E-cadherin Function Research Articles

Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions

BackgroundEpithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified.ResultsThis study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin.ConclusionsPP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.

Identification of novel small compounds that restore E-cadherin expression and inhibit tumor cell motility and invasiveness

Tumor dissemination and invasive behavior are associated with a majority of cancer-related mortality cases. Loss of E-cadherin, which is caused by several tumor-promoting factors, is associated with metastasis and poor prognosis in many neoplasms. In this study, we aimed to identify small molecule compounds that restore the expression of E-cadherin, because these molecules are most likely to suppress tumor malignancy by restoring E-cadherin function and/or by inhibiting signals that suppress E-cadherin expression. Here, we developed a fluorescence screen system based on E-cadherin expression. A pilot drug library screen revealed that methotrexate (MTX) strongly induces E-cadherin expression in a colorectal cancer cell line, SW620. From the screen for 9600 compounds, we identified 9 hit compounds, which restored the expression of E-cadherin in SW620 and/or a melanoma cell line, SK-MEL-28. We confirmed that MTX and the other identified compounds transcriptionally promote E-cadherin expression. Among these, 2 compounds suppressed migration/invasion capacity in colorectal cancer cells and 3 in melanoma cells. A compound reduced SW620 migration and invasion with subtle effects on cell viability in SW620, SK-MEL-28, and a non-tumor cell line, HaCaT, with decrease in AKT and ERK1/2 protein levels. One of the other compounds reduced SK-MEL-28 cell migration and invasion and affected the viability only of SW620 and SK-MEL-28 cells but not HaCaT cells. These results suggest that these compounds would be attractive lead molecules as anti-metastasis agents.

Gene of the month: E-cadherin (CDH1)

Epithelial-cadherin (E-cadherin; encoded by CDH1 ) is a member of the classical cadherins (the others being neural cadherin (N-cadherin) and vascular endothelial cadherin (VE-cadherin)). These single-pass transmembrane glycoproteins are expressed...

COX-2 regulates E-cadherin expression through the NF-κB/Snail signaling pathway in gastric cancer

Cyclooxygenase-2 (COX-2) participates in cancer invasion and metastasis by decreasing the expression of E-cadherin. However, the molecular mechanisms through which COX-2 regulates E-cadherin expression and function have not yet been fully elucidated. The aim of this study was to investigate the possible molecular mechanisms through which COX-2 regulates E-cadherin expression in gastric cancer. The mRNA and protein expression of COX-2, nuclear factor-κB (NF-κB), Snail and E-cadherin was detected in gastric cancer cells by quantitative PCR and western blot analysis, respectively. The expression of these genes was also detected in healthy gastric mucosa and gastric cancer tissues by immunohistochemistry. We detected various levels of COX-2, nuclear factor-κB (NF-κB), Snail and E-cadherin expression in the normal gastric mucosa and cancer tissues; however, the expression patterns differed: the increased expression of COX-2, NF-κB and Snail was observed in the gastric cancer tissues, whereas there was a considerable reduction in E-cadherin expression in the cancer tissues compared to the normal gastric mucosa. The expression patterns of COX-2, NF-κB and Snail were similar. The increased expression of COX-2 in the gastric cancer tissues closely correlated with the increased expression of NF-κB and Snail, but inversely correlated with the expression of E-cadherin. Treatment of the SGC7901 cells (which express high levels of COX-2) with celecoxib, a COX-2 inhibitor, not only led to a marked dose- and time-dependent decrease in the expression of COX-2, NF-κB and Snail, but also led to a significant increase in the expression of E-cadherin, and this was associated with a reduction in cell invasion. By contrast, the same treatment did not alter the expression of these genes in another gastric cancer cell line, MGC803 (which barely expresses COX-2). These data suggest that COX-2 regulates the expression of E-cadherin through the NF-κB and Snail signaling pathway in gastric cancer.

