Regulation Of Snail Research Articles

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells

BackgroundMaspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT). We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells.MethodsExpression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP) assays.ResultsSnail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells.ConclusionsOverall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.

Hepatitis D Virus Isolates with Low Replication and Epithelial-Mesenchymal Transition-Inducing Activity Are Associated with Disease Remission

Clearance of hepatitis D virus (HDV) viremia leads to disease remission. Large hepatitis delta antigen (L-HDAg) has been reported to activate transforming growth factor β, which may induce epithelial-mesenchymal transition (EMT) and fibrogenesis. This study analyzed serum HDV RNA "quasispecies" in HDV-infected patients at two stages of infection: before and after alanine aminotransferase (ALT) elevations. Included in the study were four patients who went into remission after ALT elevation and three patients who did not go into remission and progressed to cirrhosis or hepatocellular carcinoma. Full-length HDV cDNA clones were obtained from the most abundant HDV RNA species at the pre- and post-ALT elevation stages. Using an in vitro model consisting of Huh-7 cells transfected with cloned HDV cDNAs, the pre- or post-ALT elevation dominant HDV RNA species were characterized for (i) their replication capacity by measuring HDV RNA and HDAg levels in transfected cells and (ii) their capacity to induce EMT by measuring the levels of the mesenchymal-cell-specific protein vimentin, the EMT regulators twist and snail, and the epithelial-cell-specific protein E-cadherin. Results show that in patients in remission, the post-ALT elevation dominant HDV RNA species had a lower replication capacity in vitro and lower EMT activity than their pre-ALT elevation counterparts. This was not true of patients who did not go into remission. The expression of L-HDAg, but not small HDAg, increased the expression of the EMT-related proteins. It is concluded that in chronically infected patients, HDV quasispecies with a low replication capacity and low EMT activity are associated with disease remission.

Cadherin switch from E- to N-cadherin in melanoma progression is regulated by the PI3K/PTEN pathway through Twist and Snail

Transition of normal melanocytic cells to malignant melanoma has characteristic features of epithelial to mesenchymal transition. This includes the disruption of the adherens junctions caused by the downregulation of E-cadherin and the upregulation of N-cadherin. The cadherins have functional importance in normal skin homeostasis and melanoma development; however, the exact mechanism(s) that regulate the 'cadherin switch' are unclear. To determine the mechanistic role of the PI3K/PTEN pathway in regulating the change in cadherin phenotype during melanoma progression. Using a panel of cell lines representative of the phases of melanoma progression, we determined cellular expressions of the components of the PI3K/PTEN pathway, E- and N-cadherin, and the transcriptional regulators Twist, Snail and Slug with Western blot and immunofluorescence analysis. Transcriptional regulation of E-cadherin, N-cadherin, Twist and Snail by the PI3K/PTEN pathway was confirmed using quantitative reverse transcription-polymerase chain reaction. Loss or inactivity of PTEN correlated with the switch in cadherin phenotype during melanoma progression. PTEN-null or inactive cells exhibited high levels of phosphorylated protein kinase B (PKB)/AKT (Serine 473) (PKB-Ser473-P), undetectable levels of E-cadherin and high levels of N-cadherin. Re-introduction of PTEN or treatment with the PI3K inhibitor Wortmannin resulted in the re-expression of E-cadherin and downregulation of N-cadherin. This cadherin switch was regulated at the transcriptional level by Twist and Snail which were, in turn, transcriptionally regulated by the PI3K pathway. Although E-cadherin was re-expressed, it failed to localize to the plasma membrane. The PI3K/PTEN pathway transcriptionally regulates the 'cadherin switch' via transcriptional regulation of Twist and Snail but does not regulate the localization of E-cadherin to the plasma membrane.

