Elevated expression of chemokine C-C ligand 2 in stroma is associated with recurrent basal-like breast cancers.
Yao Min,Yu Elaine,Staggs Vincent,Fan Fang,Cheng Nikki
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Despite advances in treatment, up to 30% of breast cancer patients experience disease recurrence accompanied by more aggressive disease and poorer prognosis. Treatment of breast cancer is complicated by the presence of multiple breast cancer subtypes, including: luminal, Her2 overexpressing, and aggressive basal-like breast cancers. Identifying new biomarkers specific to breast cancer subtypes could enhance the prediction of patient prognosis and contribute to improved treatment strategies. The microenvironment influences breast cancer progression through expression of growth factors, angiogenic factors and other soluble proteins. In particular, chemokine C-C ligand 2 (CCL2) regulates macrophage recruitment to primary tumors and signals to cancer cells to promote breast tumor progression. Here we employed a software-based approach to evaluate the prognostic significance of CCL2 protein expression in breast cancer subtypes in relation to its expression in the epithelium or stroma or in relation to fibroblast-specific protein 1 (Fsp1), a mesenchymal marker. Immunohistochemistry analysis of tissue microarrays revealed that CCL2 significantly correlated with Fsp1 expression in the stroma and tumor epithelium of invasive ductal carcinoma. In the overall cohort of invasive ductal carcinomas (n=427), CCL2 and Fsp1 expression in whole tissues, stroma and epithelium were inversely associated with cancer stage and tumor size. When factoring in molecular subtype, stromal CCL2 was observed to be most highly expressed in basal-like breast cancers. By Cox regression modeling, stromal CCL2, but not epithelial CCL2, expression was significantly associated with decreased recurrence-free survival. Furthermore, stromal CCL2 (HR=7.51 P=0.007) was associated with a greater hazard than cancer stage (HR=2.45, P=0.048) in multivariate analysis. These studies indicate that stromal CCL2 is associated with decreased recurrence-free survival in patients with basal-like breast cancer, with important implications on the use of stromal markers for predicting patient prognosis.
Claudin as a target for drug development.
Takahashi A,Kondoh M,Suzuki H,Yagi K
Current medicinal chemistry
Tight junctions (TJs) play pivotal roles in the fence and barrier functions of epithelial and endothelial cell sheets. Since the 1980s, the modulation of the TJ barrier has been utilized as a method for drug absorption. Over the last decade, the structural and functional biochemical components of TJs, such as occludin and claudin, have been determined, providing new insights into TJ-based pharmaceutical therapy. For example, the modulation of the claudin barrier enhances the jejunal absorption of drugs, and claudin expression is deregulated in cancer cells. Claudin is a co-receptor for the hepatitis C virus. Moreover, claudin is modulated during inflammatory conditions. These findings indicate that claudins are promising drug targets. In this review, we discuss the seeds of claudin-based drug development, which may provide potential pharmaceutical breakthroughs in the future.
Spiral progression in the development of absorption enhancers based on the biology of tight junctions.
Kondoh Masuo,Takahashi Azusa,Yagi Kiyohito
Advanced drug delivery reviews
Epithelium covers the body and, therefore, separates the inner body from the outside environment. Passage across the epithelium is the first step in drug absorption. Tight junctions (TJs) seal the space between adjacent epithelial cells and prevent the free movement of solutes through the paracellular space. Modulation of the epithelial barrier is the most important strategy for enhancing drug absorption. Development of the strategy has accelerated with progress in understanding of the biology of the TJ seal. The first-generation absorption enhancers were screened on the basis of their absorption-enhancing activity in vivo. However, TJs were not well understood initially. The identification of TJ components, including those based on occludin and claudins, has led to the development of new strategies for drug absorption. Accumulation of knowledge of claudins has provided new insights into the paracellular transport of drugs. This review examines the relationship between advances in understanding of TJ biology and paracellular transport of drugs and discusses progress in the development of mucosal absorption enhancers.
