E-cadherin Signaling Pathways Research Articles

Moscatilin Reverses EMT Progression and its Resulting Enhanced Invasion and Migration by Affecting the TGF-β Signaling Pathway in Bladder Cancer.

Bladder cancer metastasis is an essential process in the progression of muscle-invasive bladder cancer. EMT plays a crucial role in facilitating the spread of cancer cells. Identifying compounds that can inhibit these abilities of cancer cells is a significant international endeavor. To explore the migration and invasion effect of Moscatilin on the bladder and clarify the mechanism of action Methods: The anti-bladder cancer effect of Moscatilin was observed by a cell proliferation experiment. The migration and invasion of bladder cancer cells inhibited by Moscatilin were detected by Transwell and Wound healing. The effects of Moscatilin on EMT-related proteins E-cadherin, N-cadherin, Snail1, Vimentin, and TGF-β signaling pathways were detected by Western blot, and nucleic acid levels were verified by qPCR. Our study revealed that Moscatilin reduced the viability of bladder cancer cells in vitro and impeded their migration and invasion in experimental settings. Furthermore, we observed that Moscatilin decreased the activation levels of active proteins, specifically Smad3, Samd2, and MMP2. Additionally, we found that moscatilin significantly reduced the expression level of TGF-β and was also capable of reversing the overexpression effect of TGF-β. Treatment with Moscatilin also led to significant inhibition of interstitial cell markers Ncadherin and Snail1, which are associated with EMT. These findings indicate that Moscatilin impedes the migration and invasion of bladder cancer cells by influencing cell survival, modulating TGF-β/Smad signaling, and inhibiting EMT.

A rare case of TFEB/6p21/VEGFA-amplified renal cell carcinoma diagnosed by whole-exome sequencing: clinicopathological and genetic feature report and literature review

BackgroundTFEB/6p21/VEGFA-amplified renal cell carcinoma (RCC) is rare and difficult to diagnose, with diverse histological patterns and immunohistochemical and poorly defined molecular genetic characteristics.Case presentationWe report a case of a 63-year-old male admitted in 2017 with complex histomorphology, three morphological features of clear cell, eosinophilic and papillary RCC and resembling areas of glomerular and tubular formation. The immunophenotype also showed a mixture of CD10 and P504s. RCC with a high suspicion of collision tumors was indicated according to the 2014 WHO classification system; no precise diagnosis was possible. The patient was diagnosed at a different hospital with poorly differentiated lung squamous cell carcinoma one year after RCC surgery. We exploited molecular technology advances to retrospectively investigate the patient’s molecular genetic alterations by whole-exome sequencing. The results revealed a 6p21 amplification in VEGFA and TFEB gene acquisition absent in other RCC subtypes. Clear cell, papillary, chromophobe, TFE3-translocation, eosinophilic solid and cystic RCC were excluded. Strong TFEB and Melan-A protein positivity prompted rediagnosis as TFEB/6p21/VEGFA-amplified RCC as per 2022 WHO classification. TMB-L (low tumor mutational load), CCND3 gene acquisition and MRE11A and ATM gene deletion mutations indicated sensitivity to PD-1/PD-L1 inhibitor combinations and the FDA-approved targeted agents Niraparib (Grade C), Olaparib (Grade C), Rucaparib (Grade C) and Talazoparib (Class C). GO (Gene Ontology) and KEGG enrichment analyses revealed major mutations and abnormal CNVs in genes involved in biological processes such as the TGF-β, Hippo, E-cadherin, lysosomal biogenesis and autophagy signaling pathways, biofilm synthesis cell adhesion substance metabolism regulation and others. We compared TFEB/6p21/VEGFA-amplified with TFEB-translocated RCC; significant differences in disease onset age, histological patterns, pathological stages, clinical prognoses, and genetic characteristics were revealed.ConclusionWe clarified the patient’s challenging diagnosis and discussed the clinicopathology, immunophenotype, differential diagnosis, and molecular genetic information regarding TFEB/6p21/VEGFA-amplified RCC via exome analysis and a literature review.

