Expression Levels Of Snail Research Articles

Abstract P5-08-02: PTK6 small molecule inhibitor enhances efficacies of chemotherapy in mesenchymal TNBC

Abstract Background: EMT in cancer promotes resistance to chemotherapy and radiotherapy, as well as immune suppression in the tumor microenvironment. EMT is also associated with enhanced tumor dissemination to other organs. EMT is a dynamic cell re-programming process whereby cancer cells lose epithelial markers and acquire mesenchymal markers, enhanced cell migration, and anoikis resistance. EMT is promoted by transcriptional and epigenetic regulators, as well as by signaling pathways. TNBCType and 101-gene model have identified distinct subsets of TNBC that exhibit mesenchymal gene signatures and phenotypes. This particular subset may be associated with chemotherapy resistance and metastatic recurrence in patients with TNBC. We identified protein tyrosine kinase 6 (PTK6) as a promoter of EMT in mesenchymal TNBC through its ability to prevent degradation of SNAIL, a key EMT transcriptional factor. A higher level of SNAIL expression is associated with poor TNBC patient prognosis. We investigated whether SNAIL suppression and EMT reversal by PTK6 small molecule inhibitor treatment enhance efficacy of chemotherapeutic agents that are part of standard of care treatment for patients with TNBC. Methods: Mesenchymal TNBC cell lines or organoids generated from TNBC PDX tumors were treated with varying concentrations of PTK6 small molecule inhibitor alone or in combination with chemotherapeutic agents in 3D cell cultures. The cell viability was assessed using 3D CellTiter-Glo or alamar blue. The combination Index was calculated to examine potential synergistic effects (CI<1: synergism, CI=1: additive, CI>1: antagonism). The in vivo combination effects of PTK6 inhibitor and paclitaxel were also assessed in two TNBC PDX models. Gene ontology analysis, targeted RT-PCR gene expression profiling and protein array were performed to identify potential mechanisms for chemosensitization effects of PTK6 inhibitor treatment. Results: Pre-treatment with PTK6 inhibitor increases sensitivity to paclitaxel or doxorubicin in 3D matrigel culture of TNBC cell lines, as well as in TNBC PDX organoids. The Combination Index suggested synergies between PTK6 inhibitor and chemotherapy treatment (paclitaxel or doxorubicin). While administration of PTK6 inhibitor or paclitaxel alone only modestly suppressed growth of PDX tumors in vivo, PTK6 inhibitor treatment sensitized tumors to paclitaxel treatment, as evidenced by the dramatic suppression of tumor volume and rate of growth. Gene ontology analysis identified gene sets that are significantly differentially expressed in PTK6 inhibitor-treated TNBC tumors, including extracellular matrix, cell migration, cell cycle and microtubule activity. A targeted RT-PCR profiling and protein array found that PTK6 inhibitor modulates expression of molecules that are associated with chemotherapy resistance and immune regulation; decrease in MMP3, PLEK2, osteopontin, IL-6 and CCL5 and increase in CD40. We are currently investigating whether these changes are dependent on Snail downregulation/EMT reversal caused by PTK6 inhibition. Conclusion: PTK6 inhibition may sensitize TNBC to chemotherapy treatment by suppressing Snail and reversing EMT. We will further validate these effects of PTK6 inhibitor treatment with other chemotherapies and immunotherapies. Citation Format: Ito K, Lee E, Sato K, Byerly JH, Zhu J, Irie HY. PTK6 small molecule inhibitor enhances efficacies of chemotherapy in mesenchymal TNBC [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-02.

MiR-30a regulates the proliferation and invasion of breast cancer cells by targeting Snail.

The present study aims to investigate the effect of miR-30a on the proliferative and invasive abilities of breast cancer cells, and to observe the role of miR-30a in the pathogenesis of breast cancer. With the increase of pathological grade and malignant degree of breast cancer cells, the miR-30a expression level gradually decreased (P<0.01). Transfection with miR-30a mimic significantly inhibited the proliferative and invasive ability of SK-BR-3 cells (P<0.01), while transfection with anti-miR-30a significantly improved the proliferative and invasive ability of these cells (P<0.01). It was revealed using bioinformatic methods that Snail was the functional target gene of miR-30a, and this was verified by the results of a luciferase reporter gene assay. The results of analysis of Snail expression in breast cancer tissues and breast cancer cells revealed that with the increase in pathological grade and malignant degree of breast cancer cells, Snail expression levels gradually increased (P<0.01). Western blotting revealed that miR-30a significantly inhibited Snail expression in SK-BR-3 cells, upregulated the expression of EMT-associated E-cadherin, and downregulated the expression of EMT-associated N-cadherin and Vimentin. MiR-30a was able to affect the proliferation and invasion of breast cancer cells by regulating Snail expression, and therefore has a regulatory effect on the occurrence and development of breast cancer.

