Transforming growth factor beta (TGFβ) is transiently increased in the mammary gland during involution and by radiation. While TGFβ normally has a tumour suppressor role, prolonged exposure to TGFβ can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFβ during involution to determine the persistent effects on premalignant mammary epithelium.
CDβGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFβ (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFβ and then assessed for markers of EMT and transformation.
The 14-day exposure to TGFβ induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFβ. TGFβ-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFβ-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFβ-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFβ2 and changes in extra cellular matrix.
This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.
Transforming growth factor beta; TGFβ; Epithelial to mesenchymal transition; EMT; Transdifferentiation
TGFβ induces epithelial-mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, identification of TGFβ-inducible, EMT-specific genes has met with limited success. Here, we identify a post-transcriptional pathway by which TGFβ modulates expression of EMT-specific proteins, and EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33 nucleotides (nt) TGF beta-activated translation (BAT) element in the 3’-UTR of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and repress their translation. TGFβ activation leads to phosphorylation at Ser43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI mRNAs. Modulation of hnRNP E1 expression or its post-translational modification alters TGFβ-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGFβ-inducible post-transcriptional regulon that controls EMT during development and metastatic progression of tumors.
Dysregulated repair following epithelial injury is a key forerunner of disease in many organs, and the acquisition of a mesenchymal phenotype by the injured epithelial cells (epithelial to mesenchymal transition, EMT) may serve as a source of fibrosis. The macrolide antibiotic azithromycin and the DNA synthesis inhibitor mycophenolate are in clinical use but their mechanism of action remains unknown in post-transplant bronchiolitis obliterans syndrome (BOS). Here we determined if regional variation in the EMT response to TGFβ1 underlies the bronchiolocentric fibrosis leading to BOS and whether EMT could be inhibited by azithromycin or mycophenolate.
We found that small and large airway epithelial cells from stable lung transplant patients underwent EMT when stimulated with TGFβ1, however mesenchymal protein expression was higher and loss of epithelial protein expression more complete in small airway epithelial cells. This regional difference was not mediated by changes in expression of the TGFβRII or Smad3 activation. Azithromycin potentially inhibited EMT in both small and large airway epithelial cells by inhibiting Smad3 expression, but not activation.
Collectively, these observations provide a biologic basis for a previously unexplained but widely observed clinical phenomena, and a platform for the development of new approaches to fibrotic diseases.
Bone morphogenic protein (BMP)-7 is a member of the BMP family which are structurally and functionally related, and part of the TGFβ super family of growth factors. BMP-7 has been reported to inhibit renal fibrosis and TGFβ1-induced epithelial-mesenchymal transition (EMT), in part through negative interactions with TGFβ1 induced Smad 2/3 activation. We utilized in vivo bleomycin-induced fibrosis models in the skin and lung to determine the potential therapeutic effect of BMP-7. We then determined the effect of BMP-7 on TGFβ1-induced EMT in lung epithelial cells and collagen production by human lung fibroblasts. We show that BMP-7 did not affect bleomycin-induced fibrosis in either the lung or skin in vivo; had no effect on expression of pro-fibrotic genes by human lung fibroblasts, either at rest or following exposure to TGFβ1; and did not modulate TGFβ1 -induced EMT in human lung epithelial cells. Taken together our data indicates that BMP-7 has no anti-fibrotic effect in lung or skin fibrosis either in vivo or in vitro. This suggests that the therapeutic options for BMP-7 may be confined to the renal compartment.