E-cadherin and NF-κB expression in the vagina after ovarian ischemia and reperfusion.

To investigate inflammation and cell adhesion molecules in the vagina after ovarian ischemia-reperfusion (IR) injury. 20 Wistar albino female rats were divided into two groups: control, and IR groups. In IR group, blood flow was restricted for 2hours for ovarian ischemia. Then, tissues were re-blood 2hours for reperfusion. Vagina tissues were excised and processed for histopathological analysis. Histopathological and biochemical follow-ups were performed. Both malondialdehyde and myeloperoxidase values were increased in IR group compared to control group. Glutathione content was decreased in IR group compared to control group. Epithelial degeneration, inflammation, dilatation, and nuclear factor-κB (NF-κB) expression were increased in IR group compared to control group. E-cadherin expression was significantly decreased in IR group. In the IR group, E-cadherin showed a positive reaction in adenomas, gland-like cryptic structures, cellular junctions with clustered inflammatory cells. In the IR group, NF-κB expression was increased in basement membrane, inflammatory cells, in blood vessels. Ovarian ischemia caused degeneration of epithelial cells in the vaginal region and disruptions in the cell junction complex, which leads to activation of E-cadherin and NF-κB signaling pathway and alterations in reproductive and embryonal development in the vaginal region.

CXCR7 promotes the migration of fibroblasts derived from patients with acquired laryngotracheal stenosis by NF-κB signaling.

Laryngotracheal stenosis (LTS) is a life-threatening disease that commonly results in airway obstruction in children. Traditional treatments such as laryngotracheal reconstruction and balloon dilation all have the risk of laryngotracheal restenosis. It is of great importance to spare patients the morbidity of LTS and risks of restenosis associated with these treatments. Laboratory and clinical trials have focused on fibrosis, the crucial pathological process of LTS. This study was undertaken to investigate the function of CXC chemokine receptor-7 (CXCR7) in the fibroblasts derived from LTS. RNA sequencing was performed on acquired human LTS and normal trachea tissues to analyze differentially expressed genes. Fibroblasts from LTS and normal trachea tissues were isolated and cultured. CXCR7 knockdown was performed using specific small interfering RNAs (siRNAs) and activated by CXCR7 agonist VUF11207. The assessment of cell proliferation and migration was conducted using EdU proliferation, wound healing, and transwell assays. The assessment of cell proliferation and migration was conducted using EdU proliferation, wound healing, and transwell assays. The expressions of CXCR7, E-cadherin and NF-κB signaling pathway were analyzed by quantitative polymerase chain reaction (qPCR), western blotting, immunohistochemistry, and immunofluorescence. RNA sequencing showed that CXCR7 was among the most differentially expressed genes. LTS had an increased CXCR7 expression but decreased E-cadherin expression in vivo. CXCR7 agonist stimulated the migration of LTS derived fibroblasts significantly in vitro, with no significant influence on the cell proliferation and apoptosis. CXCR7 agonist inhibited the expression of E-cadherin by activating the NF-κB signaling pathway. The effects of CXCR7 on cell migration and E-cadherin expression were blocked by CXCR7 siRNA. LTS had an increased CXCR7 expression but decreased E-cadherin expression. CXCR7 activation inhibited E-cadherin expression by NF-κB signaling pathway and thereby promoted the migration of LTS derived fibroblasts.

High expression of RRM2 as an independent predictive factor of poor prognosis in patients with lung adenocarcinoma.