PO-274 Development of an in vitro tumour model to evaluate drug efficacy and epithelial mesenchymal transition in triple negative breast cancer

IntroductionThe advent of diverse methods for 3-dimensional (3D) cell culture has allowed scientists to address some of the limitations of conventional 2D methods and have been applied to studies concerned with cancer therapy response. A major component of the tumour microenvironment overlooked during drug discovery and cancer studies is the interstitial fluid pressure (IFP).The aim of this project is to study breast cancer cell invasion and responsiveness to chemotherapeutic agents in a more physiologically relevant environment taking into account the effect of IFP and fluid flow.Material and methodsMDA-MB231 breast cancer cells were selected due to their triple negative subtype and were seeded in a dense (80 mg/mL) collagen scaffold, to form an ‘artificial cancer mass’ (ACM). The metabolic activity of the cells in 2D, 3D, static and under flow conditions: 100 and 500 µl/min; IFP: 19 mmHg were evaluated using an Alamar Blue assay. Markers of epithelial-mesenchymal transition (EMT) were determined using qRT-PCR and ΔCt values derived with reference to GAPDH. The responsiveness of the cells to doxorubicin was assessed.Results and discussionsMDA-MB231 cells exhibited a decrease in metabolic activity when cultured in 3D compared to 2D. The 3D/flow environment promoted a switch from an epithelial to a mesenchymal phenotype as evidenced by increased expression levels of Snail (2 fold); vimentin (0.6 fold); MMP1 (0.7 fold); HIF-α (0.2 fold). In contrast, incorporation of IFP in the 3D/flow system was associated with decreased expression levels of vimentin (0.25 fold) and HIF-α (0.56 fold) compared to cells grown in static 3D whilst MMP 14 expression increased (0.5 fold) and snail remained unchanged. 3D/flow was also associated with a reduction in cadherin 11 mRNA levels. Reports have suggested that cadherin-11 has a pro-apoptotic role; analysis of apoptotic markers is planned in order to investigate this finding further.Cells grown in 3D culture were less responsive to doxorubicin treatment compared with those grown in 2D monolayer. When MDA-MB231 cells were grown in 3D/flow/pressure and treated with doxorubicin 2.5 µM, the metabolic activity measured using Alamar blue was 41% lower than for MDA-MB231 cells cultured in 3D static conditions.ConclusionThe results obtained in this study show that the metabolic activity and drug responsiveness of the cancer cell line MDA-MB231 is reduced when cells are grown in 3D. The implementation of flow and IFP on 3D cell cultures promotes EMT of MDA-MB231 cells.

High snail expression predicts a poor prognosis in breast invasive ductal carcinoma patients with HER2/EGFR-positive subtypes

BackgroundHigh Snail expression is known as a poor prognostic factor in breast cancer. However, its prognostic impact for breast cancer with different molecular subtypes is still controversial. MethodsSnail expression was examined by immunohistochemistry in tissue microarray slides of 85 corresponding tumor-adjacent normal (CTAN) and 247 breast invasive ductal carcinoma (IDC) tissues. Multivariable Cox regression analysis was used to assess the impact of Snail expression on survival rate by different molecular subtypes of breast IDC patients. ResultsThe level of Snail expression in IDC tumor tissues was significantly higher than that in CTAN tissues. Moreover, high Snail expression had direct impacts on poor disease specific survival (DSS) and disease-free survival (DFS) in breast IDC patients with human epidermal growth factor receptor 2 (HER2)-positive and human epidermal growth factor receptor (EGFR)-positive statuses as well as the HER2 intrinsic subtype. Additionally, breast IDC patients with a combination of three prognostic factors, including high Snail expression and HER2-positive and EGFR-positive statuses, had much poor DSS and DFS with a statistically significant linear trend. ConclusionHigh Snail expression could predict a poor prognosis for breast IDC patients with HER2/EGFR-positive subtypes.

