Purpose: TGF-b promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). However, the underlying mechanisms causing this are not entirely clear. Long noncoding RNAs (lncRNA) have been shown to play important regulatory roles in cancer progression. The lncRNA malat1 (metastasis associated lung adenocarcinoma transcript 1) is a critical regulator of the metastasis phenotype of lung cancer cells.Experimental Design: We, therefore, investigated whether TGF-b regulates malat1 expression to promote tumor metastasis of bladder cancer. The expression levels of malat1 and EMT markers were assayed in specimens of bladder cancer. The role of malat1 in regulating bladder cancer metastasis was evaluated in cell and animal models.Results: TGF-b induces malat1 expression and EMT in bladder cancer cells. malat1 overexpression is significantly correlated with poor survival in patients with bladder cancer. malat1 and E-cadherin expression is negatively correlated in vitro and in vivo. malat1 knockdown inhibits TGF-b-induced EMT. malat1 is associated with suppressor of zeste 12 (suz12), and this association results in decrease of E-cadherin expression and increase of N-cadherin and fibronectin expression. Furthermore, targeted inhibition of malat1 or suz12 suppresses the migratory and invasive properties induced by TGF-b. Finally, we demonstrated that malat1 or suz12 knockdown inhibits tumor metastasis in animal models.Conclusion: These data suggest that malat1 is an important mediator of TGF-b-induced EMT, and suggest that malat1 inhibition may represent a promising therapeutic option for suppressing bladder cancer progression. Clin Cancer Res; 20(6); 1531-41. Ó2014 AACR.

Chemotherapy is a reasonable alternative to cystectomy in patients with invasive and advanced bladder cancer. However, bladder cancer cells often develop drug resistance to these therapies, and~50% of patients with advanced bladder cancer do not respond to chemotherapy. Recent studies have shown that long non-coding RNA (lncRNA) is involved in the development of chemoresistance. Here we investigated the role of the urothelial cancer-associated 1 (UCA1) lncRNA in cisplatin resistance during chemotherapy for bladder cancer. We showed that cisplatin-based chemotherapy results in up-regulation of UCA1 expression in patients with bladder cancer. Similarly, UCA1 levels are increased in cisplatin-resistant bladder cancer cells. Over-expression of UCA1 significantly increases the cell viability during cisplatin treatment, whereas UCA1 knockdown reduces the cell viability during cisplatin treatment. UCA1 inhibition also partially overcomes drug resistance in cisplatin-resistant T24 cells. Furthermore, we showed that UCA1 positively regulates expression of wingless-type MMTV integration site family member 6 (Wnt6) in human bladder cancer cell lines. UCA1 and Wnt6 expression is also positively correlated in vivo. Up-regulation of UCA1 activates Wnt signaling in a Wnt6-dependent manner. We finally demonstrate that UCA1 increases the cisplatin resistance of bladder cancer cells by enhancing the expression of Wnt6, and thus represents a potential target to overcome chemoresistance in bladder cancer.

Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

Mesenchymal stem cells (MSCs) derived exosomes have been shown to have protective effects on the kidney in ischemia/reperfusion-induced renal injury. However, the key components in the exosomes and their potential mechanisms for the kidney protective effects are not well understood. In our current study, we focused on the abundant proteins in exosomes derived from MSCs (MSC-exo) and found that the C-C motif chemokine receptor-2 (CCR2) was expressed on MSC-exo with a high ability to bind to its ligand CCL2. We also proved that CCR2 high-expressed MSC-exo could reduce the concentration of free CCL2 and suppress its functions to recruit or activate macrophage. Further, knockdown of CCR2 expression on the MSC-exo greatly abolished these effects. Finally, we also found that CCR2 knockdown impaired the protective effects of MSC-exo for the renal ischemia/reperfusion injury in mouse. The results indicate that CCR2 expressed on MSC-exo may play a key role in inflammation regulation and renal injury repair by acting as a decoy to suppress CCL2 activity. Our study may cast new light on understanding the functions of the MSC-exo and these receptor proteins expressed on exosomes.

