In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFβ1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFβ1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

A challenge to mimicking nature's "bottom up" approach to generate tissue is the coordination of cellular self-assembly and emergent phenotype. Here we create a biosynthetic platform to mimic native cell-cell interactions and to drive emergent tissue behavior by human multipotent cells from the periosteal niche, i.e. PDCs, whose regenerative capacity is equal or greater to those from the bone marrow niche. Western blots showed that human PDCs express proteins for both N-cadherin, a hallmark of mesenchymal condensation, as well as for ZO-1, a tight junction membrane protein conferring epithelial barrier membrane properties. Hence, we functionalized a solid supported lipid bilayer (SLB) membrane with recombinant N-cadherin and investigated the short term phenotype of PDCs seeded on unfunctionalized and N-cadherin functionalized SLBs compared to that of PDCs seeded on glass coverslips. After 24 h, SLB functionalization promoted aggregation of PDCs seeded at high density (35,000 cells/cm(2)), with no significant concomitant changes in transcription of N-cadherin (CDH2) as measured by rtPCR. In contrast, cells seeded on unfunctionalized SLBs remained non-adherent but showed a significant upregulation in transcription of N-cadherin. Furthermore, culture of PDCs at high density on N-cadherin functionalized SLBs was negatively correlated with expression of ZO-1, while culture on unfunctionalized SLBs was positively correlated with the expression of the tight junction membrane protein. High density seeding on N-cadherin functionalized and unfunctionalized SLBs places PDCs in distinct cellular contexts and relates to emergent behavior typical for mesenchymal condensation. These studies demonstrate a biosynthetic in vitro cell culture platform to elucidate and guide emergent tissue architectures by PDCs.

BACKGROUND

N-Cadherin (CDH2) is a calcium-dependent adhesion protein, whose de novo expression, re-expression, up-regulation and down-regulation in human tumors has been demonstrated. The aim of the present work was to define the prognostic role of N-Cadherin in a large series of oral squamous cell carcinomas (OSCCs).

METHODS

A total of 94 selected OSCCs were quantitatively and qualitatively analyzed by immunohistochemistry for N-Cadherin. The association between protein expression and clinico-pathological parameters was assessed by statistical analysis.

RESULTS

In neoplastic tissue, N-Cadherin levels were more evident than in normal peritumoral epithelium (p<0.05). Protein staining was mainly detected in the neoplastic cells, and only focal nuclear positivity was observed. Expression of cytoplasmic N-Cadherin correlated significantly with poor histological differentiation (p<0.05). Furthermore, we have observed significant a statistical trend for stage and a correlation with worst patient outcome, also confirmed by Kaplan-Meier estimates.

CONCLUSIONS

Our work has underlined the key-role of N-Cadherin in oral carcinogenesis and in the prognostic stratification of patients.

The etiology of chronic periodontitis clearly includes a heritable component. Our purpose was to perform a small exploratory genome-wide association study in adults ages 18-49 years to nominate genes associated with periodontal disease-related phenotypes for future consideration. Full-mouth periodontal pocket depth probing was performed on participants (N = 673), with affected status defined as two or more sextants with probing depths of 5.5 mm or greater. Two variations of this phenotype that differed in how missing teeth were treated were used in analysis. More than 1.2 million genetic markers across the genome were genotyped or imputed and tested for genetic association. We identified ten suggestive loci (p-value ≤ 1E-5), including genes/loci that have been previously implicated in chronic periodontitis: LAMA2, HAS2, CDH2, ESR1, and the genomic region on chromosome 14q21-22 between SOS2 and NIN. Moreover, we nominated novel loci not previously implicated in chronic periodontitis or related pathways, including the regions 3p22 near OSBPL10 (a lipid receptor implicated in hyperlipidemia), 4p15 near HSP90AB2P (a heat shock pseudogene), 11p15 near GVINP1 (a GTPase pseudogene), 14q31 near SEL1L (an intracellular transporter), and 18q12 in FHOD3 (an actin cytoskeleton regulator). Replication of these results in additional samples is needed. This is one of the first research efforts to identify genetic polymorphisms associated with chronic periodontitis-related phenotypes by the genome-wide association study approach. Though small, efforts such this are needed in order to nominate novel genes and generate new hypotheses for exploration and testing in future studies.