Snail/FOXK1/Cyr61 Signaling Axis Regulates the Epithelial–Mesenchymal Transition and Metastasis in Colorectal Cancer

Background/Aims: Metastasis is the primary cause of colorectal cancer (CRC)-related death. However, the molecular mechanisms underlying metastasis in CRC remain unclear. Methods: We evaluated mRNA and protein expression levels by quantitative real-time reverse transcription PCR, western blotting, immunofluorescence, tissue microarrays, and immunohistochemistry assays. We also assessed the migration and invasion abilities of CRC cells in vitro by wound healing assays, invasion and migration assays, western blot analysis, and immunofluorescence. Tumor metastasis was evaluated in nude mice in vivo. Results: A positive correlation was observed between the expression patterns of Forkhead box k1 (FOXK1) and Snail in CRC. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that Snail directly bound to and activated the human FOXK1 gene promoter. Moreover, the Snail-FOXK1 axis promote epithelial mesenchymal transition (EMT)-mediated CRC cell invasion and metastasis. FOXK1 and Snail expression levels were correlated with tumor progression and served as significant predictors of overall survival in patients with CRC. Furthermore, overexpression of FOXK1 induced the EMT by upregulating the expression of cysteine-rich angiogenic inducer 61 (Cyr61). Luciferase assays showed that Cyr61 was a direct transcriptional target of FOXK1. Down regulation of Cyr61 decreased FOXK1-enhanced “CRC cell” migration, invasion, and metastasis. Additionally, FOXK1 expression was positively correlated with Cyr61 expression and was associated with poor prognosis. Conclusions: The Snail/FOXK1/Cyr61 signaling axis regulates the EMT and metastasis of CRC.

Deubiquitinase USP9X promotes cell migration, invasion and inhibits apoptosis of human pancreatic cancer.

Ubiquitin specific peptidase9, X-linked (USP9X), a significant regulatory protease in protein ubiquitination, has been proven to act as a proto-oncogene in several types of cancers, such as cervix, colon, breast, brain and lung cancers. The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to its high invasive and metastatic abilities. Nevertheless, whether USP9X acts as a proto-oncogene or a tumor-suppressor gene in PDAC is controversial and the mechanism of metastasis remains unknown. The present study focused on the effect of USP9X on the migration, invasion and apoptosis of PADC cells. We analyzed the expression of USP9X in pancreatic cancer tissues of different pathologic grades by immunohistochemical analysis. USP9X expression in the pancreatic cancer tissues was markedly increased in contrast to that noted in the adjacent non-tumor tissues. USP9X expression in PANC-1 cells was downregulated after transfection of shRNA-USP9X. Knockdown of endogenous USP9X expression evidently inhibited the migration and invasion of PANC-1 cells, and promoted cell apoptosis. Meanwhile, expression levels of Snail, Twist, N-cadherin and vimentin were downregulated. E-cadherin expression was negatively correlated with USP9X expression and the expression of survivin was also downregulated in the PANC-1 cells. In brief, USP9X promoted the migration and invasion of PANC-1 cells probably by provoking epithelial-mesenchymal transition, and also inhibited apoptosis. We believe that USP9X is a major oncogene that may play a significant role in the treatment and prognosis of PDAC.

2,3,5,4‑tetrahydroxy diphenylethylene‑2‑O‑glucoside inhibits the adhesion and invasion of A549 human lung cancer cells.

Lung cancer is considered to be a serious disease that poses a significant threat to human health. 2,3,5,4-tetrahydroxy diphenylethylene-2-O-glucoside (THSG) is a bioactive compound derived from Polygonum multiflorum Thunb. That has been demonstrated to possess antioxidative, anti-inflammatory and antitumor activities. However, little is currently known regarding the potential anticancer effects of this compound in lung cancer. Therefore, the present study aimed to investigate the effects of THSG on the adhesion and invasion of A549 human lung cancer cells in vitro, and to identify the putative mechanisms involved. Cell Counting kit-8 assay was performed to determine A549 cell viability following treatment with various doses (0, 5, 10, 25, 50, 100, 150 and 200 µM) of THSG for 12, 24 and 48 h. In addition, cell adhesion and invasion were determined following treatment of A549 cells with 0, 10, 25 or 50 µM THSG for 1, 2 or 3 h, respectively. Reverse transcription-quantitative polymerase chain reaction analysis was performed to examine the mRNA expression levels of Snail, E-cadherin, vimentin, matrix metalloproteinase (MMP) 2 and MMP9 following THSG treatment for 12 h. Western blot analysis was conducted to detect the protein expression levels of Snail, E-cadherin, vimentin, MMP2 and MMP9 following THSG treatment for 24 h. Treatment with THSG (10, 25 and 50 µM) significantly suppressed the adhesion and invasion of A549 human lung cancer cells in a dose-dependent manner. In addition, the mRNA and protein expression levels of adhesion and invasion-associated factors were decreased significantly in A549 cells treated with THSG. In conclusion, THSG effectively suppressed the adhesion and invasion of human lung cancer cells potentially by inhibiting the expression of adhesion and invasion-related genes.