Abstract 5332: Snail transcription factor contributes to prostate cancer tumor progression via reactive oxygen species and Rac1 activation

Abstract Epithelial-mesenchymal transition (EMT) represses cell adhesion associated genes, making cells more migratory. Snail is the master gene that regulates the EMT process by down regulating tight junction proteins, such as E-cadherin, and up-regulating mesenchymal genes, such as vimentin. Cell migration can also be regulated by the Rho family of GTPases such as Rac1, which can induce reactive oxygen species (ROS), specifically superoxide, via the NADPH oxidase (Nox) pathways. ROS, specifically hydrogen peroxide has been shown to mediate EMT induced by MMP-3. The objective of this study is to determine whether Snail Transcription factor contributes to tumor progression via ROS and Rac1 and to study the effect(s) of antioxidants on Snail signaling. We utilized androgen-dependent LNCaP and androgen-independent 22Rv1 and ARCaP cell lines overexpressing Snail to examine Rac1 activity in vitro. ROS (superoxide and hydrogen peroxide) levels were examined in vitro and in vivo. Western blot analysis of EMT markers, cell migration on collagen and Real Time PCR Array to assay oxidative stress enzymes was also performed. We found that LNCaP, 22Rv1 and ARCaP cells overexpressing Snail displayed increased Rac1 activity while LNCaP and ARCaP cells with Snail overexpression expressed higher levels of superoxide and hydrogen peroxide in vitro. Furthermore, ARCaP cells overexpressing Snail injected into nude mice showed higher tumorigenicity as compared to ARCaP Neo control cells and excised tumors displayed increased levels of superoxide and hydrogen peroxide ex vivo. Real Time PCR Array revealed significant increase in key enzymes involved in oxidative stress such as aldehyde oxidase I and peroxidasin. Treatment with the hydrogen peroxide scavenger, N-acetyl cysteine (NAC), could partially revert Snail-mediated EMT in ARCaP cells as evidenced by re-expression of E-cadherin after 3 days. The muscadine grape skin extract (MSKE) antioxidant decreased ROS levels and Rac1 activity and reverted Snail-mediated EMT as seen by decreased vimentin, re-induction of E-cadherin, and decreased cell migration. This is the first report of Snail regulation of ROS by regulating oxidative stress enzymes, leading to EMT and tumor progression. We are also showing novel data that Snail can regulate cell motility via Rac1 activation. Therefore, targeting Snail-mediated EMT with antioxidants offers a promising therapy to reduce prostate cancer progression in future. Research supported by NIH Grants: 1P20MD002285 and G12RR03062. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5332. doi:1538-7445.AM2012-5332

Coexpression of Bcl-2 with epithelial–mesenchymal transition regulators is a prognostic indicator in hepatocellular carcinoma

The anti-apoptosis factor Bcl-2 is known to contribute to tumorigenesis and metastasis. Epithelial-mesenchymal transition (EMT) may also participate in tumor invasion and metastasis. This study investigated the relationship between coexpression profiles of Bcl-2 and EMT regulators in hepatocellular carcinoma (HCC) tumor samples and clinical outcome. The nuclear (Nu) and cytoplasmic (Cyt) expression of Bcl-2 and the EMT regulators Twist-1, Twist-2, and Snail were determined by immunohistochemical staining in tumor tissue isolated from 97 HCC patients. The clinical prognostic values of both individual protein expression and various expression combinations were investigated using univariate and multivariate survival analysis. Results showed that patients with nuclear expression of Bcl-2 had worse clinical outcomes than patients exhibiting cytoplasmic expression. Overall survival was significantly shorter in HCC patients individually expressing Bcl-2-Nu, Twist-1-Nu, Twist-1-Cyt, and Snail (all P<0.05). Patients coexpressing Bcl-2-Nu with Twist-1-Nu, Twist-1-Cyt, Twist-2, or Snail had even worse prognoses than those expressing no biomarker or any one biomarker alone (all P<0.05). Multivariate analysis showed that HCC patients coexpressing Bcl-2-Nu with Twist-1-Cyt had the worst prognosis. This study provides clinical evidence that nuclear expression of Bcl-2 combined with cytoplasmic expression of Twist-1 is a predictor of very poor prognosis in HCC. Coexpression profiles of Bcl-2 and EMT regulators might aid in the selection of the most efficacious therapy for patients with HCC.

