E-cadherin In Breast Cancer Research Articles

E-Cadherin Induces Serine Synthesis to Support Progression and Metastasis of Breast Cancer.

The loss of E-cadherin, an epithelial cell adhesion molecule, has been implicated in metastasis by mediating the epithelial-mesenchymal transition, which promotes invasion and migration of cancer cells. However, recent studies have demonstrated that E-cadherin supports the survival and proliferation of metastatic cancer cells. Here, we identified a metabolic role for E-cadherin in breast cancer by upregulating the de novo serine synthesis pathway (SSP). The upregulated SSP provided metabolic precursors for biosynthesis and resistance to oxidative stress, enabling E-cadherin+ breast cancer cells to achieve faster tumor growth and enhanced metastases. Inhibition of phosphoglycerate dehydrogenase, a rate-limiting enzyme in the SSP, significantly and specifically hampered proliferation of E-cadherin+ breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. These findings reveal that E-cadherin reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers. Significance: E-Cadherin promotes the progression and metastasis of breast cancer by upregulating the de novo serine synthesis pathway, offering promising targets for inhibiting tumor growth and metastasis in E-cadherin-expressing tumors.

Microrchidia 2/Histone Deacetylase 1 Complex Regulates E-cadherin Gene Expression and Function.

Although Microrchidia 2 (MORC2) is widely overexpressed in human malignancies and linked to cancer cell proliferation, metabolism, and metastasis, the mechanism of action of MORC2 in cancer cell migration and invasion is yet undeciphered. Here, we identified for the first time that MORC2, a chromatin remodeler, regulates E-cadherin expression and, subsequently regulates breast cancer cell migration and invasion. We observed a negative correlation between the expression levels of MORC2 and E-cadherin in breast cancer. Further, the overexpression of MORC2 resulted in decreased expression levels of E-cadherin. In addition, coimmunoprecipitation and chromatin immunoprecipitation assays revealed that MORC2 interacts with HDAC1 and gets recruited onto the E-cadherin promoter to inhibit its transcription, thereby suppress its expression. Consequently, knockdown of HDAC1 in MORC2-overexpressing cells led to reduced cancer cell migration and invasion. Interestingly, we noticed that MORC2 regulated glucose metabolism via c-Myc, and LDHA, also modulates the expression of E-cadherin. Collectively, these results demonstrate for the first time a mechanistic role for MORC2 as an upstream regulator of E-cadherin expression and its associated functions in breast cancer.

MicroRNA-543 inhibits the proliferation, migration, invasion, and epithelial-mesenchymal transition of triple-negative breast cancer cells via down-regulation of ACTL6A gene.

To investigate the effect of microRNA-543 (miR-543) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of triple-negative breast cancer (TNBC) cells, and the associated mechanism. Human breast cancer cells (MDA-MB-231, HCC1937, and MCF-7, ZR-75-1) and normal human breast epithelial cell line (MCF10A) were transfected with miR-543 mimics or inhibitor using lipofectamine 2000. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the mRNA and protein expression levels of miR-543, actin-like protein 6A (ACTL6A), vimentin, Snail, and E-cadherin in breast cancer cells/tissue. Cell counting kit-8 (CCK-8), wound-healing, and Transwell assays were used to measure the effect of miR-543 on TNBC cell proliferation, invasion, and migration. Overall survival was determined using data from Gene Expression Omnibus (GEO) and Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis and luciferase reporter gene assay were used to determine the regulatory effect of miR-543 on ACTL6A. The level of expression of miR-543 was significantly lower in breast cancer cells/tissue than in normal human breast epithelial cell/tissue (p < 0.05). MicroRNA-543 expression level was significantly reduced in TNBC cells/tissue, relative to the other breast cancer cells/normal breast tissue (p < 0.05). MicroRNA-543 significantly suppressed tumor growth and the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells, in mouse xenograft model (p < 0.05). miR-543 influences the biological behavior of TNBC cells by directly targeting ACTL6A gene. miR-543 could serve as a novel diagnostic and therapeutic target for TNBC.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Characterizing the invasion of different breast cancer cell lines with distinct E-cadherin status in 3D using a microfluidic system

