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.     

Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma

Background:

Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial–mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT.

Methods:

We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells.

Results:

In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells.

Conclusions:

This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC.     

Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence

Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.     

Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori-activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

Despite of the improvement in gastric cancer (GC) therapies patients still suffer from cancer recurrence and metastasis. Recently, the high ratio of these events combined with increased chemoresistance has been related to the asymptomatic Helicobacter pylori (Hp) infections. The limited efficiency of GC treatment strategies is also increasingly attributed to the activity of tumor stroma with the key role of cancer-associated fibroblasts (CAFs). In order to investigate the influence of Hp infection within stromal gastric tissue on cancer initiation and progression, we have exposed normal gastric epithelial cells to long-term influence of Hp-activated gastric fibroblast secretome. We have referred obtained results to this secretome influence on cancer cell lines. The invasive properties of cells were checked by time-lapse video microscopy and basement membrane assays. The expression of invasion-related factors was checked by RT-PCR, Western Blot, immunofluorescence and Elisa. Hp-activated gastric fibroblast secretome induced EMT type 3-related shifts of RGM1 cell phenotype; in particular it augmented their motility, cytoskeletal plasticity and invasiveness. These effects were accompanied by Snail1/Twist activation, the up-regulation of cytokeratin19/FAP/TNC/Integrin-β1 and MMPs, and by the induction of cMet^high/pEGFR^high phenotype. Mechanistic studies suggest that this microevolution next to TGFβ relies also on c-Met/EGFR signaling interplay and engages HGF-Integrin-Ras-dependent Twist activation leading to MMP and TNC upregulation with subsequent positive auto- and paracrine feedback loops intensifying this process. Similar shifts were detected in cancer cells exposed to this secretome. Collectively, we show that the secretome of Hp-infected fibroblasts induces reprogramming/microevolution of epithelial and cancer cells towards type 3 EMT-related invasive phenotype in a manner reciprocally reliant next to TGFβ on cMet/Integrin-β1/p-EGFR-dependent axis. Apparently, the phenotypical plasticity of Hp-activated fibroblast reprogrammed gastric epithelial cells determines their susceptibility to the pro-invasive signaling, which results in re-organization of gastric niches and provides the cues for GC promotion/progression.     

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients

Introduction  

Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.     

Cancer‐associated fibroblasts and epithelial‐mesenchymal transition in metastatic oral tongue squamous cell carcinoma

We examined cancer‐associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial‐mesenchymal transition (EMT) in 19 pair‐matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α‐Smooth muscle actin (α‐SMA) was studied to identify CAFs. EMT was studied with syndecan‐1, Cadherin‐11, fibroblast‐specific protein (FSP)‐1, s ecreted p rotein a cidic and r ich in c ysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E‐cadherin and Ki‐67) and their relationship to the CAFs (α‐SMA). We found that metastatic RLNs hosted CAFs similarly as in pair‐matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki‐67 positive) downregulate E‐cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan‐1, Cadherin‐11, FSP‐1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.     

Phenotype of SDHB mutation carriers in the Netherlands

SDHB mutation carriers are predisposed to developing paragangliomas (PGLs). The objective of this study was to assess genotype–phenotype correlations of a Dutch cohort of SDHB mutation carriers and assess potential differences in clinical phenotypes related to specific SDHB founder mutations. Forty-seven consecutive SDHB mutation carriers were included. Initial screening consisted of measurement of 24 h urinary excretion of catecholamines and their metabolites in duplicate, repeated annually if initial biochemical screening was negative. Whole-body imaging studies with magnetic resonance imaging (MRI) or computed tomography (CT) and/or ¹²³I-MIBG scintigraphy were performed in case of catecholamine excess, and MRI or CT scans of thorax, abdomen and pelvis were performed every 2 years regardless of catecholamine levels. Repetitive head-and-neck MRI was performed at 2 year intervals. Mean follow-up was 3.6 ± 3.6 years. Twenty-seven persons (57 %) carried the SDHB c.423+1 G>A mutation and seven persons (15 %) the SDHB c.201-4429_287-933del (exon 3 deletion) mutation. No differences were found in the clinical phenotype of carriers of these two specific SDHB mutations. By end of follow-up, 49 % of SDHB mutation carriers displayed no biochemical or radiological evidence of manifest disease, i.e. they were unaffected carriers. Three persons (6 %) had been diagnosed with a pheochromocytoma (PCC), four with a sympathetic PGL (sPGL) (9 %), 18 with a HNPGL (38 %), and two persons (4 %) had developed a malignant paraganglioma, i.e. metastatic disease. In conclusion, the two main Dutch SDHB founder mutations do not differ in clinical expression and result in a relatively mild phenotype. Over one-third of SDHB mutation carriers develop HNPGL, with sPGL/PCC in only 15 % and malignancy in only 4 %.     

Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

Background

In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44⁺/CD24^- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44⁺/CD24^- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?

Methods

Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.

Results

MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.

Conclusions

MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

     

Epithelial mesenchymal transition in colorectal cancer: Seminal role in promoting disease progression and resistance to neoadjuvant therapy

BackgroundEpithelial mesenchymal transition (EMT) may be physiological as part of embryological development, or pathological as part of cancer development. It is one of the key initiating events in the metastatic cascade. EMT has profound effects on tumour cell invasiveness, proliferation and motility. In the present article we aimed to review the potential role of EMT as a process to explain colorectal cancer progression and resistance to neoadjuvant therapy.MethodsExtensive literature searches were performed in Pubmed, EMBASE and Google Scholar databases to identify relevant articles published before March 2012.ResultsThere is adequate evidence to support the complex upstream signalling alterations needed for EMT to occur in colorectal cancers. Changes of EMT are likely to be found at the tumour invasive front: the deepest, growing tumour margin. Loss of E-cadherin at the cell membrane causes loss of cellular integrity, with subsequent migration of malignant cells and tumour budding. These processes are associated with metastases and recurrence of colorectal cancer. There is early evidence from a limited number of studies that resistance to neoadjuvant therapy in colorectal cancer is associated with changes of EMT. However, there is a lack of supporting evidence originating from human colorectal cancer tissues.ConclusionsEmerging evidence demonstrates that development of EMT in colorectal cancer leads to an aggressive phenotype that may promote metastatic spread, and augment treatment resistance during neoadjuvant therapy. A clearer understanding of the processes and role of EMT in colorectal cancer may also highlight novel therapeutic strategies.     

HPV‐avertable cancer risks in India: A pooled analysis of 9 observational studies

Recent advances in cancer stem cell biology have shown that cancer stem‐like cells with epithelial–mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem‐like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem‐like cells, but none has been identified for isolating cancer stem‐like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem‐like cancer cells with EMT phenotype, by using a specific CSV‐binding antibody, 84‐1. Using this antibody, we purified the CSV‐positive, CD133‐negative (csVim⁺CD133⁻) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim⁺CD133⁻ cells have stem‐like properties similar to csVim⁻CD133⁺ population. Our investigation further revealed that the csVim⁺CD133⁻ cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E‐cadherin. The csVimentin‐negative CD133‐positive stem cells do not have any EMT phenotypes. csVim⁺CD133⁻ cells exhibited more aggressively metastatic in livers than csVim⁻CD133⁺ cells. Our findings indicate that csVim⁺CD133⁻ cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.