Mowat-Wilson syndrome is a recently delineated multiple congenital anomaly syndrome characterized by a distinctive facial appearance in association with intellectual disability, microcephaly, agenesis of the corpus callosum, seizures, congenital heart disease, Hirschsprung disease, short stature, and genitourinary anomalies. We report a 2-year-10-month-old white female with this syndrome caused by mutations in the ZEB2 gene, and in addition a duplication of the 22q11.23, a previously undocumented occurrence.

Mowat-Wilson syndrome (MWS) is an autosomal dominant developmental disorder with mental retardation and variable multiple congenital abnormalities due to mutations of the ZEB2 (ZFHX1B) gene at 2q22. MWS was first described in 1998 and the causative gene was delineated in 2001. Since then, 115 different mutations of ZEB2 have been published in association with this syndrome in 161 individuals. However, recent reports suggest that due to the variability of the congenital abnormalities, this syndrome may still be underdiagnosed. We report two unrelated patients with MWS where the clinical diagnosis was established only after finding of disruption of the ZEB2 gene by a balanced translocation breakpoint and an interstitial microdeletion, respectively.

Aberrant expression of microRNAs (miRNAs) has been widely recognized to play critical roles in the pathogenic processes of colon cancer. However, the expression and functions of miR-3653 in colon cancer remain uncovered. This study revealed for the first time that miR-3653 expression was significantly decreased in colon cancer tissues and cell lines. MiR-3653 overexpression led to decreased migration and invasion of HCT116 cells while miR-3653 knockdown resulted in opposite influence of the metastatic behaviors of HT29 cells. qRT-PCR and western blot demonstrated that miR-3653 suppressed the epithelial-mesenchymal transition (EMT) of colon cancer cells using both gain- and loss- of function assay. Mechanically, miR-3653 was found to interact with the 3'-UTR of Zeb2 through the complementary sequences and inhibited the expression of Zeb2 in colon cancer cells. Rescue experiments demonstrated that the inhibitory effect of miR-3653 overexpression on cell metastasis and EMT was abrogated by forced expression of Zeb2. This study demonstrates that miR-3653 suppresses the metastasis and EMT of colon cells by targeting Zeb2, and serves as a promising biomarker and therapeutic target in colon cancer.

"miRNA colorectal cancer" (https://mirna-coadread.omics.si/) is a freely available web application for studying microRNA and mRNA expression and their correlation in colorectal cancer. To the best of our knowledge, "miRNA colorectal cancer" has the largest knowledge base of miRNA-target gene expressions and correlations in colorectal cancer, based on the largest available sample size from the same source of data. Data from high-throughput molecular profiling of 295 colon and rectum adenocarcinoma samples from The Cancer Genome Atlas was analyzed and integrated into our knowledge base. The objective of developing this web application was to help researchers to discover the behavior and role of miRNA-target gene interactions in colorectal cancer. For this purpose, results of differential expression and correlation analyses of miRNA and mRNA data collected in our knowledge base are available through web forms. To validate our knowledge base experimentally, we selected genes FN1, TGFB2, RND3, ZEB1 and ZEB2 and miRNAs hsa-miR-200a/b/c-3p, hsa-miR-141-3p and hsa-miR-429. Both approaches revealed a negative correlation between miRNA hsa-miR-200b/c-3p and its target gene FN1 and between hsa-miR-200a-3p and its target TGFB2, thus supporting the usefulness of the developed knowledge base.

MicroRNAs (miRNAs) exert a critical influence on physiological and pathological processes through posttranscriptionalmodification of their mRNA targets. They play important roles in tumorigenesis and are considered tobe potential diagnostic and prognostic biomarkers with various cancers. MiR-200c and miR-9 are regulatory elementsthat can have dual impacts as oncogenes and/or tumor suppressor genes. MiR-200c regulates two transcription factors,ZEB1 and ZEB2, while miR-9 is a regulatory factor for the E-cadherin protein which has a critical function in cell-celljunctions and is inhibited by two transcription factors ZEB1 and ZEB2. In this study, expression levels of miR-200cand miR-9, ZEB-1, ZEB-2 and E-cadherin were assessed in 30 non-small cell lung cancers (NSCLCs) by real-timeqPCR. MiR-9 was down-regulated significantly in tumor tissues compared to normal adjacent tissues, while there wasno significant change in expression level of miR-200c. On the other hand, ZEB1 demonstrated significant increaseand ZEB2a decrease at the mRNA level. These results indicate roles for miR-9 and ZEB1 in genesis of lung cancer,although clinico-pathological associations were not evident. Further studies are necessary to assess implications fortreatment of lung cancer.