E-cadherin is a cell-cell adhesion protein that plays a prominent role in cancer invasion. Inactivation of E-cadherin in breast cancer can arise from gene promoter hypermethylation or genetic mutation. Depending on their E-cadherin status, breast cancer cells adopt different morphologies with distinct invasion modes. The tumor microenvironment (TME) can also affect the cell morphology and invasion mode. In this paper, we used a previously developed microfluidic system to quantify the three-dimensional invasion of breast cancer cells with different E-cadherin status, namely MCF-7, CAMA-1 and MDA-MB-231 with wild type, mutated and promoter hypermethylated E-cadherin, respectively. The cells migrated into a stable and reproducible microfibrous polycaprolactone mesh in the chip under a programmed stable chemotactic gradient. We observed that the MDA-MB-231 cells invaded the most, as single cells. MCF-7 cells collectively invaded into the matrix more than CAMA-1 cells, maintaining their E-cadherin expression. The CAMA-1 cells exhibited multicellular multifocal infiltration into the matrix. These results are consistent with what is seen in vivo in the cancer biology literature. In addition, comparison between complete serum and serum gradient conditions showed that the MDA-MB-231 cells invaded more under the serum gradient after one day, however this behavior was inverted after 3 days. The results showcase that the microfluidic system can be used to quantitatively assess the invasion behavior of cancer cells with different E-cadherin expression, for a longer period than conventional invasion models. In the future, it can be used to quantitatively investigate effects of matrix structure and cell treatments on cancer invasion.

MiRNA-221-5p promotes breast cancer progression by regulating E-cadherin expression.

To elucidate the role of miRNA-221-5p in the development of breast cancer (BCa) and its underlying mechanism. The expression level of miRNA-221-5p in 52 pairs of BCa tissues and adjacent normal tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between miRNA-221-5p expression and pathological indicators of BCa was analyzed. MiRNA-221-5p expression in BCa cells was also determined by qRT-PCR. After transfection of miRNA-221-5p inhibitor in MCF-7 and SKBR3 cells, we detected the regulatory effects of miRNA-221-5p on cellular behaviors through cell counting kit-8 (CCK-8), wound healing, and transwell assay. Finally, the relationship between miRNA-221-5p and E-cadherin in BCa was elucidated. QRT-PCR results showed that the expression level of miRNA-221-5p in BCa tissues was markedly higher than that in normal tissues. Compared with BCa patients with low expression of miRNA-221-5p, those with high expression had a higher incidence of lymph node metastasis and distant metastasis. However, miRNA-221-5p expression did not correlate with age and sex of BCa patients. MiRNA-221-5p was also highly expressed in BCa cells. Transfection of miRNA-221-5p inhibitor suppressed proliferative, invasive, and migratory potentials of BCa cells. Subsequently, we verified that E-cadherin was lowly expressed in BCa cells, and negatively correlated with miRNA-221-5p. In addition, rescue experiments confirmed that transfection of si-E-cadherin reversed the inhibitory role of miRNA-221-5p knockdown in migratory and invasive potentials of BCa cells. The expression of miRNA-221-5p remained high in BCa, which was correlated with lymph node metastasis, distant metastasis, and poor prognosis of BCa. MiRNA-221-5p may promote the invasive and migratory potentials of BCa by regulating E-cadherin expression.

