Expression Levels Of Twist Research Articles

A twist tale of cancer metastasis and tumor angiogenesis.

Twist1 is an evolutionally conserved transcription factor. Originally identified in Drosophila as a key regulator for mesoderm development, it was later implicated in many human diseases, including Saethre-Chotzen syndrome and cancer. Twist1's involvement in cancer has been well recognized. Driven by hypoxia-induced factor-1 (HIF-1), Twist1 has been considered as a proto-oncogene and its overexpression has been observed in a wide variety of human cancers. High expression level of Twist1 is closely related to tumor aggressiveness and metastatic potential. In cancer cells, Twist1 has been shown to function as a key regulator of epithelial-mesenchymal transition (EMT), a critical process for metastasis initiation. Twist1 has also been implicated in maintaining cancer stemness for self-renewal and chemoresistance. This review first summarizes the roles of Twist1 in embryo development and Saethre-Chotzen syndrome followed by a discussion of Twist1's critical functions in cancer. In particular, the review focuses on the recent discovery of Twist1's capability to promote endothelial transdifferentiation of cancer cells beyond EMT.

Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901 cells

To study the role of Twist gene in gastric cancer by gene silencing, including the potential of induction of apoptosis, cell cycle arrest, and proliferation inhibition in human malignant gastric SGC7901 cells. The expression level of Twist in gastric cancer samples was measured by immunohistochemistry. The effects of Twist gene silencing were detected at both mRNA and protein levels by RT-PCR and Western blot. We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry. We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit. Cell cycle distribution was analyzed by flow cytometry. Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay. Twist protein was highly expressed in gastric cancer samples. Twist gene silencing significantly induced apoptosis, cell cycle arrest at G0/G1 phase, proliferation inhibition, and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells. Meanwhile, both caspase-3 and caspase-9 were activated. The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.

Favorable Clinical Outcome and Unique Characteristics in Association with Twist1 overexpression in De Novo Acute Myeloid Leukemia

Epithelial-mesenchymal transition (EMT) is a critical process for inducing stem-like properties of epithelial cancer cells. However, the role of EMT inducers in hematologic malignancies is unknown. Twist1, an EMT inducer necessary for cell migration, has recently been found to have transcriptionally regulatory activity on the expression of Bmi1, and these two are capable of promoting tumorigenesis in a synergized manner. Knowing that Bmi1 expression is essential for maintenance of leukemic stem cells, we speculate that Twist1 might govern the pathogenesis of acute myeloid leukemia (AML) development as well.We used AML cell lines KG1a and THP1 cells, which had low and high expression levels of Twist1, respectively to demonstrate that upregulated Twist1 increased Bmi1 expression in AML. Bmi1 overexpression endued leukemic cells a higher proliferative potential and increased resistance to apoptosis. In primary AML samples, there was strong positive correlation between the expression levels of Twist1 and Bmi1. AML Patients whose leukemic blasts harbored overexpressed Twist1 had a more aggressive clinical phenotype, but they were more likely to have a better clinical outcome after standard therapy. In vitro studies confirmed that Twist1-overexpressing leukemic cells were more susceptible to cytarabine, but not daunorubicin, cytotoxicity.Our findings suggest that, in a subset of AML patients, Twist1 plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. The result indicates that Twist1 could represent a powerful biomarker that serves as a prognostic as well as therapeutic guide for patients with AML. DisclosuresNo relevant conflicts of interest to declare.

Expressions of Twist and hypoxia inducible factor-1α in invasive ductal carcinoma of breast and their relationships with epithelial-mesenchymal transition

Objective To investigate the expression of Twist and hypoxia inducible factor-1α (HIF-1α) in invasive ductal carcinoma of breast and their relationships with epithelial-mesenchymal transition (EMT).Methods Expression levels of Twist,HIF-1α,E-cadherin and Vimentin in 90 cases of invasive ductal carcinoma tissues and corresponding adjacent tissues were detected by immunohistochemical staining of streptavid-in-peroxidase (SP) method.Results The positive expression rate of Twist,HIF-1 α,E-cadherin and Vimentin was 73.3％,68.9％,15.6％ and 64.4％ respectively in breast cancer tissues,and 7.8％,8.9％,75.6％ and 34.4％ respectively in adjacent tissues.The difference in positive rate between breast cancer tissues and adjacent tissues was statistically significant (P ＜ 0.05).The expression levels of Twist and HIF-1α were correlated with histological grade and lymph node metastasis (P ＜ 0.05).There was a positive correlation between expression levels of Twist and HIF-1α.The expression levels of Twist and HIF-1α were closely correlated with low expression of E-cadherin and high expression of Vimentin.Conclusion Twist,HIF-1α and Vimentin in breast cancer tissues showed higher expression,while E-cadherin lower expression.HIF-1α may promote the occurrence of EMT by up-regulating the expression of Twist,which makes it become one of the possible ways to promote the invasion and metastasis of breast cancer. Key words: Breast carcinoma ; Epithelial-mesenchymal transition ; Twist ; Hypoxia inducible factor-1α

