Expressions of TWIST1 and CD105 markers in colorectal cancer patients and their association with metastatic potential and prognosis.

BACKGROUND: TWIST1 and CD105, which contribute to tumor malignancy, are overexpressed in cancers. Accordingly, TWIST1 enhances epithelial-to-mesenchymal transition (EMT) and promotes the formation of cancer stem cells (CSCs). Also, CD105 is a neoangiogenesis marker in endothelial cells, which is introduced as a CSC marker in tumoral epithelial cells in several types of cancers. The present study was aimed to investigate expressions of TWIST1 and CD105 in colorectal cancer (CRC) patients. METHODS: Expressions of TWIST1 and CD105 in 250 CRC tissue samples were evaluated using immunohistochemistry on tissue microarrays (TMAs). In this regard, TWIST1 expression was investigated in the subcellular locations (cytoplasm and nucleus), while CD105 was mapped in endothelial cells and cytoplasmic tumor cells of CRC tissues. The association between the expression of these markers and clinicopathological parameters, as well as survival outcomes were analyzed. RESULTS: Results indicate a statistically significant association between higher nuclear expression levels of TWIST1 and distant metastases in CRC (P = 0.040) patients. In addition, it was shown that the increased nuclear expression of TWIST1 had a poor prognostic value for disease-specific survival (DSS) and progression-free survival (PFS) (P = 0.042, P = 0.043, respectively) in patients with CRC. Moreover, analysis of CD105 expression level has revealed that there is a statistically significant association between the increased expression of CD105 in tumoral epithelial cells and more advanced TNM stage (P = 0.050). CONCLUSIONS: Our results demonstrate that nuclear TWIST1 and cytoplasmic CD105 expressions in tumor cells had associations with more aggressive tumor behavior and more advanced diseases in CRC cases.

The epithelial-mesenchymal transition regulators Twist, Slug, and Snail are associated with aggressive tumour features and poor outcome in prostate cancer patients.

The prognostic importance of transcription factors promoting epithelial-mesenchymal transition (EMT) and angiogenesis has not been well explored in prostate cancer patients with long follow-up, nor the interplay between these factors. The objective of this study was to assess the individual protein expression and co-expression of Twist, Slug (Snai2), Snail (Snai1), and hypoxia-inducible factor-1 alpha (Hif-1α) in prostate cancer in relation to EMT, angiogenesis, hypoxia, tumour features, disease recurrence, and patient survival. Immunohistochemical staining was performed on tissue microarray sections from 338 radical prostatectomies with long follow-up. In addition, 41 cases of prostatic hyperplasia, 33 non-skeletal metastases, 13 skeletal metastases, and 33 castration-resistant prostate carcinomas were included. Our findings were validated in external gene expression data sets. Twist was overexpressed in primary prostate cancer and markedly reduced in distant metastases (p < 0.0005). Strong expression of Twist and Slug was associated with Hif-1α in localised prostate cancer (p ≤ 0.001), and strong Twist was associated with Hif-1α in castration-resistant carcinomas (p = 0.044). Twist, Slug, and increased Snail at the tumour stromal border were associated with vascular factors (p ≤ 0.045). Each of the three EMT-regulating transcription factors were associated with aggressive tumour features and shorter time to recurrence and cancer-specific death. Notably, the co-expression of factors demonstrated an enhanced influence on outcome. In the subgroup of E-cadherinlow carcinomas, strong Slug was associated with shorter time to all end points and was an independent predictor of time to multiple end points, including cancer-specific death (hazard ratio 3.0, p = 0.041). To conclude, we demonstrate an important relation between EMT, hypoxia, and angiogenesis and a strong link between the investigated EMT regulators and aggressive tumour features and poor patient outcome in prostate cancer. Despite the retrospective nature of this long-term study, our findings could have a significant impact on the future treatment of prostate cancer, where tailored therapies might be directed simultaneously against epithelial-mesenchymal phenotypes, angiogenesis, and tumour hypoxia.