Breast cancer, one of the leading causes of mortality and morbidity among females, is regulated in part by diverse classes of adhesion molecules one of which is known as cadherins. Located at adherens junctions, the members of this superfamily are responsible for upholding proper cell-cell adhesion. Cadherins possess diverse structures and functions and any alteration in their structures or functions causes impeding of normal mammary cells development and maintenance, thus leading to breast malignancy. E-, N-, P-, VE-, Proto-, desmosomal and FAT cadherins have been found to regulate breast cancer in positive as well as negative fashion, whereby both Ecadherin (CDH1) and N-cadherin (CDH2) contribute significantly towards transitioning from epithelial state to mesenchymal state (EMT) and enacting the abnormal cells to invade and metastasize nearby and distant tissues. Aberration in gene expression of cadherins can be either due to somatic or epigenetic silencing or via transcriptional factors. Besides other cadherins, E-cadherin which serves as hallmark of EMT is associated with several regulatory factors such as Snail, Slug, Twist, Zeb, KLF4, NFI, TBX2, SIX, b-Myb, COX-2, Arf6, FOXA2, GATA3 and SMAR1, which modulate E-cadherin gene transcription to promote or represses tumor invasion and colonization. Signaling molecules such as Notch, TGF-β, estrogen receptors, EGF and Wnt initiate numerous signaling cascades via these vital factors of cell programming, controlling expression of E-cadherin at transcriptional (mRNA) and protein level. Thus, interactions of cadherins with their roles in tumor suppression and oncogenic transformation can be beneficial in providing valuable insights for breast cancer diagnosis and therapeutics development.

Epithelial-mesenchymal transition (EMT) is associated with tumor malignancy. The hedgehog-EMT pathway is preferentially activated in diffuse-type gastric cancer (GC) compared with intestinal-type GC; however, histological typing is currently the only method for distinguishing these two major types of GC. We compared the gene expression profiles of 12 bone marrow-derived mesenchymal stem cell cultures and 5 diffuse-type GC tissue samples. Numerous upregulated or downregulated genes were identified in diffuse-type GC, including CDH1, CDH2, VIM, WNT4 and WNT5. Among these genes, the mRNA ratio of CDH2 to CDH1 could distinguish the 15 diffuse-type GC samples from the 17 intestinal-type GC samples. Our results suggested that the mesenchymal features were more prominent in diffuse-type GC than in intestinal-type GC, but were weaker in diffuse-type GC than in mesenchymal stem cells. Diffuse-type GC that has undergone extensive EMT, which has a poor prognosis, can be identified by quantitative PCR analysis of only two genes.

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. Although cadherins have been implicated in the development of the brain, including the visual system, in several vertebrate species, little is known of their role in zebrafish. In this study, we examined distribution of cadherin-2 (Cdh2, N-cadherin) in the visual system of developing and adult zebrafish using both immunocytochemical and in situ hybridization methods, and we compared Cdh2 distribution to that of the previously reported and closely related cadherin-4 (Cdh4, R-cadherin). As in other vertebrates, in zebrafish embryos Cdh2 was widely expressed in the early nervous system, but its expression became more restricted as development proceeded. Cdh4 was not detectable until later in development, at about the time when the first ganglion cells are generated. Cdh2 and Cdh4 were expressed in distinct regions of developing visual structures, including the lens. We hypothesize that the differential expression of these two cadherins in developing zebrafish visual structures reflects functionally different roles in the development of the vertebrate visual system.