Abstract 1138: Aberrant expression of Plastin3 (PLS3) induces liver metastasis via enhancing the epithelial-mesenchymal transition and stemness in colorectal cancer (CRC)

Abstract Background PLS3 gene, located on chromosome Xq23, encodes a protein 10 product (PLS3) that reduces F-actin disassembly and inhibits cofilin-mediated depolymerization of actin fibers. We previously reported that PLS3 is a novel marker for circulating tumor cells in colorectal cancer (CRC), and the high expression of PLS3 in tumor tissue is associated with a poor prognosis in patients with primary CRC. In this study, we investigated its role in functional significance for metastatic potential of CRC performing in vitro and in vivo studies. Method Four patients with surgically resected both primary CRC and liver metastasis were included in this study. Those tissues were performed with the PLS3-immunohistochemical examination (PLS3-IHC). In vitro, we cultured LOVO, DLD-1 and HCT116 which caused no liver metastasis by injecting them into the spleen of NOD/SCID mice. We stably enhanced the expression of PLS3 in the cells by transfecting the lentiviral vector expressing PLS3. The expression levels of EMT-related genes were examined with real time RT-PCR. The migration, invasion and sphere formation assay were also performed. In vivo, NOD/SCID mice were divided into two groups, which included mice transplanted with only PLS3-transfected cells and mice transplanted with PLS3-transfected and PLS3-attenuated cells together, by injecting into the spleen. We calculated the established primary splenic tumors and liver metastasis. PLS3-IHC was also performed to the sacrificed tissues. Result (1) In the PLS3-IHC of four CRC samples, all the liver metastasis tissues included stained areas, but only one primary tissue showed PLS3-staining. This was the evidence that cancer cells expressing PLS3 had metastatic properties. (2) PLS3-transfected cells had the significant decrease of E-cadherin, while the expression levels of TWIST, ZEB and vimentin were significantly higher in PLS3-transfected than PLS3-attenuated cells. PLS3-transfected LOVO significantly induced invasion and migration, compared to PLS3-attenuatd LOVO. In the sphere formation assay, the high expression of PLS3 significantly increased the proportion of sphere generating cells (DLD-1) (p<0.001). (3) In vivo, PLS3-transfected cells (DLD-1 and HCT116) significantly induced the growth of liver metastasis. The volumes of liver metastasis of mice transplanted with the mixtures were significantly smaller than those of only PLS3-transfected cells. The liver metastasis tissues showed only stained areas in both groups in PLS3-IHC. However, the established splenic tumor of mice transplanted with the mixtures displayed not only the stained but also the nonstained areas in PLS3-IHC. Conclusion In conclusion, our study suggests experimental evidence to support the functional significance of PLS3 to induce epithelial-mesenchymal trandition, stemness and liver metastasis of human CRC. Citation Format: Masami Ueda, Keishi Sugimachi, Junji Kurashige, Shotarou Sakimura, Hidenari Hirata, Ryutarou Uchi, Yuki Takano, Hiroki Ueo, Tae Matsumura, Yoshiaki Shinden, Hidetoshi Eguchi, Tomoya Sudo, Masaki Mori, Koshi Mimori. Aberrant expression of Plastin3 (PLS3) induces liver metastasis via enhancing the epithelial-mesenchymal transition and stemness in colorectal cancer (CRC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1138. doi:10.1158/1538-7445.AM2014-1138

Expression levels of TWIST1 are associated with the clinicopathological stage of B-cell non-Hodgkin lymphoma.

The aim of the present study was to investigate the expression level of TWIST1 in B-cell non-Hodgkin lymphoma (BNHL) and its association with the clinicopathological characteristics of BNHL. Expression levels of TWIST1 were analyzed in patients with BNHL (n=45) and lymphadenosis (n=21) using immunohistochemical staining and western blot analysis. In addition, the mRNA expression levels of TWIST1 in the peripheral blood were detected by fluorescent quantitative polymerase chain reaction. The positive rate of TWIST1 expression in the BNHL tissue was 82.2%, which was significantly higher compared with the lymphadenosis tissue (5%; P<0.05). In addition, the protein expression level of TWIST1 in the BNHL tissue was higher compared with the lymphadenosis tissue. TWIST1 expression was also higher in stage III/IV BNHL tissues than in stage I/II tissues (P<0.05). The tissues were staged following the Ann Arbor system. Furthermore, the mRNA expression level of TWIST1 in the peripheral blood of the BNHL tissue (3.03±0.03) was higher compared with the lymphadenosis tissue, and the mRNA expression level of TWIST1 was higher in stage III/IV (4.41±0.12) tissues than in stage I/II BNHL (2.03±0.08) tissues. In conclusion, TWIST1 expression was higher in the tissue and peripheral blood of patients with BNHL when compared with those with lymphadenosis. Thus, TWIST1 expression was associated with the clinicopathological stage of BNHL.

