Transition Markers E-cadherin Research Articles

DNA Methyltransferase 1 Targeting Using Guadecitabine Inhibits Prostate Cancer Growth by an Apoptosis-Independent Pathway.

Epigenetic alterations such as DNA methylation and histone modifications are implicated in repressing several tumor suppressor genes in prostate cancer progression. In this study, we determined the anti-prostate cancer effect of a small molecule drug guadecitabine (gDEC) that inhibits/depletes the DNA methylation writer DNA methyltransferase 1 (DNMT1). gDEC inhibited prostate cancer cell growth and proliferation in vitro without activating the apoptotic cascade. Molecular studies confirmed DNMT1 depletion and modulated epithelial-mesenchymal transition markers E-cadherin and β-catenin in several prostate cancer cell lines (LNCaP, 22Rv1, and MDA PCa 2b). gDEC treatment also significantly inhibited prostate tumor growth in vivo in mice (22Rv1, MDA PCa 2b, and PC-3 xenografts) without any observed toxicities. gDEC did not impact the expression of androgen receptor (AR) or AR-variant 7 (AR-V7) nor sensitize the prostate cancer cells to the anti-androgen enzalutamide in vitro. In further investigating the mechanism of cytoreduction by gDEC, a PCR array analyses of 84 chromatin modifying enzymes demonstrated upregulation of several lysine-specific methyltransferases (KMTs: KMT2A, KMT2C, KMT2E, KMT2H, KMT5A), confirmed by additional expression analyses in vitro and of harvested xenografts. Moreover, gDEC treatment increased global histone 3 lysine 4 mono-and di-methylation (H3K4me1 and H3K4me2). In sum, gDEC, in addition to directly depleting the corepressor DNMT1, upregulated KMT activating epigenetic enzymes, activating terminal epithelial program activation, and prostate cancer cell cycling exits independent of apoptosis.

Oxymatrine inhibits the development of radioresistance in NSCLC cells by reversing EMT through the DcR3/AKT/GSK-3β pathway.

Lung cancer is the leading cause of cancer-associated mortality globally. In particular, non-small cell lung cancer (NSCLC) constitutes the largest percentage of all cases of lung cancer. In clinical practice, radioresistance contributes to poor responses to radiotherapy. Therefore, the demand remains to explore potential novel and effective mechanism underlying radioresistance to improve the efficacy of radiotherapy for NSCLC. Western blotting was conducted to quantify the protein expression of epithelial-mesenchymal transition markers E-cadherin and vimentin in the A549 cell line. The proliferation of A549 cells was measured using the Cell Counting Kit-8 and colony forming assays. In addition, the apoptosis of A549 cells was analyzed by flow cytometry. Invasion and migration by NSCLC cells were quantified using Transwell and wound healing assays. Plasmids were used to overexpress decoy receptor 3 (DcR3) in A549 cells. Xenograft models were established to measure the extent of NSCLC tumor growth in vivo. Our study clarified the activation of the DcR3/protein kinase B (AKT)/glycogen synthase kinase 3β (GSK-3β) pathway in radioresistant NSCLC cells. Oxymatrine (OMT) treatment restored radiosensitivity and inhibited irradiation-induced epithelial-mesenchymal transition (EMT), invasion and migration in NSCLC cells through the DcR3/AKT/GSK-3β pathway in vitro. By contrast, OMT treatment promoted the suppressive effects of radiation on the weight and volume of the xenograft tumors in animal models. OMT suppressed the development of radioresistance in NSCLC cells by promoting radiosensitivity, through the reversal of EMT process by inhibiting the DcR3/AKT/GSK-3β pathway.

Prognostic significance of apanel of two biomarkers (E-cadherin and CD105) in laryngeal cancer.