Ribonucleotide reductase subunit M2 may play a role as a potential prognostic biomarker in several cancers. In this study, we evaluated whether RRM2 gene expression is associated with the prognosis of patients with lung adenocarcinoma (LUAD) using publicly available data from The Cancer Genome Atlas (TCGA). Wilcoxon signed-rank test and logistic regression were performed to evaluate the association between RRM2 expression and clinical features in patients with LUAD. Kaplan-Meier and Cox regression methods were used to examine the effect of RRM2 expression level in the overall survival, and a nomogram was performed to illustrate the correlation between the RRM2 gene expression and the risk of LUAD. TCGA data set was used for gene set enrichment analysis (GSEA). We also performed a further experiment in vitro to assess the effect of RRM2 expression on the proliferation and invasive abilities of LUAD cells and its key signaling pathway proteins. Our results revealed that the expression level of RRM2 in patients with LUAD was much higher than that in normal tissues (p = 3.99e-32). High expression of RRM2 was significantly associated with tumor stage (IV vs. I: OR = 3.02, p = 0.012) and TNM classification (T2 vs. T1: OR = 1.88, p = 0.001; N2 vs. N0: OR = 2.69, p < 0.001). Kaplan-Meier survival analysis showed that high expression of RRM2 was associated with a worse prognosis of LUAD compared low expression of RRM2 (p = 7.86e-04). Multivariate analysis showed that high RRM2 expression was an independent factor affecting overall survival (HR = 1.29, p < 0.001). The association between RRM2 gene expression and the risk of LUAD was presented in a nomogram. GSEA revealed that the cell cycle, p53 signaling pathway, DNA replication, small cell lung cancer, apoptosis, and pathways in cancer were differentially enriched in patients with high expression of RRM2. RRM2 over-expression promoted the proliferation and invasive abilities of LUAD cells. RRM2 over-expression increased the activation of Bcl-2 and E-cadherin signaling pathways, and reduced the activation of p53 signaling pathway. In summary, high RRM2 expression is an independent predictive factor of poor prognosis in patients with lung adenocarcinoma.

Downregulated miR-383-5p contributes to the proliferation and migration of gastric cancer cells and is associated with poor prognosis.

AimThe study aims to identify differentially expressed microRNAs (DEMs) in gastric cancer (GC) and explore the expression, prognosis and downstream regulation role of miR-383-5p in GC.MethodsThe GC miRNA-Seq and clinical information were downloaded from Firebrowse which stores integrated data sourced from The Cancer Genome Atlas database. The DEMs were identified with limma package in R software at the cut-off criteria of P < 0.05 and |log2 fold change| > 1.0 (|log2FC| > 1.0). The expression of miR-383-5p in GC cell lines and 54 paired GC tissues was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The overall survival curve of miR-383-5p and the association between its expression and clinicopathological features were explored. Wound healing and cell counting kit-8 assays were performed to investigate the capacity of miR-383-5p in cell proliferation and migration. The downstream target genes were predicted by bioinformatics tools (miRDB, TargetScan and starBase). The consensus target genes were selected for gene functional enrichment analysis by FunRich v3.0 software. The luciferase reporter assay was performed to verify the potential targeting sites of miR-383-5p on lactate dehydrogenase A (LDHA).ResultsA total of 21 down-regulated miRNAs (including miR-383-5p) and 202 up-regulated miRNAs were identified by analyzing GC miRNA-Seq data. Survival analysis found that patients with low miR-383-5p expression had a shorter survival time (median survival time 21.1 months) than those with high expression (46.9 months). The results of qRT-PCR indicated that miR-383-5p was downregulated in GC cell lines and tissues, which was consistent with miRNA-Seq data. The expression of miR-383-5p was significantly associated with tumor size and differentiation grade. Besides, overexpression of miR-383-5p suppressed GC cells proliferation and migration. A total of 49 common target genes of miR-383-5p were obtained by bioinformatics tools and gene functional enrichment analysis showed that these predicted genes participated in PI3K, mTOR, c-MYC, TGF-beta receptor, VEGF/VEGFR and E-cadherin signaling pathways. The data showed that expression of miR-383-5p was negatively correlated with target LDHA (r = −0.203). Luciferase reporter assay suggested that LDHA was a target of miR-383-5p.ConclusionThe present study concluded that miR-383-5p was downregulated and may act as a tumor suppressor in GC. Furthermore, its target genes were involved in important signaling pathways. It could be a prognostic biomarker and play a vital role in exploring the molecular mechanism of GC.

CAPE-pNO2 Inhibited the Growth and Metastasis of Triple-Negative Breast Cancer via the EGFR/STAT3/Akt/E-Cadherin Signaling Pathway.