Epithelial-mesenchymal transition (EMT) is involved in the characteristics of malignancy, such as invasion, metastasis, and chemoresistance. In biliary tract cancer (BTC), EMT is induced by transforming growth factor-beta 1 (TGF-β1). The EMT is reversible; therefore, it is conceivable that it could be related to some epigenetic changes. We focused on histone deacetylase (HDAC) inhibitors as regulators of TGF-β1 signaling, and investigated their effect on EMT and chemoresistance. We employed four BTC cell lines (MzChA-1, gemcitabine-resistant MzChA-1, TFK-1, and gemcitabine-resistant TFK-1) and used vorinostat as the HDAC inhibitor. The relative mRNA expression of an epithelial marker (CDH1) and mesenchymal markers (CDH2, vimentin, SNAI1) were measured by qRT-PCR to evaluate factors associated with EMT. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the chemoresistance of each cell line. In addition, NOD/SCID mice were used to evaluate the effect of vorinostat in vivo. In the parent MzChA-1 and TFK-1 cell lines, TGF-β1 induced EMT and chemoresistance; while vorinostat inhibited the EMT and chemoresistance induced by TGF-β1. In gemcitabine-resistant cell lines that highly expressed TGF-β1, vorinostat inhibited EMT and attenuated chemoresistance. We showed that vorinostat inhibits nuclear translocation of SMAD4 which is a signaling factor of TGF-β1, and this is one of the mechanisms by which vorinostat regulates EMT. We also showed that vorinostat attenuates the binding affinity of SMAD4 to the CDH1-related transcription factors SNAI1, SNAI2, ZEB1, ZEB2, and TWIST. Furthermore, combination therapy with vorinostat and gemcitabine improved survival time in the mice xenografted with gemcitabine resistant MzChA-1 cells. In conclusion, vorinostat regulated TGF-β1-induced EMT and chemoresistance through inhibition of SMAD4 nuclear translocation.

BACKGROUND

Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw.

METHODS

EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers.

RESULTS

We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining.

CONCLUSIONS

The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

OBJECTIVE

Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation.

METHODS

Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected.

RESULTS

Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively.

CONCLUSIONS

We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.

The recent finding that the neuronal cadherin gene CDH2 confers a highly significant risk for canine compulsive disorder led us to investigate whether missense variants within the human ortholog CDH2 are associated with altered susceptibility to obsessive-compulsive disorder (OCD), Tourette disorder (TD) and related disorders. Exon resequencing of CDH2 in 320 individuals identified four non-synonymous single-nucleotide variants, which were subsequently genotyped in OCD probands, Tourette disorder probands and relatives, and healthy controls (total N=1161). None of the four variants was significantly associated with either OCD or TD. One variant, N706S, was found only in the OCD/TD groups, but not in controls. By examining clinical data, we found there were significant TD-related phenotype differences between those OCD probands with and without the N845S variant with regard to the co-occurrence of TD (Fisher's exact test P=0.014, OR=6.03). Both N706S and N845S variants conferred reduced CDH2 protein expression in transfected cells. Although our data provide no overall support for association of CDH2 rare variants in these disorders considered as single entities, the clinical features and severity of probands carrying the uncommon non-synonymous variants suggest that CDH2, along with other cadherin and cell adhesion genes, is an interesting gene to pursue as a plausible contributor to OCD, TD and related disorders with repetitive behaviors, including autism spectrum disorders.

Tumor cells must overcome apoptosis to survive throughout metastatic dissemination and distal organ colonization. Here, we show in the Polyoma Middle T mammary tumor model that N-cadherin (Cdh2) expression causes Slug (Snai2) upregulation, which in turn promotes carcinoma cell survival. Slug was dramatically upregulated in metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in carcinoma cells suppressed lung colonization by decreasing cell survival at metastatic sites, but had no effect on tumor cell invasion or extravasation. In support of this idea, Slug inhibition by shRNA sensitized tumor cells to apoptosis by DNA damage, resulting in caspase-3 and PARP cleavage. The prosurvival effect of Slug was found to be caused by direct repression of the proapoptotic gene, Puma (Bbc3), by Slug. Consistent with a pivotal role for a Slug-Puma axis in metastasis, inhibition of Puma by RNA interference in Slug-knockdown cells rescued lung colonization, whereas Puma overexpression in control tumor cells suppressed lung metastasis. The survival function of the Slug-Puma axis was confirmed in human breast cancer cells, where Slug knockdown increased Puma expression and inhibited lung colonization. This study demonstrates a pivotal role for Slug in carcinoma cell survival, implying that disruption of the Slug-Puma axis may impinge on the survival of metastatic cells.

BACKGROUND

Radiotherapy plays a critical role in the management of non-small cell lung cancer (NSCLC). This study was conducted to identify gene expression profiles of acquired radioresistant NSCLC cell line established by fractionated ionizing radiation (FIR) by cDNA microarray.