This study aimed to evaluate the clinicopathologic significance of the combined immunohistochemical expression of the epithelial-mesenchymal transition marker E-cadherin and the angiogenesis marker CD105 in laryngeal squamous cell carcinoma and assess correlation of their expression. Eighty-five patients who underwent complete resection as primary treatment were selected for this study. E-cadherin and CD105 expression levels were determined by immunohistochemistry. The receiver operating curve approach was applied to determine the cut-off value and separate patients with high and low expression of markers. The high-risk group ("CD105 high" and "E-cadherin low" expression) showed statistically significant correlations with age less than 66 years (p = 0.039), advanced T-status (T3-4) (p = 0.046), aggressive TNM stage (stage III-IV) (p = 0.003) and locoregional recurrence of disease (p = 0.004). In the Kaplan-Meier analyses, the high-risk group had significantly worse prognoses than other risk groups (log-rank test 2 = 9.415, p = 0.024). Spearman's correlation coefficient analysis showed anonsignificant negative correlation between the expression of E-cadherin and CD105 (rho = -0.073, p = 0.505). Simultaneous consideration of E-cadherin and CD105 is asimple panel of markers to determine aggressive tumour phenotype with ahigher risk of disease recurrence in patients with laryngeal cancer.

Expression of epithelial-mesenchymal transition markers E-cadherin and ZEB1 in colorectal cancer

Purpose of the study. Evaluation of expression of the epithelial-mesenchymal transition markers E-cadherin and ZEB1 in patients with stage II-IV colorectal cancer (CRC).Materials and methods. The study included operational material obtained from 299 patients aged 42–86 years (mean age 64.2±1.7 years) with stage II-IV CRC treated at National Medical Research Centre for Oncology in 2013-2017. Stage II CRC (T3-4 N0 M0 ) was diagnosed in 110 patients, stage III (T1-4 N1-2 M0 ) – in 88 patients, stage IV (T1-4 N0-2 M1 ) – in 101 patients. Polyclonal rabbit antibodies to ZEB1 (Biorbyt Ltd., UK) and mouse monoclonal antibodies to E-cadherin (Diagnostic BioSystems, USA) were used for an IHC analysis. The intensity and degree of tumor cell staining, percentage of stained tumor cells in the sample and the number of patients with positive and negative marker expression were determined. Groups were compared using the Mann–Whitney U test and the Pearson's chi-square test.Results. Positive expression of E-cadherin was found in 64.5 % (193 of 299 patients), ZEB1 – in 80.6 % (241 of 299 patients). The number of patients with E-cadherin-positive tumors statistically significantly decreased (χ2 =15.888 at p<0.001) from stage II to stage IV, while for ZEB1, on the contrary, it statistically significantly increased (χ2 =43.912 at p><0.001) from stage II to stage IV. The mean values of expression in positively stained cells were: in stage II – E-cadherin 55.3±6.8 %, ZEB1 43.0±5.9 %; in stage III – E-cadherin 38.4±5.8 %, ZEB1 77.0±5.5 %; in stage IV – E-cadherin 14.7±4.7 %, ZEB1 76.9±3.5 %. Significant differences were observed between the mean values of ZEB1 expression in stages III and IV compared to stage II, as well as between the mean values of E-cadherin expression in stages II and III compared to stage IV (p><0.05). No significant differences were found in the mean values of ZEB1 and E-cadherin expression in stages III and IV, II and III respectively.Conclusions. The study demonstrated statistically significant relationship between tumor stages and expression of E-cadherin and ZEB1 in the epithelial-mesenchymal transition. The loss of the E-cadherin expression in tumor cells of patients from stage II to stage IV and increased expression of ZEB1 in the studied groups were statistically significant (p<0.05).

Evaluation of immunohistochemical expression of epithelial- mesenchymal transition markers E-cadherin, Twist and Snail in oral submucous fibrosis and their possible association with malignant transformation.

Background:Oral submucous fibrosis (OSMF) is a chronic, potentially malignant condition of the oral mucosa, predominantly seen in people of Asian descent. The reported malignant transformation rate of OSMF is 7%–13%. In the context of the understanding progression of OSMF, the study of prime molecular expressions is essential. Various markers have received more attention, one of them is E-cadherin. Various factors which promote epithelial–mesenchymal transition (EMT) and inhibit E-Cadherin include Snail1, Snail2, Twist and EF1/ZEB1. The intended study was undertaken to evaluate the possible role of E-cadherin and its regulatory markers Twist1 and Snail1 in OSMF.Aims and Objectives: To evaluate the expression of E-cadherin, Twist1 and Snail1 in OSMFTo evaluate their possible association with malignant transformation of OSMF. Materials and Methods:Histologically proven 20 cases of each OSMF with and without dysplasia were taken as the study group, 20 normal mucosa as control group and were subjected for immunohistochemical (IHC) expression with E-cadherin, Twist1 and snail1.Results:Immunohistochemical expression of all the three markers showed statistically significant expression of all the three markers. Intensity and percentage of staining between the groups were statistically significant for E-cadherin between normal oral mucosa (NOM) and OSMF with dysplasia (OSMFD), no significance was found between NOM and OSMF, whereas Snail1 and Twsit1 were statistically significant between NOM and OSMF and also between NOM and OSMFD. However, no significance was found for all the three markers when compared between the groups OSMF and OSMFD.Conclusion:The increased expression of Snail1 and Twist1 with concomitant loss of E -cadherin in OSMF suggests the role of the EMT.

Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through Integrin-AKT Signaling Pathway in Lung Cancer.

Fibrinogen is an extracellular matrix protein composed of three polypeptide chains with fibrinogen alpha (FGA), beta (FGB) and gamma (FGG). Although fibrinogen and its related fragments are involved in tumor angiogenesis and metastasis, their functional roles are incompatible. A recent genome-scale screening reveals that loss of FGA affects the acceleration of tumor growth and metastasis of lung cancer, but the mechanism remains elusive. We used CRISPR/Cas9 genome editing to knockout (KO) FGA in human lung adenocarcinoma (LUAD) cell lines A549 and H1299. By colony formation, transwell migration and matrix invasion assays, FGA KO increased cell proliferation, migration, and invasion but decreased the expressions of epithelial-mesenchymal transition marker E-cadherin and cytokeratin 5/8 in A549 and H1299 cells. However, administration of FGA inhibited cell proliferation and migration but induced apoptosis in A549 cells. Of note, FGA KO cells indirectly cocultured by transwells with FGA wild-type cells increased FGA in the culture medium, leading to decreased migration of FGA KO cells. Furthermore, our functional analysis identified a direct interaction of FGA with integrin α5 as well as FGA-integrin signaling that regulated the AKT-mTOR signaling pathway in A549 cells. In addition, we validated that FGA KO increased tumor growth and metastasis through activation of AKT signaling in an A549 xenograft model. IMPLICATIONS: These findings demonstrate that that loss of FGA facilities tumor growth and metastasis through the integrin-AKT signaling pathway in lung cancer.

Metadherin overexpression in perihilar cholangiocarcinoma is associated with lymph node metastasis and poor prognosis.

Metadherin (MTDH) is a protein that is also named astrocyte elevated gene-1, and is highly expressed in a number of different tumor tissues. Although the expression of MTDH is associated with tumor invasion and recurrence, the expression of this protein in perihilar cholangiocarcinoma (PCCA) and its clinical use have not yet been investigated. In the present study, the expression of MTDH in patients with PCCA was investigated in order to determine its clinicopathological use. An immunohistochemical method was used to detect MTDH expression and the epithelial-mesenchymal transition markers E-cadherin and vimentin in 66 cases of PCCA. In addition to the expression of MTDH, the clinical and pathological data and the postoperative outcomes were analyzed. The MTDH positive expression rate was 48.5% (32/66) in PCCA. A significantly higher MTDH expression level was identified in the poor tumor differentiation group compared with the well differentiation group (P=0.007). In the positive lymph node metastasis group, a significantly higher MTDH expression level was revealed compared with the negative lymph node metastasis group (P=0.023). No association was noted with regard to the expression of MTDH and the variables age, sex, tumor diameter, tumor grade and tumor classification stage. Positive MTDH expression was significantly associated with high vimentin expression (P=0.037) compared with negative vimentin expression and inversely associated with positive E-cadherin expression compared with negative E-cadherin expression (P=0.030). Survival analysis suggested that the high MTDH expression group was associated with a worse overall survival (OS) rate and recurrence free survival (RFS) rate compared with the low MTDH expression group (P<0.001 and P=0.01, respectively). Cox regression analysis indicated that the Tumor-Node-Metastasis, surgery margin and high MTDH expression were independent OS and RFS factors for PCCA. MTDH expression may serve an important function in PCCA tumor growth and metastasis. Targeting MTDH may have important therapeutic applications for patients with PCCA.

Euchromatic histone lysine methyltransferase 1 regulates cancer development in human gastric cancer by regulating E-cadherin.

