpha12 as an attractive therapeutic target.