Expression Pattern Of Epithelial-mesenchymal Transition Markers Research Articles

Calcium-Activated Chloride Channel A4 (CLCA4) Plays Inhibitory Roles in Invasion and Migration Through Suppressing Epithelial-Mesenchymal Transition via PI3K/AKT Signaling in Colorectal Cancer.

BackgroundCalcium-activated chloride channel A4 (CLCA4) is known as a tumor suppressor which contributes to the progression of a number of types of malignant tumors. However, little is known about the functional roles of CLCA4 in colorectal cancer (CRC).Material/MethodsIn this study, the expression patterns and dysregulation of mRNAs in CRC tissues were profiled by analyzing GSE21510 datasets from Gene Expression Omnibus database which contains 104 primary hepatocellular carcinoma tissues and 24 normal liver tissues, and by performing Kaplan-Meier analysis of TCGA data. Additionally, immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) were performed using clinical tissues collected at our institute. In order to explore the functional role of CLCA4, gain-of-function cell models were constructed in SW620 and LoVo cells. Wound healing assay and Transwell assay were carried out to access the cell migration and invasion ability.ResultsIt was found that CLCA4 was an independent predictor for overall survival and lymph node metastasis. Additionally, immunohistochemistry and qRT-PCR results of the clinical tissues collected as part of our study further confirmed this correlation. In vitro experiments demonstrated that over-expression of CLCA4 could inhibit cell migration and invasion by suppressing epithelial-mesenchymal transition (EMT) via PI3K/ATK signaling and change the expression patterns of EMT markers in CLCA4-gain-of-function cell models.ConclusionsCLCA4 inhibits migration and invasion by suppressing EMT via PI3K/ATK signaling and predicts favorable prognosis of CRC which may help to distinguish potential risk of lymph node metastasis in CRC.

PKM2-regulated STAT3 promotes esophageal squamous cell carcinoma progression via TGF-β1-induced EMT.

Recent studies have demonstrated pleiotropic roles of pyruvate kinase isoenzyme type M2 (PKM2) in tumor progression. However, the precise mechanisms underlying the effects of PKM2 on esophageal squamous cell carcinoma (ESCC) metastasis and transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) remain to be established. In this study, we observed upregulation of PKM2 in ESCC tissues that was markedly associated with lymph node metastasis and poor prognosis. High PKM2 expression in tumor tissues frequently coincided with the high pSTAT3Tyr705 expression and low E-cadherin expression. Furthermore, altered PKM2 expression was significantly associated with proliferation, migration, and invasion of ESCC cells, in addition to expression patterns of EMT markers (Snail, E-cadherin, and vimentin) and pSTAT3Tyr705 /STAT3 ratio. Overexpression of STAT3 significantly attenuated the effects of PKM2 knockdown on cell proliferation and motility as well as expression of pSTAT3 Tyr705 and EMT markers. Consistently, stable short hairpin RNA (shRNA)-mediated silencing of PKM2 reversed the effects of TGF-β1 treatment, specifically, upregulation of PKM2, phosphorylation of STAT3 at Tyr705, and increased EMT, migration, and invasion. We propose that PKM2 regulates cell proliferation, migration, and invasion via phosphorylation of STAT3 through TGF-β1-induced EMT. Our findings collectively provide mechanistic insights into the tumor-promoting role of PKM2, supporting its prognostic value and the therapeutic utility of PKM2 inhibitors as potential antitumor agents in ESCC.

Silencing of EPHB2 promotes the epithelial-mesenchymal transition of skin squamous cell carcinoma-derived A431 cells.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligand ephrins serve crucial roles in the interactions among epithelial cells. Eph receptor/ephrin signaling regulates cell functions, including proliferation, differentiation and migration, via these cell-cell interactions. We reported previously that EPHB2, a member of the Eph receptor family, was highly expressed in chemically induced cutaneous squamous cell carcinoma (cSCC) tissues in mice. Although the higher expression level of EPHB2 has been observed in various human cancers, its roles in the development and progression of cancers are still unclear. In the present study, the functional implications of EPHB2 in the acquisition of malignant phenotypes of cSCC cells was investigated. Silencing of EPHB2 in the human cSCC cell line A431 induced epithelial-mesenchymal transition (EMT)-like morphological changes accompanied by a significant upregulation of epithelial-mesenchymal transition-associated genes such as zinc finger E-box binding homeobox 1/2. In addition, silencing of EPHB2 suppressed anchorage-independent cell growth under 3D culture conditions. Consistent with these observations, EPHB2 exhibited higher levels of expression in tumor spheres formed under 3D culture conditions than in cells cultured in adherent form, and the expression pattern of EMT markers indicated that EMT was suppressed in tumor spheres. The results of the present study indicated that EPHB2 serves a pivotal role in promoting the anchorage-independent growth of A431 cells through the suppression of EMT.