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer

BackgroundThe epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.MethodsAgilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.ResultsMorphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects.ConclusionsThis work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Gambogic acid inhibits TNF-α-induced invasion of human prostate cancer PC3 cells in vitro through PI3K/Akt and NF-κB signaling pathways

To investigate the mechanisms underlying the inhibitory effect of gambogic acid (GA) on TNF-α-induced metastasis of human prostate cancer PC3 cells in vitro. TNF-α-mediated migration and invasion of PC3 cells was examined using migration and invasion assays, respectively. NF-κB transcriptional activity and nuclear translocation were analyzed with luciferase reporter gene assays, immunofluorescence assays and Western blots. The ability of p65 to bind the promoter of Snail, an important mesenchymal molecular marker, was detected using a chromatin immunoprecipitation (ChIP) assay. After treatment with Snail-specific siRNA, the expression of invasiveness-associated genes was measured using quantitative real-time PCR and Western blot. GA significantly inhibited the viability of PC3 cells at 1-5 μmol/L, but did not produce cytotoxic effect at the concentrations below 0.5 μmol/L. GA (0.125-0.5 μmol/L) dose-dependently inhibited the migration and invasion of PC3 cells induced by TNF-α (10 ng/mL). Moreover, the TNF-α-mediated activation of phosphatidylinositol-3-OH kinase/protein kinase B (PI3K/Akt) and NF-κB pathways was suppressed by GA (0.5 μmol/L). Furthermore, this anti-invasion effect of GA was associated with regulation of Snail. Snail expression was significantly down-regulated by treatment with GA (0.5 μmol/L) in the TNF-α-stimulated PC3 cells. GA inhibits TNF-α-induced invasion of PC3 cells via inactivation of the PI3K/Akt and NF-κB signaling pathways, which may offer a novel approach for the treatment of human prostate cancer.

Reelin Is Involved in Transforming Growth Factor-β1-Induced Cell Migration in Esophageal Carcinoma Cells

Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice.

Transcriptional Activation of ZEB1 by Slug Leads to Cooperative Regulation of the Epithelial–Mesenchymal Transition-Like Phenotype in Melanoma

The E-box-binding zinc finger transcription factors Slug and ZEB1 are important repressors of E-cadherin, contributing to epithelial-mesenchymal transition (EMT) in primary epithelial cancers. Activator or repressor status of EMT transcription factors defines consequences for tumorigenesis. We show that changes in expression levels of Slug in melanoma cell lines lead to concomitant alterations of ZEB1 expression. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays identified Slug as a direct transcriptional activator at E-boxes of the ZEB1 promoter. Transcriptional activation of ZEB1 was demonstrated to be specific for Slug, as EMT regulators Snail and Twist failed to influence ZEB1 expression. Slug and ZEB1 cooperatively repressed E-cadherin expression resulting in decreased adhesion to human keratinocytes, but promoted migration of melanoma cells. Our results show that the transcriptional activity of ZEB1 is increased by Slug, suggesting a hierarchical organized expression of EMT transcription factors through directed activation, triggering an EMT-like process in melanoma.

Mouse Snail Is a Target Gene for HIF

The transcriptional inhibitor Snail is a critical regulator for epithelial-mesenchymal transition (EMT). Although low oxygen induces Snail transcription, thereby stimulating EMT, a direct role of hypoxia-inducible factor (HIF) in this process remains to be demonstrated. Here we show that hypoxia induces the expression of Snail via HIF. In silico analysis identified a potential hypoxia-response element (HRE) close to the minimal promoter of the human and mouse genome of the snail gene. Gel shift assays demonstrated that a specific hypoxia-inducible complex is formed with the putative HRE and that the complex contains HIF proteins. ChIP assays confirmed the interaction of HIF proteins with the putative HRE in vivo. Reporter gene analyses showed that the putative HRE responds to hypoxia in its natural position as well as in front of a heterologous promoter and that the HRE is directly activated by HIF-1α or HIF-2α. HIF knockdown with siRNA at 2% oxygen and overexpression of an oxygen-insensitive HIF (HIF-ΔODD) mutant at 21% oxygen showed that HIF regulates Snail activation and subsequent cell migration. Our findings identify snail as a HIF target gene and provide novel insights into the regulation of snail and hypoxia-induced EMT.

Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-β(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker α-smooth muscle actin (α-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced α-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to α-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.

Lkb1 and Pten synergise to suppress mTOR-mediated tumorigenesis and epithelial-mesenchymal transition in the mouse bladder.

The AKT/PI3K/mTOR pathway is frequently altered in a range of human tumours, including bladder cancer. Here we report the phenotype of mice characterised by deletion of two key players in mTOR regulation, Pten and Lkb1, in a range of tissues including the mouse urothelium. Despite widespread recombination within the range of epithelial tissues, the primary phenotype we observe is the rapid onset of bladder tumorigenesis, with median onset of approximately 100 days. Single deletion of either Pten or Lkb1 had no effect on bladder cell proliferation or tumour formation. However, simultaneous deletion of Lkb1 and Pten led to an upregulation of the mTOR pathway and the hypoxia marker GLUT1, increased bladder epithelial cell proliferation and ultimately tumorigenesis. Bladder tissue also exhibited characteristic features of epithelial-mesenchymal transition, with loss of the epithelial markers E-cadherin and the tight junction protein ZO-1, and increases in the mesenchymal marker vimentin as well as nuclear localization of epithelial-mesenchymal transition (EMT) regulator Snail. We show that these effects were all dependent upon mTOR activity, as rapamycin treatment blocked both EMT and tumorigenesis. Our data therefore establish clear synergy between Lkb1 and Pten in controlling the mTOR pathway within bladder epithelium, and show that loss of this control leads to the disturbance of epithelial structure, EMT and ultimately tumorigenesis.

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1-4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation.

Epithelial-to-Mesenchymal Transition in Metaplastic Breast Carcinomas With Chondroid Differentiation

Epithelial-to-mesenchymal transition (EMT) is a key process in tumor progression and is induced by the master regulator Snail, a transcription factor that down-regulates epithelial genes, including E-cadherin, and up-regulates mesenchymal genes. Metaplastic breast carcinomas with chondroid differentiation (MBCD) have an epithelial and a mesenchymal chondroid component, giving them morphologic features reminiscent of EMT. To determine whether EMT plays a role in MBCD, we differentially analyzed Snail and E-cadherin expression in 12 tumors by immunohistochemistry. The interface between the tumor components showed a transition from inactive cytoplasmic Snail expression in the carcinoma component to active nuclear Snail expression in the metaplastic cells. Membranous E-cadherin staining was present in the epithelial and absent in the metaplastic component, showing a gradual loss of expression at the interface. E-cadherin expression was inversely correlated with active nuclear Snail expression. Our results suggest that EMT is induced in MBCD and may contribute to their frequent hematogenous metastasis.

Invasion and EMT-associated genes are up-regulated in B viral hepatocellular carcinoma with high expression of CD133-human and cell culture study

Hepatocellular carcinomas (HCCs) with expression of stem/progenitor cell markers including CD133 have been reported to have more aggressive biological behavior, and epithelial–mesenchymal transition (EMT), closely related invasion, has been suggested to generate cancer stem cells. To elucidate biological characteristics of HCCs expressing CD133, we evaluated migration assay and the mRNA expression levels of CD133, invasion-associated genes [urokinase plasminogen activator receptor (uPAR), villin 2 (VIL2), and MMP1 and MMP2], and EMT regulators (Snail, Slug, Twist, E-cadherin, and N-cadherin) by real-time PCR in HCC cell lines including HepG2, Hep3B, Huh7, PLC/RFP/6, SNU423, SNU449, and SNU475. Same genes and pathological features were also investigated in 49 samples of hepatitis B virus-related human HCCs. In all HCC cell lines studied, CD133-positive cells showed higher cell migration activity and up-regulated invasion- and EMT-associated genes with increased N-cadherin and decreased E-cadherin expressions compared to CD133-negative cells. The human HCCs were divided into the CD133-high group (top 40%) and the CD133-low group (bottom 40%) according to the level of CD133 mRNA. The CD133-high group showed relatively frequent vascular invasion and significantly higher expression of invasion-associated genes [uPAR ( p = 0.002), MMP1 ( p = 0.01), and MMP2 ( p = 0.003)] and EMT regulators [Snail ( p = 0.002) and Twist ( p = 0.0003)] compared to the CD133-low group. In conclusion, our results suggest that there is a subtype of HCC with high expression of CD133, which might have more invasive characteristics by up-regulation of invasion-associated genes and EMT-associated genes.

UV irradiation induces Snail expression by AP-1 dependent mechanism in human skin keratinocytes

Ultraviolet (UV) irradiation from the sun is the major cause of keratinocyte skin cancer. Transcription factor Snail plays an important role in epithelial-to-mesenchymal transition and epithelial tumor formation. The aims of this study are to determine the regulation of Snail expression of ultraviolet (UV) irradiation on Snail expression in human skin in vivo, and the mechanisms by which UV irradiation induces Snail expression, in human keratinocytes. Real-time RT-PCR was employed to measure Snail expression in human skin in vivo and cultured human keratinocytes. Luciferase assay and electrophoretic mobility shift assay (EMSA) were employed to investigate transcriptional regulation of the in Snail gene promoter. Ultraviolet (UV) irradiation transiently induces Snail expression in human skin in vivo and cultured human keratinocytes. Snail induction is significantly reduced by specific inhibitors of ERK, p38 or JNK, indicating each of the three major mitogen-activated protein kinase (MAPK) pathways participate in Snail regulation. AP-1 transcription factor complex, a downstream target of MAPK signaling, is required for Snail induction. Inhibition of AP-1 activity by over-expression of dominant-negative c-Jun substantially reduces Snail induction. Analyses of the Snail promoter, revealed the presence of an AP-1 binding site. EMSA assay demonstrated that UV irradiation specifically induced c-Jun binding to this AP-1 site. Mutation of the AP-1-binding site completely blocked protein binding and inhibited UV irradiation-induced Snail promoter activity. UV irradiation induces Snail gene expression in human skin keratinocytes. This induction is mediated by MAPK-AP-1 dependent signaling pathway. Elevated expression of Snail in response to chronic UV irradiation in human skin may contribute to UV irradiation-induced skin tumor development.

Differential regulation of Snail by hypoxia and hyperglycemia in human proximal tubule cells

The centrality of the transcriptional regulator Snail in epithelial-to-mesenchymal transformation (EMT), known to occur in models of diabetic nephropathy, has not been established. Transforming growth factor beta-1 (TGFβ1) is induced in diabetic nephropathy and induces both Snail and EMT. Hypoxia inducible factors (HIFs) are known to induce Snail, independent of TGFβ1. Notch induction is integral to Snail induction and EMT in tumour cells, but its role in the kidney is unknown. The present study was undertaken to determine the upstream regulators of Snail in the kidney in high glucose and hypoxic conditions. HK-2 cells were cultured in normoxic, hypoxic, high glucose and combined hypoxic/high glucose conditions. The expression of HIF1α, NotchIC, Snail, Lysyl oxidase-like 2 (Loxl2), and Hairy and Enhancer Split-1 (Hes1) were measured. We found that hypoxia increased HIF1α expression; however, concurrent exposure to high glucose blunted this effect. A similar pattern was observed in Lox12 expression, suggesting that Loxl2 was downstream of HIF1α, which was confirmed using siRNA techniques. Snail was upregulated by hypoxia and high glucose and in combination the effect was additive, suggesting independent upstream activation pathways by the two stimuli. Hes1 was upregulated by high glucose and to a lesser extent by hypoxia, but the effect of the combined stimuli was no greater than that observed with high glucose alone. NotchIC was downregulated by both hypoxia and high glucose, and in combination the effect was additive. Therefore, this study suggests that hypoxia and high glucose induce Snail expression through distinct pathways, independent of Notch signalling.