English

Abstract Introduction Hereditary diffuse gastric cancer is an autosomal inherited syndrome associated with the E-cadherin germline mutations. Different types of CDH1 germline mutations have been reported; the missense alterations are rarely identified when compared with truncating mutations. The identification of missense mutation represents a clinical burden, since its pathogenicity is still under genetic and clinical studies. The aim of this critical review was to discuss E-cadherin germline missense mutations in diffuse gastric cancer. Materials and methods In this paper, we reviewed the literature data about the CDH1 germline missense mutations identified in early-onset and hereditary diffuse gastric tumours. The roles of clinical surveillance and prophylactic gastrectomy were discussed. Mutations reported in breast lobular cancer were excluded. Results We identified a total of 31 CDH1 germline missense mutations with different pathogenic impact on E-cadherin function (pathogenic vs. neutral). The majority of these alterations were localized at the extracellular domain of the CDH1 gene. These mutations affected indifferently hereditary and sporadic diffuse gastric carcinomas. Conclusion In this critical review, we reported all missense mutations identified to date and discussed about the E-cadherin missense mutation function, clinical management for asymptomatic mutation carriers and the roles of prophylactic total gastrectomy and endoscopic surveillance.

Abstract 1094: Breast cancer metastatic competency in the hepatic niche is driven by E-cadherin re-expression.

Abstract Nearly half of breast cancer metastases will become clinically evident 5 or more years after the cancer was seemingly ablated. This implies that metastatic cancer cells survived over this extended timeframe without emerging as detectable nodules. Breast cancer metastatic dormancy has yet to be universally defined, and is not well understood as a potential means by which clinically evident metastases emerge after being undetected for years (or decades) from the primary tumor diagnosis. Various mechanistic and microenvironmental factors may work independently, collaboratively (or both) in order to allow a dormant metastatic breast cancer cell (or small cluster) to survive long term. At least one tumor dormancy hallmark is likely required: that of E-cadherin re-expression in order to form heterotypic connections between the carcinoma cells and the liver parenchyma. This re-expression is known as the mesenchymal-to-epithelial-reverting-transition (MErT). This re-expression provides not only survival signals to the breast cancer cells but may also establish cell polarity and protection from autocrine signaling (or extracellular factors) that otherwise may prompt the breast cancer cells to initiate metastatic outgrowth. The purpose of this study is to determine E-cadherin's function in the establishment of breast cancer metastases to the liver. The MDA-MB-231 human breast cancer cell line, that can undergo induced MErT ex vivo, was transfected with either exogenously expressing E-cadherin or shRNA to E-cadherin. Xenograft splenic injections of these cell line variants into mice were performed and tumors allowed to grow for 30-60 days until sacrifice. Immunohistochemistry was performed on the liver and splenic tissue sections in order to assess the number and size of metastatic depostis. Preliminary data demonstrate that cells expressing exogenously encoded E-cadherin developed large splenic tumors with fewer to no liver metastases. MDA-MB-231 wild type cells spawned liver metastases about half the time. In order to more fully probe E-cadherin's role in metastatic competency experiments are underway to develop a doxycycline-inducible E-cadherin bicistronic vector allowing for the down-regulation of E-cadherin in the primary tumor and the re-expression in the metastatic niche. Subsequent immunohistochemistry will determine the metastatic E-cadherin status across these MDA-MB-231 cell variants. These results will provide insights into the role of MErT in metastatic establishment. Citation Format: Donald P. Taylor. Breast cancer metastatic competency in the hepatic niche is driven by E-cadherin re-expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1094. doi:10.1158/1538-7445.AM2013-1094

Abstract 4064: Rac1 activation promotes invasive phenotype of side population in colon cancer cells by destabilizing the E cadherin-β-catenin ligation.