Twist expression in dentigerous cyst, odontogenic keratocyst, and ameloblastoma.

Epithelial-mesenchymal transition (EMT) is a process which is associated with a loss of intercellular adhesion, acquired mesenchymal shape, and increased motility by epithelial cells. Twist is one of the key regulators of EMT.In view of the distinct clinical behavior of odontogenic lesions, the objective of the present study was to investigate the immunohistochemical expression of Twist in these lesions. In this study, 70 formalin-fixed, paraffin-embedded tissue blocks of odontogenic lesion consisting of 16 unicystic ameloblastomas (UA), 17 solid ameloblastomas (SA), 18 odontogenic keratocysts (OKC), and 19 dentigerous cysts (DC) were reviewed using immunohistochemistry for Twist staining. In this study, Twist immunostaining was evident in all groups of the specimens except the dentigerous cyst group. Twist expression was seen in 58.8% (10/17) of SA, 50% (8/16) of UA, and 44.4% (8/18) of OKCs. 23.5% of SA, 18.8% of UA, and 16.7% of OKCs showed Twist expression in more than 50% of cells. Statistical analysis showed that Twist expression levels were significantly higher in ameloblastomas (SA and UA) and OKCs than dentigerous cysts (P = 0.002). There were no significant differences between Twist expression in SAs, UAs, and OKCs (P > 0.05). The results of this study propose that the high expression rate of Twist plays a role in the pathogenesis of ameloblastomas and OKCs and might be one of the reasons for the aggressive behavior of ameloblastomas and high recurrence of OKCs and could reinforce the classification of OKC as an odontogenic tumor.

Abstract P1-07-08: RNA based Twist1 inhibition via dendrimer complex to reduce breast cancer cell metastasis

Abstract Breast cancer remains a leading cause of cancer related deaths for women in the United States with the principal cause of mortality being metastatic disease. Mortality rates associated with metastatic breast cancer tumors are significantly elevated compared to localized disease because the most common metastatic sites (brain, bone marrow, and lungs) are difficult to target either with chemotherapy or surgical intervention. Therefore reduction of tumor cell dispersion is a key component to a reduction in mortality rates among patients with breast cancer. Epithelial-Mesenchymal Transition (EMT) is the process by which cancer cells downregulate proteins associated with cell to cell adhesion (e.g. E-cadherin) resulting in cells that are able to detach from neighboring cells, invade adjacent tissue, and eventually enter the circulatory system or lymphatics. Many breast cancers are known to commandeer the EMT process, thus allowing the cells to metastasize beyond the primary tumor. The process of EMT is tightly regulated by the transcription factor Twist1, which is often overexpressed in breast cancer. Therefore, Twist1 serves as an excellent therapeutic target whose downregulation would result in fewer metastatic cancer cells and correspondingly reduce cancer mortality. Twist1 is also a desirable target because it is almost nonexistent in adult tissues and thus its silencing would have minimal side effects, especially compared to many of the non-specific cancer treatments used today. Our lab has elected to use an RNA-based mediated gene silencing approach to decrease the expression levels of Twist1 in a highly invasive breast cancer cell line (SUM 1315). Due to their fragile nature, siRNA molecules are often difficult to deliver at therapeutic levels either in vitro or in vivo. We have overcome these delivery and degradation limitations through the optimization and use of Poly (amidoamine) (PAMAM) dendrimers. By complexing the siRNA with the PAMAM dendrimers not only protects the RNA molecules, but also facilitates their uptake into the tumor cells. We hypothesize that suppression of the activity of Twist1 via dendrimer-delivered Twist1 siRNA will inhibit metastatic behavior of aggressive breast cancer cells. Here we demonstrate successful delivery of Alexa Fluor® 488 labeled siRNA using two different dendrimers (Generation 5 and a modified Generation 3) with transfection efficiency results exceeding those of Lipofectamine 2000 and with far less toxicity to cells. We also show up to a 90% reduction (lasting at least 7 days) in Twist1 expression using Western Blot and qPCR analysis. Along with the knock down of Twist1, here we also demonstrate a significant knockdown of EMT proteins N-Cadherin and Vimentin. Functionally, luciferase promoter assays of Twist1 targets were also reduced. Furthermore, we were able to appreciate a significant phenotypic decrease in the invasive nature of the breast cancer cells using migration/invasion assays. Taken together these results demonstrate successful knockdown of Twist1 using siRNA dendrimer complexes resulting in an altered cellular phenotype and function. These data will serve as a foundation for optimization of future in vivo experiments with both orthotopic and metastatic breast cancer models. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-08.

