Association between dopamine and somatostatin receptor expression and pharmacological response to somatostatin analogues in acromegaly

Acromegaly is a hormonal disorder resulting from excessive growth hormone (GH) secretion frequently produced by pituitary adenomas and consequent increase in insulin‐like growth factor 1 (IGF‐I). Elevated GH and IGF‐I levels result in a wide range of somatic, cardiovascular, endocrine, metabolic and gastrointestinal morbidities. Somatostatin analogues (SSAs) form the basis of medical therapy for acromegaly and are currently used as first‐line treatment or as second‐line therapy in patients undergoing unsuccessful surgery. However, a considerable percentage of patients do not respond to SSAs treatment. Somatostatin receptors (SSTR1‐5) and dopamine receptors (DRD1‐5) subtypes play critical roles in the regulation of hormone secretion. These receptors are considered important pharmacological targets to inhibit hormone oversecretion. It has been proposed that decreased expression of SSTRs may be associated with poor response to SSAs. Here, we systematically examine SSTRs and DRDs expression in human somatotroph adenomas by quantitative PCR. We observed an association between the response to SSAs treatment and DRD4, DRD5, SSTR1 and SSTR2 expression. We also examined SSTR expression by immunohistochemistry and found that the immunohistochemical detection of SSTR2 in particular might be a good predictor of response to SSAs.     

Predictive and prognostic significance of tumour subtype,SSTR1-5 and e-cadherin expression in a well-defined cohort of patients with acromegaly

In somatotroph pituitary tumours, somatostatin analogue (SSA) therapy outcomes vary throughout the studies. We performed an analysis of cohort of patients with acromegaly from the Czech registry to identify new prognostic and predictive factors. Clinical data of patients were collected, and complex immunohistochemical assessment of tumour samples was performed (SSTR1-5, dopamine D2 receptor, E-cadherin, AIP). The study included 110 patients. In 31, SSA treatment outcome was evaluated. Sparsely granulated tumours (SGST) differed from the other subtypes in expression of SSTR2A, SSTR3, SSTR5 and E-cadherin and occurred more often in young. No other clinical differences were observed. Trouillas grading system showed association with age, tumour size and SSTR2A expression. Factors significantly associated with SSA treatment outcome included age, IGF1 levels, tumour size and expression of E-cadherin and SSTR2A. In the group of SGST, poor SSA response was observed in younger patients with larger tumours, lower levels of SSTR2A and higher Ki67. We observed no relationship with expression of other proteins including AIP. No predictive value of E-cadherin was observed when tumour subtype was considered . Multiple additional factors apart from SSTR2A expression can predict treatment outcome in patients with acromegaly.     

Molecular evidence and clinical importance of β‐arrestins expression in patients with acromegaly

β‐arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between β‐arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of β‐arrestins with response to first‐generation SRL and invasiveness in somatotropinomas. β‐arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real‐time RT‐PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 μg/l and normal IGF‐I levels, was assessed in 40 patients. The Knosp‐Steiner criteria were used to define invasiveness. Median β‐arrestin 1, β‐arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156 ; and 3989, respectively. There was a positive correlation between β‐arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between β‐arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between β‐arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between β‐arrestins and sst2, our data clearly indicated that no association existed between β‐arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether β‐arrestins have a role in the response to treatment with SRL in acromegaly.     

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E‐cadherin to disrupt intercellular adhesion

Mammalian and prokaryotic high‐temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell‐adhesion protein E‐cadherin. E‐cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small‐molecule inhibitor that efficiently blocks HtrA activity, E‐cadherin cleavage and intercellular entry of H. pylori.     

Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.     

E‐cadherin determines Caveolin‐1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin‐1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E‐cadherin in CAV1‐dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E‐cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co‐expression of E‐cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav‐1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E‐cadherin expression in B16F10 (E‐cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co‐expression of CAV1 and E‐cadherin in B16F10 (cav‐1/E‐cad) cells abolishes tumor formation, lung metastasis, increased Rac‐1 activity, and cell migration observed with B16F10 (cav‐1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac‐1 activation in these cells.     

Proper E‐cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer

We reported that Disabled‐2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E‐cadherin. Dab2 depletion in Caco‐2 cells led to E‐cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium‐induced colitis increased Dab2/E‐cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E‐cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E‐cadherin delocalization. Epithelial Dab2 decrement preceded that of E‐cadherin. We suggest that Dab2, by inhibiting E‐cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.     

DNA methyltransferase inhibitor RG108 and histone deacetylase inhibitors cooperate to enhance NB4 cell differentiation and E‐cadherin re‐expression by chromatin remodelling

Epigenetic silencing of cancer‐related genes by abnormal methylation and the reversal of this process by DNA methylation inhibitors represents a promising strategy in cancer therapy. As DNA methylation affects gene expression and chromatin structure, we investigated the effects of novel DNMT (DNA methyltransferase) inhibitor, RG108, alone and in its combinations with structurally several HDAC (histone deacetylase) inhibitors [sodium PB (phenyl butyrate) or BML‐210 (N‐(2‐aminophenyl)‐N′phenyloctanol diamine), and all‐trans RA (retinoic acid)] in the human PML (promyelocytic leukaemia) NB4 cells. RG108 at different doses from 20 to 100 μM caused time‐, but not a dose‐dependent inhibition of NB4 cell proliferation without cytotoxicity. Temporal pretreatment with RG108 before RA resulted in a dose‐dependent cell growth inhibition and remarkable acceleration of granulocytic differentiation. Prolonged treatments with RG108 and RA in the presence of HDAC inhibitors significantly increased differentiation. RG108 caused time‐dependent re‐expression of methylation‐silenced E‐cadherin, with increase after temporal or continuous treatments with RG108 and RA, or RA together with PB in parallel, in cell maturation, suggesting the role of E‐cadherin as a possible therapeutic marker. These processes required both PB‐induced hyperacetylation of histone H4 and trimethylation of histone H3 at lysine 4, indicating the cooperative action of histone modifications and DNA methylation/demethylation in derepression of E‐cadherin. This work provides novel experimental evidence of the beneficial role of the DNMT inhibitor RG108 in combinations with RA and HDACIs in the effective differentiation of human PML based on epigenetics.     