Epithelial-mesenchymal transition in undifferentiated carcinoma of the pancreas with and without osteoclast-like giant cells.

Undifferentiated carcinoma (UC) and undifferentiated carcinoma with osteoclast-like giant cells (UCOGC) are peculiar variants of pancreatic ductal adenocarcinoma (PDAC), characterized by hypercellularity and absence of glandular patterns. The inflammatory microenvironment is peculiar in UCOGC, since it is dominated by macrophages and osteoclast-like giant cells. However, from a molecular point of view, both UC and UCOGC are very similar to conventional PDAC, sharing alterations of the most common genetic drivers. Clinically, UC usually show a worse prognosis, whereas UCOGC may show a better prognosis if it is not associated with a PDAC component. To highlight potential biological differences between these entities, we investigated the role of the epithelial to mesenchymal transition (EMT) in UC and UCOGC. Specifically, we analyzed the immunohistochemical expression of three well-known EMT markers, namely Twist1, Snai2, and E-cadherin, in 16 cases of UCOGC and 10 cases of UC. We found that EMT is more frequently activated in UC (10/10 cases) than in UCOGC (8/16 cases; p = 0.05). Furthermore, in UCOGC, EMT was activated with a higher frequency in cases with an associated PDAC component. Snai2 was the most frequently and strongly expressed marker in both tumor types (10/10 UC, 8/16 UCOGC), and its expression was higher in UC than in UCOGC (mean immunohistochemical score: 4.8 in UC vs. 2.1 in UCOGC, p < 0.01). Our results shed new light on the biology of UC and UCOGC: EMT appeared as a more important process in UC, and Snai2 emerged as a central EMT effector in this setting.

Prognostic Value of TWIST1 and EZH2 Expression in Colon Cancer.

BACKGROUND: Colorectal carcinoma (CRC) is the third most common human cancer. Twist, a basic helix-loop-helix (bHLH) transcription factor, is an epithelial-mesenchymal transition ((EMT) inducer that has been involved in carcinogenesis and chemoresistance. Also, the enhancer of Zeste homolologue2 (EZH2), a member of the polycomb group of genes, had been associated with poor prognosis in several malignancies. OBJECTIVE: To evaluate the expression of Twist1 and EZH2 in colon carcinoma in Egyptian patients and its relation to clinicopathological parameters, prognosis, and survival. METHODS: Twist1 and EZH2 expressions were evaluated immunohistochemically in 50 cases of colorectal tumors (12 colon adenomas and 38 colon carcinomas) and 20 samples from normal colonic mucosa. RESULTS: The expression of Twist1 and EZH2 was significantly higher in colon adenoma and carcinoma than that in normal colonic mucosa (P < 0.05). Twist1 and EZH2 expressions were associated with decreased tumor differentiation, advanced stage, and lymph node metastasis. Twist1 and EZH2 expressions were significantly related to 3-year disease-free survival (P = 0.005 and 0.002 respectively) and 3-year overall survival (P = 0.045 and 0.039, respectively). CONCLUSIONS: Twist1 and EZH2 may serve as prognostic predictors for colon carcinoma and may have a potential role as therapeutic targets in patients with colon carcinoma in the future.

Fecal DNA Testing of TWIST1 Methylation Identifies Patients With Advanced Colorectal Adenoma Missed by Fecal Immunochemical Test for Hemoglobin.