Carbon Nanotube-Induced Pulmonary Granulomatous Disease: Twist1 and Alveolar Macrophage M1 Activation

Sarcoidosis, a chronic granulomatous disease of unknown cause, has been linked to several environmental risk factors, among which are some that may favor carbon nanotube formation. Using gene array data, we initially observed that bronchoalveolar lavage (BAL) cells from sarcoidosis patients displayed elevated mRNA of the transcription factor, Twist1, among many M1-associated genes compared to healthy controls. Based on this observation we hypothesized that Twist1 mRNA and protein expression might become elevated in alveolar macrophages from animals bearing granulomas induced by carbon nanotube instillation. To address this hypothesis, wild-type and macrophage-specific peroxisome proliferator-activated receptor gamma (PPARγ) knock out mice were given oropharyngeal instillation of multiwall carbon nanotubes (MWCNT). BAL cells obtained 60 days later exhibited significantly elevated Twist1 mRNA expression in granuloma-bearing wild-type or PPARγ knock out alveolar macrophages compared to sham controls. Overall, Twist1 expression levels in PPARγ knock out mice were higher than those of wild-type. Concurrently, BAL cells obtained from sarcoidosis patients and healthy controls validated gene array data: qPCR and protein analysis showed significantly elevated Twist1 in sarcoidosis compared to healthy controls. In vitro studies of alveolar macrophages from healthy controls indicated that Twist1 was inducible by classical (M1) macrophage activation stimuli (LPS, TNFα) but not by IL-4, an inducer of alternative (M2) macrophage activation. Findings suggest that Twist1 represents a PPARγ-sensitive alveolar macrophage M1 biomarker which is induced by inflammatory granulomatous disease in the MWCNT model and in human sarcoidosis.

Expression and correlation of Twist and gelatinases in breast cancer

Altered expression of Twist, matrix metalloproteinase (MMP)-2 and MMP-9 proteins has been identified in various types of human cancers. However, the correlation between Twist and these gelatinases in breast cancer remains unclear. In this study, immunohistochemical analysis of Twist, MMP-2 and MMP-9 expression was performed on tissue microarrays from 200 breast cancer cases. The association of Twist and gelatinase expression with clinicopathological factors and patient survival was analyzed. Altered expression of Twist, MMP-2 and MMP-9 proteins was observed in breast cancer tissue. The positive rates of Twist, MMP-2 and MMP-9 protein expression were 75.5, 97.0 and 96.0%, respectively. Increased expression of Twist was positively correlated with the status of axillary lymph node metastasis and higher tumor-node-metastasis (TNM) stage (P<0.01). Moreover, increased expression of Twist was correlated with poor overall survival (OS) and post-operative relapse-free survival (RFS), compared with those for the patients with reduced expression levels of Twist (P<0.05, P<0.01). The expression of MMP-2 and MMP-9 was positively correlated with Twist expression (P<0.001). Our results indicate that Twist may play an important role in the invasion, metastasis and prognosis of breast cancer. Additionally, our results suggest that Twist may be a regulator of gelatinases (MMP-2 and MMP-9).

Abstract 2692: Dendrimer delivery of RNA based Twist1 inhibitors to reduce breast cancer cell metastasis.