Overexpressed epidermal growth factor receptor (EGFR) and overactivated epithelial-mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) can enhance tumorigenesis and tumor recurrence and metastasis. Caffeic acid p-nitro-phenethyl ester (CAPE-pNO2) has various pharmacological activities in our previous research, but its effect on metastasis and growth of TNBC has not been studied. In this study, Caffeic acid phenethyl ester (CAPE) was as a positive control. in vitro, MTT, Transwell, wound healing, colony formation and cell adhesion assays were performed to examine the effect on viability, invasion, migration, colony formation and adhesion of MDA-MB-231 cells by CAPE-pNO2, the results indicated that CAPE-pNO2 significantly dose-dependently inhibited metastasis of MDA-MB-231 cells (p < 0.05). in vivo, TNBC xenograft mice were established by subcutaneously injected with MDA-MB-231 cells, and they were used to estimate the effect on metastasis and growth of CAPE-pNO2 after 38 days of treatment. HE staining and TUNEL staining were carried out in tumor tissues, results showed that CAPE-pNO2 obviously suppressed the tumor growth, induced cells apoptosis (p < 0.01) and decreased pulmonary and splenic metastatic tumor cells. The results of IHC demonstrated that the VEGFA and Ki-67 proteins expression were downregulated (p < 0.01) in tumor tissues. Furthermore, western blot analysis was used to quantify key metastasis- and growth-associated proteins expression in vitro and in vivo, the results suggested that CAPE-pNO2 downregulated the proteins expression of p-EGFR, p-STAT3, p-Akt, MMP-2, MMP-9, Survivin, and key EMT-related proteins (Vimentin and N-cadherin) (p < 0.01), and increased the expression of E-cadherin (p < 0.01) in vivo and in vitro. Besides, CAPE-pNO2 had a similar effect as erlotinib in regulating the EGFR downstream proteins in EGF-induced MDA-MB-231cells. Collectively, these results indicated that CAPE-pNO2 possessed inhibitory effect on the growth and metastasis of TNBC may via the EGFR/STAT3/Akt/E-cadherin signaling pathway, and CAPE-pNO2 is better than CAPE in inhibiting growth and metastasis.

The role of estrogen receptor α and metastasis related gene 3 in human pancreatic cancer

Objective To investigate the role of estrogen receptor α (ERα) and metastasis related gene 3 (MTA3) in epithelial-mesenchymal transition (EMT) via ERα→MTA3→Snail→E-cadherin signaling pathway and the correlation with cell apoptosis in pancreatic duct adenocarcinoma (PDAC) cells. Methods The expression of ERα MTA3 and EMT related factors Snail and E-cadherin in human PDAC tissues was detected by immunohistochemistry, and the results were further proved in human PDAC cell lines by Western blotting. Results Most of the ERα expression was detected in cytoplasm of PDAC cells. The expression of ERα had no correlation with tumor differentiation and patients’ gender. MTA3 was found in 50% of PDAC patients, Snail in 60% and E-cadherin in 80% respectively. More expression of MTA3 was found in patients without lymph node metastasis (PP: 1.39, IRS: 2.67 vs. PP: 0.81, IRS: 2.06). Snail in low-differentiated (PP: 1.61, IRS: 3.5) was found higher than low-moderate-differentiated (PP: 1.54, IRS: 2.69) and moderate-differentiated (PP: 1.40, IRS: 2.67) tissues. A significantly more expression of E-cadherin was found in better-differentiated PDAC tissues (P=0.026). The relationship of ERα→MTA3→Snail→E-cadherin signaling pathway was found in PDAC tissues, and MTA3 expression was correlated with less Snail and more E-cadherin. Further experiment in pancreatic cell lines had the same results as in human PDAC tissues. Conclusion The factors involved in ERα→MTA3→Snail→E-cadherin signaling pathway existed in pancreatic cancer tissues, and this pathway existed in a subgroup of PDAC tissues. MTA3 is an upper stream regulator of EMT, and correlated with differentiation of PADC cells. Key words: Pancreatic adenocarcinoma; Estrogen receptor α; Metastasis related gene 3; Epithelial-mesenchymal transition

SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822.