INTRODUCTION: We have reported previously that fecal DNA testing of TWIST1 methylation in combination with the fecal immunochemical test for hemoglobin (FIT) (combination test) is useful for colorectal neoplasia screening. In this study, using larger sample sizes, we studied the clinical performance of the combination test for the detection of colorectal neoplasia and, especially, advanced colorectal adenoma. METHODS: We performed a prospective study in which FIT, fecal DNA testing of TWIST1 methylation, and colonoscopy were performed on 372 patients with colorectal neoplasia and 71 subjects without colorectal neoplasia. We assessed the individual clinical performance of each of FIT and fecal DNA testing of TWIST1 methylation and of the combination test for the detection of colorectal neoplasia including advanced adenoma based on morphologic subtypes. RESULTS: The FIT alone had a sensitivity of 7.5% (3/40) for nonadvanced adenoma, 32.3% (41/127) for advanced adenoma, and 93.7% (192/205) for colorectal cancer and a specificity of 87.3% (62/71). The combination test had a sensitivity of 35.0% (14/40) for nonadvanced adenoma, 68.5% (87/127) for advanced adenoma, and 95.6% (196/205) for colorectal cancer and a specificity of 80.3% (57/71). For morphological subtypes of advanced adenoma, the sensitivity of FIT was only 28.2% (20/71) for polypoid type and 16.1% (5/31) for nonpolypoid type, whereas the combination test increased the sensitivities to 64.8% (46/71) and 71.0% (22/31), respectively. DISCUSSION: The combination of the fecal DNA test with FIT seemed to be useful to detect colorectal neoplasia and, especially, advanced adenoma of the nonpolypoid type.

Role of epithelial-mesenchymal transition factors in the histogenesis of uterine carcinomas.

Several subtypes of high-grade endometrial carcinomas (ECs) contain an undifferentiated component of non-epithelial morphology, including undifferentiated and dedifferentiated carcinomas and carcinosarcomas (CSs). The mechanism by which an EC undergoes dedifferentiation has been the subject of much debate. The epithelial-mesenchymal transition (EMT) is one of the mechanisms implicated in the transdifferentiation of high-grade carcinomas. To improve our understanding of the role of EMT in these tumors, we studied a series of 89 carcinomas including 14 undifferentiated/dedifferentiated endometrial carcinomas (UECs/DECs), 49 CSs (21 endometrial, 29 tubo-ovarian and peritoneal), 17 endometrioid carcinomas (grade 1-3), and 9 high-grade serous carcinomas of the uterus, using a panel of antibodies targeting known epithelial markers (Pan-Keratin AE1/AE3 and E-cadherin), mesenchymal markers (N-cadherin), EMT transcription factors (TFs) (ZEB1, ZEB2, TWIST1), PAX8, estrogen receptors (ER), progesterone receptors (PR), and the p53 protein. At least one of the three EMT markers (more frequently ZEB1) was positive in the sarcomatous component of 98% (n = 48/49) of CSs and 98% (n = 13/14) of the undifferentiated component of UEC/DEC. In addition, 86% of sarcomatous areas of CSs and 79% of the undifferentiated component of UEC/DEC expressed all three EMT-TFs. The expression of these markers was associated with the loss of or reduction in epithelial markers (Pan-keratin, E-cadherin), PAX8, and hormone receptors. In contrast, none of the endometrioid and serous endometrial carcinomas expressed ZEB1, while 6% and 36% of endometrioid and 11% and 25% of serous carcinomas focally expressed ZEB2 and TWIST1, respectively. Although morphologically different, EMT appears to be implicated in the dedifferentiation in both CSs and UEC/DEC. Indeed, we speculate that the occurrence of EMT in a well differentiated endometrioid carcinoma may consecutively lead to a dedifferentiated and undifferentiated carcinoma, while in a type II carcinoma, it may result in a CS.

Molecular detection of epithelial-mesenchymal transition markers in circulating tumor cells from pancreatic cancer patients: Potential role in clinical practice.

