The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.     

ShcA Protects against Epithelial–Mesenchymal Transition through Compartmentalized Inhibition of TGF-β-Induced Smad Activation

Epithelial–mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-β (TGF-β) signaling, and TGF-β drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-β also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-β type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-β-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-β receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-β/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-β receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-β receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-β signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-β-induced Smad activation through differential partitioning of receptor complexes at the cell surface.     

Role of Complement 3 in TNF-α-Induced Mesenchymal Transition of Renal Tubular Epithelial Cells In Vitro

Injured renal tubular epithelial cells (RTECs) have been recently thought to directly contribute to the accumulation of myofibroblasts in renal tubulointerstitial fibrosis through a process of epithelial to mesenchymal transition (EMT). However, the factors inducing RTECs to undergo EMT and the underlying mechanisms need to be further elucidated. This study aimed to determine the EMT-inducing activity of proinflammatory cytokine TNF-α and the role for complement 3 (C3) in this activity in an in vitro model of human RTECs (HK-2 cells). Wild type HK-2 cells were treated with TNF-α, IFN-γ or C3a; C3 siRNA- or control siRNA-carrying HK-2 cells were treated with TNF-α. Changes in the cell morphology and phenotype were assessed by microscopy, RT-PCR, western blotting, and immunostaining. TNF-α effectively induced HK-2 cells to express C3 and to transform into morphologically myofibroblast-like cells that lost E-cadherin (a classical epithelial cell marker) expression but acquired alpha-smooth muscle actin (α-SMA, a classical myofibroblast differentiation marker) expression. C3 siRNA robustly attenuated all the morphologic and phenotypic changes induced by TNF-α but the control siRNA showed no effect. Our preliminary observations suggest that TNF-α may induce EMT in RTECs through inducing C3 expression.     

Epithelial Mesenchymal Transition and Pancreatic Tumor Initiating CD44+/EpCAM+ Cells Are Inhibited by γ-Secretase Inhibitor IX

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX) to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT) and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.     

SRPX2 Enhances the Epithelial–Mesenchymal Transition and Temozolomide Resistance in Glioblastoma Cells

Glioblastoma (GBM) is the most common and most aggressive central nervous system tumor in adults. Due to GBM cell invasiveness and resistance to chemotherapy, current medical interventions are not satisfactory, and the prognosis for GBM is poor. It is necessary to investigate the underlying mechanism of GBM metastasis and drug resistance so that more effective treatments can be developed for GBM patients. sushi repeat-containing protein, X-linked 2 (SRPX2) is a prognostic biomarker in many different cancer cell lines and is associated with poor prognosis in cancer patients. SRPX2 overexpression promotes interactions between tumor and endothelial cells, leading to tumor progression and metastasis. We hypothesize that SRPX2 also contributes to GBM chemotherapy resistance and metastasis. Our results revealed that GBM tumor samples from 42 patients expressed higher levels of SRPX2 than the control normal brain tissue samples. High-SRPX2 expression levels are correlated with poor prognosis in those patients, as well as resistance to temozolomide in cultured GBM cells. Up-regulating SRPX2 expression in cultured GBM cell lines facilitated invasiveness and migration of GBM cells, while down-regulating SRPX2 through RNA interference was inhibitory. These results suggest that SRPX2 plays an important role in GBM metastasis. Epithelial to mesenchymal transition (EMT) is one of the processes that facilitate GBM metastasis and resistance to chemotherapy. EMT marker expression was decreased in SRPX2 down-regulated GBM cells, and MAPK signaling pathway marker expression was also decreased when SRPX2 is knocked down in GBM-cultured cells. Blocking the MAPK signaling pathway inhibited GBM metastasis but did not inhibit cell invasion and migration in SRPX2 down-regulated cells. Our results indicate that SRPX2 facilitates GBM metastasis by enhancing the EMT process via the MAPK signaling pathway.     

Evidence for Epithelial�CMesenchymal Transition in Adult Human Pancreatic Exocrine Cells

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial–mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and α-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues. (J Histochem Cytochem 58:807–823, 2010)     

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.     

Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.     

Hirsutella sinensis Attenuates Aristolochic Acid-Induced Renal Tubular Epithelial-Mesenchymal Transition by Inhibiting TGF-β1 and Snail Expression

Objective

To investigate the inhibitory effect of Hirsutella sinensis (HS) on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells induced by aristolochic acid (AA) and its possible mechanism.

Methods

18 male Sprague-Dawley rats were randomly and equally divided into the following 3 groups: AA group, AA+HS group and control group. Urinary protein excretion and creatinine clearance (CCr) were measured. All rats were sacrificed at the end of 12th week. The pathological examination of renal tissue was performed and the mRNA and protein expression of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), cytokeratin-18 and Snail in renal cortex were determined by real time quantitative PCR and immunohistochemical staining respectively. In addition, human renal proximal tubule epithelial cells line (HKC) was divided into the following 4 groups: AA group, AA+HS group, HS control group and control group. The above mRNA and protein expression in HKC was determined by real time quantitative PCR and Western blot respectively.

Results

(1) CCr was significantly decreased, and the urinary protein excretion and relative area of renal interstitial fibrosis were significantly increased in the rats of AA and AA+HS group compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly lightened in the rats of AA+HS group compared to those in AA group (P<0.05). (2) The mRNA and protein expression of TGF-β1, α-SMA and Snail was significantly up-regulated and the expression of cytokeratin-18 was significantly down-regulated in the rat renal cortex as well as in the cultured HKC cells in AA and AA+HS groups compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly alleviated in AA+HS group compared to those in AA group (P<0.05 or P<0.01). (3) Knockdown endogenous Snail expression by siRNA could ameliorate AA-induced EMT of HKC cells, while overexpression of Snail by plasmid transfection diminished the antagonistic effect of HS on AA-induced EMT. These results suggest Snail might be a potential target of HS effect.

Conclusion

HS is able to antagonize, to some extent, tubular EMT and renal interstitial fibrosis caused by AA, which might be related to its inhibitory effects on the TGF-β1 and Snail expression.