Abstract Breast cancer in women is a leading cause of cancer related deaths in the United States with metastatic disease being the chief cause of mortality. Metastatic breast cancer tumors are often found in their later stages making them difficult to remove or treat. Inhibition of tumor cell dispersion is therefore a key component to a reduction in mortality rates among women with breast cancer. Epithelial-Mesenchymal Transition (EMT) is the process by which cancer cells downregulate proteins associated with cell to cell adhesion (e.g. E-cadherin) resulting in cells that are able to mobilize. Breast cancer cells are known to commandeer the EMT process through overexpression of Twist1, thus allowing the cells to metastasize beyond the primary tumor. Twist1 therefore serves as an excellent therapeutic target whose disruption would result in fewer metastatic cancer cells and correspondingly reduce cancer mortality. Twist1 is also a desirable target because it is almost nonexistent in adult tissues and thus its silencing would have minimal side effects. Our lab has elected to use an siRNA mediated gene silencing approach to decrease the expression levels of Twist1 in two highly invasive breast cancer cell lines (SUM 1315 and MCF7-Tw). Due to their fragile nature, siRNA molecules are often difficult to deliver at therapeutic levels. We have overcome these delivery limitations through the optimization and use of Poly (amidoamine) (PAMAM) dendrimers. We hypothesize that suppression of the activity of Twist1 via dendrimer-delivered Twist1 siRNA will inhibit metastatic behavior of aggressive breast cancer cells. Here we demonstrate successful delivery of Cy3-labled siRNA using two different dendrimers (Generation 5 and a modified Generation 3) with transfection efficiency results exceeding those of Lipofectamine 2000 and with far less toxicity to cells. We also show up to a 90% reduction (lasting at least 4 days) in Twist1 expression using Western Blot and qPCR analysis. Phenotypically we were able to appreciate a significant decrease in the invasive nature of the breast cancer cells using migration/invasion assays. Taken together these results demonstrate successful knockdown of Twist1 using siRNA dendrimer complexes resulting in an altered cellular phenotype. These data will serve as a foundation for optimization of future in vivo experiments with both orthotopic and metastatic breast cancer models. Citation Format: James B. Finlay, Cai M. Roberts, Carlotta A. Glackin. Dendrimer delivery of RNA based Twist1 inhibitors to reduce breast cancer cell metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2692. doi:10.1158/1538-7445.AM2013-2692

Expression and Significance of Twist and E-cadherin in Ovarian Cancer Tissues

To investigate the expression of Twist and E-cadherin in ovarian cancer tissues as well as the role of epithelial-mesenchymal transformation (EMT) in ovarian cancer metastasis. The expressions of Twist and E-cadherin in 54 cases of ovarian cancer and paracancerous tissues were detected by Western blottin g and reverse transcriptase polymerase chain reaction. We used RNA interference to silence Twist expression in human ovarian cancer cell line, and detected E-cadherin expression using Western blotting. There was an increase in the relative abundance of Twist proteins and a decrease in E-cadherin in ovarian cancer compared with normal ovary tissues (P < 0.05). The expression levels of Twist and E-cadherin mRNA were 1.49 ± 0.53 and 0.82 ± 0.24 in ovarian cancer, and 1.14 ± 0.38 and 1.08 ± 0.19 in paracancerous tissues, respectively. The difference between the indicators in ovarian cancer and in paracancerous tissues was statistically significant (P < 0.05). When the Twist expression was silenced in an ovarian cancer cell line, the expression of the E-cadherin protein increased (P<0.05). The expression of Twist is upregulated, whereas that of E-cadherin is downregulated in ovarian cancer. EMT, mediated by Twist, may be correlated with ovarian cancer metastasis.

Expression of TWIST1, Snail, Slug, and NF-κB and methylation of the TWIST1 promoter in mammary phyllodes tumor

TWIST1, Slug, Snail, SIP1, and NF-κB are overexpressed in various tumors and associated with metastasis and poor prognosis. In this study, we examined their potential roles in phyllodes tumor (PT). The expression of TWIST1, Snail, Slug, SIP1, and NF-κB in benign (n = 103), borderline (n = 38), and malignant (n = 38) PTs was examined by immunostaining. The methylation status of the TWIST1 promoter was analyzed by methylation-specific PCR. We detected high expression levels of TWIST1 in 47.4% of borderline/malignant PTs and 31.1% of benign PTs, Slug in 64% of borderline/malignant PTs and 62.1% of benign PTs, epithelial SIP1 in 75.0% of borderline/malignant PTs and 86.3% of benign PTs, stromal SIP1 in 35.5% of borderline/malignant PTs and 22.3% of benign PTs, and NF-κB in 63.2% of borderline/malignant PTs and 52.4% of benign PTs. Snail expression was detected in all cases. A high expression of TWIST1 (p = 0.026) and TWIST1 promoter methylation (p = 0.000) were significantly more frequent in borderline/malignant PTs than in benign PTs. Moreover, a high expression of at least four of the five antibodies was more commonly observed in borderline/malignant PTs than in benign PTs (p = 0.026). However, no relationship was found between the expression of TWIST1 or the other proteins examined and the clinical outcome. Our results suggest that a high expression of TWIST1 and related proteins plays a pivotal role in the malignant progression of PT.