CLDN6 promotes chemoresistance through GSTP1 in human breast cancer.
Yang Minlan,Li Yanru,Shen Xiangfeng,Ruan Yang,Lu Yan,Jin Xiangshu,Song Peiye,Guo Yantong,Zhang Xiaoli,Qu Huinan,Shao Yijia,Quan Chengshi
Journal of experimental & clinical cancer research : CR
BACKGROUND:Claudin-6 (CLDN6), a member of CLDN family and a key component of tight junction, has been reported to function as a tumor suppressor in breast cancer. However, whether CLDN6 plays any role in breast cancer chemoresistance remains unclear. In this study, we investigated the role of CLDN6 in the acquisition of chemoresistance in breast cancer cells. METHODS:We manipulated the expression of CLDN6 in MCF-7 and MCF-7/MDR cells with lv-CLDN6 and CLDN6-shRNA and investigated whether CLDN6 manipulation lead to different susceptibilities to several chemotherapeutic agents in these cells. The cytotoxicity of adriamycin (ADM), 5-fluorouracil (5-FU), and cisplatin (DDP) was tested by cck-8 assay. Cell death was determined by DAPI nuclear staining. The enzyme activity of glutanthione S-transferase-p1 (GSTP1) was detected by a GST activity kit. Then lv-GSTP1 and GSTP1-shRNA plasmids were constructed to investigate the potential of GSTP1 in regulating chemoresistance of breast cancer. The TP53-shRNA was adopted to explore the regulation mechanism of GSTP1. Finally, immunohistochemistry was used to explore the relationship between CLDN6 and GSTP1 expression in breast cancer tissues. RESULTS:Silencing CLDN6 increased the cytotoxicity of ADM, 5-FU, and DDP in MCF-7/MDR cells. Whereas overexpression of CLDN6 in MCF-7, the parental cell line of MCF-7/MDR expressing low level of CLDN6, increased the resistance to the above drugs. GSTP1 was upregulated in CLDN6-overexpressed MCF-7 cells. RNAi -mediated silencing of CLDN6 downregulated both GSTP1 expression and GST enzyme activity in MCF-7/MDR cells. Overexpresssion of GSTP1 in CLDN6 silenced MCF-7/MDR cells restored chemoresistance, whereas silencing GSTP1 reduced the chemoresistance due to ectopic overexpressed of CLDN6 in MCF-7 cells. These observations were also repeated in TNBC cells Hs578t. We further confirmed that CLDN6 interacted with p53 and promoted translocation of p53 from nucleus to cytoplasm, and both the expression and enzyme activity of GSTP1 were regulated by p53. Clinicopathologic analysis revealed that GSTP1 expression was positively associated with CLDN6 in human breast cancer samples. CONCLUSION:High expression of CLDN6 confers chemoresistance on breast cancer which is mediated by GSTP1, the activity of which is regulated by p53. Our findings provide a new insight into mechanisms and strategies to overcome chemoresistance in breast cancer.
CLDN6 enhances chemoresistance to ADM via AF-6/ERKs pathway in TNBC cell line MDAMB231.