Gastric cancer (GC) is among the most aggressive types of cancer and is the second leading cause of cancer-associated mortality worldwide. The specific role of deregulated expression/activity of histone methyltransferases (HMTs) in GC is poorly understood. The present study aimed to explore the possible oncogenic role of euchromatic histone lysine methyltransferase 1 (EHMT1) in gastric carcinogenesis. It was identified that EHMT1 was highly expressed in GC tissues compared with that in adjacent non-tumor tissues, and that EHMT1 expression levels were significantly associated with tumor stage and lymph node metastasis. Through knockdown of EHMT1 in the BGC-803 cell line, EHMT1 was demonstrated to promote a malignant phenotype, and to increase the wound healing, migration and invasion abilities of GC cells. Corresponding to these in vitro results, knockdown of EHMT1 also inhibited the peritoneal metastasis of GC cells in vivo. Furthermore, EHMT1 also regulated the expression of the epithelial-mesenchymal transition marker E-cadherin in vitro and in vivo. These results indicate that EHMT1 is upregulated in GC and serves an oncogenic role in GC development by regulating E-cadherin expression.

Role of epithelial-mesenchymal transition markers E-cadherin, N-cadherin, β-catenin and ZEB2 in laryngeal squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) allows neoplastic cells to gain the invasive phenotype and become migratory, which is required for cancer progression and metastasis. In the present study, the expression of EMT-associated biomarkers and their association with clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC) was investigated. E-cadherin, N-cadherin, β-catenin and zinc finger E-box binding homeobox 2 (ZEB2) protein expression was evaluated with immunohistochemistry in a cohort of 76 patients with operable LSCC. The association between these transition markers, clinicopathological parameters and their prognostic impact in LSCC was analyzed. Immunohistochemical analysis revealed that EMT-associated proteins were differentially expressed between LSCC and adjacent non-neoplastic laryngeal tissue. Negative E-cadherin expression and positive N-cadherin, β-catenin and ZEB2 expression were associated with a later tumor (T) stage, decreasing tumor differentiation and a reduced overall survival (OS) time (OS: E-cadherin, P=0.016; N-cadherin, P=0.003; β-catenin, P=0.002; ZEB2, P=0.0003). E-cadherin/β-catenin co-expression was significantly associated with the majority of clinicopathological parameters assessed, including lymph node metastases, T stage and tumor cell differentiation (P=0.004, P=0.005, and P<0.001, respectively). Multivariate analysis indicated that T stage and the positive expression of β-catenin and ZEB2 were independent risk factors for OS in LSCC (P=0.014, P=0.025 and P=0.003, respectively). It was concluded that EMT mediates tumor progression, and reduces OS time in patients with LSCC. E-cadherin/β-catenin co-expression may be associated with clinicopathological parameters. T stage, and the positive co-expression of β-catenin and ZEB2 may be independent predictors of prognosis in LSCC.

Influence of TGF-β1 on tumor transition in oral cancer cell and BMSC co-cultures

ObjectivesTGF-β1 signaling modulates epithelial mesenchymal transitions (EMT) of head and neck squamous cell carcinoma (HNSCC). Bone marrow mesenchymal stromal cells (BMSC) are able to exert a regulating influence on the expression of markers of EMT in HNSCC cells. It was thus the aim of this study to test the hypothesis that TGF-β1 modulates the interactions of tumor transition between BMSCs and HNSCC, affecting the expression of E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin. Furthermore, we analyzed alterations in the AKT-signaling of tumor and stroma cells. Materials and methodsBMSCs were isolated from iliac bone marrow aspirates and co-cultured in trans-well permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13. Following the induction with TGF-β1 under serum free conditions the expression of Vimentin and E-Cadherin was assessed via immunofluorescence. A quantitative RT-PCR analysis of tumor transition markers E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin was performed. Changes in AKT-Signaling were identified via protein analysis. ResultsIn non-induced co-cultures, BMSC were able to suppress Vimentin in PCI-13 as a marker of tumor transition. In TGF-β1 induced co-cultures PCI-13 significantly increased the expression of Vimentin, Twist, Snail, MMP14, GSK3a, PRAS40, 4E-BP1, and AMPKa compared to monolayer controls. TGF-β1 co-cultured BMSC demonstrated a significant increase of Snail, PRAS40, mTOR, GSK3a/b, Bad, PDK1 and 4E-BP1. ConclusionsTGF-β1 was able to attenuate the modulating influence of BMSC in co-culture and drive the co-culture towards a progressive tumor transition, affecting the expression of markers of EMT, AKT-Signaling and proliferative checkpoints.

