Anti-RGS16 monoclonal antibody, clone 5F6

Chondrosarcoma is the most common primary malignant bone tumor in adults, has no effective systemic treatment, and patients with this disease have poor survival. Altered expression of microRNA (miR) is involved in tumorigenesis; however, its role in chondrosarcoma is undetermined. miR-181a is overexpressed in high-grade chondrosarcoma, is upregulated by hypoxia, and increases VEGF expression. Here, the purpose was to determine the mechanism of miR-181a regulation of VEGF, determine whether miR-181a overexpression promotes tumor progression, and to evaluate an antagomir-based approach for chondrosarcoma treatment. Therapeutic inhibition of miR-181a decreased expression of VEGF and MMP1 in vitro, and angiogenesis, MMP1 activity, tumor growth, and lungmetastasis, all by more than 50%, in a xenograft mouse model. A target ofmiR-181a is a regulator of G-protein signaling 16 (RGS16), a negative regulator of CXC chemokine receptor 4 (CXCR4) signaling. CXCR4 signaling is increased in chondrosarcoma, its expression is also increased by hypoxia, and is associated with angiogenesis and metastasis; however, receptor blockade is only partially effective. RGS16 expression is restored after miR-181a inhibition and partially accounts for the antiangiogenic and antimetastatic effects of miR-181a inhibition. These data establish miR-181a as an oncomiR that promotes chondrosarcoma progression through a new mechanism involving enhancement of CXCR4 signaling by inhibition of RGS16. Implications: Targeting miR-181a can inhibit tumor angiogenesis, growth, and metastasis, thus suggesting the possibility of antagomir-based therapy in chondrosarcoma. (C) 2015 AACR.

The expression of the negative Regulator of G protein signaling 16 (RGS16) is rapidly induced in cardiomyocytes by various stimuli. To identify the promoter of the mouse RGS16 gene, a 1.8-kb deoxyribonucleic acid fragment 5' of the RGS16-coding region was subcloned into a firefly-luciferase reporter vector and four overlapping fragments were analyzed. The luciferase production was quantified in neonatal rat cardiac myocytes (NRCM). A 0.6-kb fragment that induced a tenfold increase in luciferase activity contained the minimal promoter sequence. Its activity was twofold stimulated by fetal calf serum, endothelin-1 (ET-1), and sphingosine 1-phosphate (S1P), which stimuli also elevated the level of RGS16 protein. Stimulation of NRCM with ET-1 induced activation of the monomeric GTPases RhoA and Rac1, whereas S1P and the selective S1P1 receptor agonist SEW2871 only induced a pronounced activation of Rac1. In accordance, the treatment with the Rho-, Rac-, and Cdc42-inactivating Clostridium difficile Toxin B (TcdB) 10463 inhibited ET-1 and S1P-induced transcriptional activation. The ET-1-induced activation was insensitive to pertussis toxin but selectively suppressed by the RhoA-C-specific C2I-C3 ADP-ribosyl transferase and the ETB receptor antagonist BQ788. The S1P-induced activation was specifically inhibited by pertussis toxin and the Rac-inactivating TcdB 1470. All stimulated transcriptional activity was abolished by the negative transcription factor Yin Yang 1 (YY1), which binds to a consensus sequence within the minimal promoter. Taken together, our data show that most likely ETB- and S1P1-receptors induce RGS16 protein expression in cardiac myocytes by increasing the transcriptional activity of the rgs16 gene. This activation is mediated by heterotrimeric G proteins, Rho GTPases, and is under negative control of the transcription factor YY1.