Sanguinarine inhibits pancreatic cancer stem cell characteristics by inducing oxidative stress and suppressing sonic hedgehog-Gli-Nanog pathway.

Sonic hedgehog pathway is highly activated in pancreatic cancer stem cells (CSC) which play crucial roles in cancer initiation, progression and metastasis. However, the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics is not well understood. The objectives of this study were to examine the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics. Sanguinarine inhibited cell proliferation and colony formation and induced apoptosis through oxidative damage. Sanguinarine inhibited self-renewal capacity of pancreatic CSCs isolated from human and KrasG12D mice. Furthermore, sanguinarine suppressed epithelial-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting N-cadherin. Significant decrease in expression level of Snail, Slug and Zeb1 corroborated the suppression of EMT in sanguinarine treated pancreatic CSCS. The ability of sanguinarine to inhibit pluripotency maintaining factors and CSC markers suggest that sanguinarine can be an effective agent for inhibiting pancreatic cancer growth and development by targeting CSCs. Furthermore, sanguinarine inhibited Shh-Gli pathway leading to modulation of Gli target genes in pancreatic CSCs. Chromatin immunoprecipitation assay demonstrated that Nanog directly binds to promoters of Cdk2, Cdk6, FGF4, c-Myc and Oct4, and sanguinarine inhibits the binding of Nanog with these genes, suggesting the direct involvement of Nanog in cell cycle, pluripotency and self-renewal. To further investigate the role of Shh-Gli-Nanog pathway, we regulated Shh signaling either by Shh protein or Nanog overexpression. Enforced activation of Shh or overexpression of Nanog counteracted the inhibitory effects of sanguinarine on pancreatic CSC proliferation, suggesting the actions of sanguinarine are mediated, at least in part, through Shh-Gli-Nanog pathway. Our studies suggest that sanguinarine can be used for the treatment and/or prevention of pancreatic cancer by targeting CSCs.

Impact of Snail and E-cadherin expression in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PNETs) are a rare type of malignancy with a prognosis that is relatively good, compared with that of pancreatic adenocarcinoma. However, a number of patients with PNETs have distant metastasis and a less favorable prognosis. Epithelial-mesenchymal transition (EMT) is essential for tumor progression and metastasis. Snail and E-cadherin serve key roles in the process of EMT in numerous tumor types, including gastric and pancreatic adenocarcinoma. However, the role of EMT in PNETs remains unclear. The aim of the present study was to investigate the significance of Snail and E-cadherin expression in PNETs. Tissue samples were obtained from 40 resected PNETs. The expression of Snail and E-cadherin was evaluated in the primary tumors using immunohistochemical staining. The association between protein expression and patient clinicopathological features was then analyzed. High and low Snail expression levels were observed in 11 (27.5%) and 29 (72.5%) patients, respectively. Preserved and reduced E-cadherin expression was observed in 19 (47.5%) and 21 (52.5%) patients, respectively. Patients with low Snail expression and preserved E-cadherin expression had a significantly lower risk of vascular invasion, lymphatic invasion, lymph node metastasis and liver metastasis and a lower WHO classification, as compared with the group that included patients with high Snail and reduced E-cadherin, high Snail and preserved E-cadherin, and low Snail and reduced E-cadherin expression. In addition, the patients with low Snail expression levels and preserved E-cadherin expression had more a favorable prognosis compared with the other group. The present study indicates that EMT serves an important role in tumor progression in PNETs. Immunohistochemical evaluation of Snail and E-cadherin is useful for predicting the risk of vessel invasion and metastasis in PNETs.

