Pivotal role of MUC1 glycosylation by cigarette smoke in modulating disruption of airway adherens junctions in vitro

Cigarette smoke increases the risk of lung cancer by 20‐fold and accounts for 87% of lung cancer deaths. In the normal airway, heavily O‐glycosylated mucin‐1 (MUC1) and adherens junctions (AJs) establish a structural barrier that protects the airway from infectious, inflammatory and noxious stimuli. Smoke disrupts cell–cell adhesion via its damaging effects on the AJ protein epithelial cadherin (E‐cad). Loss of E‐cad is a major hallmark of epithelial–mesenchymal transition (EMT) and has been reported in lung cancer, where it is associated with invasion, metastasis and poor prognosis. Using organotypic cultures of primary human bronchial epithelial (HBE) cells treated with smoke‐concentrated medium (Smk), we have demonstrated that E‐cad loss is regulated through the aberrant interaction of its AJ binding partner, p120‐catenin (p120ctn), and the C‐terminus of MUC1 (MUC1‐C). Here, we reported that even before MUC1‐C became bound to p120ctn, smoke promoted the generation of a novel 400 kDa glycoform of MUC1's N‐terminus (MUC1‐N) differing from the 230 kDa and 150 kDa glycoforms in untreated control cells. The subsequent smoke‐induced, time‐dependent shedding of glycosylated MUC1‐N exposed MUC1‐C as a putative receptor for interactions with EGFR, Src and p120ctn. Smoke‐induced MUC1‐C glycosylation modulated MUC1‐C tyrosine phosphorylation (TyrP) that was essential for MUC1‐C/p120ctn interaction through dose‐dependent bridging of Src/MUC1‐C/galectin‐3/EGFR signalosomes. Chemical deglycosylation of MUC1 using a mixture of N‐glycosylation inhibitor tunicamycin and O‐glycosylation inhibitor benzyl‐α‐GalNAc disrupted the Src/MUC1‐C/galectin‐3/EGFR complexes and thereby abolished smoke‐induced MUC1‐C‐TyrP and MUC1‐C/p120ctn interaction. Similarly, inhibition of smoke‐induced MUC1‐N glycosylation using adenoviral shRNA directed against N‐acetyl‐galactosaminyl transferase‐6 (GALNT6, an enzyme that controls the initiating step of O‐glycosylation) successfully suppressed MUC1‐C/p120ctn interaction, prevented E‐cad degradation and maintained cellular polarity in response to smoke. Thus, GALNT6 shRNA represents a potential therapeutic modality to prevent the initiation of events associated with EMT in the smoker's airway. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Rac1 and Cdc42 Differentially Modulate Cigarette Smoke–Induced Airway Cell Migration through p120-Catenin–Dependent and –Independent Pathways