CLCA4 inhibits cell proliferation and invasion of hepatocellular carcinoma by suppressing epithelial-mesenchymal transition via PI3K/AKT signaling.

Calcium activated Chloride Channel A4 (CLCA4), as a tumor suppressor, was reported to contribute to the progression of several malignant tumors, yet little is known about the significance of CLCA4 in invasion and prognosis of hepatocellular carcinoma (HCC). CLCA4 expression was negatively correlated with tumor size, vascular invasion and TNM stage. Kaplan-Meier analysis showed that CLCA4 was an independent predictor for overall survival (OS) and time to recurrence (TTR). In addition, CLCA4 status could act as prognostic predictor in different risk of subgroups. Moreover, combination of CLCA4 and serum AFP could be a potential predictor for survival in HCC patients. Furthermore, CLCA4 may inhibit cell migration and invasion by suppressing epithelial-mesenchymal transition (EMT) via PI3K/ATK signaling. Knockdown of CLCA4 significantly increased the migration and invasion of HCC cells and changed the expression pattern of EMT markers and PI3K/AKT phosphorylation. An opposite expression pattern of EMT markers and PI3K/AKT phosphorylation was observed in CLCA4-transfected cells. Additionally, immunohistochemistry and RT-PCR results further confirmed this correlation. Taken together, CLCA4 contributes to migration and invasion by suppressing EMT via PI3K/ATK signaling and predicts favourable prognosis of HCC. CLCA4/AFP expression may help to distinguish different risks of HCC patients after hepatectomy.

HOXC6 predicts invasion and poor survival in hepatocellular carcinoma by driving epithelial-mesenchymal transition.

Aberrant expression of HOXC6 has been reported in several malignant tumors, yet little is known about the value of HOXC6 in invasion and prognosis of hepatocellular carcinoma (HCC). HOXC6 expression was positively correlated with high AFP level, liver cirrhosis, larger tumor, vascular invasion and BCLC stage. Kaplan-Meier analysis revealed that HOXC6 was an independent predictor for overall survival (OS) and time to recurrence (TTR). In addition, HOXC6 status could act as prognostic predictor in different risk subgroups. Moreover, HOXC6 maintained its prognostic value in different ability of invasiveness. Furthermore, combination of HOXC6 and serum AFP could be a potential predictor for survival in HCC patients. Additionally, further study showed that HOXC6 may promote invasion of HCC by driving epithelial-mesenchymal transition (EMT). Knockdown of HOXC6 significantly decreased the migration and invasion of HCC cells and changed the expression pattern of EMT markers. An opposite expression pattern of EMT markers was observed in HOXC6-transfected cells. In addition, immunohistochemistry and RT-PCR results further confirmed this correlation. In conclusion, HOXC6 contributes to invasion by inducing EMT pathway and predicts poor prognosis of HCC. HOXC6/AFP expression may help to distinguish the different risks of HCC patients after hepatectomy.

