6469

SHH

BACKGROUND & AIMS: Repair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors. METHODS: Immature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc(+/-)) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD. RESULTS: In cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc(+/-) mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD. CONCLUSIONS: Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

BACKGROUND: Lung cancer, especially non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths in the United States. The aggressiveness of NSCLC has been shown to be associated with the acquisition of epithelial-to-mesenchymal transition (EMT). The acquisition of EMT phenotype induced by TGF-beta1in several cancer cells has been implicated in tumor aggressiveness and resistance to conventional therapeutics; however, the molecular mechanism of EMT and tumor aggressiveness in NSCLC remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study we found for the first time that the induction of EMT by chronic exposure of A549 NSCLC cells to TGF-beta1 (A549-M cells) led to the up-regulation of sonic hedgehog (Shh) both at the mRNA and protein levels causing activation of hedgehog signaling. These results were also reproduced in another NSCLC cell line (H2030). Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion. The aggressiveness of these cells was attenuated by the treatment of A549-M cells with pharmacological inhibitors of Hh signaling in addition to Shh knock-down by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as ZEB1 and Fibronectin, and induction of epithelial marker E-cadherin. In addition, knock-down of Shh by siRNA significantly attenuated EMT induction by TGF-beta1. CONCLUSIONS/SIGNIFICANCE: Our results show for the first time the transcriptional up-regulation of Shh by TGF-beta1, which is mechanistically associated with TGF-beta1 induced EMT phenotype and aggressive behavior of NSCLC cells. Thus the inhibitors of Shh signaling could be useful for the reversal of EMT phenotype, which would inhibit the metastatic potential of NSCLC cells and also make these tumors more sensitive to conventional therapeutics.

epithelial-mesenchymal transition (EMT) has been reported to play an important role in distant metastasis in cancer-related disease. A large number of studies have shown that sonic hedgehog-glioma-associated oncogene 1 (Shh-Gli1) signals participate in the process of EMT; however, the role and mechanism of Shh-Gli1 signals in the progression of EMT in ovarian cancer remain largely unknown. First, we investigated the occurrence of EMT and invasion and migration ability in ovarian cancer cells stimulated by different concentration of Shh-Gli1 signals agonist purmorphamine in vitro. Then, Akt siRNA was transfected into ovarian cancer cells which already stimulated by purmorphamine to elucidate the molecular mechanism underlying the pathogenesis of EMT in ovarian cancer. Gli1 expression was significantly enhanced in ovarian cancer cells after stimulated by purmorphamine. In addition, Gli1 up-regulation promoted EMT, invasion and migration ability of ovarian cancer cells. Furthermore, we validated a cross talk between Shh-Gli1 signals and PI3K-Akt pathway in the occurrence of EMT in ovarian cancer cells. These findings revealed a novel role for Shh-Gli1 signals in EMT in ovarian cancer and provided for us a potential therapeutic target for the suppression of EMT, invasion and metastasis in ovarian cancer.

Despite the screening program, breast cancer is the commonest cause of cancer death in women in the industrialized world. In this study, we investigate the correlation among poorly differentiated carcinoma, epithelial to mesenchymal transition (EMT) phenomenon, and expression of NF-kB, Sonic Hedgehog (SHH), K-RAS, and PTX3 in breast cancer in 100 breast biopsies. Samples were classified as follows: 30 benign lesions (BL), 30 ductal infiltrating carcinomas low grade (MLG1), and 40 ductal infiltrating carcinomas high grade (MLG3). expression of vimentin, CD44, beta-catenin, NF-kB, SHH, K-RAS, CD44, and PTX3 was studied by immunohistochemistry. The different rate of cells with vimentin, nuclear beta-catenin, and CD44 expression in MLG3 as compared with MLG1 and BL suggested that the process of de-differentiation of breast cancer cells could be related to the EMT. Our results showed a significant increase in NF-kB signal in MLG3 (2.33 +/- 0.77) with respect to MLG1 (1.26 +/- 0.55) and BL (0.86 +/- 0.52). SHH expression appeared low in BL (1.00 +/- 0.41) and homogenously widespread in MLG1 (1.23 +/- 0.63) and MLG3 (1.56 +/- 0.54). An important increase in K-RAS signal was observed in MLG3 compared to that in BL (2.20 +/- 0.69 vs 0.82 +/- 0.59). As regards PTX3, we observed a strong expression in MLG3 (2.00 +/- 0.78) with respect to BL (0.58 +/- 0.55) and MLG1 (1.53 +/- 0.76). The recurring expression of NF-kB, SHH, K-RAS, and PTX3 in vimentin- and CD44-positive breast cancer cells allows to speculate that breast cells acquire the ability to express these molecules in concomitance to EMT phenomenon.

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.

The sonic hedgehog (SHH) signaling pathway plays a critical role in embryonic development, tissue regeneration and organogenesis. The activation of SHH signaling produces profibrogenic effects in various tissues, such as the liver and the biliary ducts. However, the role of SHH signaling in renal fibrogenesis remains to be elucidated. For this purpose, in the present study, we evaluated the hypothesis that activated SHH signaling promotes the acquisition of a myofibroblastic phenotype through the epithelial-mesenchymal transition (EMT), resulting in renal interstitial fibrosis (RIF). Kidney samples from rats subjected to unilateral or bilateral ureteral obstruction exhibited the enhanced expression of SHH-pathway proteins, mesenchymal markers and the decreased expression of epithelial markers. Overactive SHH signaling as well as tubular EMT and RIF in the obstructed kidneys were inhibited by recanalization of the ureter. In vitro, SHH signaling was activated during EMT induction and extracellular matrix (ECM) deposition was observed in transforming growth factor-beta1 (TGF-beta1)-treated renal tubular epithelial cells [RTECs; NRK-52E cell line]. Exogenous SHH activated SHH signaling and resulted in the upregulated expression of mesenchymal genes, the profibrogenic cytokine TGF-beta1, and the downregulated expression of epithelial markers. The blockade of SHH signaling with cyclopamine abolished SHH-mediated EMT as well as the acquisition of a myofibroblastic phenotype, and decreased TGF-beta1 expression and ECM production. Thus, taken together, these findings demonstrate that the activation of the SHH signaling pathway promotes the induction of EMT and renal tubulointerstitial fibrosis. The pharmacological inhibition of SHH signaling may potentially be of therapeutic value in the management of fibrotic kidney diseases.

The epithelial-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is crucial for the induction and progression of kidney fibrosis. However, the underlying molecular mechanisms that trigger the EMT programme have not been identified. In the present study, we demonstrate that transforming growth factor (TGF)-beta1 and activated hedgehog signaling mediate the EMT programme following kidney injury. Tissue samples from fibrotic kidneys show enhanced TGF-beta1 levels, as well as upregulated hedgehog signaling activity, during the EMT process; these levels decrease when fibrosis is reversed. Injury promotes TGF-beta1 expression and activates hedgehog signaling, thus inducing tubular EMT of TECs and extracellular matrix (ECM) accumulation in vitro. The EMT response and fibrotic appearance are also induced by enhanced TGF-beta1 levels or activated hedgehog signaling. Downregulation of TGF-beta1 inhibits aristolochic acid (AA)- and TGF-beta1-induced EMT and ECM synthesis and correlates with decreased hedgehog signaling. Similarly, inhibiting the hedgehog pathway abolishes AA- and hedgehog-mediated EMT, resulting in reduced TGF-beta1 levels. These findings highlight a key role for cross-talk between TGF-beta1 and hedgehog signaling in promoting injury-induced EMT and ECM deposition in TECs.CI - (c) 2016 Federation of European Biochemical Societies.