OGT Controls the Expression and the Glycosylation of E‐cadherin,and Affects Glycosphingolipid Structures in Human Colon Cell Lines

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer‐derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N‐glycans, alteration of O‐glycans, upregulated sialylation, and O‐GlcNAcylation. Although O‐GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O‐GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O‐GlcNAc transferase (OGT, the sole enzyme driving O‐GlcNAcylation), only slightly affects overall N‐ and O‐glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E‐cadherin (a major actor of epithelial‐to‐mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O‐GlcNAcylation in colon cancer cells.     

Circ‐AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR‐296‐3p/E‐cadherin signals

Diabetic nephropathy is a leading cause of end‐stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ‐AKT3/miR‐296‐3p/E‐cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ‐AKT3 and fibrosis‐associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT‐PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40‐MES13 cells. Luciferase reporter assays were used to investigate interactions among E‐cadherin, circ‐AKT3 and miR‐296‐3p in mouse mesangial SV40‐MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ‐AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40‐MES13 cells treated with high‐concentration (25 mmol/L) glucose. In addition, circ‐AKT3 overexpression inhibited the level of fibrosis‐associated protein, such as fibronectin, collagen type I and collagen type IV. Circ‐AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40‐MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ‐AKT3 could act as a sponge of miR‐296‐3p and E‐cadherin was the miR‐296‐3p direct target. Moreover, circ‐AKT3/miR‐296‐3p/E‐cadherin modulated the extracellular matrix of mouse mesangial cells in high‐concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ‐AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR‐296‐3p/E‐cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.     

An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases‐dependent modification of E‐cadherin

We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c‐Src‐dependent tyrosine phosphorylation of β‐catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6‐induced modification of E‐cadherin. In control cells cultured at pH 7.4, E‐cadherin staining was linear and continuous at cell–cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E‐cadherin staining, consistent with the decreased levels of E‐cadherin in membrane fractions. pH 6.6 treatment activated c‐Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E‐cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6‐induced tyrosine phosphorylation of E‐cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E‐cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6‐induced detachment of E‐cadherin from AJs was blocked by pretreatment with MG132 or NH₄Cl, indicating the involvement of ubiquitin‐proteasomal/lysosomal degradation of E‐cadherin. An early loss of p120ctn prior to E‐cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E‐cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E‐cadherin and p120ctn and a weakening of the association of E‐cadherin with p120ctn and contributing to the instability of E‐cadherin at AJs. J. Cell. Biochem. 108: 851–859, 2009. © 2009 Wiley‐Liss, Inc.     

E‐cadherin can limit the transforming properties of activating β‐catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.     

Newly formed E‐cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes1

Background information. The appropriate regulation of cell–cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E‐cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin‐mediated cell–cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E‐cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell–cell contacts has been inferred from the presence of filopodia, the typical F‐actin structure induced by Cdc42 activation, as cells approach each other to establish cell–cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. Results. In the present study, we set out to address (a) whether Cdc42 is activated by new E‐cadherin cell–cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E‐cadherin clustering decreased Cdc42‐GTP levels. In the absence of serum‐ and migration‐induced Cdc42 activation, we demonstrated that cell–cell contacts do not induce filopodia or require Cdc42 function to assemble. Conclusion. We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re‐positioning or basolateral trafficking.     

E‐, P‐, and N‐cadherin are co‐expressed in the nasopharyngeal carcinoma cell line TW‐039

The cadherin/catenin complex plays a key role in the initiation of cell‐cell recognition, and adhesion, and the elaboration of structural and functional organization in multicellular tissues and organs. It is associated with tumor metastasis and also acts as an “invasion suppressor” of cancer cells. Nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic nature. The expression of the E‐cadherin/catenin complex is down‐regulated in NPC tumor specimens. To obtain better insight into the intercellular adhesive property of NPC cells, we used immunofluorescence microscopy, immunoprecipitation, and immunoblot analysis to examine the expression of the classical cadherins and β‐catenin in a NPC cell line, TW‐039. The results demonstrate a change in the distribution of E‐cadherin from cytosolic flakes to cell‐cell contacts with increasing time in culture. Between days 1 and 5 after plating, the detergent‐insoluble fraction of E‐cadherin increased from 20% to 37% of total E‐cadherin, and that for P‐cadherin increased from 33% to 40%. By contrast, the values for β‐catenin remained unchanged (26% and 25%). Both immunofluorescence and immunoblot studies suggested that P‐cadherin may be involved in pioneer contact adhesion of TW‐039 cells. Interestingly, E‐, P‐, and N‐cadherin are co‐expressed in this cell line. Immunoprecipitation studies also showed that other members of the cadherin family may be involved in the contact adhesion of TW‐039 cells. J. Cell. Biochem. 76:161–172, 1999. © 1999 Wiley‐Liss, Inc.