KIF11 Functions as an Oncogene and Is Associated with Poor Outcomes from Breast Cancer

PurposeThe study aimed to search and identify genes that were differentially expressed in breast cancer, and their roles in cancer growth and progression.Materials and MethodsThe Gene Expression Omnibus (Oncomine) and The Cancer Genome Atlas databases (https://cancergenome.nih.gov/) were screened for genes that were expressed differentially in breast cancer and were closely related to a poor prognosis. Gene expressions were verified by quantitative real-time polymerase chain reaction, and genes were knocked down by a lentivirus-based system. Cell growth and motility were evaluated and in vivo nude mice were used to confirm the in vitro roles of genes. Markers of epithelial-to-mesenchymal transition and the associations of KIF11 with the classical cancer signaling pathways were detected by Western blot.ResultsA series of genes expressed differentially in patients with breast cancer. The prognosis associated with high KIF11 expression was poor, and the expression of KIF11 increased significantly in high stage and malignant tumor cells. Inhibiting KIF11 expression in lentivirus-suppressed cells revealed that KIF11 inhibition significantly reduced cell viability and colony formation, inhibited migration and invasion, but promoted apoptosis. The sizes and weights of KIF11-inhibited tumors in nude mice were significantly lower than in the negative controls. Western blot showed that E-cadherin in breast cancer was significantly upregulated in KIF-inhibited cells and tumor tissues, whereas N-cadherin and vimentin were significantly downregulated. BT549 and MDA231 cells with KIF11 knockdown exhibited decreased ERK, AMPK, AKT, and CREB phosphorylation.ConclusionKIF11 acts as a potential oncogene that regulates the development and progression of breast cancer.

LncRNA MEG3 inhibits cell epithelial-mesenchymal transition by sponging miR-421 targeting E-cadherin in breast cancer

BackgroundMEG3, a lncRNA, has been verified in several tumors to function as tumor suppressors including breast cancer development and progression, however, the expression pattern and underlying mechanisms of MEG3 involved in breast cancer progression is still need to be further explored. MethodsThe expression of MEG3 was confirmed in 90 cases of breast cancer tissues compared to adjacent normal tissues by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The association between clinicopathological factors and MEG3 expression was evaluated by chi-square test. Kaplan–Meier curve and log rank test was performed to assess disease-free survival (DFS) and overall survival (OS) time in patients. CCK8 and transwell invasion assays were used to assess cell proliferation and invasion capacity. Luciferase report assay and RNA pull down assay were used to detect the association between miR-421 and MEG3 in breast cancer. ResultsIn the study, we demonstrated that the expression of MEG3 was significantly down-regulated in breast cancer tissues compared to adjacent normal tissues. Reducing MEG3 expression was significantly associated with TNM stage and lymph nodes metastasis in patients. Survival analysis showed that lower MEG3 predicted a poor DFS and OS for patients. In vitro, we showed that up-regulated MEG3 inhibited cell proliferation and cell invasion capacities. We further revealed that endogenous miR-421 expression was negatively regulated by MEG3 in breast cancer cells and MEG3 regulated E-cadherin expression by sponging to miR-421 in breast cancer cells. ConclusionsOur results showed that MEG3/miR-421/E-cadherin regulatory axis may be a novel therapeutic target for breast cancer.

Abstract P6-01-17: Epithelial paradox; clinical significance of co-expression of E-cadherin and vimentin in the invasion and the metastasis of breast cancer