Abstract 2981: Regulation of Snail by DDX3 and ATM in response to DNA damage

Abstract Epithelial-mesenchymal transition (EMT) is the initial step for cancer invasion and metastasis. The hallmark of EMT is the loss of expression of E-cadherin, a cell-cell adhesion protein, allowing cancer cells to gain mobility leaving the site of primary tumor to invade adjacent tissues. Snail, as a transcription repressor, binds to the E-boxes of E-cadherin and then inhibits E-cadherin expression. Thus, Snail is essential to trigger EMT during tumor progression and it has been implicated in the acquisition of invasive growth phenotype of tumors. There is a strong correlation of Snail over-expression with tumor invasion and metastasis. However, how Snail expression is regulated is not fully known. Here we report that Snail is regulated by DDX3, a DEAD box protein family member which contributes to cancer progression. Knock-down of DDX3 by shRNA reduces the basal level of Snail, and this is associated with reduced cell proliferation and migration. Snail protein and mRNA levels are increased in the presence of the HDAC inhibitors sodium butyrate or trichostatin A, and the increases are attenuated in cells with DDX3 knocked down. Treatment of cells with a DNA damaging agent camptothecin also enhances Snail protein levels in a DDX3 dependent manner. Therefore we conclude that DDX3 is required for the basal level and the increase in Snail induced by HDAC inhibitors or camptothecin, indicating that this action of DDX3 may contribute to its promotion of the progression of some cancers. This conclusion is supported by our clinical investigations that analysis of 31 glioblastoma patient samples has revealed a strong correlation between the levels of DDX3 and Snail. Snail is a phosphor-protein, and kinases responsible for phosphorylation include Pak1 and GSK3-β. We also find Pak-1 and GSK-3 independent Snail phosphorylation in response to DNA damage. Further we find that inhibition of the activity of the ATM kinase, a critical element in DNA damage responses, eliminated DNA damage-induced Snail stabilization. Taken together, these observations have highlighted a comprehensive signaling network regulating Snail in response to DNA damage and indicated a critical role of optimal DNA damage responses in tumor invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2981.

Abstract 625: Transcription factor slug conferring resistance To EGFR tyrosine kinase inhibitor gefitinib

Abstract Purpose: Somatic EGFR mutations of non-small cell lung cancer (NSCLC) are reported to be associated with clinical responsiveness to tyrosine kinase inhibitor gefitinib. Despite the dramatic responses to gefitinib, most patients ultimately have a relapse. The underlying mechanism of the drug resistance is not well clear. Recently, many studies have shown that epithelial to mesenchymal transition (EMT) is involved in resistance to gefitinib in EGFR. Zinc finger transcription factor slug is one of the EMT regulators. In this study, we will investigate whether the slug plays a role in the gefitinib resistance. Materials and Methods: PC9/IR (gefitinib-resistant cell line) was isolated from PC9 (gefitinib-sensitive cell line) after long-term treatment of gefitinib. To identify the factors involved in gefitinib resistance, we analyzed the expression levels of EMT regulators (Slug, Snail, Twist, Zeb-1) in PC9/IR and PC9 cells using quantitative real time PCR and western blot. To investigate the association of Slug and gefitinib-induced resistance, we also engineered to stably express Slug and short hairpin RNA targeting Slug transcript in PC9 and PC9/IR, respectively. Results: The findings showed that mRNA and protein levels of Slug obviously increased in PC9/IR comparing with PC9 and expression of other EMT regulators Snail, Twist, Zeb-1 were similar between these two cell lines. After gefitinib treatment, PC9/IR expressing shRNA against Slug underwent apoptosis and activation of the intrinsic apoptosis pathway. Ectopic expression of Slug protected PC9 cells from undergoing apoptosis in response to gefitinib treatment. Knockdown the expression of slug reversed gefitinib resistance of PC9/IR cells by restoring Bim expression. Conclusions: These results suggest that expression of slug may contribute to resistance to gefitinib in PC9/IR and may be a useful target for treatment of resistance to EGFR tyrosine kinase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 625.