E-cadherin Alterations Research Articles

Numb regulates cell tension required for mammary duct elongation.

ABSTRACTThe mammary gland undergoes extensive expansion of a ductal network through the stroma during puberty and is an excellent model for understanding epithelial tube morphogenesis. To investigate a role for Numb, a multifaceted adapter protein, in epithelial tube morphogenesis, we conditionally deleted it from the mammary epithelium. We report that Numb-depletion results in altered extracellular-matrix organization, reduced cell tension, altered cell shape, and increased cell packing density, which results in a 50% reduction in mammary duct elongation. Using laser ablation in vitro and geometric-based cell force inference in vivo, we determined that Numb-deficient cells have altered cortical tension. Duct elongation defects were associated with altered E-cadherin distribution, but were independent of proliferation, apoptosis in ducts or end buds. This highlights a critical role for Numb in a mechanical mechanism that is required to maintain cell packing density during epithelial tube elongation.

Stromal Gas6 promotes the progression of premalignant mammary cells.

Tumor progression is regulated by a complex interplay between neoplastic cells and the tumor microenvironment. Tumor associated macrophages have been shown to promote breast cancer progression in advanced disease and more recently, in early stage cancers. However, little is known about the macrophage-derived factors that promote tumor progression in early stage lesions. Using a p53-null model of early stage mammary tumor progression, we found that Gas6 is highly expressed in pre-invasive lesions associated with increased infiltrating macrophages, as compared to those with few recruited macrophages. We show that F4/80+CD11b+ macrophages produce Gas6 in premalignant lesions in vivo, and that macrophage-derived Gas6 induces a tumor-like phenotype ex vivo. Using a 3-D co-culture system, we show that macrophage-derived Gas6 activates its receptor Axl and downstream survival signals including Akt and STAT3, which was accompanied by altered E-cadherin expression to induce a malignant morphology. In vivo studies demonstrated that deletion of stromal Gas6 delays early stage progression and decreases tumor formation, while tumor growth in established tumors remains unaffected. These studies suggest that macrophage-derived Gas6 is a critical regulator of the transition from premalignant to invasive cancer, and may lead to the development of unique biomarkers of neoplastic progression for patients with early stage breast cancer, including ductal carcinoma in situ.

Restoration of GATA4 expression impedes breast cancer progression by transcriptional repression of ReLA and inhibition of NF-κB signaling.

There are increasing reports of aberrant expression of GATA4, correlated with oncogenesis and malignant progression in some solid tumors, but whether GATA4 functions as an oncogenic driver or a tumor suppressor in carcinogenesis remains controversial. Because the role and mechanism of GATA4 in breast cancer (BrCa) remain poorly understood, we focused on the expression of GATA4 in BrCa cell lines and tissues and its mechanism in breast oncogenesis. Semiquantitative real-timepolymerase chain reaction (RT-PCR), quantitative RT-PCR, Western blot analysis, and immunohistochemistry were used to detect expression of GATA4 in BrCa cell lines and adjacent breast tissues. Methylation statuses of the GATA4 promoter were studied using methylation-specific PCR in BrCa cell lines.The effects of GATA4 on proliferation, invasion, and cell cycle were also analyzed. Compared with adjacent breast tissue, GATA4 expression in BrCa tissue and cell lines was obviously lower and low expression levels of GATA4 predicted poor outcome. Methylation of GATA4 occurred in almost all of BrCa cell lines . GATA4 overexpression decreased viability, invasion, migration, and epithelial-to-mesenchymal transition of MB-231 and BT549 cells, and markedly induced cell cycle arrest and apoptosis. Exogenous expression GATA4 accompanied a significant alteration of MMP2, MMP3, E-cadherin, and N-cadherin expression and induction of the caspase-8 pathway. Moreover, GATA4 could directly repress RelA (p65) transcription, reduce the nuclear phosphorylation-p65 and upregulate inhibitor kappa B expression. Altogether, GATA4 plays a tumor-suppressive role via repression of NF-κB signaling in BrCa cells. Our findings suggest that GATA4 is a potential prognostic biomarker and gene therapeutic target for human BrCa.