Neural tube defects (NTDs) are a complex trait associated with gene-environment interactions. Folic acid deficiency and planar cell polarity gene mutations account for some NTD cases; however, the etiology of NTDs is still little understood. In this study, in three Han Chinese NTD pedigrees (two with multiple affected children), with no information on folic acid deficiency or supplement, we examined genome-wide methylation profiles of each individual in these families. We further compared methylation status among cases and normal individuals within the pedigrees. A unique methylation pattern co-segregated with affected status: NTD cases had more hypermethylated than hypomethylated CpG islands; genes with different methylations clustered in pathways associated with epithelial-to-mesenchymal transition (ZEB2, SMAD6, and CDH23), folic acid/homocysteine metabolism (MTHFD1L), transcription/nuclear factors (HDAC4, HOXB7, SOX18), cell migration/motility/adhesion, insulin and cell growth, and neuron/axon development. Although the genetics of NTD are likely complex, epigenetic changes may concentrate in certain key pathways.

Mowat-Wilson syndrome is a genetic disease caused by heterozygous mutations or deletions of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. The syndrome is characterized by typical facial features, moderate-to-severe mental retardation, epilepsy and variable congenital malformations, including Hirschsprung disease, genital anomalies, congenital heart disease, agenesis of the corpus callosum, and eye defects. The prevalence of Mowat-Wilson syndrome is currently unknown, but it seems that Mowat-Wilson syndrome is underdiagnosed, particularly in patients without Hirschsprung disease. We report here the first Egyptian case of Mowat-Wilson syndrome who was conceived by intracytoplasmic sperm injection. The patient manifested bilateral sensorineural hearing loss--a new feature not previously reported in cases of Mowat-Wilson syndrome. This report describes the first Egyptian patient of Mowat-Wilson syndrome who was conceived after intracytoplasmic sperm injection, and provides a new evidence for the inclusion of deafness among the congenital defects of the syndrome.

Mowat-Wilson syndrome is a multiple congenital anomaly and intellectual disability syndrome characterized by a unique face and various other structural and functional anomalies. The condition is caused by de novo heterozygous mutations or deletions in ZEB2 gene located at 2q22. ZEB2 encodes Sip1 protein, which acts during central nervous system development as an important transcription factor. Herein, we report on 3 novel mutations in 6 patients with the syndrome, with an overview of corresponding clinical findings. Growth retardation and Hirschsprung disease were less common in the present cohort. One patient with a novel mutation p.Y489X had no associated anomalies except the characteristic facial and neurobehavioral phenotype. Reporting new patients with novel mutations would contribute to better delineation of the syndrome and would help clinicians establish formal diagnostic criteria and genotype-phenotype correlations.