Differential effects of Pax3 on expression of polysialyltransferases STX and PST in TGF-β-treated normal murine mammary gland cells.

Glycosylation of certain proteins at the mammalian cell surface is an essential event in carcinogenesis. Sialylation, one type of glycosylation, can act on multiple cell-behaviors, such as migration, growth, and malignant invasion. Two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), are responsible for synthesis of polysialic acid on neural cell adhesion molecule. We showed previously that STX and PST are oppositely expressed in normal murine mammary gland cells undergoing transforming growth factor-β-induced epithelial-mesenchymal transition. The molecular basis for regulation of STX and PST remained unclear. In the present study, we observed that transcription factor Pax3 upregulates STX expression, downregulates PST expression, and modulates upregulated expression of PSA, which attaches primarily to neural cell adhesion molecule to form PSA-NCAM. Overexpression of Pax3 in normal murine mammary gland cells transformed the expression of epithelial-mesenchymal transition markers E-cadherin and N-cadherin, and significantly promoted cell migration, but had no effect on cell proliferation.

Transition of Immunohistochemical Expression of E-Cadherin and Vimentin from Premalignant to Malignant Lesions of Oral Cavity and Oropharynx.

We sought to study the expression of epithelial-to-mesenchymal transition markers E-cadherin and vimentin in precancerous lesions of the oral cavity and oropharynx and to use the specific pattern of expression to predict invasiveness. This cross-sectional study looked at 87 cases of oral and oropharyngeal lesions obtained between December 2012 and November 2014 in the Department of Pathology, Jawaharlal Nehru Medical College, Aligarh Muslim University, India. Fifty-three biopsies from the buccal mucosa, tongue, and pharynx and 34 resected oral specimens were evaluated for premalignant and malignant lesions using hematoxylin and eosin and immunohistochemical stains. Immunohistochemical expression of epithelial marker E-cadherin and mesenchymal marker vimentin was evaluated wherever possible. Slides were examined for staining pattern (cytoplasmic or membrane), proportion, and intensity of staining of tumor cells. Patients follow-up and therapy related changes were also studied. There were 64 premalignant and 23 malignant cases in our study with 65 (74.7%) cases seen in males and 22 (25.3%) cases seen in females. The majority of malignant cases, (n = 15; 64.2%) were seen in the fifth and sixth decades of life while most of the premalignant lesions (n = 36; 56.4%) were seen in the fourth and fifth decade. Amongst the 64 premalignant oral lesions, leukoplakia comprised of 14 cases (21.9%), of which three cases had associated mild to moderate dysplasia. The majority of premalignant lesions showed strong E-cadherin expression and decreased expression of vimentin with negative and weak expression in both dysplasias and carcinoma in situ (p = 0.013). E-cadherin expression was significantly reduced in invasive carcinomas compared to dysplasias and carcinoma in situ and the difference in immunoreactivity was statistically significant (p < 0.050). Vimentin expression increased as the tumor progressed from dysplasias to carcinoma in situ to invasive carcinomas (p < 0.050). Invasiveness of cancer can be analyzed using E-cadherin and vimentin immunohistochemical stains, which can help in predicting tumor behavior. These biomolecules can also be used as biomarkers for further research on the microinvasion of oral cancers for early diagnosis.

Changes in glomerular parietal epithelial cells in mouse kidneys with advanced age