Yang Minlan,Li Yanru,Ruan Yang,Lu Yan,Lin Dongjing,Xie Yinping,Dong Bing,Dang Qihua,Quan Chengshi
Molecular and cellular biochemistry
Claudin-6 (CLDN6), a critical tight junction protein acting as a tumor suppressor in breast cancer, is also considered to be a stem cell marker. Triple-negative breast cancer (TNBC) is a subtype of claudin-low and stem cell-like breast cancer which is chemoresistant to multiple anti-cancer drugs. The aim of our study was to determine whether CLDN6 plays a role in chemoresistance of TNBC. We found that overexpression of CLDN6 in TNBC cell line MDAMB231 significantly inhibited cell growth, migration, and invasion. The expression of CLDN6 increased the IC of adriamycin (ADM) and promoted the clonogenic survival. CLDN6 inhibited ADM-induced apoptosis and senescence in MDAMB231 cells. However, P-gp, a resistance-related protein highly associated with chemoresistance, was downregulated by CLDN6 overexpression in MDAMB231 cells. Epithelial mesenchymal transition (EMT) marker E-cadherin was increased, and vimentin was decreased by CLDN6. In addition, stem cell markers OCT4, SOX2, and Nanog were dramatically increased. CLDN6 colocalized and interacted with AF-6. Overexpression of CLDN6 increased the expression of afadin (AF-6) and hampered the activation of ERK signaling. PMA, a specific ERK activator, reversed the expression of EMT and stem cell markers, and decreased chemoresistance of MDAMB231 cells to ADM with a decreased IC and an increased apoptosis resulting from CLDN6. Together, we conclude that CLDN6 enhances the chemoresistance to ADM via activating the AF-6/ERK signaling pathway and up-regulating cancer stem cell characters in MDAMB231 cells.
Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours.
AIMS:Claudins are adhesion molecules present in tight junctions. To evaluate their usefulness as differentiation markers claudins 1, 2, 3, 4, 5 and 7 were studied in 116 epithelial and 92 non-epithelial tumours. METHODS AND RESULTS:Immunoreactivity for all claudins could be seen in different carcinomas. There were, however, tumour type-specific differences in their expression. Lower expression of claudin 2 was seen in breast and prostatic carcinomas, while hepatocellular and renal carcinomas expressed lower levels of claudins 4 and 5. In contrast to epithelial tumours, lymphomas did not express claudins and most soft tissue tumours and naevocytic lesions were negative or showed weaker, mainly cytoplasmic positivity for some claudins. Of non-epithelial tumours, claudin 5 was found only in angiosarcomas and benign vascular tumours, which also showed reactivity for claudins 2, 3 and 7, but was not expressed in any other soft tissue lesions or lymphomas. CONCLUSIONS:The results show that claudins 1, 2, 3, 4, 5 and 7 can be used as markers for epithelial differentiation and to distinguish epithelial neoplasms from lymphomas and selectively also from soft tissue and naevocytic lesions. Since these claudins show type-specific differential expression in epithelial tumours, they may also be of some value in distinguishing different epithelial tumours from each other. Additionally, claudin 5 shows promise as a marker for endothelial lesions compared with other soft tissue lesions.
Claudin proteins in human cancer: promising new targets for diagnosis and therapy.
Morin Patrice J
The tight junction proteins claudins are abnormally regulated in several human cancers. In particular, claudin-3 and claudin-4 are frequently overexpressed in several neoplasias, including ovarian, breast, pancreatic, and prostate cancers. Although the exact roles of these proteins in tumorigenesis are still being uncovered, it is clear that they represent promising targets for cancer detection, diagnosis, and therapy.
Claudins 2, 3, 4, and 5 in Paget's disease and breast carcinoma.
Claudins are involved in the formation of tight junctions in epithelial and endothelial cells. The present study evaluated the expression of claudin 2, 3, 4, and 5 in 20 cases of Paget's disease (13 mammary and 7 extramammary cases), and compared the results with those of other neoplastic skin lesions, including actinic keratoses, basal cell carcinomas, and malignant melanomas. To compare claudin expression in Paget's disease and breast neoplasia, it was also studied in a large set of breast carcinomas. Membrane-bound claudin 3 and 4 expression was seen in all cases of Paget's disease, whereas claudin 5 was seen in 50% of cases and claudin 2 was seen in 32% of cases. In contrast, claudins 3, 4, and 5 were not seen in the other skin lesions, and claudin 2 was seen in most of them, suggesting an inverse expression of these claudins between Paget's disease and epidermal and nevocytic lesions. Claudin expression in breast carcinomas was claudin 2 in 52%, claudin 3 in 93%, claudin 4 in 92%, and claudin 5 in 47%. Claudins 2 and 5 were found more often in ductal carcinomas than in lobular carcinomas. Expression of claudins were frequently associated with each other. They were not associated with estrogen or progesterone receptor status or with tumor grade. No significant differences were found between claudin expression in Paget's disease and breast carcinomas. The results demonstrate that claudins could be useful in diagnosing Paget's disease and in differentiating these lesions from other epidermal lesions, such as actinic keratoses, basal cell carcinomas, and nevocytic lesions. The lack of difference in claudin expression between Paget's disease and breast tumors suggests that changes in the phenotype of claudins 2, 3, 4, and 5 are not necessary for epidermal invasion.