Expression patterns of epithelial–mesenchymal transition markers in localized prostate cancer: significance in clinicopathological outcomes following radical prostatectomy

To analyse the expression patterns of multiple molecular markers implicated in epithelial-mesenchymal transition (EMT) in localized prostate cancer (PC), in order to clarify the significance of these markers in patients undergoing radical prostatectomy (RP). Expression levels of 13 EMT markers, namely E-cadherin, N-cadherin, β-catenin, γ-catenin, fibronectin, matrix metalloproteinase (MMP) 2, MMP-9, Slug, Snail, Twist, vimentin, ZEB1 and ZEB2, in RP specimens from 197 consecutive patients with localized PC were evaluated by immunohistochemical staining. Of the 13 markers, expression levels of E-cadherin, Snail, Twist and vimentin were closely associated with several conventional prognostic factors. Univariate analysis identified these four EMT markers as significant predictors for biochemical recurrence (BR), while serum prostate-specific antigen, Gleason score, seminal vesicle invasion (SVI), surgical margin status (SMS) and tumour volume were also significant. Of these significant factors, expression levels of Twist and vimentin, SVI and SMS appeared to be independently related to BR on multivariate analysis. There were significant differences in BR-free survival according to positive numbers of these four independent factors. That is, BR occurred in four of 90 patients who were negative for risk factors (4.4%), 21 of 83 positive for one or two risk factors (25.3%) and 19 of 24 positive for three or four risk factors (79.2%). Measurement of expression levels of potential EMT markers, particularly Twist and vimentin, in RP specimens, in addition to conventional prognostic parameters, would contribute to the accurate prediction of the biochemical outcome in patients with localized PC following RP.

The Inflammatory Molecules S100A8 and TGF-b1 Activate the Epithelial-Mesenchymal Transition Process in Pancreatic Cancer Cells

Context Chronic inflammation is suggested to play a key role in cancer initiation and progression. Epithelial-mesenchymal transition (EMT) is an essential developmental program that becomes reactivated in adult tissues to promote the progression of cancer. EMT is characterized by an enhanced cell motility, the loss of the epithelial marker E-cadherin (CDH1), and the overexpression of the mesenchimal marker N-cadherin (CDH2). Master regulators of EMT are the transcription factors Snail, Slug, ZEB and Twist. Objective To ascertain whether the inflammatory molecules S100A8, S100A9 and TGF-b1, overexpressed in pancreatic cancer (PaCa), might start the EMT program. Methods BxPC3 cells were treated with 10 nM S100A8 or S100A9, 0.02 ng/mL TGF-b1 or with vehicle alone (reference) for 72 hours. The expression (mRNA) of Snail, Slug, ZEB1, ZEB2, Twist, CDH1 and CDH2 were quantified by RT-PCR (Light Cycler). N-cadherin protein expression was evaluated by immunohistochemistry (IHC). Results TGF-b1 significantly enhanced the expression of all the tested EMT markers (P=0.037 for CDH1, CDH2, Slug, ZEB1 and ZEB2; P=0.005 for Snail and Twist). S100A8 significantly enhanced the expression levels of Twist (median fold increase: 4, range: 2.6-22.3; P=0.007) while S100A9 did not (median: 2, range: 0.8-9.3, P=0.10). Both molecules reverted TGF-b1 effects on Twist expression (TGF-b1: median: 6, range: 3.6-42.6; TGF-b1 and S100A8: median: 3, range: 2.3-3.8; TGF-b1 and S100A9: median: 3.3, range: 3.1-4.2). By IHC, control cells showed an inhomogeneous non-continuous membranous N-cadherin immunostain. The treatments with both TGF-b1 and S100A8 resulted in an enforced moderate complete membranous immunostain. The combined treatment with TGF-b1 and S100A8 was less effective on protein visualization in comparison to any single treatments (weak to moderate complete immunostain). Conclusion TGF-b1 activate the transcriptional program which underlies the EMT in PaCa cells. A dual role for S100A8 in the EMT was shown: it promotes the EMT by inducing Twist mRNA and N-cadherin protein expression, but it antagonizes TGF-b1 effects on both targets. S100A9 exerts only inhibitory effects on TGF-b1.

95P Comparison of Expression Levels of Twist, Snail and Slug in Primary Tumors and Lymph Node Metastases in Breast Cancer Patients