Abstract 3057: ABCG2-positive cells derived from ABCG2-negative pancreatic cancer cells in 3D-culture conditions show high stemness and anti-cancer drug resistance

Abstract ATP-binding cassette subfamily G member 2 (ABCG2), an important multidrug resistance transporter, can mediate the efflux of various chemotherapy drugs and contribute to drug resistance in cancer cells. The correlation between ABCG2 expression and cancer stem cell (CSC) phenotypes has been examined in hepatocellular carcinoma, as well as in glioma, breast, prostate, and colon cancer; however, the results remain controversial. In this study, we compared the characteristics of low- (ABCG2-) and high-expressing (ABCG2+) pancreatic ductal adenocarcinoma (PDAC) cells using a human pancreatic cancerous cell line (PANC-1) because the role of ABCG2 in CSC-related malignant characteristics in PDAC is not well elucidated. ABCG2- and ABCG2+ PDAC cells were separated using flow cytometric cell sorting. In adherent cell culture conditions, 10% of all PANC-1 cells were ABCG2+. Using transmission electron microscopy, we found that ABCG2+ cells showed more abundant and longer microvilli on the cell surface than ABCG2- cells. Unexpectedly, ABCG2+ cells did not demonstrate significantly greater drug resistance against 5-FU, gemcitabine, and vincristine than ABCG2- cells, as assessed using a WST-8 assay. Furthermore, ABCG2- cells exhibited better sphere formation ability and higher stemness marker expression, including that of Sox2, Oct4, ALDH1, CD44v9, and Nestin, than ABCG2+ cells, as observed using qRT-PCR. Cell growth rates and motilities, examined using the Boyden chamber and scratch assays, were higher in ABCG2- cells than in ABCG2+ cells. However, the epithelial-mesenchymal transition (EMT) ability, assessed by examining the alteration of E-cadherin, N-cadherin, Snail, and Vimentin expression after TGFβ addition, was comparable between ABCG2- and ABCG2+ cells. In 3D-culture conditions using ultra low-attachment plates, ABCG2- cells formed spheres containing a large number of ABCG2+ cells, and expression of stemness markers in these spheres was higher than that of spheres derived from ABCG2+ cells. Furthermore, spheres derived from ABCG2- cells included large populations of ABCG2+ cells and exhibited high resistance against anti-cancer drugs, presumably depending on ABCG2 expression. We found that in adherent culture conditions, ABCG2+ PDAC cells do not exhibit stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2- cells after sphere formation have high stemness and anti-cancer drug resistance. This suggests that ABCG2- cells have the capacity to generate ABCG2+ cells, and the malignant potential of ABCG2+ cells in PDAC depends on the environment. The 3D-culture system was expected to mimic in vivo environments. Therefore, ABCG2+ cells in pancreatic cancer tumors may exhibit high stemness and anti-cancer drug resistance. Thus, ABCG2+ cells should be considered as novel therapeutic targets for pancreatic cancer. Citation Format: Norihiko Sasaki, Fumio Hasegawa, Masaki Michishita, Yoko Matsuda, Tomio Arai, Naoshi Ishikawa, Yoko Itakura, Junko Aida, Kaiyo Takubo, Masashi Toyoda, Toshiyuki Ishiwata. ABCG2-positive cells derived from ABCG2-negative pancreatic cancer cells in 3D-culture conditions show high stemness and anti-cancer drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3057.

A novel de novo CDH1 germline variant aids in the classification of carboxy-terminal E-cadherin alterations predicted to escape nonsense-mediated mRNA decay.

Most truncating cadherin 1 (CDH1) pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer (DGC) and lobular breast cancer (LBC). However, transcripts containing carboxy-terminal premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing because of a personal history of invasive LBC diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.Glu836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most carboxy-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 carboxy-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other carboxy-terminal truncating variants as either pathogenic or benign, a fundamental step to offering presymptomatic screening and prophylactic procedures to the appropriate patients.

Collagen IV-conveyed signals can regulate chemokine production and promote liver metastasis.