Abstract B29: The elucidation for functional roles of Metadherin in metastatic cascade of pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. At present, surgical resection is the only curative treatment for PDAC, but the majority of PDAC patients show early recurrence even after curative resection. There is a possibility of having been distant micrometastasis at early stage of carcinogenesis in PDAC. The control of PDAC metastatic progression leads to improve the prognosis of PDAC patients. Recently, it is reported that a verified mediator of distant metastasis, Metadherin (MTDH), promotes metastasis in experimental models of breast cancer, and its high expression is associated with poor prognosis in a large spectrum of cancer types. Recent evidences imply that the epithelial-mesenchymal transition (EMT) and putative cancer stem cell (CSC) functions orchestrates to play crucial roles in cancer progression. In this study, we aim to investigate the function of MTDH in PDAC progression concomitant with undergoing EMT/MET plasticity or involving putative CSC functions. Experimental methods: We investigated MTDH expression in 7 human and 6 mouse PDAC cell lines and elucidated the roles of MTDH in PDAC progression by in vitro experiments for the loss of function using siMTDH and shMTDH. In immunohistochemical (IHC) staining of MTDH in resected PDAC tissues, we analyzed the correlation between MTDH expression and the clinico-pathological parameters as well as clinical outcome of the 82 PDAC patients. Results: Western blot analyses showed higher MTDH protein expression in metastatic PDAC compared to primary PDAC cell lines. In metastatic cell lines, MTDH expression has a positive correlation with E-cadherin expression. These data suggest that MTDH might be related to mesenchymal-epithelial transition (MET) and play a crucial role in metastatic cascade of PDAC. Conversely, MTDH has a negative correlation with E-cadherin in primary PDAC cell lines. It is implicated that MTDH might play inverse functions in primary site and metastatic site. Next, to examine what is the role of MTDH in the EMT/MET process, we evaluated the expression pattern of EMT markers and MTDH in response to transient TGF-β1 treatment. We found that E-cadherin expression decreases at 8 to 24 hours after treatment (EMT initiation), and then comes back to increase of the expression at 4 days after treatment (MET induction). While MTDH and E-cadherin expression shows an inverse relation during EMT initiation, these two expressions have a positive correlation during MET period (from 7 days after treatment). Additionally, we found EMT induction by TGF-β1 is attenuated by knockdown of MTDH, These results suggest MTDH might contribute to the phenotypic switch of PDAC cells via the EMT/MET process. MTDH knockdown significantly impaired the ability of pancreatosphere formation in PDAC cells and sensitized PDAC cells to gemcitabine. In FACS analyses, we also found that the percentage of CD133-positive cancer stem cell subpopulation was significantly reduced in MTDH knockdown metastatic PDAC cells. In anoikis assay, (this assay evaluate the resistance for apoptosis after losing contact from extracelluar matrix), MTDH knockdown reduced anoikis resistance in PDAC cells. These data indicate that MTDH might foster putative CSC functions in PDAC cells. In IHC staining, high MTDH expression indicated significantly higher incidence of hematogenous metastasis (p=0.02). Furthermore, High expression group showed poorer prognosis of PDAC patients compared to Low expression group in Kaplan Meier analysis (p=0.029). Conclusions: MTDH might promote PDAC metastasis to undergo EMT in primary PDAC and MET in metastatic PDAC along with.putative CSC functions. The regulation of MTDH might develop the new strategy for PDAC treatment, and lead to improve the outcome of patients. Citation Format: Kensuke Suzuki, Shigetsugu Takano, Hideyuki Yoshitomi, Shingo Kagawa, Hiroaki Shimizu, Masayuki Otsuka, Katunori Furukawa, Tukasa Takayashiki, Satoshi Kuboki, Daisuke Suzuki, Nozomu Sakai, Hiroyuki Nojima, Masaru Miyazaki.{Authors}. The elucidation for functional roles of Metadherin in metastatic cascade of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B29.

Epithelial-Mesenchymal Transition in Chronic Rhinosinusitis: Differences Revealed Between Epithelial Cells from Nasal Polyps and Inferior Turbinates.

The pathogenesis of chronic rhinosinusitis (CRS) remains unclear to date. The tissue remodeling in nasal polyps may be the result of inflammatory mediators and may involve epithelial-mesenchymal transition (EMT) and EMT-associated features such as cell motility in nasal epithelial cells (NECs). We determined whether NEC in nasal polyps of CRS already display features of EMT in vivo or respond with EMT to growth factor stimulation in vitro. Nasal polyp tissues expressed both epithelial and mesenchymal markers. Primary NEC from inferior turbinates and nasal polyps responded to the EMT-inducing agents transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) with different expression patterns of EMT markers (E-cadherin, N-cadherin, Snail, Slug, Twist), however, only NEC from nasal polyps were susceptible to TGF-β1 and EGF-dependent cell migration. Our data suggest that a partial EMT is associated with the pathogenesis of nasal polyps in CRS patients. Furthermore, we show for the first time that epithelial cells from both nasal polyps and inferior turbinates were able to undergo an EMT-like process following exposure to TGF-β1 or EGF in vitro but that only NEC from nasal polyps responded with enhanced cell motility. Our data suggest that NEC from CRS patients have undergo partial EMT and that this process may be involved in the pathogenesis of CRS.

URG11 predicts poor prognosis of pancreatic cancer by enhancing epithelial-mesenchymal transition-driven invasion.

The poor prognosis and high recurrent rate of pancreatic cancer (PC) necessitates the discovery of new predictive markers of PC invasion and prognosis. In this study, we evaluated the expression pattern of up-regulated gene 11 (URG11) in a tissue microarray with 18 pairs of PC and adjacent normal tissues. It was shown that URG11 was significantly up-regulated in PC tissues. High expression levels of URG11 were detected in all PC specimens, but were rarely detected in adjacent non-tumorous tissues. In addition, high expression of URG11 was correlated to poor prognosis. Furthermore, it was discovered that URG11 was correlated to epithelial-mesenchymal transition (EMT) markers and clinical pathological parameters indicative of high PC invasion, while knockdown of URG11 significantly changed the expression pattern of EMT markers and decreased the invasion of PC cells. These findings indicate that URG11 might enhance the invasion of PC by inducing EMT and thus lead to poor PC prognosis. Thus, URG11 has the potential to be a new predictive biomarker of PC invasion and prognosis, which may help in the diagnosis and treatment of PC patients.