AIM: To evaluate the clinical properties of three subpopulations of circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) patients. METHODS: We identified CTCs for expression of the epithelial cell marker cytokeratin or epithelial cell adhesion molecule (EpCAM) (E-CTC), the mesenchymal cell markers vimentin and twist (M-CTC), or both (E/M-CTC) using the CanPatrol system. Between July 2014 and July 2016, 107 patients with PDAC were enrolled for CTC evaluation. CTC enumeration and classification were correlated with patient clinicopathological features and outcomes. RESULTS: CTCs were detected in 78.5% of PDAC patients. The number of total CTCs ranged from 0 to 26 across all 107 patients, with a median value of six. CTC status correlated with lymph node metastasis, TNM stage, distant metastasis, blood lymphocyte counts, and neutrophil-to-lymphocyte ratio (NLR). Kaplan-Meier survival analysis showed that patients with ≥ 6 total CTCs had significantly decreased overall survival and progression-free survival compared with patients with < 6 total CTCs. The presence of M-CTCs was positively correlated with TNM stage (P < 0.01) and distant metastasis (P < 0.01). Additionally, lymphocyte counts and NLR in patients without CTCs were significantly different from those in patients testing positive for each CTC subpopulation (P < 0.01). CONCLUSION: Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulations and provides useful evidence for determining a suitable clinical approach.

Evaluation of the correlation of MACC1, CD44, Twist1, and KiSS-1 in the metastasis and prognosis for colon carcinoma.

BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) has been reported to promote tumor cell invasion and metastasis. Cancer stem cells and epithelial-mesenchymal transition (EMT) have also been reported to promote tumor cell proliferation, invasion, and metastasis. KiSS-1, a known suppressor of metastasis, has been reported to be down-regulated in various tumors. However, the associations of MACC1, CD44, Twist1, and KiSS-1 in colonic adenocarcinoma (CAC) invasion and metastasis remain unclear. The purpose of this study is to investigate the roles of MACC1, CD44, Twist1, and KiSS-1 in CAC invasion and metastasis and their associations with each other and with the clinicopathological characteristics of CAC patients. METHODS: Immunohistochemistry and multivariate analysis were carried out to explore the expression of MACC1, CD44, Twist1, and KiSS-1 in 212 whole-CAC-tissue specimens and the corresponding normal colon mucosa tissues. Demographic, clinicopathological, and follow-up data were also collected. RESULTS: The results of this study showed MACC1, CD44, and Twist1 expression to be up-regulated, and KiSS-1 expression was down-regulated in CAC tissues. Positive expression of MACC1, CD44, and Twist1 was found to be positively correlated with invasion, tumor grades, and lymph- node-metastasis (LNM) stages and tumor-node-metastasis (TNM) stages for patients with CAC. Positive expression of KiSS-1 was inversely associated with invasion, tumor size, LNM stage, and TNM stage. The KiSS-1-positive expression group had significantly more favorable OS than did the KiSS-1-negative group. Univariate analysis indicated that overexpression of MACC1, CD44, and Twists1 was negatively associated with longer overall survival (OS) time, and there was a positive relationship between KiSS-1-positive expression and OS time for patients with CAC. Multivariate Cox analysis demonstrated that overexpression of MACC1, CD44, Twist1, and low expression of KiSS-1 and LNM and TNM stages were independent predictors of prognosis in patients with CAC. CONCLUSIONS: The results in this study indicated that levels of expression of MACC1, CD44, Twist1, and KiSS-1 are related to the duration of OS in patients with CAC. MACC1, CD44, Twist1, and KiSS-1 may be suitable for use as biomarkers and therapeutic targets in CAC.

Prognostic value of Twist, Snail and E-cadherin expression in pathological N0 non-small-cell lung cancer: a retrospective cohort study.