ABSTRACT Introduction The expression of prognostic markers including the regulators of epithelial to mesenchymal transition (EMT) can change during the course of the disease. Therefore, reassessment of these markers at the time of disease progression might help to optimize their prognostic value. In this context, characterization of metastatic tissue could be of relevance. The aim of this study was to evaluate and compare the expression of master regulators of EMT TWIST, SNAIL and SLUG in primary breast cancer tumors and their corresponding lymph node metastases. Materials and methods Fresh-frozen or formalin-fixed paraffin-embedded (FFPE) primary tumor samples and their corresponding FFPE lymph node metastases from 21, stage II-III breast cancer patients were examined. Expression of TWIST, SNAIL and SLUG was measured by RT-qPCR method using Ct approach. Based on the median gene expression level samples were classified as positive or negative for a particular marker. Results from primary tumor samples and metastases were compared. Expression levels of transcription factors (TFs) in lymph node metastases were correlated with patients' outcome. Results FFPE samples from lymph node metastases showed elevated levels of all TFs when compared to FFPE primary tumor samples. Ratios between the average expression levels were 7:1, 6:1 and 3:1 for TWIST, SNAIL and SLUG, respectively. Primary tumors paired with corresponding lymph node metastases were concordant in 52% for each gene. In lymph node metastases elevated expression of TWIST and SNAIL was associated with decreased overall survival (p = 0.045 and p = 0.022, respectively) and disease free survival (p = 0.02 and p = 0.01, respectively). Conclusions All TFs had elevated expression levels in lymph node metastases when compared to primary tumors, with TWIST being most significantly upregulated. These findings could be explicable by recent studies showing the critical role of TWIST in the development of metastases. TWIST and SNAIL expression in lymph node metastases was found to be marker of poor prognosis in accordance to the fact that EMT is thought to confer aggressive behavior of the tumor. Disclosure All authors have declared no conflicts of interest.

Abstract 1298: TWIST1 is expressed by cells with fibroblastic phenotype in colon carcinoma

Abstract Twist1 is a highly conserved transcription factor that belongs to the family of basic helix-loop-helix proteins, a family of proteins with well-described functions in cell growth and differentiation, in both vertebrates and invertebrates. TWIST1 is involved in epithelial-to-mesenchymal transition (EMT), especially in invasiveness processes. Recently we have demonstrated that the expression levels of TWIST1 in colon tumor tissues can be considered as a poor prognostic marker (Gomez I et al, Plos One 28; e18023). Following in this research line we are trying to identify the specific cells expressing TWIST1 in the tumor mass, epithelial cells or fibroblastic phenotype cells, as is expected for cells in EMT. With this purpose we have isolated epithelial cells from 52 colon tumor tissues (based on the expression of EPCAM) and fibroblasts from 18 of them (based in the expression of a fibroblastic marker, Miltenyi Biotec), after mechanical cell dissociation of the different cells populations present in the tumor mass. TWIST1 expression profile was also tested in 13 tumor colon-derived cells and in the immortalized normal fibroblasts, IMR90. First we found that while 51.9 % of tumors samples expressed TWIST1, this only occurred in 1 out of 13 colon carcinoma cell lines tested, besides the IMR90 fibroblasts. Moreover, expression of TWIST1 was never detected in the epithelial fractions but was detected in 17 out of 34 non-epithelial fractions and in 10 out of the 18 samples where fibroblasts were specifically isolated. The absence of epithelial markers, Ber-EP4 and CD326 according with our isolation system, and CK18 and E-cadherin, verified by subsequent western blot, in the cells expressing TWIST1, suggests a non-epithelial origin of this cell population. Additionally, IMR90 fibroblasts co-cultured with LS174T or SW620 colon carcinoma cells showed increased TWIST1 expression (p = 0.002; p = 0.009 respectively), which was associated to up-regulation of the myofibroblast marker α-smooth muscle actin (α-SMA). Finally, there was a correlation between TWIST1 mRNA levels and tumor desmoplasia degree among those TWIST1 positive cases. We conclude that TWIST1 expression in colon adenocarcinomas corresponds to carcinoma-associated fibroblasts and could be implicated in their activation process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1298. doi:1538-7445.AM2012-1298