Abstract Background: E-cadherin and vimentin are now regarded as major and conventional canonical markers of epithelial-mesenchymal transition (EMT). It is commonly assumed E-cadherin is uniformly lost during the process of EMT. We previously reported that the elevated expression of vimentin contributes to the aggressive phenotype in invasive breast cancer. On the other hand, the role of E-cadherin in breast cancer biology might be unclear and more complex. Although, cell cohesion during breast cancer invasion is often overlooked, accumulating evidences indicate breast tumor cells are typically cohesive and often display membrane-localized E-cadherin in both the primary tumor and distant metastases, termed collective invasion. Multiple mechanisms have emerged to address how epithelial breast tumors invade. Aims: The aim of this study is to reveal the clinical importance of the expression pattern of E-cadherin and vimentin in breast cancer. Methods: The E-cadherin and vimentin protein expression were evaluated by immunohistochemistry (IHC) in 177 invasive breast cancer samples. Among these, E-cadherin and vimentin expression were evaluated in the set of primary breast cancer and metastatic lymph nodes in 65 cases. In addition, E-cadherin and vimentin expression were analyzed by immunofluorescent staining and evaluated using confocal laser scanning microscopy to see E-cadherin and vimentin localization in the breast cancer cells. Results: The positive vimentin expression was highly correlated with poor disease-free survival (DFS) and overall survival (OS) (p=0.019 and p=0.0044), however, the E-cadherin expression alone did not correlate with prognosis. Interestingly, both E-cadherin and vimentin positive tumors had the worst DFS and OS among all breast cancer (p=0.03 and p=0.0089). Vimentin expression was highly correlated between primary tumors and metastatic lymph nodes. However, E-cadherin expression levels were significantly elevated in metastatic lymph nodes (p=0.0017). Co-expression of E-cadherin and vimentin in the metastatic lymph nodes also showed worst DFS and OS (p=0.12 and p=0.027).Immunofluorescent analysis revealed that E-cadherin and vimentin were co-localized within the same tumor cells in many of the E-cadherin high/vimentin positive tumors. Conclusions: Co-expression of E-cadherin and vimentin seems to be associated with the most aggressive phenotype and poorest prognosis in breast cancer. Moreover, co-localization of E-cadherin and vimentin within the same breast cancer cells suggests the significance of the expressions of both proteins in breast cancer invasion and metastasis. Citation Format: Yamashita N, Tokunaga E, Inoue Y, Tanaka K, Saeki H, Oki E, Maehara Y. Epithelial paradox; clinical significance of co-expression of E-cadherin and vimentin in the invasion and the metastasis of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-17.

Genomic and Prognostic associations of E-cadherin in breast cancer: an immunohistochemical study of 3273 patients, systematic review and meta-analysis

Genomic and Prognostic associations of E-cadherin in breast cancer: an immunohistochemical study of 3273 patients, systematic review and meta-analysis

Abstract P2-05-12: Epithelial paradox; clinical significance of co-expression of E-cadherin and vimentin in invasive breast cancer

Abstract Background: E-cadherin and vimentin are now regarded as major and conventional canonical markers of epithelial-mesenchymal transition (EMT). It is commonly assumed E-cadherin is uniformly lost during the process of EMT. We previously reported that the elevated expression of vimentin contributes to the aggressive phenotype in invasive breast cancer. On the other hand, the role of E-cadherin in breast cancer biology might be unclear and more complex. Although, cell cohesion during breast cancer invasion is often overlooked, accumulating evidences indicate breast tumor cells are typically cohesive and often display membrane-localized E-cadherin in both the primary tumor and distant metastases, termed collective invasion. Multiple mechanisms have emerged to address how epithelial breast tumors invade. Aims: The aim of this study is to reveal the clinical importance of the expression of E-cadherin and vimentin in breast cancer. Methods: The E-cadherin and vimentin protein expression were evaluated by immunohistochemistry (IHC) in 177 invasive breast cancer samples. Among these, E-cadherin and vimentin expression were evaluated in the set of primary breast cancer and metastatic lymph nodes in 65 cases. Results: The positive vimentin expression was highly correlated with poor disease-free survival (DFS) and overall survival (OS) (p=0.019 and p=0.0044), however, the E-cadherin expression alone did not correlate with prognosis. Interestingly, Both E-cadherin and vimentin positive tumor had the worst DFS and OS among all breast cancer (p=0.03 and p=0.0089). Vimentin expression was highly correlated between primary tumors and metastatic lymph nodes. However, E-cadherin expression levels were significantly elevated in metastatic lymph nodes (p=0.0017), Co-expression of E-cadherin and vimentin in the metastatic lymph nodes also showed worst DFS and OS (p=0.12 and p=0.027). Conclusions: Co-expression of E-cadherin and vimentin seems to be associated with the most aggressive phenotype and poorest prognosis in breast cancer, and positive E-cadherin expression may not always play roles for tumor suppression. Citation Format: Yamashita N, Tokunaga E, Inoue Y, Tanaka K, Ueo H, Saeki H, Oki E, Maehara Y. Epithelial paradox; clinical significance of co-expression of E-cadherin and vimentin in invasive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-12.