Vimentin is a potential prognostic factor for tongue squamous cell carcinoma among five epithelial-mesenchymal transition-related proteins.

We aimed to investigate the association of the expression levels of five epithelial–mesenchymal transition (EMT)-related proteins (Snail, Twist, E-cadherin, N-cadherin, and Vimentin) with tumorigenesis, pathologic parameters and prognosis in tongue squamous cell carcinoma (TSCC) patients by immunohistochemistry of tissue microarray. The expression levels of Snail, E-cadherin, N-cadherin and Vimentin were significantly different between the tumor adjacent normal and tumor tissues. In tumor tissues, lower E-cadherin and higher N-cadherin levels were associated with a higher grade of cell differentiation, advanced stage of disease, and lymph node metastasis. However, higher Vimentin expression was associated with poor cell differentiation and lymph node metastasis. Patients with low E-cadherin expression had poor disease-specific survival (DSS). Conversely, positive N-cadherin and higher Vimentin expression levels were associated with poor DSS and disease-free survival. Notably, our multivariate Cox regression model indicated that high Vimentin expression was an adverse prognostic factor for DSS in TSCC patients, even after the adjustment for cell differentiation, pathological stage, and expression levels of Snail, Twist, E-cadherin, and N-cadherin. Snail, E-cadherin, N-cadherin, and Vimentin were associated with tumorigenesis and pathological outcomes. Among the five EMT-related proteins, Vimentin was a potential prognostic factor for TSCC patients.

Abstract TMEM-039: THE ROLE OF SNAIL IN CELL DIFFERENTIATION STATUS: EPITHELIAL-MESENCHYMAL TRANSITION AND CANCER STEM CELLS

Abstract PURPOSE: The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and become mesenchymal cells with migratory and invasive properties. EMT is essential for numerous developmental processes such as gastrulation, and is involved in the initiation of metastasis for cancer progression. Cancer cells that undergo EMT are enabled to metastasize to form secondary tumors. One characteristic of advanced tumors is a low expression level of let-7, a miRNA known to be vital in both maintenances of cellular differentiation and tumor suppression. There is a gap in the literature concerning the mechanism of let-7 downregulation. Our purpose in this study is to present data that shows Snail, an EMT inducer, directly represses let-7, hence inducing cancer stem cells. EXPERIMENTAL PROCEDURES: Ovarian, pancreatic and breast cancer cell lines treated with TGFβ or EGF, inducers of EMT, were studied in our experiments. Expression of several markers at protein level was visualized with immunofluorescence and quantified with flow cytometry. Gene expression of mesenchymal, epithelial and pluripotency factors and miRNAs was detected by q-RT-PCR. Chromatin immunoprecipitation of Snail was performed in ovarian cancer lines. Let-7i promoter luciferase with and without Snail were co-transfected into OVSAHO and 293T cell lines and followed by luciferase assays for detection of bioluminescence. Snail was knocked down by lentiviral small hairpin RNA. Snail was overexpressed by estrogen receptor fusion protein. Orthotopic xenografts of patient-derived ovarian cancer cells were used to test the effect of Snail knockdown on tumor burden. RESULTS: In selected ovarian cancer lines, levels of Snail expression correlated directly with Nanog, and indirectly with let-7 expression. Snail bound promoters of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic burden was significantly reduced in cell line xenografts in which Snail was knocked down. CONCLUSIONS: Let-7 is confirmed to be negatively related to Snail. Our preliminary data also shows Snail directly represses let-7, therefore induces properties of stemness in cancer cells. Cell line and patient-derived data support the relationship between Snail expression, let-7 downregulation, and the induction of cancer stem cells. Snail knockdown appears to disrupt the cancer stem cell state and reduces disease progression. Citation Format: Hanmin Wang, Alyse Huisken-Hill, Evgeny Chirshev, Juli Unternaehrer-Hamm. THE ROLE OF SNAIL IN CELL DIFFERENTIATION STATUS: EPITHELIAL-MESENCHYMAL TRANSITION AND CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-039.