METHODS

The human lung adenocarcinoma cell line Anip973 was treated with high energy X-ray to receive 60 Gy in 4 Gy fractions. The radiosensitivity of Anip973R and its parental line were measured by clonogenic assay. Gene expression profiles of Anip973R and its parental line were analyzed using cDNA microarray consisting of 21 522 human genes. Identified partly different expressive genes were validated by quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR).

RESULTS

Fifty-nine upregulated and 43 downregulated genes were identified to radio-resistant Anip973R. Up-regulated genes were associated with DNA damage repair (DDB2), extracellular matrix (LOX), cell adhesion (CDH2), and apoptosis (CRYAB). Down-regulated genes were associated with angiogenesis (GBP-1), immune response (CD83), and calcium signaling pathway (TNNC1). Subsequent validation of selected eleven genes (CD24, DDB2, IGFBP3, LOX, CDH2, CRYAB, PROCR, ANXA1 DCN, GBP-1 and CD83) by Q-RT-PCR was consistent with microarray analysis.

CONCLUSIONS

Fractionated ionizing radiation can lead to the development of radiation resistance. Altered gene profiles of radioresistant cell line may provide new insights into mechanisms underlying clinical radioresistance for NSCLC.

Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. CDH2 (N-cadherin, a mesenchymal marker of the epithelial-mesenchymal transition) and ADAM9 (a type I transmembrane protein) are related to lung cancer brain metastasis; however, it is unclear how they interact to mediate this metastasis. Because microRNAs regulate many biological functions and disease processes (e.g., cancer) by down-regulating their target genes, microRNA microarrays were used to identify ADAM9-regulated miRNAs that target CDH2 in aggressive lung cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of miR-218. MiR-218 was generated from pri-mir-218-1, which is located in SLIT2, in non-invasive lung adenocarcinoma cells, whereas its expression was inhibited in aggressive lung adenocarcinoma. The down-regulation of ADAM9 up-regulated SLIT2 and miR-218, thus down-regulating CDH2 expression. This study revealed that ADAM9 activates CDH2 through the release of miR-218 inhibition on CDH2 in lung adenocarcinoma.

Direct cell-to-cell interactions via cell adhesion molecules, in particular cadherins, are critical for morphogenesis, tissue architecture, and cell sorting and differentiation. Partially overlapping, yet distinct roles of N-cadherin (cadherin-2) and cadherin-11 in the skeletal system have emerged from mouse genetics and in vitro studies. Both cadherins are important for precursor commitment to the osteogenic lineage, and genetic ablation of Cdh2 and Cdh11 results in skeletal growth defects and impaired bone formation. While Cdh11 defines the osteogenic lineage, persistence of Cdh2 in osteoblasts in vivo actually inhibits their terminal differentiation and impairs bone formation. The action of cadherins involves both cell-cell adhesion and interference with intracellular signaling, and in particular the Wnt/β-catenin pathway. Both cadherin-2 and cadherin-11 bind to β-catenin, thus modulating its cytoplasmic pools and transcriptional activity. Recent data demonstrate that cadherin-2 also interferes with Lrp5/6 signaling by sequestering these receptors in inactive pools via axin binding. These data extend the biologic action of cadherins in bone forming cells, and provide novel mechanisms for development of therapeutic strategies aimed at enhancing bone formation.