Clinical significance of co-expression of E-cadherin and vimentin in invasive breast cancer.

e22013 Background: E-cadherin has been categorized as a tumor suppressor and its downregulation is considered to occur in the process of epithelial-mesenchymal transition (EMT). E-cadherin and vimentin are now regarded as major and conventional canonical markers of EMT. We previously reported that the elevated expression of vimentin contributes to the aggressive phenotype in invasive breast cancer. However, the role of E-cadherin in breast cancer biology might be unclear and more complex. The aim of this study is to reveal the clinical importance of the expression of E-cadherin and vimentin in breast cancer. Methods: The E-cadherin and vimentin protein expression were evaluated by immunohistochemistry (IHC) in 176 invasive breast cancer samples. Among these, E-cadherin and vimentin expression were evaluated in the set of primary breast cancer and metastatic lymph nodes in 65 cases. Results: The positive vimentin expression was highly correlated with poor disease-free survival (DFS) and overall survival (OS) (p = 0.019 and p = 0.0044), however, the E-cadherin expression alone did not correlate with prognosis. Interestingly, Both E-cadherin and vimentin positive tumor had the worst DFS and OS among all breast cancer (p = 0.03 and p = 0.0089). Vimentin expression was highly correlated between primary tumors and metastatic lymph nodes. However, E-cadherin expression levels were significantly elevated in metastatic lymph nodes (p = 0.0017), Co-expression of E-cadherin and vimentin in the metastatic lymph nodes also showed worst DFS and OS (p = 0.12 and p = 0.027). Conclusions: Co-expression of E-cadherin and vimentin seems to be associated with the most aggressive phenotype and poor prgnosis in breast cancer, and positive E-cadherin expression may not always play roles for tumor suppression.

DACH1 inhibits SNAI1-mediated epithelial-mesenchymal transition and represses breast carcinoma metastasis.

Epithelial–mesenchymal transition (EMT) has a major role in cancer progression and metastasis. However, the specific mechanism of transcriptional repression involved in this process remains largely unknown. Dachshund homologue 1 (DACH1) expression is lost in invasive breast cancer with poor prognosis, and the role of DACH1 in regulating breast cancer metastasis is poorly understood. In this study, significant correlation between the expression of DACH1 and the morphology of breast cancer cells was observed. Subsequent investigation into the relationship between DACH1 and EMT showed that overexpression of DACH1 in ZR-75-30 cells induced a shift towards epithelial morphology and cell–cell adhesion, as well as increased the expression of the epithelial marker E-cadherin and suppressed cell migration and invasion. In contrast, silencing DACH1 in MCF-7 and T47D cells disrupted the epithelial morphology and cell–cell contact, reduced the expression of E-cadherin, and induced cell migration and invasion. DACH1 also specifically interacted with SNAI1, but not SNAI2, to form a complex, which could bind to the E-box on the E-cadherin promoter in an SNAI1-dependent manner. DACH1 inhibited the transcriptional activity of SNAI1, leading to the activation of E-cadherin in breast cancer cells. Furthermore, the level of DACH1 also correlated with the extent of metastasis in a mouse model. DACH1 overexpression significantly decreased the metastasis and growth of 4T1/Luc cells in BALB/c mice. Analysis of tissue samples taken from human breast cancers showed a significant correlation between the expression of DACH1 and E-cadherin in SNAI1-positive breast cancer. Collectively, our data identified a new mechanistic pathway for the regulation of EMT and metastasis of breast cancer cells, one that is based on the regulation of E-cadherin expression by direct DACH1–SNAI1 interaction.

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

Epithelial cadherin (E-cadherin) is involved in cell-cell adhesion through its extracellular domain, whereas the intracellular domain interacts with adaptor proteins, i.e. β-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/β-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. 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/β-catenin complex and increased (P < 0.05) percentage of cells depicting nuclear β-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 β-catenin re-localisation to the cell nucleus. Expression of SLPI was associated to a decrease in E-cadherin expression and re-localisation of E-cadherin to the cell cytoplasm and β-catenin to the cell cytoplasm and nucleus, and had pro-apoptotic and cell cycle-arrest effects.