Liver metastases remain a major cause of death from gastrointestinal tract cancers as well as from other malignancies such as breast and lung carcinomas and melanoma. Understanding the underlying biology is essential for the design of effective targeted therapies. We previously reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma (M27colIV) cells increased their metastatic ability, specifically to the liver and documented high collagen IV levels in surgical resections of liver metastases from diverse tumor types. Here, we aimed to elucidate the functional relevance of collagen IV to metastatic outgrowth in the liver. Gene expression profiling revealed in M27colIVcells significant increases in the expression of chemokines CCL5 (5.7-fold) and CCL7 (2.6-fold) relative to wild-type cells, and this was validated by qPCR and western blotting. Similarly, in human colon carcinoma KM12C and KM12SM cells with divergent liver-colonizing potentials, CCL7 and CCL5 production correlated with type IV collagen expression and the metastatic phenotype. CCL7 silencing by short hairpin RNA (shRNA) reduced experimental liver metastasis in both cell types, whereas CCL5 silencing reduced metastasis of M27colIV cells, implicating these cytokines in metastatic expansion in the liver. Subsequent functional analyses implicated both MEK/ERK and PI3K signaling upstream of CCL7 upregulation and identified CCL7 (but not CCL5) as a critical migration/invasion factor, acting via the chemokine receptor CCR3. Chemokine CCL5 was identified as a regulator of the T-cell immune response in the liver. Loss of CCL7 in KM12SM cells was also associated with altered E-cadherin and reduced vimentin and Snail expression, implicating it in epithelial-to-mesenchymal transition in these cells. Moreover, in clinical specimens of colon cancer liver metastases analyzed by immunohistochemistry, CCL5 and CCL7 levels paralleled those of collagen IV. The results identify the chemokines CCL5 and CCL7 as type IV collagen-regulated genes that promote liver metastasis by distinct and complementary mechanisms.

Characterizing Metastatic HER2-Positive Gastric Cancer at the CDH1 Haplotype.

The CDH1 gene, coding for the E-cadherin protein, is linked to gastric cancer (GC) susceptibility and tumor invasion. The human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in a portion of GC. HER2 is an established therapeutic target in metastatic GC (mGC). Trastuzumab, in combination with various chemotherapeutic agents, is a standard treatment for these tumors leading to outcome improvement. Unfortunately, the survival benefit is limited to a fraction of patients. The aim of this study was to improve knowledge of the HER2 and the E-cadherin alterations in the context of GC to characterize subtypes of patients that could better benefit from targeted therapy. An association between the P7-CDH1 haplotype, including two polymorphisms (rs16260A-rs1801552T) and a subset of HER2-positive mGC with better prognosis was observed. Results indicated the potential evaluation of CDH1 haplotypes in mGC to stratify patients that will benefit from trastuzumab-based treatments. Moreover, data may have implications to understanding the HER2 and the E-cadherin interactions in vivo and in response to treatments.

T-cell immunoglobulin mucin-3 as a potential inducer of the epithelial-mesenchymal transition in hepatocellular carcinoma.

T-cell immunoglobulin mucin (TIM)-3 is an important member of the TIM gene family, which was thought to contribute to the progression of numerous types of cancer, including hepatocellular carcinoma (HCC); however, the mechanism underlying TIM-3 functions in HCC progression has not yet been extensively investigated. The present study aimed to investigate the function of TIM-3 in the metastasis of HCC and to determine whether the alteration of TIM-3 expression levels regulated the epithelial-mesenchymal transition (EMT) occurrence of HCC, using epithelial (E)-cadherin, neuronal (N)-cadherin, matrix metallopeptidase-9 (MMP-9), Twist 1, Slug, Snail, and Smad as EMT biomarkers. The results demonstrated that upregulation of TIM-3 using TIM-3 lentiviral activation particles (5 µl) increased cell migration and invasion, which was decreased in TIM-3 short interfering RNA-infected cells (10 µM, 3 µl) correspondingly. SMMC-7721 HCC cells were used as the control. EMT was aggravated in TIM-3 upregulated SMMC-7721 cells, which was attenuated in the TIM-3 interference group, accompanied by an alteration of E-cadherin, N-cadherin, MMP-9, Twist 1, Slug, Snail and Smad expression levels. The data presented suggests that TIM-3 serves an essential role in the metastasis of HCC, the mechanism of which was associated with EMT occurrence. Interference of TIM-3 is expected to be an effective means to prevent and control EMT, and further the metastasis of HCC.

FSCN1 is upregulated by SNAI2 and promotes epithelial to mesenchymal transition in head and neck squamous cell carcinoma.