The adherens junction protein p120-catenin (p120ctn) shuttles between E-cadherin–bound and cytoplasmic pools to regulate E-cadherin/catenin complex stability and cell migration, respectively. When released from the adherens junction, p120ctn promotes cell migration through modulation of the Rho GTPases Rac1, Cdc42, and RhoA. Accordingly, the down-regulation and cytoplasmic mislocalization of p120ctn has been reported in all subtypes of lung cancers and is associated with grave prognosis. Previously, we reported that cigarette smoke induced cytoplasmic translocation of p120ctn and cell migration, but the underlying mechanism was unclear. Using primary human bronchial epithelial cells exposed to smoke-concentrated medium (Smk), we observed the translocation of Rac1 and Cdc42, but not RhoA, to the leading edge of polarized and migrating human bronchial epithelial cells. Rac1 and Cdc42 were robustly activated by smoke, whereas RhoA was inhibited. Accordingly, siRNA knockdown of Rac1 or Cdc42 completely abolished Smk-induced cell migration, whereas knockdown of RhoA had no effect. p120ctn/Rac1 double knockdown completely abolished Smk-induced cell migration, whereas p120ctn/Cdc42 double knockdown did not. These data suggested that Rac1 and Cdc42 coactivation was essential to smoke-promoted cell migration in the presence of p120ctn, whereas migration proceeded via Rac1 alone in the absence of p120ctn. Thus, Rac1 may provide an omnipotent therapeutic target in reversing cell migration during the early (intact p120ctn) and late (loss of p120ctn) stages of lung carcinogenesis.Cigarette smoke contains >4000 active constituents, ≥60 of which are established carcinogens and/or mutagens.¹ With a 20-fold greater risk of lung cancer and accounting for 87% of lung cancer–related deaths,² smoking continues to represent the single most important carcinogenic exposure. Because treatment of lung cancer is largely ineffective, recent research has been focused on efforts to identify and reverse early events leading to the initiation of lung cancer by smoke.³ Emerging evidence suggests that smoke mediates epithelial-mesenchymal transition (EMT) and pretumor cell migration by disrupting cell-cell adhesion in polarized mucosal epithelia.^{4, 5} During EMT, cells switch from a polarized immobile epithelial phenotype to a highly motile fibroblast phenotype.⁶ Unregulated EMT confers epithelial cells with stem cell–like properties capable of self-renewal, metastasis, and resistance to apoptosis.^{6, 7} Little is known about how smoke mediates EMT during the early stages of lung cancer.E-cadherin (E-cad)–based adherens junctions (AJs) interact with catenins to modulate cell-cell adhesion.⁸ Structural analysis by X-ray crystallography revealed that p120-catenin (p120ctn) binds to the juxtamembrane domain of E-cad, where it regulates stability and turnover of E-cad by concealing the juxtamembrane domain residues implicated in endocytosis and ubiquitination of E-cad.^{9, 10} The disruption of p120ctn leads to E-cad degradation, a major hallmark of EMT and malignancy.⁸ Accumulating evidence suggests that p120ctn shuttles between E-cad–bound and cytoplasmic pools. When bound to E-cad, p120ctn stabilizes the AJ and acts as a tumor and/or metastasis suppressor.¹¹ When released from the AJ, p120ctn can promote EMT and cell migration through the degradation of E-cad and the modulation of Rho GTPase activity, respectively.^{8, 11, 12, 13, 14, 15, 16, 17} Accordingly, membrane loss, down-regulation, and cytoplasmic mislocalization of E-cad and p120ctn have been reported in most epithelial cancers, including all subtypes of lung cancers, and are frequently associated with a grave prognosis.^{18, 19}In lung cancer, ectopic cytoplasmic expression of p120ctn and E-cad has been associated with elevated expression of Rho GTPases.¹⁹ Rac1, Cdc42, and RhoA shuttle between their inactive GDP– and active GTP–bound forms to regulate the dynamics of the actin cytoskeleton, cell motility, cadherin-dependent adhesion, and cell proliferation.^{20, 21, 22} Lamellipodia, filopodia, and stress fibers are regarded as typical phenotypes of activated Rac1, Cdc42, and RhoA, respectively.²³ Active Rac1 and Cdc42 drive protrusion formation at the leading edge of a migrating leukocyte, whereas active RhoA aggregates at the rear and sides of the cell, preventing protrusion formation.²¹ p120ctn can act as a guanine nucleotide dissociation inhibitor to inhibit RhoA through preferential interaction and sequestration of RhoA in its GDP-bound form.¹² Alternatively, p120ctn indirectly activates Rac1 and Cdc42 through its interaction with Vav2, a guanine nucleotide exchange factor that promotes the exchange of GDP with GTP.^{13, 14}We sought to investigate the role of p120ctn in regulating Rho GTPase activity in the initiating stages of cigarette smoke–induced cell migration. Given the opposing roles of membrane versus cytoplasmic p120ctn in carcinogenesis, this study was performed in primary human bronchial epithelial (HBE) cells with intact AJs. Realizing that cancer is a multistep process requiring numerous chemically mediated insults, we mimicked the exposure of airway epithelial cells to smoke using an established model of smoke-conditioned medium (Smk).^{24, 25} Primary HBE cells exposed to Smk medium underwent malignant transformation in 8 days, demonstrating rapid proliferation, anchorage-independent growth, and tumorigenesis in nude mice.²⁶ With this approach, we discovered p120ctn-dependent and p120ctn-independent pathways mediating cell migration provoked by cigarette smoke. In the presence of p120ctn, coactivation of Rac1 and Cdc42 was essential to promote cell migration, whereas in the absence of p120ctn, activation of Rac1 alone induced migration. These data reveal new details regarding the molecular events promoting cell migration in the earliest stages of cigarette smoke–induced tumorigenesis and open the way for novel approaches to the prevention of lung cancer in smokers.     