Expression of protein regenerating liver-3 (PRL-3) and E-Cadherin in metastatic breast cancer

<h3>Background</h3> Breast cancer is the most common malignant neoplasm worldwide. Recently, PRL-3 (phosphatase of regenerating liver-3/PTP4A3) was reported to participate in the progression of breast cancer, but the mechanism still needs clarification. <h3>Aims</h3> To investigate the expression of PRL-3 and E-Cadherin in breast cancer and metastatic tumor and correlate with the clini-copathologic parameters. <h3>Methods</h3> Expressions of PRL-3 and E-Cadherin in 114 primary breast cancer specimens and 73 lymph node metastasis were detected by immunohistochemistry. <h3>Results</h3> Among primary breast cancers examined, there were 11 (9.6%) low grade, 70 (61.4%) moderate grade and 33 (28.9%) high grade malignancy. There was significant correlation between PRL-3 expression and histological grading of breast cancer (<i>p</i>=0.044) and the presence of LN metastasis (<i>p</i><0.001). The significant correlation was also found between expression of E-cadherin and the presence of LN metastasis (<i>p</i>=0.001). The expression of PRL-3 in primary site was significantly correlated with metastatic site (<i>p</i><0.001). Then, the result showed that the higher the PRL-3 expression the lower the expression of E-cadherin in breast cancer. <h3>Conclusions</h3> These results strongly suggest that PRL-3 plays a role in the progression and metastasis of breast cancer. PRL-3 may down-regulate the expression of E-cadherin and induce metastatic ability in breast cancer.

Abstract P3-05-08: Analysis of the expression of claudin-3, -4, -7 and E-cadherin in breast cancer: are they surrogates for the claudin-low subtype?

Abstract Introduction: The molecular architecture of tight junctions (TJ) has been a subject of extensive studies in a number of malignancies. Among the TJ components, the members of the protein family claudins (CLDN) have been found to be differentially expressed correlating with histological grade (HG) in a number of tissues. Furthermore, the expression of these junctional proteins (JP) have recently been implied in the identification of the claudin-low subtype by gene expression analysis. Material and methods: 261 breast cancer specimens diagnosed between 1999 and 2002 were collected and evaluated histopathologically and immunophenotypically by a single specialist. Tissue microarrays were constructed using a TMA-builder instrument (Histopathology Ltd., Pecs, Hungary). To identify the eventual prognostic role of the TJ proteins CLDN3, −4 and −7, and adherent junction protein E-cadherin (ECDH) their immunohistochemical (IHC) expression was analysed by 3 investigators separately, and a consensus score was reached. The IHC results were compared to the patients' follow-up data applying Kaplan-Meier analysis supported by log-rank test. Furthermore, we aimed at identifying the worse prognosis tumour group based on clustering of the expression of the above mentioned JPs. Results: The expression of CLDN3 correlated with vascular invasion (VI)/p = 0.029/, necrosis (NC)/p = 0.001/, HG/p = 0.001/ and mitotic index (MI)/p = 0.022/. CLDN4 and CLDN7 correlated with NC/p = 0.001, p = 0.012, respectively/ and HG/p = 0.034, p = 0.001, respectively/, while ECDH correlated with HG/p = 0.001/ and MI/p = 0.001/. In single protein analysis, groups displaying gradually increasing CLDN3, −7 and ECDH/p = 0.017/expression (split at mean expression ± SD) showed a trend of bearing poorer prognostic features. CLDN4, on the other hand, showed different tendency: gradually increasing expression also correlated with worse relapse-free survival (RFS), while CLDN4 negative/low tumors had the poorest prognosis/p = 0.034/. In multiple protein analysis, groups were created using k-means clustering and the following cohorts were created - appearing in prognostic sequence starting with the longest RFS: ECDH-low groups with 1) low/n=31/ and 2) high/n=42/ CLDN3 expression; 3) CLDN7-low (CLDN3, -4 and ECDH-high) group/n=35/, 4) “all-high” expression group/n=70/; 5) CLDN3-low (CLDN4, -7 and ECDH-high) group/n=68/ and 6) “all-low” group/n=15/. The latter was characterised by the CLDN4-low feature as compared to the other groups. Conclusion: Expression of JPs can have prognostic relevance in breast carcinomas. The groups with low expression of ECDH have good prognosis, while gradually increasing JP expression outlines poor prognostic tumours. Strikingly the drop in all JPs expression - most likely resembling the claudin-low subtype - specifies the subgroup with the poorest outcome, which still remains an infrequent disease entity. Grant support: TÁMOP-4.2.2/B-10/1-2010-0013. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-08.

