Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Induces the Oncogenic miR-17-92 Cluster and Down-Regulates TGF-β Signaling

KSHV is a DNA tumor virus that causes Kaposi’s sarcoma. Upon KSHV infection, only a limited number of latent genes are expressed. We know that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Aberrant miRNA expression contributes to the development of many types of cancer. Array-based miRNA profiling revealed that all six miRNAs of the oncogenic miR-17-92 cluster are up-regulated in KSHV infected endothelial cells. Among candidate KSHV latent genes, we found that vFLIP and vCyclin were shown to activate the miR-17-92 promoter, using luciferase assay and western blot analysis. The miR-17-92 cluster was previously shown to target TGF-β signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17-92 cluster to strongly inhibit the TGF-β signaling pathway by down-regulating SMAD2. Moreover, TGF-β activity and SMAD2 expression were fully restored when antagomirs (inhibitors) of miR-17-92 cluster were transfected into cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics to produce vFLIP or vCyclin knock-out viruses, and studied the effects in infected TIVE cells. Infection with wildtype KSHV abolished expression of SMAD2 protein in these endothelial cells. While single-knockout mutants still showed a marked reduction in SMAD2 expression, TIVE cells infected by a double-knockout mutant virus were fully restored for SMAD2 expression, compared to non-infected TIVE cells. Expression of either vFLIP or vCycIin was sufficient to downregulate SMAD2. In summary, our data demonstrate that vFLIP and vCyclin induce the oncogenic miR-17-92 cluster in endothelial cells and thereby interfere with the TGF-β signaling pathway. Manipulation of the TGF-β pathway via host miRNAs represents a novel mechanism that may be important for KSHV tumorigenesis and angiogenesis, a hallmark of KS.     

Endothelial Lineage Differentiation from Induced Pluripotent Stem Cells Is Regulated by MicroRNA-21 and Transforming Growth Factor β2 (TGF-β2) Pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.     

TGF-β1 Reduces miR-29a Expression to Promote Tumorigenicity and Metastasis of Cholangiocarcinoma by Targeting HDAC4

Transforming growth factor β1 (TGF-β1) and miRNAs play important roles in cholangiocarcinoma progression. In this study, miR-29a level was found significantly decreased in both cholangiocarcinoma tissues and tumor cell lines. TGF-β1 reduced miR-29a expression in tumor cell lines. Furthermore, anti-miR-29a reduced the proliferation and metastasis capacity of cholangiocarcinoma cell lines in vitro, overexpression of miR-29a counteracted TGF-β1-mediated cell growth and metastasis. Subsequent investigation identified HDAC4 is a direct target of miR-29a. In addition, restoration of HDAC4 attenuated miR-29a-mediated inhibition of cell proliferation and metastasis. Conclusions: TGF-β1/miR-29a/HDAC4 pathway contributes to the pathogenesis of cholangiocarcinoma and our data provide new therapeutic targets for cholangiocarcinoma.     

Biomarkers of TGF-β Signaling Pathway and Prognosis of Pancreatic Cancer

Background

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.     

SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

Transforming growth factor-β (TGF-β) mediates growth-inhibitory effects on most target cells via activation of the canonical SMAD signaling pathway. This growth-inhibitory activity may be coupled with cellular differentiation. Our studies demonstrate that TGF-β1 inhibits proliferation of primary, non-transformed human lung fibroblasts in association with the induction of myofibroblast differentiation. Differentiated myofibroblasts maintain the capacity to proliferate in response to exogenous mitogenic stimuli and are resistant to serum deprivation-induced apoptosis. These proliferative and anti-apoptotic properties of myofibroblasts are related, in part, to the down-regulation of caveolin-1 (Cav-1) by TGF-β1. Cav-1 down-regulation is mediated by early activation of p38 MAPK and does not require SMAD signaling. In contrast, myofibroblast differentiation is dependent on activation of the SMAD pathway, but not on p38 MAPK. Thus, combinatorial signaling by TGF-β1 of myofibroblast differentiation and down-regulation of Cav-1 by SMAD and p38 MAPK pathways, respectively, confer proliferative and apoptosis-resistant properties to myofibroblasts. Selective targeting of this SMAD-independent, p38-MAPK/Cav-1-dependent pathway is likely to be effective in the treatment of pathological conditions characterized by TGF-β signaling and myofibroblast activation.     

Functional Implications of MicroRNA-215 in TGF-β1-Induced Phenotypic Transition of Mesangial Cells by Targeting CTNNBIP1