Snail Enhances Glycolysis in the Epithelial-Mesenchymal Transition Process by Targeting FBP1 in Gastric Cancer

Background: Snail is a key regulator of epithelial-mesenchymal transition (EMT) in cancer. However, the regulatory role and underlying mechanisms of Snail in gastric cancer metabolism are unknown. In this study, we characterized the regulation of aerobic glycolysis by Snail in gastric cancer. Methods: The impact of Snail on glucose metabolism was studied in vitro. Combining maximum standardized uptake value (SUVmax), which was obtained preoperatively via a PET/CT scan, with immunohistochemistry staining, we further analyzed the correlation between SUVmax and Snail expression in gastric cancer tissues. Results: Increased expression of Snail promoted lactate production, glucose utilization, and decreased FBP1 expression at both mRNA and protein level. The expression level of Snail was positively associated with SUVmax in gastric cancer patients (P=0.022). Snail and FBP1 expression were inversely correlated at both mRNA and protein level (P=0.002 and P=0.015 respectively) in gastric cancer tissues. Further studies demonstrated that Snail inhibited the FBP1 gene expression at the transcriptional level. Restoring FBP1 expression reversed the effects of glycolysis and EMT induced by Snail in gastric cancer cells. Conclusions: Our results thus reveal that Snail serves as a positive regulator of glucose metabolism through regulation of the FBP1 in gastric cancer. Disrupting the Snail-FBP1 signaling axis may be effective to prevent primary tumor EMT and glycolysis process.

HPIP promotes epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation.

Hematopoietic PBX interacting protein (HPIP), a scaffold protein, is known to regulate the proliferation, migration and invasion in different cancer cell types. The aim of this study was to assess the role of HPIP in ovarian cancer cell migration, invasion and epithelial-mesenchymal transition (EMT), and to unravel the mechanism by which it regulates these processes. HPIP expression was assessed by immunohistochemistry of tissue microarrays containing primary ovarian tumor samples of different grades. OAW42, an ovarian carcinoma-derived cell line exhibiting a high HPIP expression, was used to study the role of HPIP in cell migration, invasion and EMT. HPIP knockdown in these cells was achieved using a small hairpin RNA (shRNA) approach. Cell migration and invasion were assessed using scratch wound and transwell invasion assays, respectively. The extent of EMT was assessed by determining the expression levels of Snail, Vimentin and E-cadherin using Western blotting. The effect of HPIP expression on AKT and MAPK activation was also investigated by Western blotting. Cell viabilities in response to cisplatin treatment were assessed using a MTT assay, whereas apoptosis was assessed by determining caspase-3 and PARP cleavage in ovarian carcinoma-derived SKOV3 cells. We found that HPIP is highly expressed in high-grade primary ovarian tumors. In addition, we found that HPIP promotes the migration, invasion and EMT in OAW42 cells and induces EMT in these cells via activation of the PI3K/AKT pathway. The latter was found to lead to stabilization of the Snail protein and to repression of E-cadherin expression through inactivation of GSK-3β. We also found that HPIP expression confers cisplatin resistance to SKOV3 cells after prolonged exposure and that its subsequent knockdown decreases the viability of these cells and increases caspase-3 activation and PARP proteolysis in these cells following cisplatin treatment. From these results we conclude that HPIP expression is associated with high-grade ovarian tumors and may promote their migration, invasion and EMT, a process that is associated with metastasis. In addition, we conclude that HPIP may serve as a potential therapeutic target for cisplatin resistant ovarian tumors.

EGF induces epithelial-mesenchymal transition through phospho-Smad2/3-Snail signaling pathway in breast cancer cells

Epithelial-mesenchymal transition (EMT) can contribute to tumor invasion, metastasis, and resistance to chemotherapy or hormone therapy. EMT may be induced by a variety of growth factors, such as epidermal growth factor (EGF). Most studies regarding EMT have focused on TGF-β-Smads signaling. The mechanism of EGF-induced EMT via activation of the Smad2/3 in breast cancer cells, MCF-7 and MDA-MB-231, remains unclear. The expression levels of Snail, vimentin, and fibronectin were increased by EGF treatment in a time-dependent manner, while the expression level of E-cadherin was decreased. EGF-induced nuclear co-localization of phospho-Smad2/3 and Snail and cancer cell migration were inhibited by pretreatment with an ERK1/2 inhibitor, PD98059 and a phospho-Smad2 inhibitor, SB203580. Knockdown of Smad2/3 expression suppressed EGF-induced expressions of Snail, vimentin, fibronectin, and cancer cell invasion, suggesting an acquisition of the mesenchymal and migratory phenotype in less aggressive MCF-7 cells. Moreover, MDA-MB-231 cells were shown that EGF-induced EMT, and cell invasion through ERK1/2-phospho-Smad2/3-Snail signaling pathway. We have discovered that EGF-stimulated activation of Smad2/3 upregulated several key EMT markers, inhibited E-cadherin expression, promoted EMT, enhanced migration and invasion in MCF-7 and MDA-MB-231 breast cancer cells. Identification of this molecular mechanism may provide new molecular targets for the development of therapies for metastatic breast cancer.