Lung cancer is one of the major causes of cancer-related deaths worldwide. Notably, miR-155-5p is one of the most amplified miRNAs in non-small cell lung carcinoma (NSCLC). However, the role of miR-155-5p in lung cancer metastasis has not been fully evaluated. In the present study, miR-155-5p mimic and inhibitor were used to investigate the effects of miR-155-5p on the metastasis of human lung carcinoma A549 cells. The study indicated that transfection of miR-155-5p mimic significantly suppressed cell proliferation, migration and invasion of A549 cells, whereas its inhibition significantly promoted cell proliferation, migration and invasion of A549 cells, suggesting a potential therapeutic application of miR-155-5p in controlling lung cancer metastasis. Moreover, transfection of miR-155-5p mimic suppressed the expression of Smad2/3, ZEB1, ZEB2 and N-cadherin and induced that of E-cadherin, whereas its inhibition significantly upregulated the expression of Smad2/3, ZEB1, ZEB2 and N-cadherin and downregulated that of E-cadherin. Collectively, the findings suggest that miR-155-5p suppresses the proliferation, migration and invasion of A549 cells. Therefore, loss of miR-155-5p may serve an essential role in tumorigenesis and tumour progression in lung cancers.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Whereas miR-200 family is known to be involved in the epithelial-to-mesenchymal transition (EMT), a crucial biological process observed in normal and pathological contexts, it has been largely unclear how far the functional levels of these tiny RNAs alone can propagate the molecular events to accomplish this process within several days. By developing a potent inhibitor of miR-200 family members (TuD-141/200c), the expression of which is strictly regulatable by the Tet (tetracycline)-On system, we found using a human colorectal cell line, HCT116, that several direct gene target mRNAs (Zeb1/Zeb2, ESRP1, FN1and FHOD1) of miR-200 family were elevated with distinct kinetics. Prompt induction of the transcriptional suppressors, Zeb1/Zeb2 in turn reduced the expression levels of miR-200c/-141 locus, EpCAM, ESRP1 and E-Cad. The loss of ESRP1 subsequently switched the splicing isoforms of CD44 and p120 catenin mRNAs to mesenchymal type. Importantly, within 9 days after the release from the inhibition of miR-200 family, all of the expression changes in the 14 genes observed in this study returned to their original levels in the epithelial cells. This suggests that the inherent epithelial plasticity is supported by a weak retention of key regulatory gene expression in either the epithelial or mesenchymal states through epigenetic regulation.

The pathogenesis of sarcomatous component in spindle cell carcinoma (SpCC) of the esophagus is unclear. To investigate the involvement of epithelial-mesenchymal transition (EMT) in sarcomatous differentiation, we performed immunohistochemistry for Slug, Twist, ZEB1, and ZEB2, transcription factors associated with EMT and E-cadherin, in 14 cases of SpCC of the esophagus. In order to verify the neoplastic nature of sarcomatous components, TP53 mutation status and protein expression were examined in each case. Nuclear ZEB1 expression was extensive in the sarcomatous component, greater than invasive front of carcinoma components (P < 0.0001). Membranous E-cadherin expression was mostly lost in sarcomatous cells in all cases (P < 0.0001). The p53 expression pattern was almost concordant between the two areas in all cases. TP53 mutation analysis revealed that seven cases harbored identical mutations in both components. One case had mutations only in the sarcomatous component. It is noteworthy that none of them harbored mutation in exon 5, unlike conventional esophageal squamous cell carcinoma. These findings show that ZEB1 are widely expressed in the sarcomatous area of SpCC of the esophagus, suggesting the involvement of EMT. The avoidance of exon 5 in terms of TP53 mutation may also be a feature of the tumor.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and metastasis in various malignancies. ZEB2/SIP1 is an important EMT regulator and down-regulates E-cadherin expression. The present study was planned to explore status of EMT-associated markers ZEB2/SIP1 and E-cadherin in eyelid sebaceous gland carcinoma (SGC) and to correlate with clinicopathological high-risk features. Expressions of ZEB2 and E-cadherin were evaluated by immunohistochemistry in 65 cases of histopathologically proven eyelid SGC. The results were correlated with clinicopathological high-risk features and survival of the patients to determine the prognostic significance of ZEB2, E-cadherin, and various high-risk features. Cytoplasmic overexpression of ZEB2 and membranous loss of E-cadherin were seen in 68% and 66% of cases of eyelid SGC, respectively. ZEB2 overexpression was significantly associated with E-cadherin loss (P = .002). Overexpression of ZEB2 also showed significant association with lymph node metastasis (P = .046), orbital invasion (P = .049), large tumor size (P = .018), and advanced tumor stages (P = .036). Survival analysis revealed that patients with ZEB2 overexpression had poor survival. ZEB2 overexpression and orbital invasion were found to be independent prognostic indicators (univariate analysis). However, multivariate analysis showed that ZEB2 (hazard ratio, 0.094; 95% confidence interval, 00.012-0.709; P = .022) was the best poor prognostic indicator of eyelid SGC. Our study demonstrates the role of both ZEB2 and E-cadherin in the promotion of EMT in eyelid SGC. The outcome of this study also points toward ZEB2 as an independent prognostic marker as well as a potential therapeutic target in eyelid SGC.

Introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to 'reprogram' somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process.

A miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR.

Co-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4.

Via targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.

The aim of this study was to investigate the effect of Astragaloside IV (AS-IV) on renal fibrosis in vivo and in vitro, and further to explore the underlying mechanism. To investigate the effect of AS-IV treatment on renal fibrosis in vivo, mouse renal fibrosis model was established by performing unilateral ureteral occlusion (UUO). The mice in the intervention group of AS-IV were given AS-IV 20 mg/(kg/d) on the day after surgery for 7 consecutive days. Then renal sections were stained with hematoxylin and eosin (H&E) to evaluate the degree of fibrosis. For in vitro study, human kidney tubular epithelial cells induced by (TGF-β1) were performed to research the protective role of AS-IV in anti-fibrosis. Results form the in vivo study showed that AS-IV treatment in UUO mice significantly reduced parenchymal loss and tubular atrophy, indicating that AS-IV treatment attenuated renal fibrosis caused by UUO. TGF-β1 treatment significantly increased the expression of α-SMA, vimentin, collagen I, miR-192 and decreased E-cadherin expression in HK-2 cells, suggesting that TGF-β1 stimulated renal tubulointerstitial fibrosis. Moreover, in TGF-β1 stimulated HK-2 cells, AS-IV clearly inhibited the expression levels of α-SMA, vimentin, collagen I, and miR-192 in a dose-dependent fashion while increased the expression level of E-cadherin in the same manner, indicating that AS-IV functioned the inhibitory role in renal tubulointerstitial fibrosis. Interestingly, we noted that ZEB2 was a direct target of miR-192. The effects of AS-IV on the expression of α-SMA, vimentin, collagen I and E-cadherin were inhibited by miR-192 mimic and aggravated by miR-192 inhibitor. Taken together, our results provided evidence that AS-IV could effectively protect kidney against epithelial fibrosis, and this renoprotective effect involved miR-192. Therefore, AS-IV might be considered as a potential and promising candidate drug for the treatment of renal epithelial fibrosis.

Abnormal expression levels of microRNA-204 (miR-204) have been identified in various types of human cancer. However, the expression and functions of miR-204, and the underlying molecular mechanism involved in the initiation and progression of hepatocellular carcinoma (HCC), require further investigation. The results of the present study demonstrated that miR-204 is downregulated in HCC tissues and cell lines. Notably, zinc finger E-box binding homeobox 2 (ZEB2) was identified as a direct target of miR-204 in HCC. In addition, miR-204 negatively regulates ZEB2 expression level in HCC cells at the post-transcriptional level. In functional studies, the overexpression of miR-204 inhibited the proliferation, migration and invasion of HCC cells. Furthermore, the knockdown of ZEB2 may mimic the functions of miR-204 in HCC cells, suggesting that ZEB2 is a direct functional target of miR-204. In conclusion, the results of the present study indicated that miR-204 suppresses the tumor growth, migration and invasion of HCC cells by directly targeting ZEB2, and may serve as a novel therapeutic target for HCC.

Several Fusarium species produce the polyketide mycotoxin zearalenone (ZEA), a causative agent of hyperestrogenic syndrome in animals that is often found in F. graminearum-infected cereals in temperate regions. The ZEA biosynthetic cluster genes PKS4, PKS13, ZEB1 and ZEB2 encode a reducing polyketide synthase, a non-reducing polyketide synthase, an isoamyl alcohol oxidase and a transcription factor respectively. In this study, the production of two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum via an alternative promoter was characterized. ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggers the induction of both ZEB2L and ZEB2S transcription. ZEB2L and ZEB2S interact with each other to form a heterodimer that regulates ZEA production by reducing the binding affinity of ZEB2L for the ZEB2L gene promoter. Our study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production; this regulatory mechanism is similar to that observed in higher eukaryotes.