The response of cells to mechanical inputs is a key determinant of cell behavior. In response to external forces, E-cadherin initiates signal transduction cascades that allow the cell to modulate its contractility to withstand the force. Much attention has focused on identifying the E-cadherin signaling pathways that promote contractility, but the negative regulators remain undefined. In this study, we identify SHP-2 as a force-activated phosphatase that negatively regulates E-cadherin force transmission by dephosphorylating vinculin Y822. To specifically probe a role for SHP-2 in E-cadherin mechanotransduction, we mutated vinculin so that it retains its phosphorylation but cannot be dephosphorylated. Cells expressing the mutant vinculin have increased contractility. This work provides a mechanism for inactivating E-cadherin mechanotransduction and provides a new method for specifically targeting the action of phosphatases in cells.

Regulation of HC11 mouse breast epithelial cell differentiation by the E-cadherin/Rac axis

It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E-cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E-cadherin signaling pathway, we introduced a mutationally activated form of Rac, RacV12, into the mouse breast epithelium-derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated RacV12 is able to increase differentiation, presumably reinforcing the E-cadherin/Rac differentiative signal. However, high RacV12 expression blocked differentiation concomitant with E-cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.

MicroRNA-203 suppresses invasion of gastric cancer cells by targeting ERK1/2/Slug/ E-cadherin signaling.

Growing evidence suggests that microRNA plays an essential role in the development and metastasis of many tumors, including gastric cancer (GC). Expression of microRNA-203 (miR-203) has been reported to decrease in GC. In addition, overexpression of miR-203 inhibits grow of GC cells in vitro and in vivo. However, whether miR-203 affects cell migration and invasion of GC remains unclear. This study aimed to reveal the role of miR-203 on migration and invasion of GC, and its potential mechanisms. Synthetic pre-miR-203 (miR-203), anti-miR-203 and scrambled negative control RNAs was transfected into the gastric cancer SGC7901 cells to generate miR-203 or anti-miR-203-transfected stable clones. The roles of miR-203 on cell invasion and motility were then analyzed by Transwell migration assay and Wound healing assay in vitro. Using siRNA to targeting ERK1/2, Slug, and E-cadherin or Slug cDNA transfection (to increase Slug expression) to examine the miR-203 signaling pathway. We also examined the efficacies of miR-203 or anti-miR-203 on peritoneal metastasis of SGC7901 cells in the nude mouse model. Overexpression of miR-203 inhibits SGC7901 cell invasion and motility, followed by decreased phospho-ERK1/2 (pERK1/2) and Slug expression, as well as increased E-cadherin expression. Re-expression of Slug in miR-203/SGC7901cells decreased E-cadherin expression and restored the invasive phenotypes. Targeting E-cadherin in miR-203/SGC7901cells also restored the invasive phenotypes. Inhibition of miR-203 promotes SGC7901 cell invasion and motility, followed by increased phospho-ERK1/2 (pERK1/2) and Slug expression, as well as decreased E-cadherin expression. Targeting ERK1/2 or Slug in anti-miR-203/SGC7901cells increased E-cadherin expression and reversed the invasive phenotypes. In addition, targeting ERK1/2 decreased Slug and increased the E-cadherin expression. Significantly, we found that miR-203 could exert marked inhibition of the peritoneal metastasis of SGC7901 in nude mice in vivo. Targeting miR-203 could exert marked promotion of the peritoneal metastasis of SGC7901 in nude mice in vivo. miR-203/ERK1/2/Slug/E-cadherin signaling pathway plays an essential role on SGC7901 cell invasion and motility. miR-203 can be novel modalities to prevent peritoneal metastasis of invasive cancers such as gastric cancer.

Higher EZH2 expression is associated with extramedullary infiltration in acute myeloid leukemia.