Gemcitabine is one of the most widely used drugs for the treatment of advanced Non-small cell lung cancer (NSCLC), but modest objective response rate of patients to gemcitabine makes it necessary to identify novel biomarkers for patients who can benefit from gemcitabine-based therapy and to improve the effect of clinical therapy. In this work, 3 NSCLC cell lines displaying different sensitivities to gemcitabine were applied for mRNA and microRNA (miR) expression chips to figure out the biomarkers for gemcitabine sensitivity. Genes whose expression increased dramatically in sensitive cell lines were mainly enriched in cell adhesion (NRP2, CXCR3, CDK5R1, IL32 and CDH2) and secretory granule (SLC11A1, GP5, CD36 and IGF1), while genes with significantly upregulated expression in resistant cell line were mainly clustered in methylation modification (HIST1H2BF, RAB23 and TP53) and oxidoreductase (TP53I3, CYP27B1 and SOD3). The most intriguing is the activation of Wnt/β-catenin signaling in gemcitabine resistant NSCLC cell lines. The miR-155, miR-10a, miR-30a, miR-24-2* and miR-30c-2* were upregulated in sensitive cell lines, while expression of miR-200c, miR-203, miR-885-5p, miR-195 and miR-25* was increased in resistant cell line. Genes with significantly altered expression and putatively mediated by the expression-changed miRs were mainly enriched in chromatin assembly (MAF, HLF, BCL2, and IGSF3), anti-apoptosis (BCL2, IGF1 and IKBKB), protein kinase (NRP2, PAK7 and CDK5R1) (all the above genes were upregulated in sensitive cells) and small GTPase mediated signal transduction (GNA13, RAP2A, ARHGAP5 and RAB23, down-regulated in sensitive cells). Our results might provide potential biomarkers for gemcitabine sensitivity prediction and putative targets to overcome gemcitabine resistance in NSCLC patients.

Epithelial ovarian carcinoma spreads via shedding of cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent intraperitoneal tumor cell:mesothelial cell adhesion as a key early event in metastatic seeding. Evaluation of human tumor extracts and tissues confirms that well-differentiated ovarian tumors express abundant E-cadherin (Ecad), whereas advanced lesions exhibit upregulated N-cadherin (Ncad). Two expression patterns are observed: "mixed cadherin," in which distinct cells within the same tumor express either E- or Ncad, and "hybrid cadherin," wherein single tumor cell(s) simultaneously expresses both cadherins. We demonstrate striking cadherin-dependent differences in cell-cell interactions, MCA formation, and aggregate ultrastructure. Mesenchymal-type Ncad+ cells formed stable, highly cohesive solid spheroids, whereas Ecad+ epithelial-type cells generated loosely adhesive cell clusters covered by uniform microvilli. Generation of "mixed cadherin" MCAs using fluorescently tagged cell populations revealed preferential sorting into cadherin-dependent clusters, whereas mixing of cell lines with common cadherin profiles generated homogeneous aggregates. Recapitulation of the "hybrid cadherin" Ecad+/Ncad+ phenotype, via insertion of the CDH2 gene into Ecad+ cells, resulted in the ability to form heterogeneous clusters with Ncad+ cells, significantly enhanced adhesion to organotypic mesomimetic cultures and peritoneal explants, and increased both migration and matrix invasion. Alternatively, insertion of CDH1 gene into Ncad+ cells greatly reduced cell-to-collagen, cell-to-mesothelium, and cell-to-peritoneum adhesion. Acquisition of the hybrid cadherin phenotype resulted in altered MCA surface morphology with increased surface projections and increased cell proliferation. Overall, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects ultimate metastatic success.

OBJECTIVE

Gastric cancer (GC) is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcrip- tion-3 (STAT3) signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell (ESCs) pluripotency. Here, we have investi- gated the activation status of STAT3 in GC stem-like cells (GCSLCs).

METHODS

In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, character- ized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation.

RESULTS

Spheroid cells showed higher potential for spheroid formation than the pa- rental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition (EMT) related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated (P<0.05), but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel (DTX) when compared with parental cells (P<0.05) according to the MTS assay. Al- though immunostaining and Western blotting showed expression of the STAT3 pro- tein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 (p-STAT3) in spheroid structures relative to the parent cells accord- ing to flow cytometry analysis (P<0.05).

CONCLUSIONS

The present findings point to STAT3 over activation in GCSLCs. Com- plementary experiments are required to extend the role of STAT3 in stemness fea- tures and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy.