Kidney aging is accompanied by characteristic changes in the glomerulus, but little is known about the effect of aging on glomerular parietal epithelial cells (PECs), nor if the characteristic glomerular changes in humans and rats also occur in very old mice. Accordingly, a descriptive analysis was undertaken in 27-mo-old C57B6 mice, considered advanced age. PEC density was significantly lower in older mice compared with young mice (aged 3 mo), and the decrease was more pronounced in juxtamedullary glomeruli compared with outer cortical glomeruli. In addition to segmental and global glomerulosclerosis in older mice, staining for matrix proteins collagen type IV and heparan sulfate proteoglycan were markedly increased in Bowman's capsules of older mouse glomeruli, consistent with increased extracellular matrix production by PECs. De novo staining for CD44, a marker of activated and profibrotic PECs, was significantly increased in aged glomeruli. CD44 staining was more pronounced in the juxtamedullary region and colocalized with phosphorylated ERK. Additionally, a subset of aged PECs de novo expressed the epithelial-to-mesenchymal transition markers α-smooth muscle and vimentin, with no changes in epithelial-to-mesenchymal transition markers E-cadherin and β-catenin. The mural cell markers neural/glial antigen 2, PDGF receptor-β, and CD146 as well as Notch 3 were also substantially increased in aged PECs. These data show that mice can be used to better understand the aging kidney and that PECs undergo substantial changes, especially in juxtamedullary glomeruli, that may participate in the overall decline in glomerular structure and function with advancing age.

MicroRNA-429 suppresses cell proliferation, epithelial-mesenchymal transition, and metastasis by direct targeting of BMI1 and E2F3 in renal cell carcinoma

BackgroundMicroRNA-429 (miR-429), a short noncoding RNA belonging to the miR-200 superfamily, plays a crucial role in tumorigenesis and tumor progression. It also acts as a modulator of epithelial-to-mesenchymal transition, a cell development regulating process that affects tumor development and metastasis. The aim of this study was to investigate the potential role of miR-429 in regulating growth and metastasis of renal cell carcinoma. MethodsmiR-429 expression was stably up-regulated or down-regulated in the renal cell carcinoma ACHN and A498 cell lines, and cell proliferation and metastasis were assessed. ResultsmiR-429 overexpression inhibited cell proliferation, colony formation, migration, and invasion. Suppression of endogenous miR-429 promoted cell growth and metastasis. miR-429 was shown to directly target the 3' untranslated regions of B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and E2F transcription factor 3 (E2F3) transcripts, regulating their expression, as well as that of the downstream epithelial-to-mesenchymal transition markers E-cadherin, N-cadherin, vimentin, p14, and p16. ConclusionsThese results revealed a tumor suppressive role for miR-429 in renal cell carcinoma through directly targeting BMI1 and E2F3.

Co-targeting c-Met and COX-2 Leads to Enhanced Inhibition of Lung Tumorigenesis in a Murine Model with Heightened Airway HGF

The hepatocyte growth factor (HGF)/c-Met pathway is often dysregulated in non-small-cell lung cancer (NSCLC). HGF activation of c-Met induces cyclooxygenase-2 (COX-2), resulting in downstream stimulation by prostaglandin E2 of additional pathways. Targeting both c-Met and COX-2 might lead to enhanced antitumor effects by blocking signaling upstream and downstream of c-Met. Effects of crizotinib or celecoxib alone or in combination were tested in NSCLC cells in vitro and in mice transgenic for airway expression of human HGF. Proliferation and invasion of NSCLC cells treated with a combination of crizotinib and celecoxib were significantly lower compared with single treatments. Transgenic mice showed enhanced COX-2 expression localized to preneoplastic areas following exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, which was not present without carcinogen exposure. This shows that COX-2 activity is present during lung tumor development in a high HGF environment. After 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment, a significant decrease in the number of lung tumors per animal was observed after 13-week treatments of crizotinib, celecoxib, or the combination compared with placebo (p < 0.001). With combination treatment, the number of tumors was also significantly lower than single agent treatment (p < 0.001). In the resulting lung tumors, P-c-Met, COX-2, prostaglandin E2, and P-MAPK were significantly downmodulated by combination treatment compared with single treatment. Expression of the epithelial-mesenchymal transition markers E-cadherin and snail was also modulated by combination treatment. In the presence of high HGF, dual inhibition of c-Met and COX-2 may enhance antitumor effects. This combination may have clinical potential in NSCLCs with high HGF/c-Met expression or epithelial-mesenchymal transition phenotype.

Prognostic significance of the epithelial-to-mesenchymal transition markers e-cadherin, vimentin and twist in bladder cancer.