OBJECTIVES: Lung cancer is the leading cause of cancer-related death worldwide. The 5-year survival rate for patients after curative surgery with pathological N0 non-small-cell lung cancer (NSCLC) is as low as 56%, which is due to recurrence and metastasis. Emerging evidence suggests that epithelial-mesenchymal transition is important for cancer metastasis. Twist and Snail are epithelial-mesenchymal transition regulators that induce metastasis by down-regulating E-cadherin. The aim of this study was to evaluate the prognostic value of Twist, Snail and E-cadherin expression in patients with resectable pathological N0 NSCLC. METHODS: The expression levels of Twist, Snail and E-cadherin in 78 patients with resected pathological N0 NSCLC were assessed using immunohistochemistry. The association between the expression of Twist/Snail/E-cadherin and overall survival (OS) and recurrence-free survival (RFS) was investigated. RESULTS: High expression of Twist, Snail and E-cadherin was detected in 18%, 21% and 53% of NSCLC samples, respectively. High expression of Twist and Snail and low expression of E-cadherin were associated with worse RFS [hazard ratio (HR) 2.29, 95% confidence interval (CI) 1.07-4.87, P = 0.026; HR 2.54, 95% CI 1.24-5.20, P = 0.008 and HR 2.41, 95% CI 1.23-4.73, P = 0.007, respectively] and worse OS (HR 2.26, 95% CI 1.01-5.04, P = 0.040; HR 2.56, 95% CI 1.20-5.43, P = 0.011 and HR 2.42, 95% CI 1.18-4.95, P = 0.012, respectively). Co-expression of at least 2 markers from the combination of high Twist/high Snail/low E-cadherin expression predicted poor RFS and OS (HR 4.12, 95% CI 2.08-8.16, P < 0.001 and HR 4.28, 95% CI 2.08-8.77, P < 0.001, respectively), and it was an independent predictor of RFS and OS (HR 3.99, 95% CI 1.89-8.44, P < 0.001 and HR 4.16, 95% CI 1.88-9.18, P < 0.001, respectively). CONCLUSIONS: Co-expression of at least 2 markers from the combination of high Twist/high Snail/low E-cadherin expression was a significant prognostic predictor in patients with pathological N0 NSCLC.

The MRPS18-2 protein levels correlate with prostate tumor progression and it induces CXCR4-dependent migration of cancer cells.

We have earlier found abnormal expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) in endometrial cancer, compared to the expression in hyperplasia and in normal endometrium. Here we report that expression of S18-2 was increased with disease progression in clinical specimens of prostate cancer (PCa). The level of induction of epithelial to mesenchymal cell transition (EMT) correlated with the expression level of S18-2 in PCa cell lines. Moreover, cells acquired increased ability of migration upon S18-2 overexpression, as was evaluated in zebrafish embryo model and in trans-well assay. We found that this is due to increased CXCR4 cell surface expression. Neutralizing CXCR4 protein or abrogating S18-2 expression in cells significantly reduced their migratory ability directed toward CXCL12. The mRNA expression of TWIST2, encoding one of transcription factors that induce EMT upon CXCR4 increase, positively correlated with the S18-2 protein level. Together, these data suggest that the S18-2 protein induces EMT through the TWIST2/E-cadherin signalling and, consequently, CXCR4-mediated migration of PCa cells.

Use of GATA3 and TWIST1 Immunofluorescence Staining to Assess In Vitro Syncytial Fusion Index.

In human placenta, the multinucleated syncytiotrophoblast (ST) allows all the exchanges between the maternal and fetal circulation and is also the site of placental hormonal functions. Absence or disturbances of ST formation are associated with a defect or pathologies of pregnancy such as preeclampsia (PE) and intrauterine growth retardation (IUGR). All along pregnancy, the ST is regenerated by fusion of underlying mononucleated villous cytotrophoblasts (VCT). The protocol described here provides details on how GATA3 or TWIST1 immunostaining and analysis can be used to easily assess the in vitro differentiation of human placental cytotrophoblast.

Immature CML cells implement a BMP autocrine loop to escape TKI treatment.

The BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b+ cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance.

Expression of Epithelial-Mesenchymal Transition Regulating Transcription Factors in Head and Neck Squamous Cell Carcinomas.

