Corrigendum: FXYD5/Dysadherin, a biomarker of endometrial cancer myometrial invasion and aggressiveness: its relationship with TGF-ß1 and NF-κB pathways.

[This corrects the article DOI: 10.3389/fonc.2019.01306.].

Ephrin-B1 Is a Novel Biomarker of Bladder Cancer Aggressiveness. Studies in Murine Models and in Human Samples.

Bladder cancer (BC) is the ninth most common cancer worldwide, but molecular changes are still under study. During tumor progression, Epithelial cadherin (E-cadherin) expression is altered and ß-catenin may be translocated to the nucleus, where it acts as co-transcription factor of tumor invasion associated genes. This investigation further characterizes E-cadherin and ß-catenin associated changes in BC, by combining bioinformatics, an experimental murine cell model (MB49/MB49-I) and human BC samples. In in silico studies, a DisGeNET (gene-disease associations database) analysis identified CDH1 (E-cadherin gene) as one with highest score among 130 BC related-genes. COSMIC mutation analysis revealed CDH1 low mutations rates. Compared to MB49 control BC cells, MB49-I invasive cells showed decreased E-cadherin expression, E- to P-cadherin switch, higher ß-catenin nuclear signal and lower cytoplasmic p-Ser33-ß-catenin signal, higher Ephrin-B1 ligand and EphB2 receptor expression, higher Phospho-Stat3 and Urokinase-type Plasminogen Activator (UPA), and UPA receptor expression. MB49-I cells transfected with Ephrin-B1 siRNA showed lower migratory and invasive capacity than control cells (scramble siRNA). By immunohistochemistry, orthotopic MB49-I tumors had lower E-cadherin, increased nuclear ß-catenin, lower pSer33-ß-catenin cytoplasmic signal, and higher Ephrin-B1 expression than MB49 tumors. Similar changes were found in human BC tumors, and 83% of infiltrating tumors depicted a high Ephrin-B1 stain. An association between higher Ephrin-B1 expression and higher stage and tumor grade was found. No association was found between abnormal E-cadherin signal, Ephrin-B1 expression or clinical-pathological parameter. This study thoroughly analyzed E-cadherin and associated changes in BC, and reports Ephrin-B1 as a new marker of tumor aggressiveness.

FXYD5/Dysadherin, a Biomarker of Endometrial Cancer Myometrial Invasion and Aggressiveness: Its Relationship With TGF-ß1 and NF-κB Pathways.

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models. Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-ß1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid. Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-ß1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-ß1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-ß1 mRNA levels and shorter survival rates. Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-ß1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.

Characterization of the molecular changes associated with the overexpression of a novel epithelial cadherin splice variant mRNA in a breast cancer model using proteomics and bioinformatics approaches: identification of changes in cell metabolism and an increased expression of lactate dehydrogenase B.

BACKGROUND: Breast cancer (BC) is the most common female cancer and the leading cause of cancer death in women worldwide. Alterations in epithelial cadherin (E-cadherin) expression and functions are associated to BC, but the underlying molecular mechanisms have not been fully elucidated. We have previously reported a novel human E-cadherin splice variant (E-cadherin variant) mRNA. Stable transfectants in MCF-7 human BC cells (MCF7Ecadvar) depicted fibroblast-like cell morphology, E-cadherin wild-type downregulation, and other molecular changes characteristic of the epithelial-to-mesenchymal transition process, reduced cell-cell adhesion, and increased cell migration and invasion. In this study, a two-dimensional differential gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS) protein identification and bioinformatics analyses were done to characterize biological processes and canonical pathways affected by E-cadherin variant expression. RESULTS: By 2D-DIGE and MS analysis, 50 proteins were found differentially expressed (≥ Δ1.5) in MCF7Ecadvar compared to control cells. Validation of transcript expression was done in the ten most overexpressed and underexpressed proteins. Bioinformatics analyses revealed that 39 of the 50 proteins identified had been previously associated to BC. Moreover, metabolic processes were the most affected, and glycolysis the canonical pathway most altered. The lactate dehydrogenase B (LDHB) was the highest overexpressed protein, and transcript levels were higher in MCF7Ecadvar than in control cells. In agreement with these findings, MCF7Ecadvar conditioned media had lower glucose and higher lactate levels than control cells. MCF7Ecadvar cell treatment with 5 mM of the glycolytic inhibitor 2-deoxy-glucose led to decreased cell viability, and modulation of LDHB expression in MCF7Ecadvar cells with a specific small interfering RNA resulted in decreased cell proliferation. Finally, a positive association between expression levels of the E-cadherin variant and LDHB transcripts was demonstrated in 21 human breast tumor tissues, and breast tumor samples with higher Ki67 expression showed higher LDHB mRNA levels. CONCLUSIONS: Results from this investigation contributed to further characterize molecular changes associated to the novel E-cadherin splice variant expression in BC cells. They also revealed an association between expression of the novel variant and changes related to BC progression and aggressiveness, in particular those associated to cell metabolism.