Helicobacter pylori alters the distribution of ZO-1 and p120ctn in primary human gastric epithelial cells

Helicobacter pylori infection is related to the development of diverse gastric pathologies, possibly by affecting epithelial junctional complexes that define cell polarity and play an essential role in transepithelial transport and cell-cell adhesion. Using primary gastric epithelial cell cultures, effects of H. pylori on the expression and localization of tight/adherence junction proteins and the resulting morphological changes and migratory capabilities were studied under in vivo-like conditions. Gastric epithelial cells were isolated from biopsies or gastrectomies and maintained in Quantum286 on collagen I-coated culture dishes or cover-slips. Cell cultures were characterized and further analyzed by western blot and immunofluorescent staining for ZO-1, p120ctn, and H. pylori CagA. Morphological changes and migratory response were monitored by time-lapse digital image microscopy. ZO-1 and p120ctn protein expression levels remain unaffected by H. pylori infection. Immunocytochemistry on H. pylori-infected primary cell monolayers focally showed disruption of intercellular ZO-1 staining and accumulation of ZO-1 in small vesicles. H. pylori infection recruited non-phosphorylated p120ctn to perinuclear vesicles. The fraction of phosphorylated p120ctn increased and could be detected in the nucleus, at the cell membrane, and at the leading edge of migrating cells. These alterations, triggered by H. pylori infection, are associated with an elongation phenotype and increased migration.     

Membrane β‐catenin and adherens junctions in early gonadal patterning

Background: Mechanisms involved in early patterning of the mammalian gonad as it develops from a bipotential state into a testis or an ovary are as yet not well understood. Sex‐specific vascularization is essential in this process, but more specific mechanisms required to, for example, establish interstitial vs. cord compartments in the testis or ovigerous cords in the ovary have not been reported. Adherens junctions (AJs) are known for their roles in morphogenesis; we, therefore, examined expression of AJ components including β‐catenin, p120 catenin, and cadherins for possible involvement in sex‐specific patterning of the gonad. Results: We show that, at the time of early gonadal sex differentiation, membrane‐associated β‐catenin and p120 catenin colocalize with cell‐specific cadherins in both sex‐nonspecific and sex‐specific patterns. These expression patterns are consistent with an influence of AJs in overall patterning of the testis vs. ovary through known AJ mechanisms of cell–cell adhesion, cell sorting, and boundary formation. Conclusions: Together these complex and dynamic patterns of AJ component expression precisely mirror patterning of tissues during gonadogenesis and raise the possibility that AJs are essential effectors of patterning within the developing testis and ovary. Developmental Dynamics 241:1782–1798, 2012. © 2012 Wiley Periodicals, Inc.     

The force‐sensing device region of α‐catenin is an intrinsically disordered segment in the absence of intramolecular stabilization of the autoinhibitory form

Mechanotransduction by α‐catenin facilitates the force‐dependent development of adherens junctions (AJs) by recruiting vinculin to reinforce actin anchoring of AJs. The α‐catenin mechanotransducing action is facilitated by its force‐sensing device region that autoinhibits the vinculin‐binding site 1 (VBS1). Here, we report the high‐resolution structure of the force‐sensing device region of α‐catenin, which shows the autoinhibited form comprised of helix bundles E, F and G. The cryptic VBS1 is embedded into helix bundle E stabilized by direct interactions with the autoinhibitory region forming helix bundles F and G. Our molecular dissection study showed that helix bundles F and G are stable in solution in each isolated form, whereas helix bundle E that contains VBS1 is unstable and intrinsically disordered in solution in the isolated form. We successfully identified key residues mediating the autoinhibition and produced mutated α‐catenins that display variable force sensitivity and autoinhibition. Using these mutants, we demonstrate both in vitro and in vivo that, in the absence of this stabilization, the helix bundle containing VBS1 would adopt an unfolded form, thus exposing VBS for vinculin binding. We provide evidence for importance of mechanotransduction with the intrinsic force sensitivity for vinculin recruitment to adherens junctions of epithelial cell sheets with mutated α‐catenins.     