Mesangial cell (MC) phenotypic transition is crucial for the progression of diabetic nephropathy. A major stimulus mediating high glucose-induced MC phenotypic transition is TGF-β1. Our current study focuses on microRNA-215 (miR-215) and investigates its role in TGF-β1-mediated MC phenotypic transition. Using real-time quantitative PCR (qRT-PCR) and northern blotting, we determined that the miR-192/215 family is dramatically upregulated under diabetic conditions both in vitro and in vivo. Gain- and loss-of-function approaches demonstrated that miR-215 inhibition significantly inhibited TGF-β1-induced mouse mesangial cell (MMC) phenotypic transition, whereas miR-215 upregulation promoted MMC phenotypic transition. Interestingly, these changes were not detected in cells that were treated with TGF-β1 and miR-192 mimics or inhibitors. These results suggest that miR-215 participates in TGF-β1-induced MMC phenotypic transition. Luciferase reporter assays were used to identify whether catenin-beta interacting protein 1 (CTNNBIP1) is a direct target of miR-215, which was predicted by bioinformatic analysis. Mechanistic studies revealed that CTNNBIP1 suppresses Wnt/β-catenin signaling and that miR-215 promotes β-catenin activation and upregulates α-SMA and fibronectin expression in TGF-β1-treated MMCs by targeting CTNNBIP1. In addition, in vivo miR-215 silencing with a specific antagomir significantly increased CTNNBIP1 protein expression, resulting in reduced β-catenin activity and decreased α-SMA and fibronectin expression in db/db mouse kidney glomeruli. Taken together, our findings indicate that miR-215 plays an essential role in MC phenotypic transition by regulating the CTNNBIP1/β-catenin pathway, which is related to the pathogenesis of diabetic nephropathy.     

An Integrated Expression Profiling Reveals Target Genes of TGF-β and TNF-α Possibly Mediated by MicroRNAs in Lung Cancer Cells

EMT (epithelial-mesenchymal transition) is crucial for cancer cells to acquire invasive phenotypes. In A549 lung adenocarcinoma cells, TGF-β elicited EMT in Smad-dependent manner and TNF-α accelerated this process, as confirmed by cell morphology, expression of EMT markers, capacity of gelatin lysis and cell invasion. TNF-α stimulated the phosphorylation of Smad2 linker region, and this effect was attenuated by inhibiting MEK or JNK pathway. Comprehensive expression analysis unraveled genes differentially regulated by TGF-β and TNF-α, such as cytokines, chemokines, growth factors and ECM (extracellular matrices), suggesting the drastic change in autocrine/paracrine signals as well as cell-to-ECM interactions. Integrated analysis of microRNA signature enabled us to identify a subset of genes, potentially regulated by microRNAs. Among them, we confirmed TGF-β-mediated induction of miR-23a in lung epithelial cell lines, target genes of which were further identified by gene expression profiling. Combined with in silico approaches, we determined HMGN2 as a downstream target of miR-23a. These findings provide a line of evidence that the effects of TGF-β and TNF-α were partially mediated by microRNAs, and shed light on the complexity of molecular events elicited by TGF-β and TNF-α.     

MicroRNA-153 Inhibits Osteosarcoma Cells Proliferation and Invasion by Targeting TGF-β2

Increasing evidence indicates that microRNAs (miRNAs), a class of small noncoding RNAs, participate in almost every step of cellular processes. MiRNAs are aberrantly expressed in human cancers and contribute to cancer development and progression. Study of miRNAs may provide a new clue for understanding the mechanism of carcinogenesis and a new tool for cancer treatment. In the present study, miR-153 was downregulated in human osteosarcoma tissues and cell lines. Introduction of miR-153 mimics into the MG-63 cells inhibited cell proliferation and invasion. Our results further revealed that transforming growth factor beta 2 (TGF-β2) was negatively regulated by miR-153. Furthermore, overexpression of miR-153 decreased p-SMAD2, p-SMAD3, epidermal growth factor receptor (EGFR) and insulin-like growth factor binding protein-3 (IGFBP-3) expressions, which were the downstream signaling molecules of TGF-β. Furthermore, miRNA-153 suppressed TGF-β-mediated MG-63 proliferation and migration. Therefore, our results suggest that miR-153 may act as a tumor suppressor in osteosarcoma through targeting TGF-β2.     

miRNA 17 Family Regulates Cisplatin-Resistant and Metastasis by Targeting TGFbetaR2 in NSCLC

MicroRNAs (miRNAs) have been proven to play crucial roles in cancer, including tumor chemotherapy resistance and metastasis of non-small-cell lung cancer (NSCLC). TGFβ signal pathway abnormality is widely found in cancer and correlates with tumor proliferation, apoptosis and metastasis. Here, miR-17, 20a, 20b were detected down-regulated in A549/DDP cells (cisplatin resistance) compared with A549 cells (cisplatin sensitive). Over-expression of miR-17, 20a, 20b can not only decrease cisplatin-resistant but also reduce migration by inhibiting epithelial-to-mesenchymal transition (EMT) in A549/DDP cells. These functions of miR-17, 20a, 20b may be caused at least in part via inhibition of TGFβ signal pathway, as miR-17, 20a, 20b are shown to directly target and repress TGF-beta receptor 2 (TGFβR2) which is an important component of TGFβ signal pathway. Consequently, our study suggests that miRNA 17 family (including miR-17, 20a, 20b) can act as TGFβR2 suppressor for reversing cisplatin-resistant and suppressing metastasis in NSCLC.     

Association of v-ErbA with Smad4 Disrupts TGF-β Signaling

Disruption of the transforming growth factor-β (TGF-β) pathway is observed in the majority of cancers. To further understand TGF-β pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-β responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV). Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-β–induced cell growth inhibition and that dysregulation of TGF-β signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-β–induced growth inhibition and that TGF-β sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-β signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia.     

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.     

Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II

We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells.