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1.  Snail1 expression in colorectal cancer and its correlation with clinical and pathological parameters 
BMC Cancer  2013;13:145.
Snail1 is a transcription regulator of E-cadherin. The loss of E-cadherin seems to be a crucial step in the process of Epithelial-mesenchymal transition (EMT). EMT initiates invasion and proliferation in many tumours. Overexpression of Snail1 is known to be associated with poor outcome in several solid tumours. The aim of this study was to analyse its expression profile and prognostic significance in colorectal cancer.
Tissue microarrays (TMA) containing paraffin-embedded primary colorectal cancer (CRC) tissue samples from 251 patients were used in this study. The expression of Snail1 and E-cadherin was assessed by immunohistochemistry in different tumour compartments, corresponding lymph node metastases and normal colonic mucosa. Intensity of staining was classified according to the Remmele score (standardized scoring system) as well as the semiquantitative score established by Blechschmidt et al.
Snail1 expression was observed in 76% of the CRC. Loss of E-cadherin was noted in 87% of the CRC. Snail1 positive tumours were significantly correlated with Snail1 positive lymph node metastases (p=0.03). There was no significant correlation between loss of E-cadherin and Snail1 expression, or between N-stage or grading and Snail1 expression. Kaplan-Meier survival analysis identified no prognostic impact of Snail1 expression on overall survival.
Snail1 expression was detectable in most of the CRC but showed no significant association with E-cadherin loss, clinical pathological characteristics or overall survival. The observed loss of E-cadherin could be explained by effects of other important EMT pathways, such as the Wnt-signalling cascade.
PMCID: PMC3617032  PMID: 23522088
Snail1; E-cadherin; Colorectal carcinoma; Prognostic factor; EMT
2.  Nuclear expression of Snail1 in borderline and malignant epithelial ovarian tumours is associated with tumour progression 
BMC Cancer  2009;9:289.
Transcription factor Snail1 has a central role in induction of epithelial-mesenchymal transition (EMT). The aim of the present study was to elucidate the expression of Snail1 protein during epithelial ovarian tumourigenesis and to study the association of Snail1 expression with clinicopathological factors and prognosis.
Epithelial and stromal fibroblast-like fusiform cells of 14 normal ovarian samples, 21 benign, 24 borderline and 74 malignant epithelial ovarian tumours were studied for Snail1 protein using immunohistochemistry.
Nuclei of surface peritoneal cells of normal ovaries (n = 14) were regarded as negative for Snail1. Nuclear expression of Snail1 protein in epithelial ovarian tumours was increased during tumour progression from precursor lesions into carcinomas both in epithelial (p = 0.006) and stromal cells (p = 0.007). Nuclei of benign tumours (n = 21) were negative for Snail1. In borderline tumours (n = 24) occasional positive epithelial cells were found in 2 (8%) samples and in 3 (13%) samples stromal cells were focally positive for Snail1. In carcinomas (n = 74) focal Snail1 staining in epithelial cells was present in 17 (23%) tumours, and in stromal cells in 18 (24%) tumours. Nuclear expression of Snail1 in epithelial or stromal cells was not associated with clinicopathological factors or prognosis.
Nuclear Snail1 expression seems to be related to tumour progression, and expression in borderline tumours indicates a role for Snail1 in early epithelial ovarian tumour development. Snail1 also appears to function more generally in tissue remodelling as positive staining was demonstrated in stromal cells.
PMCID: PMC3087336  PMID: 19695091
3.  Prognostic significance of snail expression in hilar cholangiocarcinoma 
Many patients with hilar cholangiocarcinoma (HC) have a poor prognosis. Snail, a transcription factor and E-cadherin repressor, is a novel prognostic factor in many cancers. The aim of this study was to evaluate the relationship between snail and E-cadherin protein expression and the prognostic significance of snail expression in HC. We examined the protein expression of snail and E-cadherin in HC tissues from 47 patients (22 males and 25 females, mean age 61.2 years) using immunohistochemistry and RT-PCR. Proliferation rate was also evaluated in the same cases by the MIB1 index. High, low and negative snail protein expression was recorded in 18 (38%), 17 (36%), and 12 (26%) cases, respectively, and 40.4% (19/47) cases showed reduced E-cadherin protein expression in HC samples. No significant correlation was found between snail and E-cadherin protein expression levels (P = 0.056). No significant correlation was found between snail protein expression levels and gender, age, tumor grade, vascular or perineural invasion, nodal metastasis and invasion, or proliferative index. Cancer samples with positive snail protein expression were associated with poor survival compared with the negative expresser groups. Kaplan-Meier curves comparing different snail protein expression levels to survival showed highly significant separation (P < 0.0001, log-rank test). With multivariate analysis, only snail protein expression among all parameters was found to influence survival (P = 0.0003). We suggest that snail expression levels can predict poor survival regardless of pathological features and tumor proliferation. Immunohistochemical detection of snail protein expression levels in routine sections may provide the first biological prognostic marker.
PMCID: PMC3854268  PMID: 22570087
Hilar cholangiocarcinoma; Prognostic marker; Snail; E-cadherin; Immunohistochemistry
4.  Roles of transcriptional factor Snail and adhesion factor E-cadherin in clear cell renal cell carcinoma 
The aim of this study was to investigate the roles of the transcription factor Snail and adhesion factor epithelial-cadherin (E-cadherin) in clear cell renal cell carcinoma (CCRCC) and evaluate their correlation with tumor pathological grading, clinical stage, invasion and metastases. The expression of Snail and E-cadherin protein in 69 samples of CCRCC tissue, 58 samples of para-cancerous mucosa and 10 samples of normal renal tissue were detected using the immunohistochemical streptavidin-peroxidase method. The positivity rate of Snail in CCRCC was 82.61%, which was significantly higher than that in para-cancerous mucosa (43.10%, P<0.001). The positivity rate of E-cadherin in CCRCC was 31.88%, which was significantly lower than that in para-cancerous mucosa (91.38%, P<0.001). The expression of E-cadherin and Snail correlated significantly with tumor differential degree, clinical stage and the depth of tumor invasion and distant metastasis (P<0.05). There was a negative correlation between the expression of E-cadherin and Snail in CCRCC. The overexpression of Snail and reduced expression of E-cadherin may be important biological markers for the invasion and metastasis of CCRCC. The combined detection of E-cadherin and Snail has far-reaching significance for the prediction of CCRCC invasion and metastasis.
PMCID: PMC3829762  PMID: 24255679
clear cell renal cell carcinoma; E-cadherin; Snail; epithelial-mesenchymal transition
5.  Nuclear Snail1 and nuclear ZEB1 protein expression in invasive and intraductal human breast carcinomas 
Human Pathology  2011;42(8):1125-1131.
Snail1 and ZEB1 are transcriptional repressors that drive tumor initiation and metastasis in animal models. Snail1 and ZEB1 are frequently coexpressed in tumor cell lines, suggesting that these factors may cooperate to promote tumor progression. However, coexpression of these transcriptional repressors in primary human cancer specimens has not been investigated. Previous studies assessed expression in primary breast cancers of Snail1 messenger RNA, which does not reflect Snail1 activity because Snail1 is subject to posttranslational modifications that inhibit its nuclear localization/activity. In the current study, using breast tumor cell lines of known Snail1 and ZEB1 expression status, we developed immunohistochemistry protocols for detecting nuclear Snail1 and nuclear ZEB1 proteins. Using these protocols, we assessed nuclear Snail1 and nuclear ZEB1 expressions in primary human breast cancers of varying subtypes (n = 78). Nuclear Snail1 and estrogen receptor α expression were inversely associated in primary breast cancers, and nuclear Snail1 was expressed in approximately 80% of triple-negative breast cancers (lacking estrogen receptor α, progesterone receptor, and human epidermal growth factor receptor 2 overexpression). In contrast, nuclear ZEB1 was expressed at a significantly lower frequency in these breast cancers. Notably, nuclear Snail1 protein was detected in 45% of ductal carcinoma in situ specimens (n = 29), raising the important possibility that nuclear Snail1 expression in early stage breast lesions may predict future development of invasive breast cancer. Collectively, our studies demonstrate frequent expression of nuclear Snail1, but not nuclear ZEB1, in invasive, triple-negative breast cancers as well as in intraductal carcinomas.
PMCID: PMC3315281  PMID: 21315410
Snail1; ZEB1; Breast cancer; Estrogen receptor; Ductal carcinoma in situ
6.  Novel Snail1 Target Proteins in Human Colon Cancer Identified by Proteomic Analysis 
PLoS ONE  2010;5(4):e10221.
The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT.
Methodology/Principal Findings
We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4) that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and the Splicing factor proline/glutamine-rich (SFPQ), was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3ε, 14-3-3τ, 14-3-3ζ and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3σ and Snail1 expression in human colorectal tumors.
We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.
PMCID: PMC2857666  PMID: 20421926
7.  Bmi-1 expression predicts prognosis in squamous cell carcinoma of the tongue 
British Journal of Cancer  2010;102(5):892-897.
The prognosis of squamous cell carcinoma of the oral tongue is poor and it would be beneficial to find prognostic markers to better adjust treatment. Bmi-1 controls cell cycle and self-renewal of tissue stem cells, transcription factor c-myc affects cell proliferation and apoptosis, and Snail regulates epithelial–mesenchymal transition. The expression of these markers has been connected to prognosis in many cancer types.
Bmi-1, c-myc, and Snail expressions were studied in our material consisting of 73 primarily T1N0M0 oral tongue carcinoma patients. We compared the immunoexpressions of Bmi1, c-myc, and Snail with clinical parameters including the degree of histological differentiation, tumour size, TNM classification, depth of invasion, and resection margins. In addition, survival analyses were performed, comparing disease-free survival time with the registered protein expression of the markers mentioned above.
A significant correlation between Bmi-1 protein expression and recurrence (log-rank test, P=0.005) was detected. Snail and c-myc expression did not correlate with prognosis. Snail expression correlated with histopathological grade (Fisher's exact test, P=0.007) and with the invasion depth of tumours (χ2-test, P=0.037).
Negative Bmi-1 immunoexpression might serve as a marker of poor prognosis in oral tongue carcinoma patients.
PMCID: PMC2833245  PMID: 20145620
Bmi-1; c-myc; Snail; oral cancer; prognosis; immunohistochemistry
8.  TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study 
BMC Cancer  2007;7:156.
Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM) on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta) signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited.
Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4) in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2) vs. high-grade (i.e. grade 3 and 4)), lymph node metastasis, distant metastasis, 5 year cancer related survival) using Chi-square or Fisher's exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed.
High-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and shorter survival. Stromal TGF-beta receptor 2 expression was an independent prognostic factor for cancer related survival.
Histological phenotype and clinical behaviour of colon cancer is not only influenced by mutational incidents in tumour cells but also affected by interaction of tumour tissue with inflammatory cells like macrophages and associated stroma and TGF-beta signalling is one important part of this crosstalk. Further studies are needed to elucidate the exact mechanisms.
PMCID: PMC1988827  PMID: 17692120
9.  High levels of cleaved caspase-3 in colorectal tumour stroma predict good survival 
British Journal of Cancer  2013;108(10):2097-2105.
The primary aim was to determine the prognostic significance of apoptosis in colorectal tumour cells and tumour-associated stroma. A secondary aim was to determine whether apoptosis was related to immune surveillance.
Immunohistochemistry was performed using monoclonal antibodies recognising cleaved caspase-3 (CC3), cleaved poly (ADP-ribose) polymerase (PARP), p53, Bcl2, MHC-II, B cells (CD16), macrophages (CD68) and T cells (CD3), on a tissue microarray of 462 colorectal tumours.
Kaplan–Meier analysis demonstrated that patients with high expression of CC3 in the tumour or CC3 or cleaved PARP in tumour-associated stroma have a good prognosis. This suggests that tumour stroma is promoting tumourigenesis and that high levels of death within the stroma breaks this link. CC3 levels in the tumour correlated with cleaved PARP and MHC-II expression but not with CD16, CD68, CD3, p53 or Bcl2 expression. CC3 levels on tumour-associated stroma also correlated with cleaved PARP and MHC-II expression but not with CD16, CD68, CD3, p53 or Bcl2 expression. Tumour cells express MHC-II in response to IFN-γ, suggesting that this may be one of the initiators of apoptosis within the good prognosis tumours. Although 73% of the MHC-II-positive tumour had high levels of apoptosis, many tumours had high levels of apoptosis in the absence of MHC-II, implying that this is only one of many causes of apoptosis within tumours. On multivariate analysis, using Cox's proportional hazards model, tumour stage, vascular invasion and expression of CC3 in tumour-associated stroma were shown to be independent markers of prognosis.
This study shows that a high level of apoptosis within colorectal tumour-associated stroma is an independent marker of good prognosis.
PMCID: PMC3670501  PMID: 23591201
Apoptosis; cleaved caspase-3; cleaved PARP; MHC-II; colorectal cancer; tumour-associated stroma
10.  Expression of epithelial to mesenchymal transition-related markers in lymph node metastases as a surrogate for primary tumor metastatic potential in breast cancer 
Breast cancers are phenotypically and genotypically heterogeneous tumors containing multiple cancer cell populations with various metastatic potential. Aggressive tumor cell subpopulations might more easily be captured in lymph nodes metastases (LNM) than in primary tumors (PT). We evaluated mRNA and protein levels of master EMT regulators: TWIST1, SNAIL and SLUG, protein levels of EMT-related markers: E-cadherin, vimentin, and expression of classical breast cancer receptors: HER2, ER and PgR in PT and corresponding LNM. The results were correlated with clinicopathological data and patients outcomes.
Formalin-fixed paraffin-embedded samples from PT and matched LNM from 42 stage II-III breast cancer patients were examined. Expression of TWIST1, SNAIL and SLUG was measured by reverse-transcription quantitative PCR. Protein expression was examined by immunohistochemistry on tissue microarrays. Kaplan-Meier curves for disease-free survival (DFS) and overall survival (OS) were compared using F-Cox test. Hazard ratios (HRs) with 95% confidence intervals (95% CI) were computed using Cox regression analysis.
On average, mRNA expression of TWIST1, SNAIL and SLUG was significantly higher in LNM compared to PT (P < 0.00001 for all). Gene and protein levels of TWIST1, SNAIL and SLUG were highly discordant between PT and matched LNM. Increased mRNA expression of TWIST1 and SNAIL in LNM was associated with shorter OS (P = 0.04 and P = 0.02, respectively) and DFS (P = 0.02 and P = 0.01, respectively), whereas their expression in PT had no prognostic impact. Negative-to-positive switch of SNAIL protein correlated with decreased OS and DFS (HR = 4.6; 1.1-18.7; P = 0.03 and HR = 3.8; 1.0-48.7; P = 0.05, respectively).
LNM are enriched in cells with more aggressive phenotype, marked by elevated levels of EMT regulators. High expression of TWIST1 and SNAIL in LNM, as well as negative-to-positive conversion of SNAIL confer worse prognosis, confirming the correlation of EMT with aggressive disease behavior. Thus, molecular profiling of LNM may be used as surrogate marker for aggressiveness and metastatic potential of PT.
PMCID: PMC3524044  PMID: 23157797
Breast cancer; Primary tumor; Lymph node metastasis; Gene expression; Immunohistochemistry; Biomarkers; Epithelial-mesenchymal transition
11.  14-3-3 binding sites in the Snail protein are essential for Snail-mediated transcriptional repression and epithelial-mesenchymal differentiation 
Cancer research  2010;70(11):4385-4393.
The Snail transcription factor is a repressor and a master regulator of epithelial-mesenchymal transition events (EMT) in normal embryonic development and during tumor metastases. Snail directly regulates genes affecting cell adhesion, motility and polarity. Invasive tumor cells express high levels of Snail and it is a marker for aggressive disease and poor prognosis. Transcriptional repression and EMT induction by Snail requires binding to its obligate corepressor, the LIM protein Ajuba. It is unclear how this complex is assembled and maintained on Snail target genes. Here we define functional 14-3-3 binding motifs in Snail and Ajuba which selectively bind 14-3-3 protein isoforms. In Snail, a NH2-terminal motif in the repression domain cooperates with a COOH-terminal, high affinity motif for binding to 14-3-3 proteins. Coordinate mutation of both motifs abolishes 14-3-3 binding and inhibits Snail-mediated gene repression and EMT differentiation. Snail, 14-3-3 proteins, and Ajuba form a ternary complex which is readily detected via ChIP at the endogenous E-cadherin promoter. Collectively, these data show that 14-3-3 proteins are new components of the Snail transcriptional repression machinery and mediate its important biological functions.
PMCID: PMC2894621  PMID: 20501852
Snail; 14-3-3; Ajuba; epithelial-mesenchymal transition; transcriptional repression
12.  Expression of the zinc-finger transcription factor Snail in adrenocortical carcinoma is associated with decreased survival 
British Journal of Cancer  2008;99(11):1900-1907.
In this study, we evaluate whether Snail is expressed in adrenocortical cancer (ACC) and if its expression is related to patient outcome. One of the best known functions of the zinc-finger transcription factor Snail is to induce epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT plays a pivotal role in tumour progression and metastatic spread. Snail and E-cadherin expression were assessed by immunohistochemistry in 26 resected ACCs and real-time quantitative RT–PCR expression analysis was performed. Data were correlated with clinical outcome and in particular with overall patient survival. Seventeen of 26 (65%) ACC tumour samples expressed Snail when assessed by immunohistochemistry. Snail expression was neither detected in normal adrenocortical tissue, nor in benign adrenocortical adenomas. Expression levels were confirmed on the mRNA level by Real-Time–PCR. Survival rates were significantly decreased in Snail-positive tumours compared to Snail-negative tumours: 10 out of 16 vs one out of eight patients succumbed to disease after a median follow up of 14.5 and 28.5 months, respectively (P=0.03). Patients with Snail-expressing ACCs presented in advanced disease (11 out of 12 vs 6 out of 14, P=0.01) and tend to develop distant metastases more frequently than patients with negative staining (7 out of 11 vs two out of eight, P=0.19). In conclusion, we describe for the first time that Snail is expressed in a large subset of ACCs. Furthermore, Snail expression is associated with decreased survival, advanced disease and higher risk of developing distant metastases.
PMCID: PMC2600683  PMID: 19018264
adrenocortical carcinomas; Snail; survival
13.  The Use of Automated Quantitative Analysis to Evaluate Epithelial-to-Mesenchymal Transition Associated Proteins in Clear Cell Renal Cell Carcinoma 
PLoS ONE  2012;7(2):e31557.
Epithelial-to-mesenchymal transition (EMT) has recently been implicated in the initiation and progression of renal cell carcinoma (RCC). Some mRNA gene expression studies have suggested a link between the EMT phenotype and poorer clinical outcome from RCC. This study evaluated expression of EMT-associated proteins in RCC using in situ automated quantitative analysis immunofluorescence (AQUA) and compared expression levels with clinical outcome.
Methods/Principal Findings
Unsupervised hierarchical cluster analysis of pre-existing RCC gene expression array data (GSE16449) from 36 patients revealed the presence of an EMT transcriptional signature in RCC [E-cadherin high/SLUG low/SNAIL low]. As automated immunofluorescence technology is dependent on accurate definition of the tumour cells in which measurements take place is critical, extensive optimisation was carried out resulting in a novel pan-cadherin based tumour mask that distinguishes renal cancer cells from stromal components. 61 patients with ccRCC and clinical follow-up were subsequently assessed for expression of EMT-associated proteins (WT1, SNAIL, SLUG, E-cadherin and phospho-β-catenin) on tissue microarrays. Using Kaplan-Meier analysis both SLUG (p = 0.029) and SNAIL (p = 0.024) (log rank Mantel-Cox) were significantly associated with prolonged progression free survival (PFS). Using Cox regression univariate and multivariate analysis none of the biomarkers were significantly correlated with outcome. 14 of the 61 patients expressed the gene expression analysis predicted EMT-protein signature [E-cadherin high/SLUG low/SNAIL low], which was not found to be associated to PFS when measured at the protein level. A combination of high expression of SNAIL and low stage was able to stratify patients with greater significance (p = 0.001) then either variable alone (high SNAIL p = 0.024, low stage p = 0.029).
AQUA has been shown to have the potential to identify EMT related protein targets in RCC allowing for stratification of patients into high and low risk groups, as well the ability to assess the association of reputed EMT signatures to progression of the disease.
PMCID: PMC3283650  PMID: 22363672
14.  Expression of transcription factors snail, slug, and twist in human bladder carcinoma 
Slug, Snail, and Twist are transcription factors that regulate the expression of tumor suppressors such as E-cadherin. In this study, we aimed to examine the expression of these transcription factors in human bladder carcinoma.
We first investigated expression of Slug, Snail, Twist and E-cadherin in five bladder Carcinoma cell lines by reverse transcription-polymerase chain reaction and western blotting. Furthermore, we investigated Slug, Snail, and Twist and E-cadherin expression by immunohistochemistry with bladder carcinoma (tumor, n = 120; background, n = 42).
Expression of Slug mRNA and protein was detected in all cell lines, Twist was clearly expressed in two out of five bladder carcinoma cell lines, Snail was not expressed, and E-cadherin was detected in 3 cell lines. 44.2% (53/120) of human bladder Carcinoma tissues and 38%(16/42) background tissue showed an expression of Twist; 62.5%(75/120) of human bladder Carcinoma tissues and 40%(17/42) background tissue showed an expression of Slug, 15.8% (19/120) of human bladder Carcinoma tissues and 76%(32/42) background tissue showed an expression of Snail, and 25.8% (31/120) cases were negative for E-cadherin expression in carcinoma tissues. Expression of Slug and Twist shows increased levels in tumors, whereas Snail seems reduced. Statistically significant correlations were found between Twist, Slug, and E-cadherin expression. Immunohistochemistry analysis showed that Twist was elevated with increasing tumor stage (P = 0.001), the grade (P < 0.001), the progression (P = 0.035). Slug was elevated and Snail was reduced with increasing nodal involvement (tumor-node-metastasis status) (P = 0.004, P = 0.01). E-cadherin was reduced in expression corresponding with tumor grade (P < 0.01). Positive Twist, Slug and E-cadherin expression clearly predicted poorer PFS (P = 0.042, P = 0.014, P = 0.001). In the multivariate analysis, only Snail and E-cadherin expression were independent prognostic factors for OS (P = 0.002, P < 0.001).
These data demonstrate that Twist, Snail and Slug have inappropriate expression in bladder carcinoma and that this may play a part in the progression of human bladder carcinoma.
PMCID: PMC2942802  PMID: 20809941
15.  Snail controls the mesenchymal phenotype and drives erlotinib resistance in Oral epithelial and HNSCC cells 
The presence of regional metastases in HNSCC patients is a common and adverse event associated with poor prognosis. Understanding the molecular mechanisms that mediate HNSCC metastasis may enable identification of novel therapeutic targets. Our recent work on human HNSCC tissues underlies Snail’s role as a molecular prognostic marker for HNSCC. Snail positivity is significantly predictive of poorly differentiated, lymphovascular invasive, as well as regionally metastatic tumors. We recently reported the role of Snail in the inflammation-induced promotion of EMT in HNSCC. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the capacity of Snail to drive EMT in human oral epithelial cell lines, and its ability to confer drug resistance.
Snail was overexpressed HNSCC and oral epithelial cell lines. AIG assays, wound healing assays, invasion & migration assays, spheroid modeling, and cell survival assays were performed.
The overexpression of Snail in human HNSCC and oral epithelial cell lines drives EMT. The sole transfection of Snail confers the expression of a mesenchymal molecular signature including down-regulation of the epithelial adherens, such as E-cadherin and β-catenin, and induction of mesenchymal markers, Snail overexpressing cell lines demonstrate rapid growth in Anchorage-independent growth assays; a decreased capacity to form tight spheroids; increased resistance to erlotinib; and have an increased capacity for invasion.
Snail controls the mesenchymal phenotype and drives erlotinib resistance in HNSCC cells. Snail may prove to be a useful marker in predicting EGFR inhibitor responsiveness.
PMCID: PMC4167686  PMID: 22568942
16.  Expression of Snail transcription factor in prostatic adenocarcinoma in Egypt: correlation with Maspin protein expression and clinicopathologic variables 
Background: Snail transcription factor and Maspin tumor suppressor serpin are involved in the regulation of progression, invasion and metastasis of many human malignancies. However, there is very limited data in the literature about their role in prostatic adenocarcinoma. The present study was designed to investigate Snail and Maspin expression, their interrelationship and their relationship to different clinicopathologic variables in clinically detectable prostatic adenocarcinoma. Material and methods: Tissue sections from 110 resected prostatic lesions distributed as 80 cases of prostatic adenocarcinoma and 30 cases of benign prostatic hyperplasia (BPH) were evaluated for Snail and Maspin proteins expression by immunohistochemistry. Results: Snail protein expression was detected in 53.8% of prostatic adenocarcinomas versus none of BPH cases (p = < 0.001). A significant positive correlation of Snail expression to cancer grade (p = 0.015), lymph node metastasis (p = 0.026) and pTNM stage (p = 0.036). Maspin expression was detected in 36.6% of prostatic adenocarcinomas versus 93.3% of BPH cases (p = < 0.001). A significant negative correlation of Maspin expression to cancer grade (p = 0.007) and lymphovascular invasion (p = 0.017). Also detected was a significant negative relationship between Snail and Maspin expression in cancer cases under investigation (p = 0.002). Conclusion: Snail immunohistochemical expression can be promising as a potential prognostic biomarker in prostatic adenocarcinoma since it was significantly associated with clinicopathologic variables of progressive disease. A potential role for Snail in regulating Maspin expression is suggested based on the finding of negative association between Snail and Maspin expression in prostatic adenocarcinoma.
PMCID: PMC3726971  PMID: 23923074
Benign prostatic hyperplasia; immunohistochemistry; Maspin; prostatic adenocarcinoma; Snail; clinicopathologic variables
17.  Analysis of Snail-1, E-Cadherin and Claudin-1 Expression in Colorectal Adenomas and Carcinomas 
We report the expression of Snail-1, E-cadherin and claudin-1 by indirect immunohistochemistry in colonic neoplasia. Snail-1 is a zinc finger transcription factor expressed in cells that already have undergone almost complete epithelial-mesenchymal transition (EMT) and have already evaded from the tumor. The main mechanism by which Snail induces EMT is downregulation of E-cadherin, of which expression was shown to be frequently downregulated in many different types of tumors, where it accompanies the invasiveness and metastatic behavior of malignant cells. Moreover, Snail-1 may downregulate the expression of claudin-1, a cell-cell adhesion protein which plays a likely role in progression and dissemination during tumorigenesis. Snail-1 was expressed in both carcinoma and adenoma cells with histologically normal epithelium in the mucosa, adjacent to the tumors, without significant differences, and predominant strong intensity of staining. Statistically significant differences were revealed between normal and tumorous epithelium (p = 0.003) at the subcellular level, where the shift of the protein to the cytoplasm with combined cytoplasmic/nuclear or pure cytoplasmic expression was observed. E-cadherin expression was present in 100% of cases of both adenocarcinomas and adenomas, with prevailing strong membranous immunoreactivity and no differences between protein expression in tumors and normal mucosa. Predominating strong positivity of claudin-1 was detected in tumor cells of adenocarcinomas and adenomas. Marked differences were seen in protein localization, where membranous staining, typical for nontumorous epithelium, changed to combined membranous/cytoplasmic expression in adenocarcinomas (p = 0.0001) and adenomas (0.0002), in which cytoplasmic shift was associated with a higher degree of dysplasia. Furthermore, membranous/cytoplasmic localization was more frequent in the carcinoma group (87%) in comparison with adenomas (51%) (p = 0.0001). We conclude that dystopic subcellular localizations of Snail-1 and claudin-1 may participate in changes of cellular morphology and behavior which might be associated with altered effectory pathways of proteins and thus substantially contribute to the cancer development.
PMCID: PMC3291982  PMID: 22408413
Snail-1; E-cadherin; claudin-1; adenocarcinoma; adenoma; immunohistochemistry
18.  Snail promotes CXCR2 ligand dependent tumor progression in NSCLC 
As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition (EMT), invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of non-small cell lung cancer (NSCLC).
Experimental Design
Immunohistochemistry was performed to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis (TMA) was utilized to correlate these findings with survival. NSCLC cell lines gene-modified to stably over-express Snail were evaluated in vivo in two severe combined immunodeficiency (SCID) murine tumor models. Differential gene expression between Snail over-expressing and control cell lines was evaluated using gene expression microarray analysis.
Snail is up-regulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (p<0.026). In a heterotopic model, mice bearing Snail over-expressing tumors developed increased primary tumor burden (p=0.008). In an orthotopic model, mice bearing Snail over-expressing tumors also demonstrated a trend toward increased metastases. In addition, Snail over-expression led to increased angiogenesis in primary tumors as measured by MECA-32 (p<0.05) positivity and CXCL8 (p=0.002) and CXCL5 (p=0.0003) concentrations in tumor homogenates. Demonstrating the importance of these pro-angiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression.
Snail up-regulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands.
PMCID: PMC2783274  PMID: 19887480
Snail; lung cancer; angiogenesis; CXCL8; CXCL5
19.  Activation of the ATM-Snail pathway promotes breast cancer metastasis 
The DNA damage response (DDR) is critical for the maintenance of genetic stability and serves as an anti-cancer barrier during early tumorigenesis. However, the role of the DDR in tumor progression and metastasis is less known. Here, we demonstrate that the ATM kinase, one of the critical DDR elements, is hyperactive in late stage breast tumor tissues with lymph-node metastasis and this hyperactivity correlates with elevated expression of the epithelial–mesenchymal transition marker, Snail. At the molecular level, we demonstrate that ATM regulates Snail stabilization by phosphorylation on Serine-100. Using mass spectrometry, we identified HSP90 as a critical binding protein of Snail in response to DNA damage. HSP90 binds to and stabilizes phosphorylated Snail. We further provide in vitro and in vivo evidence that activation of ATM-mediated Snail phosphorylation promotes tumor invasion and metastasis. Finally, we demonstrate that Snail Serine-100 phosphorylation is elevated in breast cancer tissues with lymph-node metastasis, indicating clinical significance of the ATM-Snail pathway. Together, our findings provide strong evidence that the ATM-Snail pathway promotes tumor metastasis, highlighting a previously undescribed role of the DDR in tumor invasion and metastasis.
PMCID: PMC3464396  PMID: 22923499
ATM; snail; metastasis
20.  Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation 
BMC Cancer  2009;9:235.
A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC.
PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.
When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.
ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.
PMCID: PMC2717979  PMID: 19604397
21.  Expression of e-cadherin, n-cadherin and snail and their correlation with clinicopathological variants: an immunohistochemical study of 132 invasive ductal breast carcinomas in Egypt 
Clinics  2011;66(10):1765-1771.
To evaluate the expression of the cell adhesion molecules E-cadherin and N-cadherin and the transcription factor Snail in invasive ductal breast carcinomas and to determine their relationships with clinicopathological features.
Immunohistochemistry was used to examine E-cadherin, N-cadherin, and Snail protein expression in 132 invasive breast carcinomas.
The expression of E-cadherin was decreased (negative or weak) in 37.1% of invasive carcinomas, while N-cadherin and Snail overexpression were detected in 51.9% and 40.9% of carcinomas, respectively. Low E-cadherin expression was significantly correlated with poorly differentiated carcinoma (53.1%), positive node status (80.9%), poor Nottingham Prognostic Index (64.7%), and the presence of estrogen and progesterone receptors. Overexpression of N-cadherin and Snail were also significantly correlated with poorly differentiated carcinoma, positive node status, and poor Nottingham Prognostic Index but were correlated with the absence of hormone receptors. Loss of E-cadherin immunoexpression was strongly associated with the presence of membranous N-cadherin (87.8%) and nuclear Snail (69.4%).
Loss of E-cadherin and overexpression of N-cadherin and Snail in breast carcinomas may play a central role in the development of invasive ductal breast carcinoma. These biomarkers may provide a valuable reference for the study of invasive ductal carcinoma progression and to characterize the biological behavior of the tumor. In the future, increased N-cadherin and decreased E-cadherin expression may be used as indicators of the progression and prognosis of invasive ductal carcinoma.
PMCID: PMC3180143  PMID: 22012049
E-cadherin; Immunohistochemistry; Invasive ductal carcinoma; N-cadherin; Snail
22.  Expression of Snail2 in long bone osteosarcomas correlates with tumour malignancy 
Tumour Biology  2011;32(3):515-526.