Loss of E-Cadherin Expression Is Associated with a Poor Prognosis in Stage III Colorectal Cancer

Purpose: The epithelial-mesenchymal transition (EMT) is known to be associated with tumor progression, invasion and metastasis in colorectal cancer (CRC). Materials and Methods: Tissue samples obtained from 409 patients with stage III CRC treated from 2006 to 2007 were examined by immunohistochemistry to reveal the expression levels of E-cadherin, fibronectin, vimentin and α-smooth muscle actin (SMA). Results: Among the 409 patients, 402 cases (98.3%) showed positive E-cadherin expression. Positive E-cadherin expression was associated with well or moderately differentiated cell types and a stable microsatellite status. In multivariate analysis, a preoperative carcinoembryonic antigen level >5 ng/ml (p = 0.021), advanced N stage (p = 0.017), positive vascular invasion (p = 0.048), positive perineural invasion (p = 0.002) and negative E-cadherin expression (p = 0.002, relative risk = 5.098, 95% CI = 1.801-14.430) were poor prognostic factors affecting disease-free survival. The declining E-cadherin expression was associated with a poor outcome in terms of overall survival in univariate (p = 0.016) but not in multivariate analyses (p = 0.303, relative risk = 1.984, 95% CI = 0.539-7.296). Fibronectin, vimentin and α-SMA were of no prognostic value in this study. Conclusion: The expression pattern of EMT markers in stage III CRC suggests that declining E-cadherin expression is a possible immunohistochemical predictor of patient prognosis.

Musashi2 predicts poor prognosis and invasion in hepatocellular carcinoma by driving epithelial–mesenchymal transition

The high incidence of recurrence and the poor prognosis of hepatocellular carcinoma (HCC) necessitate the discovery of new predictive markers of HCC invasion and prognosis. In this study, we evaluated the expression pattern of two members of a novel oncogene family, Musashi1 (MSI1) and Musashi2 (MSI2) in 40 normal hepatic tissue specimens, 149 HCC specimens and their adjacent non-tumourous tissues. We observed that MSI1 and MSI2 were significantly up-regulated in HCC tissues. High expression levels of MSI1 and MSI2 were detectable in 37.6% (56/149) and 49.0% (73/149) of the HCC specimens, respectively, but were rarely detected in adjacent non-tumourous tissues and were never detected in normal hepatic tissue specimens. Nevertheless, only high expression of MSI2 correlated with poor prognosis. In addition, MSI2 up-regulation correlated with clinicopathological parameters representative of highly invasive HCC. Further study indicated that MSI2 might enhance invasion of HCC by inducing epithelial–mesenchymal transition (EMT). Knockdown of MSI2 significantly decreased the invasion of HCC cells and changed the expression pattern of EMT markers. Moreover, immunohistochemistry assays of 149 HCC tissue specimens further confirmed this correlation. Taken together, the results of our study demonstrated that MSI2 correlates with EMT and has the potential to be a new predictive biomarker of HCC prognosis and invasion to help guide diagnosis and treatment of post-operative HCC patients.

Correlation of epithelial-mesenchymal transition markers with clinicopathologic parameters in adenocarcinomas and squamous cell carcinoma of the lung.

Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial cell junction proteins and the gain of mesenchymal markers. The aim of this study was to analyze the associations between the EMT-related markers vimentin, E-cadherin, β-catenin, slug, snail, and twist1 and clinicopathologic parameters as well as epidermal growth factor receptor (EGFR) gene copy number and protein expression in non-small cell lung carcinoma (NSCLC). Fifty-nine squamous cell carcinomas (SCC) and 43 adenocarcinomas (AD) were immunohistochemically examined for respective EMT markers and for EGFR, using the EGFR PharmDx kit (Dako) for protein expression and automated silver enhanced in situ hybridization (SISH) for copy number. Vimentin expression in tumor epithelia was significantly higher in AD samples than in SCC samples (P=0.015). Among AD samples, vimentin expression was positively correlated with histologic grade (2 vs. 3; P=0.021) and exhibited a tendency toward a positive correlation with pTNM stage (I vs. II-IV; P=0.052). EGFR gene copy number was positively correlated with EGFR protein expression among both AD samples (P=0.008) and SCC samples (P=0.042), with EGFR protein expression being significantly higher in SCC samples compared with AD (P=0.038). Among AD samples, EGFR protein expression was associated with higher cytoplasmic expression of β-catenin (P=0.031). Among SCC samples, EGFR protein expression was negatively correlated with nuclear expression of β-catenin (P=0.033) but positively with nuclear slug (P=0.021). The expression pattern of EMT markers in AD suggests that vimentin is a possible immunohistochemical predictor of tumor progression.