In this study, we investigated whether there is any association between the expression of FSCN1 and SNAI2 and the possible underlying mechanisms in head and neck squamous cell carcinoma (HNSC). In addition, we also investigated whether FSCN1 modulates epithelial-to-mesenchymal transition (EMT) in HNSC cells. Microarray data of dysregulated genes in HNSC were searched in GEO datasets. The association between FSCN1 expression and the 5-year/10-year overall survival (OS), as well as the correlation between the expression of FSCN1 and SOX2, MYBL2, SNAI2, STAT1, and SOX4, was analyzed based on data in TCGA HNSC cohort (TCGA-HNSC). The binding site of SNAI2 in FSCN1 promoter was verified using luciferase reporter assay. SCC9 and SCC15 cells were transfected with pCMV-SNAI2 or pCMV-FSCN1 expression vector or the empty control. Alteration of E-cadherin, Claudin 1, Vimentin, and N-cadherin was then quantified. Our results showed that FSCN1 is significantly upregulated in HNSC tissues compared with the normal control tissues. High FSCN1 expression is associated with worse 5-year and 10-year OS among the HNSC patients. Bioinformatic prediction showed a highly possible SNAI2 binding site in FSCN1 promoter and following luciferase reporter assay verified this site. SNAI2 overexpression significantly increased FSCN1 expression at both mRNA and protein level. FSCN1 overexpression reduced the expression of E-cadherin and Claudin 1, but increased the expression of Vimentin and N-cadherin in SCC9 and SCC-15 cells. Therefore, we infer that FSCN1 is a downstream effector of SNAI2 in promoting EMT in HNSC cells.

Impact of Small Molecules on β-Catenin and E-Cadherin Expression in HPV16-positive and -negative Squamous Cell Carcinomas.

The validation of potential molecular targets in head and neck squamous cell carcinoma (SCC) is mandatory. β-Catenin and E-cadherin are crucial for cancer progression through epithelial-mesenchymal transition. We analyzed the effect of the tyrosine kinase inhibitors nilotinib, dasatinib, erlotinib and gefitinib on β-catenin and E-cadherin expression in SCC with respect to human papillomavirus (HPV) status. Expression of β-catenin and E-cadherin in cell lines UMSCC 11A, UMSCC 14C and CERV196 under the influence of tyrosine kinase inhibitors were analyzed by enzyme-linked immunosorbent assay. All agents reduced β-catenin and E-cadherin expression of HPV16-negative cells. Increased E-cadherin expression was observed after treatment with gefitinib and dasatinib in HPV16-positive cells. All substances, nilotinib, dasatinib, erlotinib and gefitinib have a significant impact on β-catenin and E-cadherin expression in both HPV16-positive and HPV16-negative cells in vitro. Alterations of β-catenin and E-cadherin could provide novel insights for future targeted therapies of head and neck SCC.

The biologic behavior of squamous cervical carcinoma after neoadjuvant therapy (NAT) according to immunohistochemical expression of E-cadherin and CD44v6

The efficiency of neoadjuvant therapy in cervical carcinoma has been well demonstrated, although the cellular mechanisms of different response to this treatment have not been thoroughly investigated. The present study consists of 24 patients with cervical cancer, including 12 patients in stages IB-IIIB with first-line surgical treatment and 12 patients in stages IIB-IIIB, with neoadjuvant therapy followed by radical hysterectomy. The aim of our study was to assess the correlations between the alterations in E-cadherin and CD44v6 immunoexpression in cervical carcinoma, as a tool of evaluation the response to neoadjuvant therapy and its prognostic significance. The intensity of CD44v6 immunoexpression was higher in more aggressive tumors and E-cadherin immunoexpression was approximately constant among the cases with neoadjuvant therapy. Our results demonstrate that the evaluation of CD44v6 immunoexpression in cervical carcinomas is useful for the assessment of tumor response to neoadjuvant therapy and of tumor aggressiveness. The high level of E-cadherin immunoexpression in tumors with neoadjuvant therapy reflects its involvement in the prevention of HPV oncoproteins action, with benefits on the outcome. A larger group of patients and a panel of antibodies, including CD4 and COX2, could provide a better characterization of the tumor response to neoadjuvant therapy, with a positive prognosis impact.

Iron Overload Causes Alterations of E-Cadherin in the Liver

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Allosteric Regulation of E-Cadherin Adhesion

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

Effects of non-protein-type amino acids of fine particulate matter on E-cadherin and inflammatory responses in mice

