SERPINB3 induces epithelial–mesenchymal transition

Epithelial–mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over‐expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial–mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E‐cadherin and an increase of β‐catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti‐protease activity of the protein, as documented by the results obtained with an active loop‐deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti‐SERPINB3 antibody in a dose‐dependent manner and SERPINB3‐transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial–mesenchymal transition, acting at both the autocrine and the paracrine level. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

I131 reinforces antitumor activity of metuximab by reversing epithelial–mesenchymal transition via VEGFR‐2 signaling in hepatocellular carcinoma

CD147 is highly expressed in hepatocellular carcinoma (HCC) and associated with the invasion and metastasis of HCC. The efficacy of I¹³¹‐metuximab (I¹³¹‐mab), a newly developed agent that targets CD147, as a radio‐immunotherapy for local HCC, has been validated in clinical practice. However, the synergistic anticancer activity and molecular mechanism of different conjugated components within I¹³¹‐mab remain unclear. In this study, the cytological experiments proved that I¹³¹‐mab inhibited the proliferation and invasion of HCC cells. Mechanically, this inhibition effect was mainly mediated by the antibody component part of I¹³¹‐mab, which could reverse the epithelial–mesenchymal transition of HCC cells partially by suppressing the phosphorylation of VEGFR‐2. The inhibitory effect of I¹³¹ on HCC cell proliferation and invasion is limited, whereas, when combined with metuximab, I¹³¹ significantly enhanced the sensitivity of HCC cells to CD147‐mab and consequently reinforced the anticancer effects of CD147‐mab, suggesting that the two components of I¹³¹‐mab exerted synergistic anti‐HCC capability. Furthermore, the experiments using SMMC‐7721 human HCC xenografts in athymic nude mice showed that I¹³¹‐mab and CD147‐mab significantly inhibited the growth of xenograft tumors and that I¹³¹‐mab was more effective than CD147‐mab. In conclusion, our results elucidated the mechanism underlying the anti‐HCC effects of I¹³¹‐mab and provided a theoretical foundation for the clinical application of I¹³¹‐mab.     

Oestrogen‐induced epithelial–mesenchymal transition of endometrial epithelial cells contributes to the development of adenomyosis

Adenomyosis is an oestrogen‐dependent disease caused by a downward extension of the endometrium into the uterine myometrium. Epithelial–mesenchymal transition (EMT) endows cells with migratory and invasive properties and can be induced by oestrogen. We hypothesized that oestrogen‐induced EMT is critical in the pathogenesis of adenomyosis. We first investigated whether EMT occurred in adenomyotic lesions and whether it correlated with serum 17β‐oestradiol (E2) levels. Immunohistochemistry was performed on adenomyotic lesions and corresponding eutopic endometrium samples from women with adenomyosis. Endometria from women without endometrial disorders were used as a control. In the epithelial component of adenomyotic lesions, vimentin expression was up‐regulated and E‐cadherin expression was down‐regulated compared to the eutopic endometrium, suggesting that EMT occurs in adenomyosis. In adenomyosis, the serum E2 level was negatively correlated with E‐cadherin expression in the epithelial components of the eutopic endometrium and adenomyotic lesions, suggesting the involvement of oestrogen‐induced EMT in endometrial cells. In oestrogen receptor‐positive Ishikawa endometrial epithelial cells, oestrogen induced a morphological change to a fibroblast‐like phenotype, a shift from epithelial marker expression to mesenchymal marker expression, increased migration and invasion, and up‐regulation of the EMT regulator Slug. Raloxifene, a selective oestrogen receptor modulator, abrogated these effects. To determine the role of oestrogen‐induced EMT in the implantation of ectopic endometrium, we xenotransplanted eutopic endometrium or adenomyotic lesions from adenomyosis patients into ovariectomized SCID mice. The implantation of endometrium was oestrogen‐dependent and was suppressed by raloxifene. Collectively, these data highlight the crucial role of oestrogen‐induced EMT in the development of adenomyosis and suggest that raloxifene may be a potential therapeutic agent for adenomyosis patients. 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.     