BACKGROUND/AIM: The transcription factors Twist, Snail, Slug, ZEB1 and ZEB2 regulate epithelial-mesenchymal transition (EMT) and their expression has been associated with a poor prognosis in several cancer entities. The aim of this analysis was to investigate in parallel the expression of all of these transcription factors in head and neck squamous cell carcinomas (HNSCCs) in order to gain insight into their possible co-expression. MATERIALS AND METHODS: Tumor tissue samples were immunohistochemically stained using antibodies against these transcription factors. The staining intensity and cellular distribution of the immunoreactivity was recorded. RESULTS: In general, transcription factor immunoreactivity was noted in the nucleus of both cancer and stromal cells. The highest immunoreactivity was observed for Twist. Snail, Slug, ZEB1 and ZEB2 showed a much lesser immunoreactivity in cancer cells and they were expressed independently from each other. CONCLUSION: Twist is the major transcription factor active in HNSCC; the other transcription factors of EMT seem to be of less importance in this tumor entity.

Correlation Between Expression of Twist and Podoplanin in Ductal Breast Carcinoma.

BACKGROUND/AIM: As a result of activation of transcription factors engaged in epithelial-mesenchymal transition (EMT), such as Twist, inhibition of epithelial markers and an increased expression of mesenchymal markers are observed. One of the specific markers of cancer-associated fibroblasts is podoplanin (PDPN) - a mucin-type membrane glycoprotein. The aim of this work was to study the localisation and intensity of expression of Twist and PDPN on the mRNA and protein level in cases of invasive ductal breast carcinoma (IDC), and its association with patients' clinico-pathological data. MATERIALS AND METHODS: The study included archival material in a form of 80 paraffin IDC blocks and 11 IDC fragments frozen in liquid nitrogen. Immunohistochemical expression of Twist and PDPN was evaluated using light microscope and semiquantitative scale for evaluation of nuclear expression or immunoreactive scale (IRS) for evaluation of cytoplasmic expression. Material was isolated from frozen IDC fragments using laser micro-dissection (from cancer and stromal cells, separately) and was used to perform real-time PCR. RESULTS: Twist expression was higher in stromal cells in comparison to cancer cells. Analysis of patients' survival rate showed, that higher expression of Twist in cancer cells was associated with shorter overall survival time and shorter event-free survival time. The expression of PDPN was also higher in stromal cells in comparison with cancer cells. In addition, positive correlation was observed between expression of Twist and PDPN in stromal cells of IDC (r=0.267; p<0.05). CONCLUSION: The relationship between the higher expression of Twist in both cancer and stromal cells and shorter patients' survival indicates Twist as a potential useful prognostic marker in IDC. Positive correlation of Twist and PDPN expression may indicate the role of PDPN in EMT in IDC.

Evaluation of expression of genes CADM1, TWIST1 and CDH1 by immunohistochemestry in melanocytic lesions.

AIM: Malignant melanoma is an aggressive disease and its incidence is increasing worldwide. Genetic predisposition and exposure to environmental factors, especially sunlight, are risk factors. Histopathologic distinction between nevi and melanomas can be difficult. It is anticipated that the evaluation of the immunohistochemical expression of some genes could contribute to the differential diagnosis of questionable histologically lesions. The objective of this study was to investigate wether the evaluation of the immunohistochemical expression of genes CADM1, TWIST1 and CDH1 (E-cadherin), that take part in mechanisms of cell adhesion and epithelial-mesenchymal transition, contributes to the differential diagnosis of melanocytic lesions difficult to diagnose. MATERIALS AND METHODS: Retrospective cross-sectional study based on immunohistochemistry analysis of samples of 30 dysplastic compound melanocytic nevi, 30 melanomas less than 1.0mm thick and 30 melanomas more than 1.0mm thick, diagnosed between 2013 and 2016. A score was used to evaluate color intensity and percentage of cells stained. RESULT: There were significant reductions in the expression of the genes CADM1 and CDH1 in melanomas (below and above 1.00mm of thickness) and in melanomas more than 1.0mm thick, respectively, compared to dysplastic compund melanocytic nevi. There was also lower expression of the genes CADM1 and CDH1 in melanomas greater than 1.0mm thick compared to melanomas less than 1.0mm. The gene TWIST1 showed no significant difference in expression between groups. CONCLUSION: These findings allow us to conclude that the immunohistochemical expression of CADM1 has the potential to contribute as an auxiliary tool to the differential diagnosis between nevi and melanomas.