Accumulating evidence indicates that enhancer of zeste homolog 2 (EZH2) promotes the metastatic ability of solid tumors, but the role of EZH2 in extramedullary infiltration (EMI) in acute myeloid leukemia (AML) has not been thoroughly explored. In the present study, we investigated the possible association between EZH2 and EMI. We found that the messenger RNA (mRNA) and protein expression levels of EZH2 in AML patients were both significantly higher than in idiopathic thrombocytopenic purpura (ITP) patients. Furthermore, a positive correlation between EZH2 mRNA expression and percentage of peripheral blood blasts wa s found in AML patients (r = 0.404, p = 0.009). The migratory capacities of Kasumi-1 and HL-60, which both show a high level of EZH2 expression, were markedly higher than those of U937 and KG-1α. In contrast, silencing of EZH2 resulted in reduction in proliferation and migration ability and an increase in apoptosis. The latter observation was accompanied by reduced expression of associated proteins p-ERK, p-cmyc, and matrix metalloproteinase 2 (MMP-2) and an increase in epithelial cadherin (E-cadherin). These data suggest that higher expression of EZH2 may be associated with extramedullary infiltration in acute myeloid leukemia and affect pathogenesis via activation of the p-ERK/p-cmyc/MMP-2 and E-cadherin signaling pathways.

Abstract 2394: Cancer-associated fibroblasts in invasive lobular breast carcinoma

Abstract Cancer-associated fibroblasts (CAFs) can promote breast cancer progression and therapy response through paracrine and juxtacrine signaling pathways. CAFs are abundantly observed in especially the invasive lobular carcinoma (ILC) subtype of breast cancer. Surprisingly, there is nothing known about the function of these CAFs in ILC. Given the fact that human ILC is generally resistant to standard chemotherapy, we believe that the cause of this therapy resistance lies into the active crosstalk between CAFs and tumor cells leading to a more progressive and resistant phenotype. Therefore, we aim to discover the exact function of CAFs in ILC as well as their drugable targets. In order to reliably study the role of CAFs in ILC, we generated a genetically engineered mouse model for ILC based on the tissue-specific loss of E-cadherin and PI3K signaling pathway activation, two hallmarks of human ILC. This clinically relevant mouse model spontaneously developed tumors that grow as typically invasive ‘indian files’ into a rigid extracellular matrix (ECM) that is very rich of CAFs. Uniquely, these tumors closely resemble human classical ILC, including its growth rate. Based on a mammary gland transplantation setting using fluorescent labeled hosts, we have shown that fibroblasts are recruited by ILC, both in vivo and in vitro, suggesting ILC-secreted mediators. Interestingly, in vitro inhibition of PDGFRβ (expressed by fibroblasts) can at least in part inhibit this recruitment. We are currently investigating whether inhibition of PDGFRβ in vivo can prevent the recruitment of fibroblasts and subsequently tumor development or progression. In parallel, we have isolated fibroblasts and epithelial cells from primary mouse ILCs and wild-type mammary glands to screen for other important players in the tumor-stroma crosstalk using RNA-seq and secretome proteomics. We will subsequently assess their functional significance during ILC development, progression and therapy response. Citation Format: Mirjam C. Boelens, Ellen Wientjens, Eva Schut, Sjoerd Klarenbeek, Karin E. de Visser, Jos Jonkers. Cancer-associated fibroblasts in invasive lobular breast carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2394. doi:10.1158/1538-7445.AM2015-2394

Adam10 mediates the choice between principal cells and intercalated cells in the kidney.

A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane-anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2)(+) principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1.

Bufalin attenuates the stage and metastatic potential of hepatocellular carcinoma in nude mice