δ‐Catenin promotes malignant phenotype of non‐small cell lung cancer by non‐competitive binding to E‐cadherin with p120ctn in cytoplasm

As a member of the catenin family, little is known about the clinical significance and possible mechanism of δ‐catenin expression in numerous tumours. We examined the expression of δ‐catenin by immunohistochemistry in 115 cases of non‐small cell lung cancer (NSCLC) (including 65 cases with follow‐up records and 50 cases with paired lymph node metastasis lesions). The mRNA and protein expression of δ‐catenin was also detected in 30 cases of paired lung cancer tissues and normal lung tissues by RT‐PCR and western blotting, respectively. Co‐immunoprecipitation was used to examine whether δ‐catenin competitively bound to E‐cadherin with p120ctn in lung cancer cells or not. The effects of δ‐catenin on the activity of small GTPases and the biological behaviour of lung cancer cells were explored by pull‐down assay, flow cytometry, MTT, and Matrigel invasive assay. The results showed that the mRNA and protein expression of δ‐catenin was increased in lung cancer tissues; the positive expression rate of δ‐catenin was significantly increased in adenocarcinoma, stage III–IV, paired lymph node metastasis lesions, and primary tumours with lymph node metastasis (all p < 0.05); and the postoperative survival period of patients with δ‐catenin‐positive expression was shorter than that of patients with δ‐catenin‐negative expression (p < 0.05). No competition between δ‐catenin and p120ctn for binding to E‐cadherin in cytoplasm was found in two lung cancer cell lines. By regulating the activity of small GTPases and changing the cell cycle, δ‐catenin could promote the proliferation and invasion of lung cancer cells. We conclude that δ‐catenin is an oncoprotein overexpressed in NSCLC and that increased δ‐catenin expression is critical for maintenance of the malignant phenotype of lung cancer. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

β‐Catenin activation contributes to the pathogenesis of adenomyosis through epithelial–mesenchymal transition

Adenomyosis is defined by the presence of endometrial glands and stroma within the myometrium. Despite its frequent occurrence, the precise aetiology and physiopathology of adenomyosis is still unknown. WNT/β‐catenin signalling molecules are important and should be tightly regulated for uterine function. To investigate the role of β‐catenin signalling in adenomyosis, the expression of β‐catenin was examined. Nuclear and cytoplasmic β‐catenin expression was significantly higher in epithelial cells of human adenomyosis compared to control endometrium. To determine whether constitutive activation of β‐catenin in the murine uterus leads to development of adenomyosis, mice that expressed a dominant stabilized β‐catenin in the uterus were used by crossing PR‐Cre mice with Ctnnb1^f(ex3)/+ mice. Uteri of PR^cre/+ Ctnnb1^f(ex3)/+ mice displayed an abnormal irregular structure and highly active proliferation in the myometrium, and subsequently developed adenomyosis. Interestingly, the expression of E‐cadherin was repressed in epithelial cells of PR^cre/+ Ctnnb1^f(ex3)/+ mice compared to control mice. Repression of E‐cadherin is one of the hallmarks of epithelial–mesenchymal transition (EMT). The expression of SNAIL and ZEB1 was observed in some epithelial cells of the uterus in PR^cre/+ Ctnnb1^f(ex3)/+ mice but not in control mice. Vimentin and COUP‐TFII, mesenchymal cell markers, were expressed in some epithelial cells of PR^cre/+ Ctnnb1^f(ex3)/+ mice. In human adenomyosis, the expression of E‐cadherin was decreased in epithelial cells compared to control endometrium, while CD10, an endometrial stromal marker, was expressed in some epithelial cells of human adenomyosis. These results suggest that abnormal activation of β‐catenin contributes to adenomyosis development through the induction of EMT. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mucinous breast carcinoma with a lobular neoplasia component: A subset with aberrant expression of cell adhesion and polarity molecules and lack of neuroendocrine differentiation