The goal of this study was to utilize long-term patient follow-up to determine whether epithelial-to-mesenchymal transition (EMT)-related markers can predict bladder cancer patient survival and progression of disease. This study included 121 patients with bladder cancer. Sixty-four of these patients presented with non-muscle invasive (NMI, stage T1) bladder cancer and 57 with muscle invasive (MI, stage T2, T3). The patients were diagnosed and treated between May 1998 and July 2012. The EMT markers E-cadherin, Twist, and Vimentin were detected via immunohistochemistry. Univariate and multivariate/Cox analyses were then utilized to determine whether these EMT markers could be useful prognostic markers for predicting bladder cancer patient outcomes. Analysis of the 121 bladder cancer patients in this study revealed that the frequency of E-cadherin expression was 59.5% (72/121), Twist was 54.5% (66/121), and Vimentin was 24.8% (30/121). Twist and Vimentin were found to have statistically significant correlations with grade, recurrence, and progression but not with stage, whereas E-cadherin was associated with stage but not with the other parameters. In the univariate analysis, grade (p = 0.02) was the only significant predictor for progression-free survival (PFS). Stage, grade, and expression of E-cadherin, Vimentin and Twist were included in the multivariate analysis of predicting PFS. In this analysis, grade (p = 0.01) and Vimentin expression (p = 0.001) were found to be significant prognostic factors in predicting PFS. Grade and Vimentin are potential independent indicators in predicting bladder cancer progression and survival.

Expression and roles of the epithelial mesenchymal transition markers E-cadherin and vimentin in hepatocellular carcinoma

To determine the differential protein expressions of epithelial mesenchymal transition (EMT) markers E-cadherin and vimentin in hepatocellular carcinorma (HCC) and to investigate their correlation to the molecular mechanisms of metastasis to explore their potential utility as prognostic indicators of HCC. Tumor tissues and patient-matched adjacent non-tumor tissues were collected from individuals diagnosed with HCC. E-cadherin and vimentin protein expressions in the tissue specimens were quantified by western blot with densitometry of fluorescence emission and comparatively analyzed to determine the associations with molecular and clinical features. The expressions of E-cadherin and vimentin, as well as the other EMT-related protein Twist, were also detected in the tissue specimens by immunohistochemistry. Statistical analyses were carried out by paired-samples t-test, Mann-Whitney test, and Spearman rank correlation analysis. E-cadherin expression was significantly lower in tumor tissues (0.082 +/- 0.063 vs. adjacent non-tumor tissues: 0.226 +/- 0.215, t = -4.050, P less than 0.01), lower in patients with portal vein tumor thrombus (vs. non-thrombic HCC patients, P = 0.001), and correlated with TNM stage (III/IV > I/II, P = 0.003). Vimentin expression was significantly higher in tumor tissues (vs. adjacent non-tumor tissues, P = 0.002), negatively correlated with E-cadherin expression (t = -0.509, P = 0.004), and closely associated with some clinical parameters, such as portal vein tumor thrombus (P less than 0.01), TNM stage (P less than 0.01), and Milan criteria (P = 0.005). Immunohistochemistry showed that E-cadherin expression was very weak in tumors but very strong in the cell membranes of non-tumor tissues, and that vimentin and Twist expressions were strong in tumors but undetectable in non-tumor tissue. Expression levels of the EMT markers E-cadherin and vimentin in HCC are related to clinical parameters, including portal vein tumor thrombus and TNM stage, and may represent useful prognostic markers of metastasis.

MiR-18a promotes malignant progression by impairing microRNA biogenesis in nasopharyngeal carcinoma

Dysregulation of microRNA (miRNA) biogenesis is implicated in cancer development and progression. Dicer and Drosha are established regulators of miRNA biogenesis. In this study, we used a miRNA array to evaluate the miRNA expression profiles in nasopharyngeal carcinoma (NPC) samples. The significance analysis of microarrays showed a global downregulation of miRNA expression in NPC samples compared with normal nasopharyngeal epithelial tissues. Notably, miR-18a, a member of the oncogenic miR-17-92 cluster, was upregulated in the NPC samples and cell lines. Clinical parameter studies showed that higher levels of miR-18a correlated with NPC advanced stage, lymph node metastasis, Epstein-Barr virus infection and a higher death rate from NPC, indicating oncogenic roles in NPC development. The expression levels of miR-18a and Dicer1 were inversely related in NPC tissues. Further studies demonstrated that miR-18a negatively regulated Dicer1 by binding to the 3' untranslated regions of Dicer1. In vitro and in vivo biological function assays showed that miR-18a promoted the growth, migration and invasion of NPC cells by regulating Dicer1 expression, which caused the global downregulation of miRNA expression levels including miR-200 family and miR-143. Furthermore, we found that the epithelial mesenchymal transition marker E-cadherin and the oncogene K-Ras were aberrantly expressed after miR-18a transduction, and these alterations were directly induced by downregulation of the miR-200 family and miR-143. Collectively, our findings indicate that miR-18a plays an oncogenic role in the development of NPC by widespread downregulation of the miRNome and could be a potential therapeutic target for NPC.