Abstract [Background] Side population (SP) cells are small fraction of cancer cells that have tumorigenic activity, chemotherapy resistance and metastatic potential. However, it remains to be clarified what characteristics SP cells possess. Here we report SP cells possess more invasive property than non-SP cells, occurring coincident with enhanced activation of a member of Rho-GTPase, Rac1. [Methods] Thirteen colon cancer cell lines were assessed for Rac1 activation by pull-down assay and the percentage of SP cell fraction within total number of cells by the flow cytometry-based side population (SP) technique to get invasive phenotype. Invasion assay was performed to evaluate SP cells’ behavior. Cell adhesion associated proteins such as E-cadherin and β-catenin and actin cytoskeleton were evaluated by immunocytochemistry [Results] We identified positive correlation between active Rac1 expression and SP cell frequency. In addition, Enhanced Rac1 activation and SP cell frequency were associated with poorly differentiated histology. Isolated SP cells expressed more active Rac1 as compared to non-SP cells, being associated with increased invasive activity in SP cells. Mesenchymal and amoeboid types of migration are based on reorganization of the actin cytoskeleton, but their requirements for Rho and Rac signaling differ. With respect to mesenchymal migration, membrane protrusions at the leading edge are formed in a Rac-dependent manner. Expression of a dominant-negative form of Rac in elongated cells inhibits the formation of such protrusive membrane structures, causing decreased cell migration. In contrast, Rho signaling is not essential for mesenchymal migration. With respect to amoeboid migration, the actin cytoskeleton is reorganized along the plasma membrane, causing dynamic membrane blebbing along the cell surface. Suppression of Rac1 activity by either dominant-negative Rac1 or dominant negative WAVE, a downstream mediator of Rac1 signaling reduced SP cell invasion activity. Over expression of constitutively active mutant of Rac1 enhanced SP cell motility. Furthermore, the E-cadherin attenuation and β-catenin enhancement were confirmed by immunohistocytochemistry after Rac1 inhibition. [Conclusions] We identified that Rac1 would appear to be a key molecule to mediate SP cells’ motility. We also implicate that suppression of Rac1 activity leads to reduction in number of invasive SP cells through restored E-cadherin function. Thus present study provides a concept for a new therapeutic strategy that targeting Rac1 molecule would be effective to suppress colon cancer invasive cell. Citation Format: Shinsuke Funakoshi, Toshifumi Azuma, Hajime Higuchi, Yasuo Hamamoto, Masayuki Adachi, Ayano Kabashima-Niibe, Akinori Hashiguchi, Hiromasa Takaishi, Toshifumi Hibi. Rac1 activation promotes invasive phenotype of side population in colon cancer cells by destabilizing the E cadherin-β-catenin ligation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4064. doi:10.1158/1538-7445.AM2013-4064

Abstract 5377: Profiling of aurora kinase signaling in microdissected gastric cancer samples indicates a significant increase in histone deacetylase 4 (HDAC4).

Abstract Introduction & Methods: Gastric cancer represents one of the leading causes of cancer related death in the world second only to lung cancer resulting in 800,000 deaths per year. Aurora kinases, a family of serine/threonine kinases essential for cytokinesis and chromosome segregation, are frequently over-expressed in various cancer types. Moreover, Aurora kinase has recently been shown to directly regulate epigenetic changes (Guise et al., Mol Cell Proteomics. 2012 Nov;11(11):1220-9). Aurora kinase signaling may also impact E-cadherin function via a GSK3-α/β induced increase in β-catenin. E-cadherin deregulation has been implicated in gastric cancer, especially in diffuse types where it is mutated in many cases. We have profiled Aurora kinase signaling pathway activation and its effects on epigenetic and cytoskeletal changes in gastric cancer. Human gastric cancer tumor samples (n=10; 4 diffuse, 5 intestinal, 1 undifferentiated adenocardinoma) and patient matched normal mucosa were procured by laser capture microdissection. Reverse phase protein microarrays were used to quantify total and post-translationally modified proteins in cell signaling pathways directly and indirectly related to Aurora kinase. Results: We found a significant increase in histone deacetylase 4 levels (HDAC4, p<0.0001) concomitant with increased phosphorylation of Aurora kinase (p<0.1) and a decrease in phosphorylated GSK3-α/β (p<0.05) in gastric tumor epithelia compared to patient matched normal mucosa. Gastric cancer samples of diffuse type showed a significantly reduced expression of E-Cadherin (p<0.05) as compared to intestinal type gastric cancer. In contrast, patient samples with lymph node metastasis (n=6) were associated with an increased level of phospho-GSK3-α/β in both normal mucosa (p<0.05) and matched tumor epithelia (p<0.1). Conclusions: To our knowledge, this is the first time that increased levels of HDAC4 have been reported in gastric cancer. The demonstrated increase in Aurora kinase phosphorylation in gastric tumor epithelia may provide a mechanistic link to the observed HDAC4 upregulation. Citation Format: Lorenzo Colarossi, Lorenzo Memeo, Lance A. Liotta, Virginia Espina, Claudius Mueller. Profiling of aurora kinase signaling in microdissected gastric cancer samples indicates a significant increase in histone deacetylase 4 (HDAC4). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5377. doi:10.1158/1538-7445.AM2013-5377

Smad Mediated Regulation of Inhibitor of DNA Binding 2 and Its Role in Phenotypic Maintenance of Human Renal Proximal Tubule Epithelial Cells

The basic-Helix-Loop-Helix family (bHLH) of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2) has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC), TGFβ1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFβ1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFβ1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFβ1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFβ1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFβ1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFβ1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion

Known proteins associated with the cell-adhesion protein E-cadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed E-cadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for E-cadherin-dependent adhesion and suggested that it plays a role in coordination of the cell-cell and cell-substrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin.