Colon cancer (CC) likes many epithelial-derived cancers, resulting from a complex tumorigenic process. However, the exactly mechanisms of development and progression of CC are still unknown. In this study, integrated analysis in the GSE33113 and Fudan University Shanghai Cancer Center Hospital datasets revealed that WISP1 expression was significantly increased in CC cases, positivity correlated with the advanced pathologic stage and a poor prognosis was more likely in CC patients with higher levels of WISP1. Downregulation of WISP1 inhibited cell proliferation and invasion through increasing apoptosis and blocking cell cycle at G1 phase in CC LOVO and RKO cells. Besides, Gene set enrichment analysis (GSEA) revealed that relative genes involved in the Cell adhesion molecules and Cytokine-cytokine receptor interaction pathways were enriched in WISP1-higher expression patients. Western blot analysis showed that Cell adhesion molecules pathway associated genes (ICAM- 1, VCAM-1, SDC2 and CDH2) and Cytokine-cytokine receptor interaction pathway associated genes (VEGFC, CCL18, CXCR4 and TGFBR1) were also modulated by WISP1 downregulation. Then, we found that the protein β-catenin was identified as a binding partner of WISP1 and mediated the functions of WISP1 through promoting cell proliferation and invasion in LOVO and RKO cells. Further in vivo tumor formation study in nude mice indicated that inhibition of WISP1 delayed the progress of tumor formation and inhibited PCNA expression. These results indicate that WISP1 could act as an oncogene and may serve as a promising therapeutic strategy for colon cancer.

Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum-free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die-offs even after long-term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT-PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time-dependent manner. Without any neural induction, spheroid-derived cells spontaneously converted into neuron-like cells with positive staining of neural markers HuC/D and P75 under the serum-free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration.

Appropriate localization of neurons within the brain is a crucial component of the establishment of neural circuitry. In the zebrafish hindbrain, the facial branchiomotor neurons (FBMNs) undergo a chain-like tangential migration from their birthplace in rhombomere (r) 4 to their final destination in r6/r7. Here, we report that ablation of either the cell body or the trailing axon of the leading FBMN, or 'pioneer' neuron, blocks the migration of follower FBMNs into r5. This demonstrates that the pioneer neuron and its axon are crucial to the early migration of FBMNs. Later migration from r5 to r6 is not dependent on pioneer neurons but on the medial longitudinal fasciculus (MLF), a bundle of axons lying ventral to the FBMNs. We find that MLF axons enter r5 only after the pioneer neuron has led several followers into this region; the MLF is then contacted by projections from the FBMNs. The interactions between FBMNs and the MLF are important for migration from r5 to r6, as blocking MLF axons from entering the hindbrain can stall FBMN migration in r5. Finally, we have found that the adhesion molecule Cdh2 (N-cadherin) is important for interactions between the MLF and FBMNs, as well as for interactions between the trailing axon of the pioneer neuron and follower FBMNs. Interestingly, migration of pioneer neurons is independent of both the MLF and Cdh2, suggesting pioneer migration relies on independent cues.

Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO(2) reduction and cleavage/synthesis of acetyl-CoA. Methanosarcina acetivorans C2A contains two very similar copies of a six-gene operon (cdh genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in M. acetivorans, mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of cdh disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are bona fide CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of cdh1, suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.

TGFBR1*6A is a common hypomorphic variant of the type I transforming growth factor (TGF)-beta receptor (TGFBR1), which transduces TGF-beta growth inhibitory signals less effectively than TGFBR1. Recent studies suggest that TGFBR1*6A confers a selective growth advantage to both normal appearing and cancerous epithelial cells in the presence of TGF-beta. We have previously shown that TGFBR1*6A is somatically acquired in head and neck and colon cancer (10). Using microdissected tissues, we show that TGFBR1*6A is somatically acquired by stromal and epithelial cells adjacent to colorectal and head and neck tumors. Somatic acquisition of the TGFBR1*6A allele is not accompanied by acquisition of other tumor-specific mutations. Furthermore, lymphocytes located within the stroma or the normal appearing epithelium do not have evidence of TGFBR1*6A acquisition. The highest TGFBR1*6A/TGFBR1 allelic ratio is observed at the tumor's edge, and traces of TGFBR1*6A are detected as far as 2 cm away from the tumor, which is suggestive of centrifugal spread of cells that harbor TGFBR1*6A. Assessment of CDH1 and CDH2 expression does not indicate epithelial-mesenchymal transformation. The results suggest that TGFBR1*6A somatic acquisition is a critical event in the early stages of cancer development that is associated with field cancerization. They also represent the first human report of somatically acquired altered stromal TGF-beta signaling during oncogenesis and the first report of a concordant mutation in the stromal and epithelial compartments in colon cancer.