E-cadherin: A determinant molecule associated with ovarian cancer progression, dissemination and aggressiveness.

Ovarian cancer (OC) is the fifth cancer death cause in women worldwide. The malignant nature of this disease stems from its unique dissemination pattern. Epithelial-to-mesenchymal transition (EMT) has been reported in OC and downregulation of Epithelial cadherin (E-cadherin) is a hallmark of this process. However, findings on the relationship between E-cadherin levels and OC progression, dissemination and aggressiveness are controversial. In this study, the evaluation of E-cadherin expression in an OC tissue microarray revealed its prognostic value to discriminate between advanced- and early-stage tumors, as well as serous tumors from other histologies. Moreover, E-cadherin, Neural cadherin (N-cadherin), cytokeratins and vimentin expression was assessed in TOV-112, SKOV-3, OAW-42 and OV-90 OC cell lines grown in monolayers and under anchorage-independent conditions to mimic ovarian tumor cell dissemination, and results were associated with cell aggressiveness. According to these EMT-related markers, cell lines were classified as mesenchymal (M; TOV-112), intermediate mesenchymal (IM; SKOV-3), intermediate epithelial (IE; OAW-42) and epithelial (E; OV-90). M- and IM-cells depicted the highest migration capacity when grown in monolayers, and aggregates derived from M- and IM-cell lines showed lower cell death, higher adhesion to extracellular matrices and higher invasion capacity than E- and IE-aggregates. The analysis of E-cadherin, N-cadherin, cytokeratin 19 and vimentin mRNA levels in 20 advanced-stage high-grade serous human OC ascites showed an IM phenotype in all cases, characterized by higher proportions of N- to E-cadherin and vimentin to cytokeratin 19. In particular, higher E-cadherin mRNA levels were associated with cancer antigen 125 levels more than 500 U/mL and platinum-free intervals less than 6 months. Altogether, E-cadherin expression levels were found relevant for the assessment of OC progression and aggressiveness.

Identification of a Novel Human E-Cadherin Splice Variant and Assessment of Its Effects Upon EMT-Related Events.

Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368-1386, 2017. © 2016 Wiley Periodicals, Inc.

Secretory Leukocyte Protease Inhibitor (SLPI) expression downregulates E-cadherin, induces ß-catenin re-localisation and triggers apoptosis-related events in breast cancer cells.

BACKGROUND INFORMATION: Epithelial cadherin (E-cadherin) is involved in cell-cell adhesion through its extracellular domain, whereas the intracellular domain interacts with adaptor proteins, i.e. ß-catenin, links E-cadherin to the actin cytoskeleton and participates in signal transduction events. E-cadherin protects mammary epithelial cells from apoptosis and its loss during tumour progression has been documented. Secretory Leukocyte Protease Inhibitor (SLPI) has anti- and pro-tumourigenic activities but its role in breast cancer has not been fully elucidated. Notwithstanding its relevance, how SLPI affects E-cadherin in breast cancer is still unknown. This study evaluated the effect of SLPI upon E-cadherin/ß-catenin expression and apoptosis-related markers in murine (F3II) and human (MCF-7) breast tumour cells either treated with exogenous recombinant human SLPI (rhSLPI) or stably transfected with a plasmid encoding its sequence. RESULTS: Addition of rhSLPI to F3II cells caused a decrease (P < 0.05) in E-cadherin transcript and protein levels. Similar results were observed in SLPI-stable F3II transfectants (2C1), and treatment of 2C1 cells with a siRNA toward SLPI restored E-cadherin to control levels. SLPI-expressing cells showed disruption of E-cadherin/ß-catenin complex and increased (P < 0.05) percentage of cells depicting nuclear ß-catenin localisation. Associated to these changes, 2C1 cells showed increased Bax/Bcl-2 ratio and p21 protein levels, decreased c-Myc protein levels and decreased Cyclin D1 and Claudin-1 transcript levels. No differences in N- and P-cadherin were observed between SLPI-transfected cells and controls. Addition of rhSLPI to MCF-7 cells or stable transfection with SLPI caused a decrease (P < 0.05) in E-cadherin expression (transcript/protein) and its redistribution to the cytoplasm, as well as ß-catenin re-localisation to the cell nucleus. CONCLUSIONS: Expression of SLPI was associated to a decrease in E-cadherin expression and re-localisation of E-cadherin to the cell cytoplasm and ß-catenin to the cell cytoplasm and nucleus, and had pro-apoptotic and cell cycle-arrest effects.