Aim: To explore the biological functions and clinicopathologic significance of the long noncoding RNA KTN1-AS1 in head and neck squamous cell carcinoma (HNSCC). Materials & methods: We assessed the effects of KTN1-AS1 and identified the target miRNA by bioinformatics analysis, luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. The clinicopathologic features of KTN1-AS1 and its target miRNA were analyzed in HNSCC. Results:KTN1-AS1, a competing endogenous RNA, promoted cell proliferation, migration, invasion and epithelial-mesenchymal transition by sponging miR-153-3p in HNSCC. Dysregulation of SNAI1 and ZEB2 mediated the effect of KTN1-AS1 due to miR-153-3p exhaustion. The KTN1-AS1 and miR-153-3p combination can accurately diagnose HNSCC. Conclusion: The KTN1-AS1 and miR-153-3p combination could be a valuable diagnostic and prognostic predictor for HNSCC.

Zeb2 (Sip1, Zfhx1b) is a transcription factor that plays essential role in neuronal development. Sip1 mutation in humans was shown to cause Mowat-Wilson syndrome, a syndromic form of Hirschprung's disease. Affected individuals exhibit multiple severe neurodevelopmental defects. Zeb2 can act as both transcriptional repressor and activator. It controls expression of a wide number of genes that regulate various aspects of neuronal development. This review addresses the molecular pathways acting downstream of Zeb2 that cause brain development disorders.

Mouse utricle sensory epithelial cell-derived progenitor cells (MUCs), which have hair cell progenitor and mesenchymal features via epithelial-to-mesenchymal transition (EMT) as previously described, provide a potential approach for hair cell regeneration via cell transplantation. In this study, we treated MUCs with trichostatin A (TSA) to determine whether histone deacetylase inhibitor is able to stimulate the expression of epithelial genes in MUCs, an essential step for guiding mesenchymal-like MUCs to become sensory epithelial cells. After 72 h of TSA treatment, MUCs acquired epithelial-like features, which were indicated by increased expression of epithelial markers such as Cdh1, Krt18, and Dsp. Additionally, TSA decreased the expression of mesenchymal markers, including Zeb1, Zeb2, Snai1, and Snai2, and prosensory genes Lfng, Six1, and Dlx5. Moreover, the expression of the hair cell genes Atoh1 and Myo6 was increased in TSA-treated MUCs. We also observed significantly decreased expression of Hdac2 and Hdac3 in TSA-treated MUCs. However, no remarkable change was detected in protein expression using immunofluorescence, indicating that TSA-induced HDAC inhibition may contribute to the initial stage of the mesenchymal-to-epithelial phenotypic change. In the future, more work is needed to induce hair cell regeneration using inner ear tissue-derived progenitors to achieve an entire mesenchymal-to-epithelial transition.

Vasohibin-2 (VASH2) is a homolog of VASH1, an endothelium-derived angiogenesis inhibitor. Vasohibin-2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial-mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor-β (TGF-β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF-β type I receptor, namely activin receptor-like kinase 5. Transforming growth factor-β1-induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF-β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF-β1-induced reduced expression of epithelial markers including E-cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF-β1-induced EMT. In accordance with these results, the effects of TGF-β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF-β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.

The bidirectional regulation of epithelial-mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.

The anchorage-independent colony growth of cancer cells is reportedly correlated with the tumor-forming activity; however, the correlation between the morphophenotype of each colony and the tumor-forming activity has not been clarified. To assess this problem, we cultured single A549 cells (human lung adenocarcinoma cell line) in growth medium in individual wells (n = 426) for 14 days under anchorage-independent conditions and analyzed the resulting growth characteristics. The single A549 cells formed various sizes of floating colonies. The proportion of large colonies (>400 μm) was 3.8% and this proportion increased dramatically with the exogenous addition of EGF (21.6%) or HGF (27.6%). Morphologically, the floating colonies could be divided into: (ii) Type A, spheroid colony; and (ii) Type B, dispersed villous colony. The Type B colonies expressed significantly higher levels of epithelial-mesenchymal transition (EMT)-related mRNAs (Snail 1, ZEB 1, and ZEB2) than the Type A colonies. Furthermore, the subcutaneous injection of a single cell-derived colony with a large size and a Type B morphology resulted in more efficient tumor formation. The present results indicated that the morphophenotypes of floating colonies derived from single cancer cells have a critical impact on tumor-forming activity.