Exposure to particulate matter less than 2.5μm (PM2.5) in size is an urgent issue for the protection of human health. Chemicals with PM2.5 collected during a period of intensive haze episodes in Beijing (BJ), Xian (XA) and Hong Kong (HK) were characterised for organic carbon (OC), elemental carbon (EC), total carbon (TC) and free amino acids. BALB/c mice underwent aspiration exposure of 50 or 150μg of PM2.5/mouse (BJ, XA and HK) on days 1 and 7 and were euthanised on day 14. The effects of these exposures on E-cadherin and inflammatory responses in the mouse lungs were analysed. The PM2.5 chemicals consisted of significant amounts of OC: 36.6±17.2μg/m3 for BJ, 38.8±3.8μg/m3 for XA and 7.2±1.4μg/m3 for HK. A total of 23 free amino compounds for the PM2.5 samples were analysed: 4075±1578pmol/m3 for BJ, 4718±2190pmol/m3 for XA and 1145±213pmol/m3 for HK. Exposure to PM2.5 resulted in the suppression of E-cadherin levels in the lung tissues and increased IFN-γ, IL-2, IL-4, IL-6 and IL-10 in the bronchoalveolar lavage fluid. The alterations in E-cadherin, IFN-γ, IL-6 and IL-10 were associated with OC, TC and some amino acids, particularly non-protein-type amino acids. These data emphasised the deleterious health effects of PM2.5.

Multi-transcript profiling in archival diagnostic prostate cancer needle biopsies to evaluate biomarkers in non-surgically treated men.

BackgroundMost biomarkers in prostate cancer have only been evaluated in surgical cohorts. The value of these biomarkers in a different therapy context remains unclear. Our objective was to test a panel of surgical biomarkers for prognostic value in men treated by external beam radiotherapy (EBRT) and primary androgen deprivation therapy (PADT).MethodsThe Fluidigm® PCR array was used for multi-transcript profiling of laser microdissected tumours from archival formalin-fixed diagnostic biopsies of patients treated by EBRT or PADT. Cases were matched for disease characteristics and had known 5 year biochemical relapse outcomes (n = 60). Results were validated by immunohistochemistry in a custom needle biopsy tissue microarray. Six biomarkers previously tested only in surgical cohorts were analysed (PTEN, E-Cadherin, EGFR, EZH2, PSMA, MSMB). Transcript and protein expression was correlated with clinical outcome analysed using Kruskal Wallis, Fisher’s test and Cox proportional hazard model.ResultsAltered expression of E-Cadherin (p = 0.008) was associated with early relapse after EBRT. In PADT treated men however only altered MSMB transcript was prognostic for early relapse (p = 0.001). The remaining biomarkers however did not demonstrate prognostic ability in either cohort. In a separate tissue array we validated altered E-Cadherin protein as a predictor of early relapse after EBRT (n = 47) (HR 0.34, CI p = 0.02) but not in PADT treated men (n = 63).ConclusionWe demonstrate proof of principle of multiple transcript profiling in archival diagnostic biopsies of non-surgically treated men for biomarker discovery. We identify a role for E-Cadherin as a novel biomarker of early relapse following EBRT.

Contribution of cyclin D1 (CCND1) and E-cadherin (CDH1) alterations to colorectal cancer susceptibility: a case-control study.

Cyclin D1 (CCND1) and E-cadherin (CDH1) are two important genes of the β-catenin/LEF pathway that is involved in tumorigenesis of various cancers including colorectal cancer (CRC). However, studies of the association between genetic variants of these two genes and CRC have shown conflicting results. We conducted a genetic association study in South Indian population (cases, 103; controls, 107) to assess the association of CCND1 870G/A and CDH1 -160C/A single nucleotide polymorphisms (SNPs) with CRC risk. Genotyping of SNPs was performed by PCR sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand the role of CCND1 and CDH1 in the pathophysiology of CRC, the expression pattern was evaluated in analogous tumor and adjacent normal tissues from 23 CRC patients by Western blot analysis. The frequencies of CCND1 870A/A (P = 0.045) genotype, CDH1 -160A allele (P = 0.042), and 870A/-160A haplotype (P = 0.002) were significantly higher in patients as compared with controls. Strong LD was observed between 870G/A and -160C/A SNPs in cases (D' = 0.76) as compared to controls (D' = 0.32). Furthermore, elevated CCND1 and diminished CDH1 expression was observed in tumor tissue as compared with analogous normal tissue of CRC patients. Interestingly, advanced-stage tumors showed wider expression alterations than in early-stage tumors. In conclusion, CCND1 870G/A and CDH1 -160C/A SNPs may modify the risk of CRC susceptibility in South Indian population. In addition, elevated CCND1 and diminished CDH1 expression appears to be useful prognostic markers for CRC.

Brain metastases from lung cancer show increased expression of DVL1, DVL3 and beta-catenin and down-regulation of E-cadherin.