Sex differences in the pituitary transforming growth factor-ß1 system: studies in a model of resistant prolactinomas.

Dopamine and estradiol interact in the regulation of lactotroph cell proliferation and prolactin secretion. Ablation of the dopamine D2 receptor gene (Drd2(-/-)) in mice leads to a sexually dimorphic phenotype of hyperprolactinemia and pituitary hyperplasia, which is stronger in females. TGF-ß1 is a known inhibitor of lactotroph proliferation. TGF-ß1 is regulated by dopamine and estradiol, and it is usually down-regulated in prolactinoma experimental models. To understand the role of TGF-ß1 in the gender-specific development of prolactinomas in Drd2(-/-) mice, we compared the expression of different components of the pituitary TGF-ß1 system, including active cytokine content, latent TGF-ß-binding protein isoforms, and possible local TGF-ß1 activators, in males and females in this model. Furthermore, we evaluated the effects of dopamine and estradiol administration to elucidate their role in TGF-ß1 system regulation. The expression of active TGF-ß1, latent TGF-ß-binding protein isoforms, and several putative TGF-ß1 activators evaluated was higher in male than in female mouse pituitary glands. However, Drd2(-/-) female mice were more sensitive to the decrease in active TGF-ß1 content, as reflected by the down-regulation of TGF-ß1 target genes. Estrogen and dopamine caused differential regulation of several components of the TGF-ß1 system. In particular, we found sex- and genotype- dependent regulation of active TGF-ß1 content and a similar expression pattern for 2 of the putative TGF-ß1 activators, thrombospondin-1 and kallikrein-1, suggesting that these proteins could mediate TGF-ß1 activation elicited by dopamine and estradiol. Our results indicate that (1) the loss of dopaminergic tone affects the pituitary TGF-ß1 system more strongly in females than in males, (2) males express higher levels of pituitary TGF-ß1 system components including active cytokine, and (3) estradiol negatively controls most of the components of the system. Because TGF-ß1 inhibits lactotroph proliferation, we propose that the higher levels of the TGF-ß1 system in males could protect or delay the development of prolactinomas in Drd2(-/-) male mice.

Histone deacetylase inhibitors impair NK cell viability and effector functions through inhibition of activation and receptor expression.

HDACi are being used as a novel, therapeutic approach for leukemias and other hematological malignancies. However, their effect on immune cells remains ill-defined, as HDACi may impair immune surveillance. In this work, we demonstrate that TSA, VPA, and NaB inhibited IFN-γ production by CD56(dim) and CD56(bright) NK cells and NK cell-mediated cytotoxicity against K562 target cells. HDACi promoted minor NK cell apoptosis but inhibited nuclear mobilization of NF-κB p50, which was accompanied by a robust down-regulation of NKG2D and NKp46 on resting NK cells and of NKG2D, NKp44, NKp46, and CD25 on cytokine-activated NK cells. Decreased CD25 expression promoted a weakened IFN-γ secretion upon restimulation of NK cells with IL-2, whereas reduced expression of NKG2D and NKp46 was accompanied by an impaired NKG2D- and NKp46-dependent cytotoxicity. Moreover, NK cells from normal mice treated in vivo with TSA displayed a diminished expression of NK1.1, NKG2D, and NKp46 and secreted reduced amounts of IFN-γ upon ex vivo stimulation with cytokines. Thus, our preclinical results indicate that HDACi exert deleterious effects on NK cell function, which may weaken immune surveillance and facilitate relapse of the malignant disease in HDACi-treated patients.

Expression analysis of epithelial cadherin and related proteins in IBH-6 and IBH-4 human breast cancer cell lines.

Epithelial cadherin (E-cadherin) is a 120 kDa cell-cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as beta-catenin. Loss of E-cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down-regulation of E-cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH-6 and IBH-4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E-cadherin and related proteins in IBH-6 and IBH-4 cell lines was evaluated. In IBH-6 and IBH-4 cell extracts, only an 89 kDa E-cadherin form (Ecad89) was detected, which is truncated at the C-terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E-cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH-6 and IBH-4 cells showed an intracellular scattered E-cadherin localization. beta-catenin accompanied E-cadherin localization, and actin stress fibers were identified in both cell types. E-cadherin mRNA levels were remarkably low in IBH-6 and IBH-4 cells. The E-cadherin mRNA and genomic sequence encoding exons 14-16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up-regulation of Twist mRNA was found in both cell lines. In conclusion, IBH-6 and IBH-4 breast cancer cells show down-regulation of E-cadherin expression with aberrant protein localization, and up-regulation of Twist; these features can be related to their invasive/metastatic characteristics.