Expression of KiSS-1, mucins 1 and E-cadherin in breast cancer and their correlations with clinicopathological features

Objective To investigate the expression of KiSS-1, mucins 1 (MUC1) and E-cadherin (E-cad) in human breast cancer and their correlations with clinicopathological features. Methods The expression of KiSS-1, MUC1 and E-cad in 106 specimens of breast cancer was detected by immunohistochemistry, and their correlations to the clinicopathological features of breast cancer were analyzed. The correlations of KiSS-1 expression to MUC1 and E-cad were analyzed by Spearman' s rank correlation. Results The expression of KiSS-1, MUC1 and E-cad had some relations with clinical stages ( Ⅰ and Ⅱ 60. 66％/39. 34％/70. 49％, Ⅲ and Ⅳ 8. 89％/71.11％/48. 89％ ), lymph node metastasis ( N0 58.97％/38.46％/74. 36％ ; N1 26. 87％/61.19％/53.73％ ) of breast cancer (x2 =10. 715, 5. 112, 4. 422 and 29. 259, 10. 487, 5.095,P all ＜0. 05), but they had no correlation with age, menopause, T stages and M stages (P all ＞ 0. 05). The expression of KiSS-1 and E-cad were decreased gradually with the progress of lymph node metastasis and clinical stages, and MUC1 expression increased. The expression of E-cad (well 75.00％ ; moderate 66. 67％ ; poor 28.58％ ) was decreased gradually with the progress of histological grade (x2 =12. 391 ,P ＜0. 05), and MUC1 ( well 32. 14％ ; moderate 56. 14％ ; poor 71.43％ ) expression increased (x2 =16. 631 ,P ＜ 0. 05). The negative correlation was observed between KiSS-1 expression and MUC1 expression ( r =-0. 714, P ＜ 0. 05 ), and positive correlation between KiSS-1 and E-cad in breast cancer (r =0. 763,P ＜0. 05). Conclusion The expressions of KiSS-1, MUC1 and E-cad may reflect the metastasis and clinical stages of human breast cancer. The interaction of these factors plays an important role in regulation of the invasion and metastasis of breast cancer cells, and it is possible for KiSS-1 to play an important role as a tumor metastasis inhibition protein. Key words: Breast neoplasm; KiSS-1 ; Mucins1 ; E-cadherin

An immunohistochemically positive E-cadherin status is not always predictive for a good prognosis in human breast cancer

Background:In primary breast cancers dichotomic classification of E-cadherin expression, according to an arbitrary cutoff, may be inadequate and lead to loss of prognostic significance or contrasting prognostic indications. We aimed to assess the prognostic value of high and low E-cadherin levels in a consecutive case series (204 cases) of unilateral node-negative non-lobular breast cancer patients with a 8-year median follow-up and that did not receive any adjuvant therapy after surgery.Methods:Expression of E-cadherin was investigated by immunohistochemistry and assessed according to conventional score (0, 1+, 2+, 3+). Multiple correspondence analysis was used to visualise associations of both categorical and continuous variables. The impact of E-cadherin expression on patients outcome was evaluated in terms of event-free survival curves by the Kaplan–Meier method and proportional hazard Cox model.Results:Respect to intermediate E-cadherin expression values (2+), high (3+) or low (0 to 1+) E-cadherin expression levels had a negative prognostic impact. In fact, both patients with a low-to-nil (score 0 to 1+) expression level of E-cadherin and patients with a high E-cadherin expression level (score 3+) demonstrated an increased risk of failure (respectively, hazard ratio (HR)=1.71, confidence interval (CI)=0.72–4.06 and HR=4.22, CI=1.406–12.66) and an interesting association with young age.Conclusions:The findings support the evidence that high expression values of E-cadherin are not predictive for a good prognosis and may help to explain conflicting evidence on the prognostic impact of E-cadherin in breast cancer when assessed on dichotomic basis.