E-cadherin–integrin crosstalk in cancer invasion and metastasis

E-cadherin is a single-pass transmembrane protein that mediates homophilic cell-cell interactions. Tumour progression is often associated with the loss of E-cadherin function and the transition to a more motile and invasive phenotype. This requires the coordinated regulation of both E-cadherin-mediated cell-cell adhesions and integrin-mediated adhesions that contact the surrounding extracellular matrix (ECM). Regulation of both types of adhesion is dynamic as cells respond to external cues from the tumour microenvironment that regulate polarity, directional migration and invasion. Here, we review the mechanisms by which tumour cells control the cross-regulation between dynamic E-cadherin-mediated cell-cell adhesions and integrin-mediated cell-matrix contacts, which govern the invasive and metastatic potential of tumours. In particular, we will discuss the role of the adhesion-linked kinases Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK), and the Rho family of GTPases.

E-cadherin Controls Bronchiolar Progenitor Cells and Onset of Preneoplastic Lesions in Mice

Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body.associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways.

Biphasic Papillary and Lobular Breast Carcinoma With PIK3CA and IDH1 Mutations

Morphologic "special types" of breast carcinomas have been recognized for many years, and their molecular and genetic properties have not been specifically studied until recently. Lobular carcinoma lacks functional E-cadherin expression but shares molecular similarities with low-grade invasive ductal carcinomas. Papillary carcinoma is relatively rare, and molecular features are just being elucidated. We report a case of concurrent invasive lobular and papillary carcinoma, the latter with extensive nodal involvement. Multiplex screening for activating point mutations identified different point mutations in the distinct morphologic components: lobular PIK3CA H1047R, papillary; PIK3CA Q546P, and IDH1 R132H. These molecular data favor coincidental "collision tumors" over clonal evolution. The IDH1 R132H point mutation is common in gliomas and acute myelogenous leukemia, but this has not been previously reported in breast carcinoma. The characterization of activating point mutations in morphologic special types of breast carcinoma may suggest avenues amenable to targeted therapy.

E-cadherin and adherens-junctions stability in gastric carcinoma: Functional implications of glycosyltransferases involving N-glycan branching biosynthesis, N-acetylglucosaminyltransferases III and V

BackgroundE-cadherin is a cell–cell adhesion molecule and the dysfunction of which is a common feature of more than 70% of all invasive carcinomas, including gastric cancer. Mechanisms behind the loss of E-cadherin function in gastric carcinomas include mutations and silencing at either the DNA or RNA level. Nevertheless, in a high percentage of gastric carcinoma cases displaying E-cadherin dysfunction, the mechanism responsible for E-cadherin dysregulation is unknown. We have previously demonstrated the existence of a bi-directional cross-talk between E-cadherin and two major N-glycan processing enzymes, N-acetylglucosaminyltransferase-III or -V (GnT-III or GnT-V). MethodsIn the present study, we have characterized the functional implications of the N-glycans catalyzed by GnT-III and GnT-V on the regulation of E-cadherin biological functions and in the molecular assembly and stability of adherens-junctions in a gastric cancer model. The results were validated in human gastric carcinoma samples. ResultsWe demonstrated that GnT-III induced a stabilizing effect on E-cadherin at the cell membrane by inducing a delay in the turnover rate of the protein, contributing for the formation of stable and functional adherens-junctions, and further preventing clathrin-dependent E-cadherin endocytosis. Conversely, GnT-V promotes the destabilization of E-cadherin, leading to its mislocalization and unstable adherens-junctions with impairment of cell–cell adhesion. ConclusionsThis supports the role of GnT-III on E-cadherin-mediated tumor suppression, and GnT-V on E-cadherin-mediated tumor invasion. General significanceThese results contribute to fill the gap of knowledge of those human carcinoma cases harboring E-cadherin dysfunction, opening new insights into the molecular mechanisms underlying E-cadherin regulation in gastric cancer with potential translational clinical applications.

