Miamen, Alexander G.

Abstract Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvβ3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo. The TGFβ1/SMAD pathway was identified as a critical signaling axis between SULF2 and upregulation of POSTN transcription. In clinical HCC specimens, elevated levels of SULF2 correlated with increased microvascular density, POSTN levels, and relatively poorer patient survival. Together, our findings define an important axis controlling angiogenesis in HCC and a mechanistic foundation for rational drug development. Cancer Res; 77(3); 632–45. ©2016 AACR.

In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1‐Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1‐Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild‐type mice. Lung metastases were found in 75% of Sulf1‐Tg mice but not in wild‐type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor‐β (TGF‐β)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo . This effect of SULF1 on the TGF‐β/SMAD pathway is functional; overexpression of SULF1 promotes TGF‐β‐induced gene expression and epithelial–mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF‐β from the cell surface. We also show that SULF1 expression decreases the interaction between TGF‐β1 and its heparan sulfate proteoglycan sequestration receptor, TGFβR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence‐free survival (hazard ratio 4.1, 95% confidence interval 1.9‐8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF‐β expression and with several TGF‐β‐related epithelial–mesenchymal transition genes in human HCC. Conclusion : Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF‐β pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC. (H epatology 2015;61:1269–1283)

Abstract The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1–dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1–mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.