We investigated whether some mucinous carcinomas (MUCs) are associated with lobular neoplasia (LN) components, and if so, whether this subset has any distinct biological properties. MUC specimens from 41 patients were stratified into pure and mixed types. The LN components adjacent to MUC lesions were examined histopathologically. We also tested immunohistochemically for E‐cadherin, β‐catenin, and the neuroendocrine markers chromogranin A and synaptophysin; and compared results between MUCs with and without LN. Of 41 patients with MUC, LN was detected in 12 patients (29%); LN alone was the noninvasive component in 8 patients (20%). Decreased E‐cadherin and β‐catenin expression in the MUC component was detected in 2 (17%) and 7 (58%) cases, respectively, of MUC with LN, compared with 0% (P = 0.080) and 21% (P = 0.018) in MUCs without LN. Neuroendocrine factors were frequently detected in MUCs with LN (42%) and without LN (52%), but tended to be less frequent in MUCs with only LN components (25%) than in other MUCs (55%; P = 0.133). MUCs associated with LN components appear to be a biologically characteristic subset that frequently shows decreased cell–cell adhesion, cell polarity molecules and lack of neuroendocrine differentiation.     

Requirement of the F‐actin‐binding activity of l‐afadin for enhancing the formation of adherens and tight junctions

The apical junctional complex consists of adherens junctions (AJs) and tight junctions (TJs) in polarized epithelial cells, which are attached to each other to form a sheet. Actin filaments (F‐actin) are associated with AJs and TJs and required for the formation and maintenance of this complex. l‐Afadin is an F‐actin‐binding protein, which is localized at AJs through binding to the cell adhesion molecule nectin, and regulates the formation of AJs and TJs. However, the role of the F‐actin‐binding activity of l‐afadin for the formation of the apical junctional complex remains unknown. We generated here the cultured EpH4 mouse mammary epithelial cells in which afadin was genetically ablated. In the Ca²⁺ switch assay, the formation of both AJs and TJs was markedly impaired in the afadin‐deficient cells. Re‐expression of l‐afadin in the afadin‐deficient cells fully restored the formation of both AJs and TJs, but the re‐expression of the l‐afadin mutant lacking the FAB domain did not completely restore the formation of AJs or TJs. These results indicate that the F‐actin‐binding activity of l‐afadin is required for enhancing the formation of both AJs and TJs.     

P120 连环蛋白参与细胞炎症反应的研究进展

P120 连环蛋白(p120ctn)属于犰狳重复结构域蛋白亚家族的原型成员,与内皮钙黏蛋白(E-cadherin)胞质域 结合,促进内皮细胞间黏附连接的完整性,是内皮细胞和其他极化贴壁细胞黏附连接必不可少的组成部分。此外,p120ctn 还 在胚胎发育、细胞增殖与极性、肿瘤细胞迁移和恶性肿瘤进展中起着重要的作用。近年来,研究认为p120ctn 可能是一种内源 性抗炎介质,在细胞先天免疫和感染相关的炎症反应中扮演重要作用,但相关机制仍不清楚。本文就p120ctn 与免疫炎症反 应的相关研究作一综述。     

Nectin and junctional adhesion molecule are critical cell adhesion molecules for the apico‐basal alignment of adherens and tight junctions in epithelial cells

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial‐like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin‐based cell–cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)‐based cell–cell adhesion, and cadherin and claudin were recruited to the nectin‐3‐ and JAM‐based cell–cell adhesion sites to form AJ‐like and TJ‐like domains, respectively. This inversed alignment of the AJ‐like and TJ‐like domains was reversed by complementary expression of CPMs Par‐3, atypical protein kinase C, Par‐6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico‐basal alignment of TJs and AJs in epithelial cells.     

Increased expression of delta-catenin/neural plakophilin-related armadillo protein is associated with the down-regulation and redistribution of E-cadherin and p120ctn in human prostate cancer