Prognostic Value of Twist-1, E-cadherin and EZH2 in Prostate Cancer: An Immunohistochemical Study.

OBJECTIVE: There is an urgent need to identify molecular biomarkers rather than clinical markers to distinguish aggressive prostate cancer from the indolent majority for proper treatment and accurate prediction of prognosis. We aimed to investigate the immunohistochemical expression of epithelial-mesenchymal transition (EMT)-related molecules (Twist-1 and E-cadherin) and the stem cell marker EZH2 in prostate cancer and to assess their ability to identify the high-risk patients, in a trial to explore their prognostic implications. MATERIAL AND METHOD: Immunohistochemical expression of Twist-1, E-cadherin and EZH2 in 50 specimens of prostate cancer and 20 cases of benign prostatic hyperplasia were studied. The relationship between their expression and the clinicopathological variables, biochemical recurrence, and biochemical progression-free survival were investigated. RESULTS: Our results revealed that high Twist-1, as well as high EZH2 expression, was strongly associated with higher pre-treatment PSA level, Gleason score ≥7, advanced tumor stage, lymph node involvement, distant metastasis and biochemical progression. Aberrant E-cadherin expression was significantly associated with higher pre-treatment PSA level, Gleason score ≥7, advanced tumor stage, lymph node involvement, and distant metastasis. A significant positive correlation between Twist-1 and EZH2 expression was found (p < 0.001), while E-cadherin expression showed a negative correlation with both markers (p < 0.001). A significant association was found between high Twist-1, high EZH2& aberrant E-cadherin expression, and shorter biochemical progression-free survival. CONCLUSION: The high Twist-1 expression, aberrant E-cadherin and high EZH2 expression in primary prostate cancer are considered as adverse prognostic markers of an aggressive tumor with high metastatic potential. Assessment of their expression level would contribute to the accurate prediction of biochemical progression.

Tumor budding at the invasive front of colorectal cancer may not be associated with the epithelial-mesenchymal transition.

Tumor budding is thought to reflect the epithelial-mesenchymal transition (EMT). However, the molecular mechanism linking tumor buds and the EMT remains unclear. Here, we examined the induction of tumor budding and EMT and their association with EMT-related proteins (ZEB1, TWIST, SNAIL, and SLUG) in colorectal cancer (CRC). Immunohistochemical expression of pan-cytokeratin was examined for identification of tumor budding in 101 CRCs. Grading of tumor budding was classified into low- and high-grade groups. Tissue microarray was conducted to identify tumor budding sites. The expression of E-cadherin, ZEB1, TWIST, SNAIL, and SLUG was examined in areas of tumor budding and the surrounding tumor stroma using a double-immunostaining method. Specifically, pan-cytokeratin and EMT-related proteins were assessed by double immunostaining. Low or no expression of E-cadherin was found in areas of tumor budding. Moreover, ZEB1, TWIST, SNAIL, and SLUG were not expressed in regions of tumor budding. However, the expression level of ZEB1 in the stromal cells surrounding tumor budding was significantly more frequent than that of TWIST, SNAI, and SLUG. In addition, the expression of EMT-related proteins in surrounding stromal cells was significantly greater in areas of high-grade tumor budding than in low-grade areas. Our present results suggest that EMT-related proteins play a minor role in forming tumor buds. In addition, our findings suggest the existence of subtypes of stromal cells in CRC with phenotypical and functional heterogeneity.

DNMT3A mutation leads to leukemic extramedullary infiltration mediated by TWIST1.