1472 EXPRESSION OF EPITHELIAL-MESENCHYMAL TRANSITION MARKERS IN LOCALIZED PROSTATE CANCER: IMPACT ON CLINICOPATHOLOGICAL OUTCOMES IN PATIENTS UNDERGOING RADICAL PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Localized VII1 Apr 20121472 EXPRESSION OF EPITHELIAL-MESENCHYMAL TRANSITION MARKERS IN LOCALIZED PROSTATE CANCER: IMPACT ON CLINICOPATHOLOGICAL OUTCOMES IN PATIENTS UNDERGOING RADICAL PROSTATECTOMY Hideaki Miyake, Hosny Behnsawy, and Masato Fujisawa Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Hosny BehnsawyHosny Behnsawy Kobe, Japan More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1993AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Radical prostatectomy (RP) is an effective therapeutic modality for men with clinically organ-confined prostate cancer; however, approximately 30% of patients will develop a postoperative biochemical recurrence (BR). The objective of this study was to evaluate the expression patterns of multiple molecular markers involved in the process of epithelial-mesenchymal transition (EMT) in prostate cancer to clarify the significance of these markers as predictors for BR in patients undergoing RP. METHODS This study included a total of 197 consecutive patients with clinically organ-confined prostate cancer who underwent RP between 2002 and 2008 without any neoadjuvant therapies. Expression levels of 13 EMT markers, including E-cadherin, N-cadherin, β-catenin, γ-catenin, fibronectin, matrix metalloproteinase (MMP)-2, MMP-9, Snail, Slug, Twist, vimentin, ZEB1 and ZEB2, in RP specimens obtained from these 197 patients were measured by immunohistochemical staining. These findings were analyzed according to several clinicopathological parameters. RESULTS Of the 13 markers, expression levels of E-cadherin, Snail, Twist and vimentin were significantly associated with several conventional prognostic factors. Univariate analysis identified these four EMT markers as significant predictors for BR as well, while serum prostate-specific antigen (PSA), Gleason score, seminal vesicle invasion (SVI), surgical margin status (SMS), lymph node metastasis and tumor volume were also significant. Of these significant factors, expression levels of Twist and vimentin, SVI, and SMS appeared to be independently related to BR by multivariate analysis. Furthermore, there were significant differences in BR-free survival according to positive numbers of these four independent factors; that is, BR occurred in 5 of 90 patients who were negative for risk factor (5.6%), 22 of 83 positive for a single or two risk factor (26.5%), and 19 of 24 positive for three or four risk factors (79.2%). CONCLUSIONS These findings suggest that consideration of expression levels of potential EMT markers in RP specimens, in addition to conventional prognostic parameters, would contribute to accurate prediction of BR following RP for localized prostate cancer. Moreover, combined evaluation of Twist and vimentin expression, SVI, and SMS would be particularly useful for further refinement of the system in predicting biochemical outcome following RP. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e597 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hideaki Miyake Kobe, Japan More articles by this author Hosny Behnsawy Kobe, Japan More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Involvement of the transcription factor twist in phenotype alteration through epithelial–mesenchymal transition in lung cancer cells

Epithelial-mesenchymal transition (EMT), which involves the persistent loss of epithelial markers and expression of mesenchymal markers, is assumed to have a critical role in not only tissue development during embryogenesis but also central mechanisms that enhance the invasive and metastatic ability of cancer cells. Twist has been identified to play an essential role in EMT-mediated tumor invasion and metastasis. Although recent studies suggest that twist expression levels in tissue specimens of lung cancer might be associated with prognosis, the expression of twist in lung cancer cells itself and its effect have not been fully evaluated. Here, we evaluated twist expression and its effect on phenotype alteration in lung cancer cell lines. Twist expression varied among human lung cancer cell lines. The lung cancer cell lines with high twist expression also tended to show a high vimentin/E-cadherin ratio, which was supported by a migration assay, in which high twist expression gave rise to high cell motility. Furthermore, in comparison to control cells, the lung cancer cells with ectopic expression of twist showed a significant phenotype alteration through EMT and an increasing ability to migrate in vitro, in part, due to a tenfold increase in matrix metalloproteinases activity and almost a 60% increase in modulation of focal adhesion kinase activity, although a contribution of microRNA appeared unlikely in our study. Our present analysis of twist expression in lung cancer provide clues to comprehensive understanding of the mechanisms, by which metastasis often develops in lung cancer.

TWIST1 Is Expressed in Colorectal Carcinomas and Predicts Patient Survival

TWIST1 is a transcription factor that belongs to the family of basic helix-loop-helix proteins involved in epithelial-to-mesenchymal transition and invasion processes. The TWIST1 protein possesses oncogenic, drug-resistant, angiogenic and invasive properties, and has been related with several human tumors and other pathologies. Colorectal cancer is one of the tumors in which TWIST1 is over-expressed, but its involvement in the clinical outcome of the disease is still unclear. We tested, by RT-PCR, the expression levels of TWIST1 in normal and tumor paired-sample tissues from a series of 151 colorectal cancer patients, in order to investigate its prognostic value as a tumor marker. TWIST1 expression was restricted to tumor tissues (86.1%) and correlated with lymph node metastasis (LNM). Adjusted analysis showed that the expression levels of TWIST1 correlated with overall survival (OS) and disease-free survival (DFS). Importantly, TWIST1 expression levels predicted OS specifically at stages I and II. Moreover, patients with stage II tumors and high TWIST1 levels showed even shorter survival than patients with stage III tumors. These results suggest that TWIST1 expression levels could be a tumor indicator in stage II patients and help select patients at greater risk of poor prognosis who might benefit from adjuvant chemotherapy.