Development of the secondary palate in mammals is a complex process under the control of numerous growth and differentiation factors that regulate key processes such as cell proliferation, synthesis of extracellular matrix molecules, and epithelial-mesenchymal transdifferentiation. Alterations in any one of these processes either through genetic mutation or environmental insult have the potential to lead to clefts of the secondary palate. Members of the TGFβ family of cytokines are crucial mediators of these processes and emerging evidence supports a pivotal role for members of the Wnt family of secreted growth and differentiation factors. Previous work in this laboratory demonstrated cross-talk between the Wnt and TGFβ signaling pathways in cultured mouse embryonic palate mesenchymal cells. In the current study we tested the hypothesis that unique gene expression profiles are induced in murine embryonic palate mesenchymal cells as a result of this cross-talk between the TGFβ and Wnt signal transduction pathways.
embryo; palate; TGFβ; Wnt; microarray; gene expression
Epithelial-mesenchymal-transition (EMT) is a fundamental cellular process that is critical for normal development and tumor metastasis. The transforming growth factor beta (TGFβ) is a potent inducer of EMT like effects, but the mechanisms that regulate TGFβ-induced EMT remain incompletely understood. Using the widely employed NMuMG mammary epithelial cells as a model to study TGFβ-induced EMT, we report that TGFβ downregulates the levels of the SUMO E3 ligase PIAS1 in cells undergoing EMT. Gain and loss of function analyses indicate that PIAS1 acts in a SUMO ligase dependent manner to suppress the ability of TGFβ to induce EMT in these cells. We also find that TGFβ inhibits sumoylation of the PIAS1 substrate SnoN, a transcriptional regulator that antagonizes TGFβ-induced EMT. Accordingly, loss of function mutations of SnoN sumoylation impair the ability of SnoN to inhibit TGFβ-induced EMT in NMuMG cells. Collectively, our findings suggest that PIAS1 is a novel negative regulator of EMT and reveal that inhibition of the PIAS1-SnoN sumoylation pathway represents a key mechanism by which TGFβ induces EMT, with important implications in normal development and tumor metastasis.
The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT.
As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining.
Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific sialylated structures. These results provide leads for targeting aggressive and drug resistant forms of pancreatic cancer cells.
Epithelial–mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor β (TGFβ) is a well-studied inducer of EMT. We demonstrate that TGFβ potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGFβ receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGFβ-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGFβ-induced EMT. Interestingly, HAS2 suppression completely abolished the TGFβ-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGFβ-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGFβ-induced EMT.
EMT; growth factors; TGFβ; hyaluronan synthase 2; migration
Epithelial–mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFβ signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFβ3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFβ and EMT.
Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.
Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.
TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.
Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.
Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTC) that are locally invasive and have well-defined foci of poorly differentiated carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of 8 paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by IHC, as vimentin expression was increased and E-cadherin lost in PDTC compared to adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for TGFβ in mediating this process. Accordingly, TGFβ induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFβ-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required MAPK pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFβ-induced EMT, through a MAPK-dependent process.
The 1029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val12 ras. These cell lines respond to TGFβ by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFβ. In vitro, vimentin expression is induced in all the cell lines by TGFβ treatment, while cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFβ are mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFβ treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFβ, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.
EMT; TGFβ; vimentin; suppression; metastasis
Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.
The transforming growth factorβ (TGFβ) superfamily regulates a broad spectrum of biological responses throughout embryonic development and adult life, including cell proliferation and differentiation, epithelial-to-mesenchymal transition, apoptosis, and angiogenesis. TGFβ members initiate signaling by bringing together a complex of serine/threonine kinase receptors that transmit signals through intracellular Smad proteins. Genetic alterations in numerous components of the TGFβ signaling pathway have been associated with several human cancers. In addition, tight regulation of TGFβ signaling is pivotal to the maintenance of homeostasis and the prevention of carcinogenesis. The ubiquitin/proteosome system is one mechanism by which cells regulate the expression and activity of effectors of the TGFβ signaling cascade. Mounting evidence also suggests that disruption of the ubiquitin-dependent degradation of components of the TGFβ pathway leads to the development and progression of cancer. Therefore, understanding how these two pathways intertwine will contribute to the advancement of our knowledge of cancer development.