delta-Catenin, or neural plakophilin-related armadillo protein, is a unique armadillo domain-containing protein in that it is neural-specific and primarily expressed in the brain. However, our recent analysis of the human genome revealed a consistent association of delta-catenin messenger RNA sequences with malignant cells, although the significance of these findings was unclear. In this study, we report that a number of delta-catenin epitopes were expressed in human prostate cancer cells. Western blot and tissue microarray revealed a close association between increased delta-catenin expression and human primary prostatic adenocarcinomas. The analyses of 90 human prostate cancer and 90 benign prostate tissue samples demonstrated that an estimated 85% of prostatic adenocarcinomas showed enhanced delta-catenin immunoreactivity. delta-Catenin expression increased with prognostically significant increased Gleason scores. By analyzing the same tumor cell clusters using consecutive sections, we showed that an increased delta-catenin immunoreactivity was accompanied by the down-regulation and redistribution of E-cadherin and p120ctn, major cell junction proteins whose inactivation is frequently associated with cancer progression. Furthermore, overexpression of delta-catenin in tumorigenic CWR-R1 cells that are derived from human prostate cancer xenograft resulted in reduced immunoreactivity for E-cadherin and p120ctn at the cell-cell junction. This is the first study comparing overexpression of delta-catenin with the E-cadherin/catenin system in cancer and shows that delta-catenin may be intimately involved in regulating E-cadherin/p120ctn cell-cell adhesion in prostate cancer progression.     

p120-catenin表达下调可促进肺癌细胞的生长

目的 研究p120-catenin( p120ctn)对肺癌细胞是否有调节作用,探讨其对细胞周期是否具有调节功能.方法 应用siRNA方法建立p120ctn基因沉默的多种癌细胞系BEI,SPC和LTE的细胞克隆后,采用流式细胞术和裸鼠移植实验在活体内外观察p120ctn对肺癌细胞的生长是否具有调节作用.结果 我们发现在...     

Epithelial–mesenchymal transition and clinicopathological correlation in craniopharyngioma

Qi S‐T, Zhou J, Pan J, Zhang C, Silky C & Yan X‐R (2012) Histopathology 61 , 711–725
Epithelial–mesenchymal transition and clinicopathological correlation in craniopharyngioma Aims: To assess the immunophenotypic changes associated with epithelial–mesenchymal transition (EMT) in craniopharyngioma, especially at the tumour invasive front, and to correlate the findings with clinicopathological features and patient outcomes. Methods and results: Forty‐two craniopharyngiomas were investigated for the presence of EMT markers (vimentin, E‐cadherin and β‐catenin) by immunohistochemistry and western blot. The relationships between expression of these markers and various clinicopathological indicators and clinical outcomes of the tumours were analysed. There were statistically significant differences in the expression of vimentin and E‐cadherin–β‐catenin between adamantinomatous and papillary variants. The expression of vimentin and E‐cadherin (but not that of β‐catenin) in whole tumour sections was associated with tumour recurrence, and with postoperative weight and hypothalamic disturbances; the expression of vimentin and E‐cadherin–β‐catenin at the tumour invasive front was also associated with tumour recurrence, postoperative weight, and hypothalamic disturbances. The results from western blotting closely matched those of immunohistochemistry. Conclusions: Our study demonstrates, for the first time, the potential prognostic implications of vimentin, E‐cadherin and β‐catenin expression in craniopharyngiomas. EMT may represent a crucial mechanism in the progression of craniopharyngiomas.     

Abnormalities of E- and P-cadherin and catenin (beta-, gamma-catenin,and p120ctn) expression in endometrial cancer and endometrial atypical hyperplasia

Abnormal expression of cadherins and catenins plays a critical role in the initiation and progression of multiple human tumours. This study aimed to evaluate the immunoreactivity of E- and P-cadherin, beta- and gamma-catenin, and p120ctn in premalignant and malignant endometrial lesions and to correlate their membranous expression with clinicopathological features. In addition, we examined whether or not LOH and promoter hypermethylation of the CDH1 gene were associated with E-cadherin expression and clinicopathological variables. Finally, we studied the frequency of beta-catenin mutations in premalignant endometrial lesions. Immunohistochemical staining was performed in 21 atypical endometrial hyperplasias (AEHs), 95 endometrioid carcinomas (EECs), and 33 non-endometrioid carcinomas (NEECs). Reduced E-cadherin expression was observed in 57.8% of the cases, being more frequent in NEECs (87.1%, p = 0.001) and carcinomas of more advanced stage (85.7% of stage III-IV carcinomas, p = 0.01). LOH of CDH1 gene was found in 57.1% of NEECs but only in 22.5% of EECs (p = 0.011) and showed a trend towards association with reduced E-cadherin expression (p = 0.089). CDH1 promoter hypermethylation was found in 21.2% of endometrial carcinomas but was not associated with clinicopathological or immunohistochemical variables. Reduced expression of beta- and gamma-catenin and p120ctn was found in 76.1%, 94.3%, and 63.6% of the cases, respectively, being more frequent in lesions with reduced E-cadherin expression. In addition, beta-catenin, but not gamma-catenin or p120ctn expression, was associated with the histology of the lesion, since it was reduced in 35% of AEHs, 80.3% of EECs, and 96.9% of NEECs (p = 0.000). Mutations in exon 3 of the beta-catenin gene, associated with beta-catenin nuclear expression, were detected in 3 (14.0%) AEH, a frequency similar to that previously reported in this series of ECs. Finally, upregulation of P-cadherin was observed in 28.6% of cases. This alteration was associated with the histology of the lesion, since it was found in 9.5% of AEHs, 27.7% of EECs, and 46.2% of NEECs (p = 0.021).     