BACKGROUND: DNMT3A mutations are frequently discovered in acute myeloid leukemia (AML), associated with poor outcome. Recently, a relapse case report of AML extramedullary disease has showed that AML cells harboring DNMT3A variation were detected in the cerebral spinal fluid. However, whether a causal relationship exists between DNMT3A mutation (D3Amut) and extramedullary infiltration (EMI) is unclear. METHODS: We took advantage of DNMT3A (R882C) mutation-carrying AML cell strain, that is, OCI-AML3, assessing its migration ability in vitro and in vivo. By RNA interfering technology and a xenograft mouse model, we evaluated the effect of DNMT3A mutation on cell mobility and explored the possible mechanism. RESULTS: OCI-AML3 displayed extraordinary migration ability in vitro and infiltrated into meninges of NOD/SCID mice after intravenous transfusion. We found that this leukemic migration or infiltration capacity was significantly compromised by the knockdown of DNMT3A mutant. Notably, TWIST1, a critical inducer of epithelial-mesenchymal transition, which underlies the metastasis of carcinomas, was highly expressed in association with R882 mutations. Abrogation of TWIST1 in DNMT3A mutated cells considerably weakened their mobility or infiltration. CONCLUSIONS: Our results demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1.

Impact of Tight Junction Protein ZO-1 and TWIST Expression on Postoperative Survival of Patients with Hepatocellular Carcinoma.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection. SUMMARY: The mRNA expression of 15 genes related to EMT was assessed by quantitative real-time polymerase chain reaction in cancerous tissues from 72 patients who underwent hepatic resection of HCC between January 2005 and December 2010 at our hospital. The upregulation of TWIST and the downregulation of tight junction protein ZO-1 (TJP1) were significantly associated with shorter RFS as well as OS. Increased levels of TWIST and decreased levels of TJP1 should be predictive markers for poor prognosis in patients with HCC after hepatectomy; those could serve as potential biomarkers for the treatment of HCC. Key Messages: A low level of TJP1 and high level of TWIST expression were prognostic factors predicting HCC after hepatic resection.

Role of TWIST2, E-cadherin and Vimentin in epithelial ovarian carcinogenesis and prognosis and their interaction in cancer progression.

UNLABELLED: Globally, most patients are at late-stage when they have been diagnosed with ovarian cancer. Investigating the potential mechanisms involved in tumor progression and prognosis is essential for improving treatment options, outcomes, and survival. OBJECTIVE: This study elucidated the clinico-pathological significance of TWIST2 and the relationship of TWIST2, E-cadherin, and Vimentin expression in the progression and prognosis of epithelial ovarian cancer (EOC). MATERIALS AND METHODS: Immunohistochemical staining was used to quantify the expression and relevance of TWIST2, E-cadherin, and Vimentin in 103 ovarian specimens, including 30 cases of benign ovarian tumors, 30 cases of borderline ovarian tumors, and 43 cases of EOC. RESULTS: The expression of TWIST2 in the cytoplasm may help to maintain characteristics of epithelial cancer cells with E-cadherin normal membranous expression, while nuclear TWIST2 induces tumor translation front with membranous expression of Vimentin, which eventually promotes cancer metastasis. Moreover, the upregulation of TWIST2 was also related to the aberrant expression of E-cadherin and the increased expression of Vimentin, which were reported as important indicators of epithelial-mesenchymal transition (EMT). DISCUSSION: The data suggested that co-expression of TWIST2/Vimentin was an independent prognostic indicator for both overall survival and disease-free survival by multivariate Cox proportional hazards model. TWIST2 regulates EMT by depriving the epithelial cell phenotype of E-cadherin and endowing the mesenchymal cell phenotype with Vimentin, which may be involved in the progression and prognosis of ovarian cancer, and TWIST2/Vimentin co-expression might be a novel indicator with prognostic potential in EOC patients.