Malignant phenotype of renal cell carcinoma cells is switched by Ukrain administration in vitro

We investigated whether Ukrain modulates the malignant phenotype of clear cell renal cell carcinoma (ccRCC) cells Caki-1, Caki-2, and ACHN treated with four doses (5, 10, 20, and 40 μmol/l) for 24 and 48 h. The epithelial-to-mesenchymal transition markers E-cadherin, β-catenin, and vimentin were analyzed by immunofluorescence as well as actin and tubulin; matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was analyzed by SDS-zymography, intracellular and secreted SPARC levels by western blot, and cell cycle by flow cytometry. Ukrain did not induce E-cadherin/β-catenin immunoreactivity at the cell-cell boundary, although it determined the actin cortical expression in Caki-2 and ACHN, and did not affect vimentin organization; however, in some Caki-1 and ACHN cells the perinuclear concentration of vimentin was consistent with its downregulation. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was significantly downregulated 48 h after 20 μmol/l Ukrain administration. At this time point, Ukrain significantly decreased migration and invasion, and downregulated SPARC levels in cell supernatants at all doses in Caki-2, and at 20 μmol/l in Caki-1 and ACHN cells. Concomitantly, SPARC was upregulated in all ccRCC cells, suggesting that Ukrain could also affect cell proliferation by cell cycle inhibition, as supported by the cell cycle analysis, as SPARC also acts as a cell cycle inhibitor. Our results suggest that Ukrain may switch the epithelial-to-mesenchymal transition-related phenotype of ccRCC cells, and targets the two major aspects involved in RCC progression, such as tumor invasion/microenvironment remodeling and cell proliferation.

Abstract C47: Dual kinase inhibition of EGFR and HER2 can overcome resistance to an EGFR-targeting agent

Abstract EGFR and HER2 expression are correlated with poor prognosis in several cancers including bladder cancer. The EGFR-targeting agent cetuximab is currently in phase II clinical trials for the treatment of bladder cancer, but has only been modestly effective as a single agent in most solid tumor types. In colon cancer, lack of cetuximab efficacy has been attributed to compensatory activating mutations in Kras. The incidence of Kras mutations is rare in bladder carcinomas. There is a need for preclinical models with which to study resistance to EGFR-targeting agents. Here, we generated four cetuximab-resistant clones of a cetuximab-sensitive transitional bladder cancer cell line in vivo by exposing cetuximab-sensitive bladder cancer cell line xenografts to slowly increasing concentrations of cetuximab. We verified this resistance in vivo by demonstrating growth of cetuximab-resistant xenografts in the presence of high doses of cetuximab, as well as establishing differential sensitivity to cetuximab between sensitive and resistant xenografts in vivo. We validated the cetuximab-resistant phenotype of these cells in vitro by showing decreased response to cetuximab in an invasion assay. We examined the levels of total and phosphorylated EGFR, HER2, HER3, AKT, MAPK, and the total protein levels of epithelial-to-mesenchymal transition markers E-cadherin and Vimentin in the isogenic cetuximab-sensitive and cetuximab-resistant cells. Increased phosphorylation of the ERBB family members HER2 and HER3 was detected in cetuximab-resistant clones compared with their cetuximab-sensitive parental cell lines. Treatment of the cetuximab-resistant cells with an irreversible dual kinase inhibitor targeting EGFR and HER2 (BIBW2992), successfully inhibited their growth. These data suggest that activation of HER2 and HER3 may contribute to cetuximab resistance where targeting these other HER family members may enhance the efficacy of EGFR blockade. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C47.