Expression of epidermal growth factor-like domain 7 may be a predictive marker of the effect of neoadjuvant chemotherapy for locally advanced uterine cervical cancer.

Neoadjuvant chemotherapy (NAC) followed by hysterectomy may be effective for the treatment of locally advanced uterine cervical cancer and improve patient prognosis. It is important to identify markers that are able to predict whether NAC may be successful. Epidermal growth factor-like domain 7 (EGFL7) regulates vascular sprouting in blood vessel formation. In numerous types of human cancer, EGFL7 is upregulated and inhibits endothelial cell adhesion molecules, decreasing vascular tightness and, thus, increasing vascular permeability. It is considered that the overexpression of EGFL7 is able to inhibit drug delivery, resistance to apoptosis and the epithelial-to-mesenchymal transition. In the current study, 63 patients with locally advanced uterine cervical cancer were reviewed and classified as stage IIIA-IIIB using the International Federation of Gynecology and Obstetrics criteria. These patients (aged <70 years) were treated at Osaka City University Medical School Hospital, Japan, between 1995 and 2010. Tumor tissue samples were obtained by biopsy prior to NAC. The tissue samples were classified as group 1 or 2 depending on the efficacy of NAC. Surgery and radiotherapy were administered in group 1 (n=36), for which NAC was effective. In the patients of group 2 NAC was not effective, and radiotherapy alone was administered (n=27). The expression of EGFL7 and Snail was examined in paraffin-embedded tissue sample sections using the avidin-biotin peroxidase complex method. The results indicated that EGFL7 expression levels were significantly higher in group 2, as compared with group 1 (P<0.001). A similar result was observed for the expression levels of Snail (P=0.001). Group 1 exhibited significantly longer overall survival times compared with group 2 (P=0.001). The patients were also classified into a low EGFL7 expression level group (weighted score of ≤6) and a high EGFL7 expression level group (weighted score of ≥8). NAC was observed to be significantly more effective in the low EGFL7 expression level group (P<0.001), as compared with the high expression level group. The results suggest that the expression levels of EGFL7 may be a potential predictive marker of the efficacy of NAC for the treatment of locally advanced uterine cervical cancer.

Cytokeratin 18 is necessary for initiation of TGF-β1-induced epithelial-mesenchymal transition in breast epithelial cells.

During epithelial-mesenchymal transition (EMT), epithelial cells lose key phenotypic markers (e.g., E-cadherin and cytokeratin 18) and acquire mesenchymal markers (e.g., N-cadherin and vimentin). Although the loss of cytokeratin 18 is a hallmark of EMT, the regulatory role of cytokeratin 18 in EMT is not yet fully understood. Here, we report that cytokeratin 18 is involved in the regulation of transforming growth factor-beta1 (TGF-β1)-induced EMT in breast epithelial cells. When MCF10A cells were treated with TGF-β1 for 24h, considerable morphological changes, indicative of the early stages of EMT (e.g., loss of cell-cell contact), were observed and cytokeratin 18 was downregulated. However, E-cadherin levels were not altered until a later time point. This suggests that cytokeratin 18 may play an active role during the earlier stages of EMT. Consistent with this notion, siRNA-mediated knockdown of cytokeratin 18 delayed TGF-β1-mediated EMT, and the associated downregulation of E-cadherin reduced the phosphorylation/nuclear localization of smad 2/3 and decreased the expression levels of snail and slug (which inhibit E-cadherin expression in epithelial cells as an early response to TGF-β1). Taken together, these results suggest that cytokeratin 18 critically contributes to initiating TGF-β1-induced EMT via the smad 2/3-mediated regulation of snail and slug expression in breast epithelial cells.

Gastrokine-2 suppresses epithelial mesenchymal transition through PI3K/AKT/GSK3β signaling in gastric cancer.