Inhibition of CCN6 (Wnt-1-Induced Signaling Protein 3) Down-Regulates E-Cadherin in the Breast Epithelium through Induction of Snail and ZEB1

The cysteine-rich protein CCN6 [or Wnt-1-induced signaling protein 3 (WISP3)] exerts tumor-suppressive effects in aggressive inflammatory breast cancer. Loss of CCN6 is associated with poorly differentiated phenotypes and increased invasion. Here, we show that reduction of CCN6 expression occurs in 60% of invasive breast carcinomas and is associated with axillary lymph node metastases. Furthermore, low CCN6 expression in invasive carcinoma tissue samples correlates with reduced expression of E-cadherin. In vitro, RNA interference knockdown of CCN6 in two benign human mammary epithelial cell lines (HME and MCF10A) decreased expression of E-cadherin protein and mRNA and reduced activity of the E-cadherin promoter; this reduction was dependent on intact E-box elements. CCN6 knockdown in HME cells resulted in up-regulation of the E-cadherin transcriptional repressors Snail and ZEB1 and enhanced their recruitment and binding to the E-cadherin promoter as analyzed by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail blocked the down-regulation of E-cadherin caused by CCN6 inhibition. These data show, for the first time, that CCN6 expression is reduced or lost in a substantial number of invasive breast carcinomas and that CCN6 modulates transcriptional repressors of E-cadherin. Together, our results lead to a new hypothesis that Snail and ZEB1 are downstream of CCN6 and play a critical role in CCN6-mediated regulation of E-cadherin in breast cancer.

Expression of survivin and E-cadherin in breast cancer

Objective: Survivin is a member of the inhibitor of apoptosis (IAP) family, and is involved in the regulation of cell division. E-cadherin functionally belongs to transmembrane glycoproteins family, it is responsible for intercellular junction mechanism that is crucial for the mutual association of vertebrate cells. These genes are thought to be associated with cancer aggression. This study was to investigate the relationship between surviving gene, E-cadherin expression and invasion clinicopathological features of breast cancer. Methods: The expression of surviving gene and E-cadherin were detected by SP immunohistochemical technique in tissues of 66 breast cancer, 20 breast fibroadenoma and 20 adjacent breast tissue. Results: The positive rate of surviving gene expression in breast cancer was 42.2%, significantly higher (P=0.025) than those in breast fibroadenoma (35.0%), and adjacent breast tissue (10.0%). The positive rate of E-cadherin in the groups of adjacent breast tissue, breast fibroadenoma and breast cancer were 100%, 100% and 42.4%, there was significant difference between the group of benign and malignant tumor (P=0.005). The positive rate of surviving in breast cancer with local lymph node metastasis was significant higher than that in breast cancer without lymph node metastasis (P=0.01), and E-cadherin in breast cancer with local lymph node metastasis was significant lower than that without lymph node metastasis (P=0.01). There was no significant difference among the groups of pathological types and TNM stages in the expression of surviving (P=0.966 & P=0.856), but there was significant difference in the expression of E-cadherin among these groups (P=0.01 & P=0.023). Conclusion: The loss or decrease of E-cadherin expression may promote the exfoliation of cancerous cells from original tissues, and surviving gene may promote the viability of the exfoliated cancer cells and the formation of new metastasis focus. These 2 factors cooperate with each other in the process of metastasis and invasion. They have a close relation with the breast cancer clinicopathological features.