Up-regulated cytoplasmic expression, with reduced membranous distribution, of the src substrate p120(ctn) in gastric carcinoma.

p120(ctn) is a substrate of the tyrosine kinase pp60 src. Tyrosine kinases such as src localize to the adherens junctions and phosphorylate junctional proteins in both normal and transformed cells.(1) p120(ctn) forms a complex with E-cadherin at the adherens junction and is phosphorylated by ligands such as epidermal growth factor receptor as well as pp60 src. Phosphorylation of p120(ctn) has been shown to correlate with cell transformation. The aim of this study was to investigate in vivo expression of p120(ctn) in gastric carcinoma and to examine any relationship to pathological characteristics and patient survival. Immunohistochemical staining for p120(ctn) was performed in 68 gastric carcinoma specimens (19 diffuse, 49 intestinal type), in 22 lymph node metastases, and in gastric mucosal biopsies from 16 patients with gastric dysplasia and ten healthy controls. Up-regulation of p120(ctn) cytoplasmic staining was seen in six (37 per cent) of the gastric dysplasia cases and in 45 (66 per cent) tumours (89 per cent of diffuse and 57 per cent of intestinal tumours). Loss of membranous distribution of staining for p120(ctn) was seen in 22 (32 per cent) tumours (52 per cent of diffuse and 24 per cent of intestinal tumours). The staining pattern in the primary tumour showed no correlation with tumour type, grade, or stage, or patient survival. Of 22 lymph node metastases examined, 13 (60 per cent) showed loss of membranous staining. In conclusion, staining for p120(ctn) in gastric carcinoma and dysplasia revealed marked up-regulation of cytoplasmic staining, sometimes associated with reduced membranous expression. Up-regulation of expression of p120(ctn) has not previously been described in human epithelial malignancy. The significance of these findings is uncertain, but they may reflect a change in tyrosine kinase signal transduction pathways, and a role for p120(ctn) in ligand-induced mitogenic signalling and cell transformation.     

Epithelial–mesenchymal transition markers in malignant ovarian germ cell tumors

The purpose of this study was to determine the expression and potential clinical role of epithelial‐to‐mesenchymal transition (EMT)‐related factors in malignant ovarian germ cell tumors (MOGCT). Protein expression of E‐cadherin, N‐cadherin, P‐cadherin, Zeb1, HMGA2, and vimentin by immunohistochemistry was analyzed in 42 MOGCT from patients treated in Norway during the period 1981–2001. Expression was analyzed for association with clinicopathologic parameters. E‐cadherin (p = 0.016) and HMGA2 (p = 0.002) expression was significantly higher in immature teratomas and yolk sac tumors compared with dysgerminomas. Vimentin (p < 0.001) and Zeb1 (p = 0.029) staining was significantly higher in immature teratomas compared with yolk sac tumors and dysgerminomas, whereas no significant differences were observed for N‐cadherin and P‐cadherin. EMT‐associated markers were not significantly related to clinicopathologic parameters including age, tumor diameter, and FIGO stage. In conclusion, based on this limited series, EMT‐associated markers are not associated with clinical parameters in MOGCT, in contrast to ovarian carcinoma. EMT‐related proteins are differentially expressed among various MOGCT subtypes, suggesting differences in biological characteristics associated with invasion and metastasis.