EGFR and HER2 receptor kinase signaling mediate epithelial cell invasion by Candida albicans during oropharyngeal infection

The fungus Candida albicans is the major cause of oropharyngeal candidiasis (OPC). A key feature of this disease is fungal invasion of oral epithelial cells, a process that can occur by active penetration and fungal-induced endocytosis. Two invasins, Als3 and Ssa1, induce epithelial cell endocytosis of C. albicans, in part by binding to E-cadherin. However, inhibition of E-cadherin function only partially reduces C. albicans endocytosis, suggesting that there are additional epithelial cell receptors for this organism. Here, we show that the EGF receptor (EGFR) and HER2 function cooperatively to induce the endocytosis of C. albicans hyphae. EGFR and HER2 interact with C. albicans in an Als3- and Ssa1-dependent manner, and this interaction induces receptor autophosphorylation. Signaling through both EGFR and HER2 is required for maximal epithelial cell endocytosis of C. albicans in vitro. Importantly, oral infection with C. albicans stimulates the phosphorylation of EGFR and HER2 in the oral mucosa of mice, and treatment with a dual EGFR and HER2 kinase inhibitor significantly decreases this phosphorylation and reduces the severity of OPC. These results show the importance of EGFR and HER2 signaling in the pathogenesis of OPC and indicate the feasibility of treating candidal infections by targeting the host cell receptors with which the fungus interacts.

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, β-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein.

Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesis†

Disruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discovered several new transcripts. One, named CDH1a, with high expression in spleen and absent from normal stomach, was demonstrated to be translated into a novel isoform, differing from canonical E-cadherin in its N-terminal, as determined by mass spectrometry. Quantitative and functional assays showed that when overexpressed in an E-cadherin negative context, CDH1a replaced canonical protein interactions and functions. However, when co-expressed with canonical E-cadherin, CDH1a increased cell invasion and angiogenesis. Further, interferon-induced gene IFITM1 and IFI27 levels were increased upon CDH1a overexpression. Effects on invasion and IFITM1 and IFI27 expression were reverted upon CDH1a-specific knockdown. Importantly, CDH1a was de novo expressed in gastric cancer cell lines. This study presents a new mechanism by which E-cadherin functions are impaired by cis-regulatory mechanisms possibly with the involvement of inflammatory machinery. If confirmed in other cancer models, our data enclose potential for designing targeted therapies to rescue E-cadherin function.

Hereditary Diffuse Gastric Cancer: A Family Diagnosis and Treatment

Hereditary diffuse gastric cancer (HDGC) is a rare cancer representing approximately 2% of all gastric cancers. It is caused by CDHI gene mutations, inherited in an autosomal dominant fashion, that affect the function of E-cadherin. Approximately 38% of HDGC families have a CDHI gene mutation. With an estimated 75% penetrance rate, carriers are at high risk for HDGC. We describe the case of a Caucasian male of German-Russian ancestry, carrying a CDHI gene mutation, who survived for 18 months after being diagnosed with HDGC. The results of genetic testing undergone by his family members are also reported, along with a review of the current literature. Since surveillance methods for HDGC are ineffective and unreliable, total prophylactic gastrectomy is advised for individuals with the gene mutation. Additionally, a diagnosis of HDGC should lead to genetic evaluation of family members followed by preventative measures.

The RhoGAP protein Deleted in Liver Cancer 3 (DLC3) is essential for adherens junctions integrity

Epithelial cell–cell contacts are mediated by E-cadherin interactions, which are regulated by the balanced local activity of Rho GTPases. Despite the known function of Rho at adherens junctions (AJs), little is known about the spatial control of Rho activity at these sites. Here we provide evidence that in breast epithelial cells the Deleted in Liver Cancer 3 (DLC3) protein localizes to AJs and is essential for E-cadherin function. DLC3 is a still poorly characterized RhoA-specific GTPase-activating protein that is frequently downregulated in various types of cancer. We demonstrate that DLC3 depletion leads to mislocalization of E-cadherin and catenins, which was associated with impaired cell aggregation and increased migration. This is explained by aberrant local Rho signaling because ROCK inhibition restored cell–cell contacts in DLC3 knockdown cells. We thus identify DLC3 as a novel negative regulator of junctional Rho and propose that DLC3 loss contributes to carcinogenesis by compromising epithelial integrity.

Glucocorticoids and histone deacetylase inhibitors cooperate to block the invasiveness of basal-like breast cancer cells through novel mechanisms

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.