BackgroundAdvanced hepatocellular carcinoma (HCC) patients undergo significant tumor growth and metastasis. Here, we investigated bufalin for treating HCC, which exhibits anti-tumor activities in many tumor cell lines.MethodIn our experiment, HCCLM3-R cells were injected into nude mice to form subcutaneous human HCC tumors that were implanted into the liver to establish orthotopic transplantation tumor models. Bufalin was injected intraperitoneally at 1 or 1.5 mg/kg. LY294002 (100 mg/kg), a potent inhibitor of Akt which reduced the levels of pAkt in HCCLM3 cell lines, was injected intraperitoneally into one group thrice weekly. The control was injected with an equal volume of saline. Morphological alterations were evaluated in the liver and lung by stereomicroscopy, the apoptotic rate was measured by TUNEL staining, and expression of AKT/GSK3β/β-catenin/E-cadherin signaling pathway-related proteins was detected by immunohistochemistry (IHC) and western blot analysis.ResultsThese results suggested that the sizes and qualities of orthotopic transplanted tumors as well as pulmonary metastasis decreased markedly at the highest bufalin dose compared with that in the control. Orthotopic transplanted tumor tissues were necrotic in bufalin-treated groups and the apoptotic cell number was markedly higher at the highest bufalin dose compared with that in the control. Certain changes of expression of AKT/GSK3β/β-catenin/E-cadherin signaling pathway-related proteins were in tumor tissues, which were related to the bufalin dose. Similar results were observed in the LY294002-treated group.ConclusionBased on the above, one can draw conclusions that bufalin has significant anti-tumor activities and reduces the metastatic potential in an orthotopic transplantation tumor model of human HCC. Inhibition of AKT/GSK3β/β-catenin/E-cadherin signaling pathways by bufalin may show therapeutic effects in advanced HCC patients.

Bufalin, a component in Chansu, inhibits proliferation and invasion of hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma (HCC) is a common and aggressive cancer, and the treatment options are limited for patients with advanced HCC. Bufalin, the major digoxin-like component of the traditional Chinese medicine Chansu, exhibits significant anti-tumor activities in many tumor cell lines. In the present study, we investigated the effect of bufalin on the inhibition of an AKT-related signaling pathway, and examined the relationship between regulatory proteins and anti-tumor effects in hepatoma cells.MethodsProliferation, wound healing, transwell-migration/invasion and adhesion assays were performed in HCCLM3 and HepG2 cell lines. The protein levels of pAKT, AKT, pGSK3β, GSK3β, pβ-catenin, β-catenin, E-cadherin, MMP-9, and MMP-2 were measured by western blot analysis. E-Cadherin and β-catenin expression levels were also evaluated by immunofluorescence.ResultsBufalin inhibited hepatoma cell proliferation, migration, invasion and adhesion. In addition, treatment with bufalin significantly decreased the levels of pAKT, pGSK3β, MMP-9, and MMP-2, while increasing the levels of GSK3β and E-cadherin and suppressing the nuclear translocation of β-catenin.ConclusionsBufalin is a potential anti-HCC therapeutic candidate through its inhibition of the AKT/GSK3β/β-catenin/E-cadherin signaling pathway. Further studies with bufalin are warranted in patients with HCC, especially those with the disease at advanced stages.

Abstract P5-04-05: E-Cadherin is Required for In Vivo Growth of Inflammatory Breast Cancer: Importance of Mesenchymal-Epithelial Transition (MET) and Role of HIF1α

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and deadly form of breast cancer and is phenotypically distinct from other forms of locally advanced breast cancer. The 5-year survival for IBC patients (40%) has not improved even with the implementation of multi-disciplinary care and targeted therapies (e.g. anti-HER2 or anti-estrogen therapies). Lymph node involvement is common at first diagnosis and the accelerated metastasis program that characterizes IBC account for a disproportionate number of the 40,000 women who will die from breast cancer. The most common molecular marker for IBC is the cell adhesion surface glycoprotein, E-Cadherin. Classically, E-Cadherin is thought to be a tumor suppressor and its loss is associated with the epithelial-mesenchymal transition (EMT) and acquisition of an invasive phenotype. The importance of EMT in metastasis is well accepted in numerous tumor models although the presence of EMT in patient samples is rarely demonstrated. Given the propensity of IBC to metastasize, one would expect a strong EMT signature in IBC cell lines, however the persistent robust expression of E-Cadherin in IBC patient tumors and in IBC cell lines, with a lack of expression of ZEB1, a transcriptional repressor of E-cadherin and diminished expression of genes within the transforming growth factor beta signaling pathway, suggests otherwise. Methods: To further define the role of E-Cadherin in IBC, we used the SUM149 IBC cell line to manipulate E-Cadherin via shRNA knockdown and by overexpression of ZEB1 a known transcriptional repressor of E-cadherin. Results: While the lack of E-Cadherin or ZEB1 overexpression in SUM149 cells led to an EMT phenotype in vitro, E-Cadherin was required for in vivo growth of SUM149 tumor cells. Gene profiling identified novel E-Cadherin signaling pathways including an hypoxia gene signature through HIF1a as well as cytokine responsive pathways. The requirement of HIF-1a for in vivo growth of SUM149 cells was confirmed by shRNA knockdown of HIF-1a. Conclusions: The absolute requirement for E-Cadherin and maintenance of the mesenchymal-epithelial transition in IBC tumor growth is demonstrated. Furthermore, a novel functional link between E-Cadherin and HIF-1a and in their critical role in survival of IBC tumors is identified for the first time and warrants further investigation. Supported in part by The Susan G Komen Organization Promise Grant KG081287 (FMR and MC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-05.