Tumour budding in colorectal carcinoma

The term tumour budding denotes that at the invasion front of colorectal adenocarcinomas tumour cells, singly or in small aggregates, become detached from the neoplastic glands. This morphological feature is increasingly being recognized as a strong and robust adverse prognostic factor. Biologically, tumour budding is closely related to the epithelial-mesenchymal transition. In this review the morphological features of tumour budding are discussed, as observed by the surgical pathologist reporting colorectal carcinoma resection specimens. The morphological features are put into context with the rapidly expanding knowledge of the epithelial-mesenchymal transition in general, and the molecular pathology of colorectal carcinoma in particular. Finally, a systematic analysis of the relevant published clinicopathological studies emphasizes the potential of tumour budding as a prognostic factor for routine surgical pathology.     

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.     

Muscleblind‐like 1 is a negative regulator of TGF‐β‐dependent epithelial–mesenchymal transition of atrioventricular canal endocardial cells

The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind‐like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor β‐dependent increase in epithelial–mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development. Developmental Dynamics 238:3266–3272, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of protein arginine methyltransferase‐5 in oral squamous cell carcinoma and its significance in epithelial‐to‐mesenchymal transition

Protein arginine methyltransferases (PRMT) 5, a member of type II arginine methyltransferases, catalyzes the symmetrical dimethylation of arginine residues on histone and non‐histone substrates. Although the overexpression of PRMT5 has been reported in various cancers, its role in oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, we immunohistochemically examined the expression of PRMT5 in surgically resected oral epithelial dysplasia (OED, n = 8), oral intraepithelial neoplasia (OIN)/carcinoma in situ (CIS) (n = 11) and OSCC (n = 52) with or without contiguous OED lesions. In the normal epithelium, PRMT5 was weakly expressed in the cytoplasm of basal layer cells. In OED, OIN/CIS, and OSCC, its expression consistently and uniformly increased in the cytoplasm of dysplastic and cancer cells. Moreover, nuclear and cytoplasmic localization was detected in the invasive front of cancer cells, particularly in cases showing poor differentiation or aggressive invasion patterns. The concomitant nuclear and cytoplasmic expression of PRMT5 correlated with the loss of E‐cadherin and cytokeratin 17, and the upregulation of vimentin, features that are both indicative of epithelial‐to‐mesenchymal transition. PRMT5 may play a role from early oncogenesis through to the progression of OSCC, particularly in the aggressive mode of stromal invasion.     

Micropapillary variant of urothelial carcinoma of the urinary bladder; a clinicopathological and immunohistochemical study

AIMS: To investigate whether prognosis in micropapillary urothelial carcinoma is related to the proportion of the micropapillary component (MPC), and to identify the immunohistochemical features of MPC. METHODS AND RESULTS: This study presents a clinicopathological analysis of 20 patients with micropapillary urothelial carcinoma of the bladder with cystectomy specimens for evaluation. Tumours were stratified on the extent of MPC: focal, <10%; moderate, 10-50%; extensive, >50%; and this was correlated with tumour stage and prognosis. Sixteen males and four females were aged 56-81 years (mean 69 years). All cases had high-grade morphology in the micropapillary carcinoma and typical urothelial carcinoma. All cases with extensive MPC (n = 4) were of a high pathological stage (pT3 or pT4) and died of disease (DOD) or other causes. Eighty percent with moderate MPC (eight of 10 cases) were pT3 or pT4 and 50% DOD or are alive with disease. Eighty-four percent with focal MPC (five of six cases) were pT1 or pTa. In high-stage cases, the most invasive component was MPC. High-stage cases had an 85% risk of being advanced at presentation with micropapillary carcinoma. All pT2 or lower stage cases had micropapillary carcinoma on prior transurethral resections of bladder tumour (TURB). High-stage carcinomas had 30% and 54%, respectively, of surface MPC and urothelial carcinoma in situ, in comparison with 85% and 28% in lower stage carcinomas. Immunohistochemical staining was similarly positive in MPC and typical urothelial carcinoma with cytokeratin (CK)7, CK20, epithelial membrane antigen, carcinoembryonic antigen and cytokeratin 34betaE12. CA125 staining was seen only in MPC in 43% of cases. CONCLUSIONS: Micropapillary urothelial carcinoma is a high-grade carcinoma in which the prognosis is related to the proportion and location of the MPC. Cases with moderate or extensive MPC are at high risk of being advanced at presentation. Cases with <10% MPC and surface MPC have a high chance of detection at an early stage. The morphology and immunohistochemical profile of the MPC suggest that it is a form of glandular differentiation in urothelial carcinoma.