Reduced expression of claudin-2 is associated with high histological grade and metastasis of feline mammary carcinomas.
Flores A R,Rêma A,Carvalho F,Faustino A,Dias Pereira P
Journal of comparative pathology
Claudins (CLDNs) are a family of tight junction (TJ) proteins that play an important role in maintaining cell polarity, in controlling paracellular ion flux and in regulating cell proliferation and differentiation. There is a growing body of evidence that associates changes in CLDN expression with the development of human breast cancer. In the present study CLDN-2 expression was examined immunohistochemically in samples of normal feline mammary tissue (n = 5) and mammary carcinomas (n = 52), including metastatic lesions (n = 29). Seventy-seven percent of carcinomas showed reduced CLDN-2 expression compared with that observed in normal mammary gland. Reduced expression of CLDN-2 was significantly associated with a high histological grade of carcinoma (P = 0.011), with 88.6% of grade II/III carcinomas showing decreased expression. Furthermore, CLDN-2 down-regulation was significantly associated with metastatic disease (P = 0.0027), with 93.1% of cases with signs of metastasis showing decreased expression of this protein. CLDN-2 may constitute a molecular marker for identification of a subgroup of feline mammary carcinomas characterized by high histological grade and the development of metastasis.
Down-regulation of claudin-2 in breast carcinomas is associated with advanced disease.
Kim T H,Huh J H,Lee S,Kang H,Kim G I,An H J
AIMS:Claudin 2 (CLDN2) is a family of integral membrane tight junctions. The aim was to determine the influence of CLDN2 expression on tumour behaviour and its role in breast carcinogenesis. METHOD AND RESULTS:Thirty-seven invasive breast carcinomas and corresponding normal breast tissues were examined for CLDN2 protein and mRNA expression using Western blotting and semiquantitative reverse transcriptase-polymerase chain reaction. The expression of CLDN2 protein in 118 cases of breast carcinoma was further studied with immunohistochemistry and related to various clinicopathological parameters. CLDN2 protein expression was significantly down-regulated (0.4-fold) in tumours compared with corresponding normal breast tissue (P < 0.0001). Down-regulation of CLDN2 was significantly associated with lymph node metastasis (P = 0.047) by Western blot analysis, and with high clinical stage (P = 0.040) by immunohistochemistry. The expression levels of CLDN2 mRNA in high clinical stages (stages II and III) were lower than those in low clinical stage (stage I) and normal tissue, but not statistically significantly so. CONCLUSIONS:These results suggest that CLDN2 is implicated in the progression as well as the development of breast carcinoma, indicating that CLDN2 is a possible tumour suppressor gene product.
Clinicopathological significance of immunoexpression of claudin-1 and claudin-7 in feline mammary carcinomas.
Flores A Rute,Rêma A,Carvalho F,Lopes G,Faustino A,Dias Pereira P
Journal of comparative pathology
Claudins (CLDNs) are tight junction proteins that have a role in regulating cell adhesion and polarity, paracellular permeability, proliferation and differentiation. Several immunohistochemical studies have shown reduced expression of CLDN-1 and CLDN-7 in human and canine mammary carcinomas, suggesting that these proteins may participate in mammary carcinogenesis, invasion and metastasis. The present study characterizes expression of CLDN-1 and CLDN-7 in feline mammary carcinomas (n = 52) and their metastases (n = 29). There was an inverse association between CLDN-7 expression and histological grade of tumour. Reduced expression of CLDN-7 was significantly associated with decreased tubule formation, high proliferative activity and metastasis. No significant associations were found between CLDN-1 expression and any of these features. Evaluation of expression of CLDN-7, but not CLDN-1, may therefore provide prognostic information, assisting in the diagnosis of a subgroup of aggressive feline mammary carcinomas that share some features with the recently described 'claudin-low' subgroup of human breast cancer.