Shear stress, especially low shear stress (LowSS), plays an important role in vascular remodeling during atherosclerosis. Endothelial cells (ECs), which are directly exposed to shear stress, convert mechanical stimuli into intracellular signals and interact with the underlying vascular smooth muscle cells (VSMCs). The interactions between ECs and VSMCs modulate the LowSS-induced vascular remodeling. With the use of proteomic analysis, the protein profiles of rat aorta cultured under LowSS (5 dyn/cm 2 ) and normal shear stress (15 dyn/cm 2 ) were compared. By using Ingenuity Pathway Analysis to identify protein–protein association, a network was disclosed that involves two secretary molecules, PDGF-BB and TGF-β1, and three other linked proteins, lamin A, lysyl oxidase, and ERK 1/2. The roles of this network in cellular communication, migration, and proliferation were further studied in vitro by a cocultured parallel-plate flow chamber system. LowSS up-regulated migration and proliferation of ECs and VSMCs, increased productions of PDGF-BB and TGF-β1, enhanced expressions of lysyl oxidase and phospho-ERK1/2, and decreased Lamin A in ECs and VSMCs. These changes induced by LowSS were confirmed by using PDGF-BB recombinant protein, siRNA, and neutralizing antibody. TGF-β1 had similar influences on ECs as PDGF-BB, but not on VSMCs. Our results suggest that ECs convert the LowSS stimuli into up-regulations of PDGF-BB and TGF-β1, but these two factors play different roles in LowSS-induced vascular remodeling. PDGF-BB is involved in the paracrine control of VSMCs by ECs, whereas TGF-β1 participates in the feedback control from VSMCs to ECs.

Heterodimeric integrin receptors for extracellular matrix (ECM) play vital roles in bidirectional signaling during tissue development, organization, remodeling, and repair. The ␤ integrin subunit cytoplasmic domain is essential for transmission of many of these signals and overexpression of an unpaired ␤ tail in cultured cells inhibits endogenous integrins. Unlike vertebrates, which have at least nine ␤ subunit genes, the nematode Caenorhabditis elegans expresses only one ␤ subunit (␤pat-3), and a null mutation in this gene causes embryonic lethality. To determine the functions of integrins during larval development and in adult tissues, we have taken a dominant negative approach by expression of an HA-␤tail transgene composed of a hemagglutinin (HA) epitope tag extracellular domain connected to the ␤pat-3 transmembrane and cytoplasmic domains. Expression of this transgene in muscle and gonad, major sites of integrin expression, caused a variety of phenotypes dependent on the level of transgene expression. Abnormalities in body wall and sex muscles led to uncoordinated movement and egg-laying defects. Significant anomalies in migration and pathfinding were caused by tissue-specific expression of HA-␤tail in the distal tip cells (DTC), the cells that direct gonad morphogenesis. A pat-3 gene with Tyr to Phe mutations in the cytoplasmic domain was able to rescue pat-3 null animals but also showed DTC migration defects. These results show that ␤pat-3 plays important roles in post-embryonic organogenesis and tissue function. The extracellular matrix (ECM)1 is a complex network of glycoproteins and proteoglycans that determines tissue organization and function through interactions with cell surface receptors. Mechanical and chemical signals provided by the ECM are transduced primarily through integrin adhesion receptors (1-3). Integrins are transmembrane heterodimers composed of ␣ and ␤ subunits that play a central role in integrating bidirectional information between the ECM and the inside of the cell. Binding of integrins to ECM ligands leads to receptor clustering at focal adhesions where connections to the actin cytoskeleton are formed (4). In vitro binding studies and expression of mutant integrins have implicated specific ␤ cytoplasmic tail sequences in the linkage to actin filaments (5).In the nematode Caenorhabditis elegans, two ␣ (PAT-2 and INA-1) integrins and one ␤ (PAT-3) integrin are expressed in many tissues, including muscles, neurons, and gonad (6 -8). In body wall muscles, ␤pat-3 integrins are localized to muscle attachment sites called dense bodies (9). Attachment of muscle cells to the nearby hypodermis is essential for muscle filament assembly and function and attachment defects caused by ␣pat-2 and ␤pat-3 loss-of-function alleles give a paralyzed and arrested at two-fold (Pat) embryonic phenotype (10). In addition to integrins, many cytoskeletal and signaling molecules such as DEB-1/vinculin (11), talin (12), and UNC-97/PINCH (13) are colocalized to muscle dense bodies showing their similarity to ...

The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-beta1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-beta1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-beta1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.

Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active β-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation-associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical β-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/β-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/β-catenin signaling, which may offer prognostic and therapeutic opportunities.