Epithelial-mesenchymal transition (EMT) plays an important role in metastasis of gastric cancer. Our previous study showed that Gastrokine-2 (GKN2) can inhibit the metastasis of SGC-7901 and AGS cells. Herein, we further explored the role of GKN2 in epithelial mesenchymal transition of gastric cancer cells and the underlying mechanisms. We found that overexpression of GKN2 can lower the protein expression level of Snail and markedly elevate E-cadherin protein level in SGC7901 and AGS cells. Further data showed that knockdown of snail can inhibit the migration and invasion of SGC-7901 and AGS cells. It is known that Snail can be phosphorylated by GSK3β, a downstream protein of PI3K/AKT pathway. We then test protein expression of p-GSK3β(Ser-9), the downstream protein of PI3K/AKT, which was significantly decreased under the circumstance of GKN2 overexpression. Moreover, LY294002, a PI3K inhibitor, can reverse the protein expression change of E-cadherin and snail induced by siGKN2. Taken together, these findings suggested that GKN2 suppressed epithelial mesenchymal transition of gastric cancer cells by downregulation of snail through PI3K/AKT/GSK3β signaling pathway.

TLR7 agonist induced repression of hepatocellular carcinoma via the TLR7-IKK-NF-κB-IL6 signaling pathway.

Toll-like receptors (TLRs) are key members of innate immunity, involved in the defense against diseases, and evidence has revealed that TLR4/5 is involved in the carcinogenesis of hepatic cancer. TLR7 belongs to the TLR family, and its roles in immune-associated hepatic diseases have been well characterized; however, the consequences of agonist targeting of TLR7 in hepatic cancer have not previously been reported. The present study aimed to investigate the effects and underlying mechanisms of Imiquimod, a TLR7 agonist, on hepatic carcinogenesis by affecting the self-renewal of hepatic cancer stem cells. To detect the effects of this TLR7 agonist on hepatic cancer cells an MTT assay, mammosphere formation assay, ALDEFLUOR™ fluorescence-based stem cell sorting was used, and the potential signaling involved in the mechanism was investigated by western blot analysis. The TLR7 agonist Imiquimod demonstrated inhibitory effects on the cell proliferation and mammosphere formation of hepatic cells and stem cells, and decreased stem cell number (P<0.01). These effects may be achieved via the TLR7/IκB kinase/nuclear factor-κB/interleukin-6 signaling pathway, with decreased levels of Snail expression. The present study demonstrated the effects and mechanisms of the TLR7 agonist on hepatic cancer occurred via suppression of the self-renewal of cancer stem cells, indicating novel potential functions of the TLR7 agonist in the treatment of HCC.

HIF-1α regulates EMT via the Snail and β-catenin pathways in paraquat poisoning-induced early pulmonary fibrosis.

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.

Heterophyllin B inhibits the adhesion and invasion of ECA-109 human esophageal carcinoma cells by targeting PI3K/AKT/β-catenin signaling.

The present study aimed to measure the effect of heterophyllin B (HB) on the adhesion and invasion of ECA-109 human esophageal carcinoma cells, and examine the possible mechanism involved. A Cell Counting kit 8 assay was performed to determine the cell viability. Cell adhesion and invasion were determined following treatment of the ECA-109 cells with HB (0, 10, 25 and 50 µM) for 24 h. The levels of phosphorylated (p-)ATK and p-phosphoinositide 3-kinase (PI3K), and the protein levels of β-catenin were measured using western blot analysis. The mRNA and protein expression levels of E-cadherin, vimentin, snail, matrix metalloproteinase (MMP)2 and MMP9 were detected using reverse trancsription-quantitative polymerase chain reaction and western blot analyses, respectively. HB (10, 25 and 50 µM) significantly suppressed the adhesion and invasion of the ECA-109 human esophageal carcinoma cells in a dose-dependant manner. The expression levels of p-ATK, p-PI3K and β-catenin were markedly decreased. The expression of E-cadherin was promoted, whereas the expression levels of snail, vimentin, MMP 2 and MMP 9 were decreased significantly in the ECA-109 cells treated with HB. In addition, HB inhibited the adhesion and invasion induced by PI3K activating peptide in the ECA-109 cells, and the protein expression levels were also adjusted. These results suggested that HB effectively suppressed the adhesion and invasion of the human esophageal carcinoma cells by mediating the PI3K/AKT/β-catenin pathways and regulating the expression levels of adhesion- and invasion-associated genes.