TGFβ; Smad; Smurf; ubiquitination; cancer
TGFβ induces lens epithelial cells to undergo epithelial mesenchymal transition (EMT) and many changes with characteristics of fibrosis including posterior capsular opacification (PCO). Consequently much effort is directed at trying to block the damaging effects of TGFβ in the lens. To do this effectively it is important to know the key signaling pathways regulated by TGFβ that lead to EMT and PCO. Given that Wnt signaling is involved in TGFβ-induced EMT in other systems, this study set out to determine if Wnt signaling has a role in regulating this process in the lens. Using RT-PCR, in situ hybridization and immunolocalization this study clearly shows that Wnts 5a, 5b, 7b, 8a, 8b and their Frizzled receptors are upregulated in association with TGFβ-induced EMT and cataract development. Both rat in vitro and mouse in vivo cataract models show similar profiles for the Wnt and Frizzled mRNAs and proteins that were assessed. Currently it is not clear if the canonical β-catenin/TCF signaling pathway, or a non-canonical pathway, is activated in this context. Overall, the results from the current study indicate that Wnt signaling is involved in TGFβ-induced EMT and development of fibrotic plaques in the lens.
Lens epithelium; Anterior subcapsular cataract; Wnt; TGFβ; Posterior capsular opacification
During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms’ tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.
Tubulointerstitial renal fibrosis, characterized as a progressive detrimental connective tissue deposition on the kidney parenchyma, appears to be a harmful process leading inevitably to renal function deterioration, independently of the primary renal disease which causes the original kidney injury. Epithelial to Mesenchymal Transition (EMT) of tubular epithelial cells which are transformed to mesenchymal fibroblasts migrating to adjacent interstitial parenchyma constitutes the principal mechanism of renal fibrosis along with local and circulating cells. Proteinuria as well as hypoxia is included among the main mechanisms of EMT stimulation. TGFβ-1 through the SMAD pathway is considered as the main modulator regulating the EMT molecular mechanism, probably in cooperation with hypoxia inducible factors. Hepatocyte Growth Factor (HGF) and Bone Morphogenetic Factor-7 (BMF-7) are inhibitory to EMT molecules which could prevent in experimental and clinical level the catastrophic process of interstitial fibrosis. Interesting data emerge indicating that HGF and BMF-7 administration prevents the peritoneal fibrosis of mesothelial cells.
cytokines; growth factors; fibrosis; hypoxia; angiotensin II; epithelial mesenchymal transition; TGF-β; proteinuria
Breast cancer recurrence is believed to be caused by a sub-population of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSCs) by epithelial to mesenchymal transition (EMT), although the physiological process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is demonstrated by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed down regulated expression of genes encoding Claudin 3, 4 and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer.
The development of interstitial fibrosis and tubular atrophy is a common complication after kidney transplantation and is associated with reduced long-term outcome. The hallmark of tubulointerstitial fibrosis is an increase in extracellular matrix resulting from exaggerated activation of fibroblasts/myofibroblasts, and tubular atrophy is characterized by a decrease in tubular diameter and loss of function. Atrophic epithelial cells may undergo epithelial-to-mesenchymal transition (EMT) with potential differentiation into interstitial fibroblasts. One potential driver of EMT in developing interstitial fibrosis and tubular atrophy is chronic hypoxia.
The expression of 46 EMT-related genes was analyzed in an in vitro hypoxia model in renal proximal tubular epithelial cells (RPTEC). Furthermore, the expression of 342 microRNAs (miR) was evaluated in hypoxic culture conditions.
Hypoxic RPTEC expressed markers of a more mesenchymal phenotype and showed an increased expression of matrix metalloproteinase-2 (MMP2). MMP2 expression in RPTEC correlated inversely with a decreased expression of miR-124, which was found to have a putative binding site for the MMP2 transcript. Overexpression of miR-124 inhibited MMP2 protein translation. Hypoxia was associated with increased migration/proliferation of RPTEC which was reversed by miR-124.
These results indicate that hypoxia promotes a mesenchymal and migratory phenotype in renal epithelial cells, which is associated with increased MMP2 expression. Hypoxia-dependent MMP2 expression is regulated via a reduced transcription of miR-124. Overexpression of miR-124 antagonizes hypoxia-induced cell migration. Further research is needed to elucidate the functional role of miR-124 and MMP2 in the development of fibrosis in renal transplant degeneration.
Hypoxia; Fibrosis; Matrix metalloproteinase-2; miR-124
Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.