The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p = 0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p = 0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain.

Frameshift mutations of cadherin genes DCHS2, CDH10 and CDH24 genes in gastric and colorectal cancers with high microsatellite instability.

Cadherins (CDHs) are important in maintenance of cell adhesion and polarity, alterations of which contribute to tumorigenesis. Alterations of E-cadherin, a prototype CDH, have been reported in many cancers. However, alterations of unconventional CDHs, including CDH10, CDH24 and DCHS2 are largely unknown in cancers. Aim of this study was to explore whether CDH10, CDH24 and DCHS2 genes are mutated in gastric (GC) and colorectal cancers (CRC). In a public database, we found that CDH10, CDH24 and DCHS2 genes had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the mutations in 89 GC and 131 CRC (high MSI (MSI-H) or stable MSI/low MSI (MSS/MSI-L)) by single-strand conformation polymorphism analysis and DNA sequencing. We found six DCHS2, one CDH10 and one CDH24 frameshift mutations in them. All of the mutations were detected in cancers with MSI-H and there was a statistical difference in the frameshift mutation frequencies between the cancers with MSI-H (8/105) and MSS/MSI-L (0/115). The DCHS2 frameshift mutations were found in 8.8% and 4.2% of GC and CRC with MSI-H respectively. Our results show that unconventional CDH10, CDH24 and DCHS2 genes harbored frameshift mutations. These mutations might inactivate the cell adhesion-related functions and could be a feature of GC and CRC with MSI-H.

Cell fusion mediates dramatic alterations in the actin cytoskeleton, focal adhesions, and E‐cadherin in trophoblastic cells

The syncytiotrophoblast of the human placenta is a unique epithelia structure with millions of nuclei sharing a common cytoplasm. The syncytiotrophoblast forms by cell-cell fusion of cytotrophoblasts (CTB), the mononuclear precursor cells. The trophoblastic BeWo cell line has been used as a surrogate for CTB since they can be induced to fuse, and subsequently display numerous syncytiotrophoblast differentiation markers following syncytial formation. In this study, we have focused on alterations in the cell-adhesion molecule E-cadherin, actin cytoskeleton, and focal adhesions following BeWo cell fusion, since these entities may be interrelated. There was a dramatic reorganization of the distribution of E-cadherin as well as a reduction in the amount of E-cadherin following cell fusion. Reorganization of the actin cytoskeleton was also observed, which was associated with a change in the globular actin (G-actin)/filamentous actin (F-actin) ratio. Concomitantly, the morphology of focal adhesions was altered, but this occurred without a corresponding change in the levels of focal adhesion marker proteins. Thus, extensive remodeling of the actin cytoskeleton and focal adhesions accompanies cell fusion and differentiation and appears related to alterations in E-cadherin in trophoblastic cells.

Increased expression of S100A6 promotes cell proliferation and migration in human hepatocellular carcinoma

High levels of S100A6 have been associated with poor outcome in some types of human cancers, but the role of S100A6 in the molecular pathogenesis of these cancers is largely unknown. This study was performed to explore the expression and functional roles of S100A6 in hepatocellular carcinoma (HCC). The expression level of S100A6 in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry analysis. The potential functions of S100A6 in tumorigenesis and metastasis were analyzed by cell proliferation, migration, and invasion assays in human liver cancer cells. Moreover, through expression and purification of S100A6 recombinant protein tagged with cell-penetrating peptide, we analyzed its complex extracellular/intracellular effects in a S100A6-silenced cellular model. As a result, the expression of S100A6 was up-regulated in human HCC compared with adjacent peritumoral tissues. S100A6 silencing inhibited the growth and motility of HCC cells, while intracellular re-expression of S100A6 could rescue the proliferation and migration defects. Intracellular over-expression of S100A6 resulted in down-regulation of E-cadherin expression and promoted nuclear accumulation of β-catenin. Moreover, we found that the enhanced cell proliferation and motility after S100A6 stimulation were dependent on the activation of PI3K/AKT pathway. These results suggest that S100A6 may be involved in promotion and progression of human liver cancer. S100A6 is overexpressed in human hepatocellular carcinoma clinical specimens. S100A6 promotes proliferation and migration of human hepatoma cells. Overexpression of S100A6 results in alteration of E-cadherin and β-catenin. The multi-effects of S100A6 may be mediated in part by PI3K/AKT pathway activation.