Transforming growth factor β-induced peritoneal fibrosis is mouse strain dependent*

Encapsulating peritoneal sclerosis (EPS) is a rare but devastating complication of peritoneal dialysis. The etiology is unclear, but genetic predisposition may be a contributing factor. We used adenovirus-mediated gene transfer of transforming growth factor (TGF) β1 to the peritoneum in four genetically distinct laboratory mouse strains to assess differences in fibrogenic response. Mice from four genetic backgrounds (C57BL/6J, DBA/2J, C3H/HeJ and SJL/J) received an intraperitoneal injection of an adenovirus expressing TGFβ1 (AdTGFβ1) or control adenovirus (AdDL) and were assessed 4 and 10 days after infection. Submesothelial thickening, angiogenesis and gene expression were quantified from peritoneal tissue. Protein was extracted from omental tissue and assessed for collagen, E-cadherin and TGFβ signaling pathway proteins. There was a graded response among the mouse strains to the peritoneal overexpression of TGFβ1. TGFβ1 induced a significant fibrogenic response in the C57BL/6J mice, whereas the SJL/J mice were resistant. The DBA/2J and the C3H/HeJ mice had intermediate responses. A similar graded response was seen in collagen protein levels in the omental tissue and in fibrosis-associated gene expression. TGFβ type 1 receptor and SMAD signaling pathways appeared to be intact in all the mouse strains. There were significant differences in mouse strain susceptibility to peritoneal fibrosis, suggesting that genetic factors may play a role in the development of peritoneal fibrosis and possibly EPS. As early TGFβ1 signaling mechanisms appear to be intact, we hypothesize that fibrosis resistance in the SJL/J mice lies further down the wound-healing cascade or in an alternate, non-SMAD pathway.

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT‐like phenotype change and MMP‐9‐mediated degradation of E‐cadherin

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

β-catenin signaling involves HGF-enhanced HepG2 scattering through activating MMP-7 transcription

It is well accepted that cell scattering (dispersion of clustered cells into single cells) is the initial step of tumor metastasis, and the downregulation of E-cadherin is associated with metastatic potential of tumor cells; however, the molecular mechanisms underlying loss of E-cadherin during tumor development are still poorly understood. Here, we report that hepatocyte growth factor (HGF) induced E-cadherin downregulation and cell scattering are attributed to the activation of Wnt/beta-catenin signaling and transcriptional activation of matrix metalloproteinase MMP-7. Furthermore, the increased MMP-7 is secreted into the medium and cleaves the ectodomain of E-cadherin. Inhibition of HGF signal by siRNA of c-Met, blocking the beta-catenin transcriptional activity through a dominant negative form of TCF4, MMP-7 knockdown by siRNA or suppression of MMP-7 enzymatic activity with a neutralization antibody allowed inhibition of HGF-induced loss of E-cadherin and HepG2 scattering. Our data presented here revealed the intrinsic mechanism of HGF activated Wnt/beta-catenin signaling regulation of HepG2 cell scattering through MMP-7 transcription activation and E-cadherin degradation. The results suggest that the blocking of HGF/c-Met/beta-catenin/MMP-7/E-cadherin signaling pathway might present a practical therapeutic target for interference with hepatocellular carcinoma metastasis.