PI3K; MAPK; TGFβ; EMT; metastasis
Aberrant regulatory DNA methylation patterns have been associated with breast cancer progression. While most efforts have been focused on describing the gene targets for DNA methylation, the molecular events that define the activity of the DNA methylation machinery have remained elusive. Here we describe the use of a breast cancer cell line model system to investigate the mechanisms that regulate epigenetic alterations of gene expression patterns responsible for epithelial-mesenchymal transition (EMT), a critical step during conversion to malignant breast cancer. We found that breast cancer cells which have undergone EMT exhibit overactive TGFβ signaling and loss of expression of genes including CDH1, CGN, CLDN4 and KLK10 mediated by DNA hypermethylation of their corresponding promoter regions. Consistent with the notion that activated TGFβ-Smad signaling is involved in “epigenetic memory” to maintain epigenetically silenced state of critical genes, disruption of Smad signaling due to Smad7 overexpression or depletion of Smad2, but not Smad4, in mesenchymal-like breast cancer cells resulted in DNA demethylation and re-expression of the corresponding genes. This reversal of epigenetic changes was accompanied with the acquisition of epithelial morphology and suppression of invasive properties of breast cancer cells. Furthermore, disruption of TGFβ signaling caused a corresponding decrease in DNMT1 binding activity suggesting that failure to maintain methylation of the newly synthesized DNA is the likely cause of demethylation. In summary, our studies reveal for the first time, that hyperactive TGFβ-TGFβR-Smad2 signaling axis is involved in the maintenance of epigenetic silencing of critical target genes to facilitate breast cancer progression.
TGFβ; Smad; DNA methylation; EMT; breast cancer; epigenetic memory
Fibrosis affects an extensive range of organs and is increasingly acknowledged as a major component of many chronic disorders. It is now well accepted that the elevated expression of certain inflammatory cell–derived cytokines, especially transforming growth factor β (TGFβ), is involved in the epithelial-to-mesenchymal transition (EMT) leading to the pathogenesis of a diverse range of fibrotic diseases. In lens, aberrant TGFβ signaling has been shown to induce EMT leading to cataract formation. Sproutys (Sprys) are negative feedback regulators of receptor tyrosine kinase (RTK)-signaling pathways in many vertebrate systems, and in this study we showed that they are important in the murine lens for promoting the lens epithelial cell phenotype. Conditional deletion of Spry1 and Spry2 specifically from the lens leads to an aberrant increase in RTK-mediated extracellular signal-regulated kinase 1/2 phosphorylation and, surprisingly, elevated TGFβ-related signaling in lens epithelial cells, leading to an EMT and subsequent cataract formation. Conversely, increased Spry overexpression in lens cells can suppress not only TGFβ-induced signaling, but also the accompanying EMT and cataract formation. On the basis of these findings, we propose that a better understanding of the relationship between Spry and TGFβ signaling will not only elucidate the etiology of lens pathology, but will also lead to the development of treatments for other fibrotic-related diseases associated with TGFβ-induced EMT.
Tissue-specific stem cells are considered to have a limited differentiation potential. Recently, this notion was challenged by reports that showed a broader differentiation potential of neural stem cells, in vitro and in vivo, although the molecular mechanisms that regulate plasticity of neural stem cells are unknown. Here, we report that neural stem cells derived from mouse embryonic cortex respond to Lif and serum in vitro and undergo epithelial to mesenchymal transition (EMT)-mediated dedifferentiation process within 48 h, together with transient upregulation of pluripotency markers and, more notably, upregulation of mesendoderm genes, Brachyury (T) and Sox17. These induced putative mesendoderm cells were injected into early gastrulating chick embryos, which revealed that they integrated more efficiently into mesoderm and endoderm lineages compared to non-induced cells. We also found that TGFβ and Jak/Stat pathways are necessary but not sufficient for the induction of mesendodermal phenotype in neural stem cells. These results provide insights into the regulation of plasticity of neural stem cells through EMT. Dissecting the regulatory pathways involved in these processes may help to gain control over cell fate decisions.
In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.