Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFb/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFb-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFb on the expression of epithelial-mesenchymal transitionrelated factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFb/Smad signaling pathway via TRIM33 in ccRCC.Implications: This study suggests that miR-629 has biomarker potential through its ability to regulate TGFb/Smad signaling and accelerate ccRCC cell motility and invasion.

Vascular normalization in tumors may improve drug delivery and anti-tumor immunity. Angiogenesis inhibitors induce hypoxia, which may facilitate malignant progression; therefore, we investigated other methods to promote vascular maturation. Here, we show that lysophosphatidic acid (LPA) enhances blood flow by promoting fine vascular networks, thereby improving vascular permeability and suppressing tumor growth when combined with anti-cancer drug treatment. Six different G protein-coupled receptors have been identified as LPA receptors (LPA1-6). In studies using mutant mice, we found that LPA4 is involved in vascular network formation. LPA4 activation induces circumferential actin bundling beneath the cell membrane and enhances linear adherens junction formation by VE-cadherin in endothelial cells. Therefore, we conclude that activation of LPA4 is a promising approach for vascular regulation.

Clear cell renal cell carcinoma (ccRCC) is the most common histologically defined subtype of renal cell carcinoma (RCC). To define the molecular mechanism in the progression of ccRCC, we focused on LOX-like protein 2 (LOXL2), which is critical for the first step in collagen and elastin cross-linking. Using exon array analysis and quantitative validation, LOXL2 was shown to be significantly upregulated in clinical specimens of human ccRCC tumor tissues, compared with adjacent noncancerous renal tissues, and this elevated expression correlated with the pathologic stages of ccRCC. RNAi-mediated knockdown of LOXL2 resulted in marked suppression of stress-fiber and focal adhesion formation in ccRCC cells. Moreover, LOXL2 siRNA knockdown significantly inhibited cell growth, migration, and invasion. Mechanistically, LOXL2 regulated the degradation of both integrins a5 (ITGAV5) and b1 (ITGB1) via protease-and proteasome-dependent systems. In clinical ccRCC specimens, the expression levels of LOXL2 and integrin a5 correlated with the pathologic tumor grades. In conclusion, LOXL2 is a potent regulator of integrin a5 and integrin b1 protein levels and functions in a tumor-promoting capacity in ccRCC.Implications: This is the first report demonstrating that LOXL2 is highly expressed and involved in ccRCC progression by regulating the levels of integrins a5 and b1. Mol Cancer Res; 12(12); 1807-17. Ó2014 AACR. IntroductionRenal cell carcinoma (RCC) is the leading cause of death among urological malignancies, and clear cell renal cell carcinoma (ccRCC) is the most common histologic subtype of RCC. Early-stage ccRCC is usually curable by clinical surgery, but a large number of early-stage ccRCC cases are asymptomatic, with approximately one-third of all patients presenting with locally metastatic cancer at the time of diagnosis (1). Therefore, a better understanding of the molecular mechanisms of ccRCC progression is crucial for the discovery of novel prognostic markers and targeted therapies.A genetic hallmark of ccRCC is the inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL functions as a part of an E3 ubiquitin ligase complex that

The structure and function of tumor blood vessels profoundly affects the tumor microenvironment. Signals mediated through the lysophosphatidic acid receptor 4 (LPA4) promote vascular network formation to restore normal vascular barrier function in subcutaneous tumors and thus improve drug delivery. However, the characteristics of the vasculature vary by organ and tumor types, and how drug delivery and leukocyte trafficking are affected by modification of vascular function by LPA in different cancers is unclear. Here, we show that LPA4 activation promotes the formation of fine vascular structures in brain tumors. RhoA/ROCK signaling contributed to LPA-induced endothelial cell-cell adhesion, and RhoA/ROCK activity following LPA4 stimulation regulated expression of VCAM-1. This resulted in increased lymphocyte infiltration into the tumor. LPA improved delivery of exogenous IgG into brain tumors and enhanced the anticancer effect of antiprogrammed cell death-1 antibody therapy. These results indicate the effects of LPA on vascular structure and function apply not only to chemotherapy but also to immunotherapy.Significance: These findings demonstrate that lysophosphatidic acid, a lipid mediator, promotes development of a fine capillary network in brain tumors by inducing tightening of endothelial cell-to-cell adhesion, facilitating improved drug delivery, and lymphocyte penetration.

Galectin-3 (Gal-3; gene LGALS3) is a member of the b-galactoseebinding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates positively with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the number of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of b3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.