Snail2 is a marker of malignancy in epithelial tumours; however, in sarcomas, it is not known if this protein is present. Here we examine the expression of Snail2 in one type of sarcoma, osteosarcoma, and explore its relationship to tumour grade, subtype and anatomical location in cases of long bone and cranial bone osteosarcoma. Long bone osteosarcomas typically have a much greater metastatic capability and a poorer prognosis. We find that Snail2 is expressed in the three main subtypes of long bone osteosarcoma—osteoblastic, chondroblastic and fibroblastic. Regression analysis showed that Snail 2 expression was statistically correlated with tumour grade (p = 0.014) in all of these subtypes. Snail2 was only expressed in high-grade cranial bone osteosarcomas, suggesting a link between Snail2 expression and metastasis. This is the first time Snail2 has been associated with any sarcoma, and this study shows that Snail2 may be a useful prognostic marker for this disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s13277-010-0146-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3109975  PMID: 21207222
Osteosarcoma; Snail2; Runx2; Prognostic marker
23.  Expression of cyclooxygenase-2 (COX-2) in tumour and stroma compartments in cervical cancer: clinical implications 
British Journal of Cancer  2002;87(10):1145-1152.
This study aims at investigating the relationship between cyclooxygenase-2 expression in tumour vs stroma inflammatory compartment and its possible clinical role. The study included 99 stage IB-IV cervical cancer patients: immunostaining of tumour tissue sections was performed with rabbit antiserum against cyclooxygenase-2. CD3, CD4, CD8, CD25, Mast Cell Tryptase monoclonal antibodies were used to characterise stroma inflammatory cells in nine cervical tumours. An inverse relation was found between cyclooxygenase-2 levels (cyclooxygenase-2 IDV) of tumour vs stroma compartment (r=−0.44, P<0.0001). The percentage of cases showing high tumour/stromal cyclooxygenase-2 IDV ratio was significantly higher in patients who did not respond to treatment (93.3%) with respect to patients with partial (60.5%), and complete (43.7%) response (P= 0.009). Cases with a high tumour/stroma cyclooxygenase-2 IDV ratio had a shorter overall survival rate than cases with a low tumour/stroma cyclooxygenase-2 IDV (P<0.0001). In the multivariate analysis advanced stage and the status of tumour/stroma cyclooxygenase-2 IDV ratio retained an independent negative prognostic role. The proportion of CD3+, CD4+, and CD25+ cells was significantly lower in tumours with high tumour/stroma cyclooxygenase-2 IDV ratio, while a higher percentage of mast cells was detected in tumours showing high tumour/stroma cyclooxygenase-2 IDV ratio. Our study showed the usefulness of assessing cyclooxygenase-2 status both in tumour and stroma compartment in order to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant therapy and unfavourable prognosis.
British Journal of Cancer (2002) 87, 1145–1152. doi:10.1038/sj.bjc.6600578
© 2002 Cancer Research UK
PMCID: PMC2376190  PMID: 12402155
cervical cancer; COX-2; prognosis; chemotherapy response
24.  Snail promotes an invasive phenotype in lung carcinoma 
Respiratory Research  2012;13(1):104.
Snail is a transcriptional factor which is known to influence the epitheliomesenchymal transition (EMT) by regulating adhesion proteins such as E-cadherin and claudins as well as matrix metalloproteases (MMP).
To evaluate the functional importance of snail, a transciptional factor involved in EMT in lung tumors, we investigated its expression in a large set of lung carcinomas by immunohistochemistry. Expression of snail and effects of snail knockdown was studied in cell lines.
Nuclear snail expression was seen in 21% of cases this being strongest in small cell lung carcinomas (SCLC). There was significantly greater snail expression in SCLC compared to squamous cell or adenocarcinoma. Positive snail expression was associated with poor survival in the whole material and separately in squamous cell and adenocarcinomas. In Cox regression analysis, snail expression showed an independent prognostic value in all of these groups. In several cell lines knockdown of snail reduced invasion in both matrigel assay and in the myoma tissue model for invasion. The influence of snail knockdown on claudin expression was cell type specific. Snail knockdown in these cell lines modified the expression of MMP2 and MMP9 but did not influence the activation of these MMPs to any significant degree.
The results show that snail plays an important role in the invasive characteristics of lung carcinoma influencing the survival of the patients. Snail knockdown might thus be one option for targeted molecular therapy in lung cancer. Snail knockdown influenced the expression of claudins individually in a cell-line dependent manner but did not influence MMP expressions or activations to any significant degree.
PMCID: PMC3546026  PMID: 23157169
Lung; Carcinoma; Snail; Claudin; MMP
25.  A novel molecular pathway for Snail-dependent, SPARC-mediated invasion in non-small cell lung cancer pathogenesis 
Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non-small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to upregulation of Secreted Protein, Acidic and Rich in Cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates TGF-β, ERK1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-βR2 are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulated SPARC expression. Inhibition of MEK phosphorylation downregulated SPARC. MiR-29b is downregulated in Snail overexpressing cell lines, while overexpression of miR-29b inhibited SPARC expression. In addition, miR-29b was upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis.
PMCID: PMC3919444  PMID: 24253315
NSCLC; Snail; SPARC; invasion; parallel progression

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