Immunoexpression of claudins 4 and 7 among invasive breast carcinoma subtypes: A large diagnostic study using tissue microarray.
Logullo Angela Flávia,Pasini Fatima Solange,Nonogaki Suely,Rocha Rafael Malagoli,Soares Fernando Augusto,Brentani Maria Mitzi
Molecular and clinical oncology
Molecular phenotyping and tissue microarray (TMA) studies have identified distinct invasive breast carcinoma subtypes: Luminal A, luminal B, enriched with overexpressed human epidermal growth factor receptor 2 (HER-2) and triple-negative, i.e., negative for HER-2, as well as for estrogen and progesterone receptor (ER and PR, respectively) expression. These subtypes are useful in clinical management, since they bear distinct prognoses and predictive responses to targeted therapy. However, although molecular profiling provides important prognostic indicators, breast cancer risk stratification remains a challenge in triple-negative cases. What is referred to as claudin-low subtype was identified as a triple-negative subset that is associated with more aggressive tumor behavior and worse prognosis. However, the immunohistochemical expression of claudins has not yet been standardized. Our objective was to verify whether the immunoexpression of claudins 4 and 7 (the main claudins specifically expressed in human breast tissue) in TMA is associated with survival and prognosis in luminal A, HER-2 and triple-negative molecular subtypes. In this diagnostic study, we investigated ER/PR receptor status, HER-2, claudin 4 and 7 expression and stem cell CD44/24 profiles, and verified the association with prognosis and survival outcomes in 803 invasive breast carcinoma cases arranged in four TMAs. Among these, 503 (62.6%) were positive for claudin 4 and 369 (46.0%) for claudin 7. Claudin 4 exhibited the lowest expression in luminal A and triple-negative subtypes, and the highest frequency of expression in HER-2-enriched subtypes, whereas claudin 7 staining was not associated with any subtype. The stem cell phenotype was not associated with subgroups or claudins 4 and 7. Claudin immunoexpression profile was not able to distinguish between patients with better or worse prognosis, and it was not correlated to triple-negative cases. Therefore, it may be concluded that the immunoexpression of claudins 4 and 7, individually or within the usual immunohistochemical context (ER, PR and HER-2), does not provide additional prognostic information on breast cancer subtypes.
CLDN3 expression and significance - breast carcinoma versus ovarian carcinoma.
Ionescu Popescu Carmen,Liliac Ludmila,Ceauşu Raluca Amalia,Balan Raluca,Grigoraş Adriana,Căruntu Irina Draga,Amălinei Cornelia
Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie
Claudins (CLDNs) are transmembrane proteins, as normal constituents of the architecture of tight junctions. Recent studies support their involvement in carcinogenesis, as changes in CLDNs structure result in alterations in tight junctions' structure and function, facilitating malignant transformation. We aimed CLDN3 investigation in both breast and ovarian carcinoma, targeting the identification of its expression differences. The immunohistochemical assessment was performed on 20 cases of breast carcinomas (Group 1) and 19 cases of epithelial ovarian carcinomas (Group 2). Firstly, the specific panel for the molecular classification was applied for specimens of the first group. Then, all the specimens were immunostained for CLDN3 and a semi-quantitative evaluation was made, based on the percentage of positive cells and the intensity of staining. In Group 1, in the ER positive category, CLDN3 was overexpressed in five cases (four cases of luminal A and one case of luminal B subtype, respectively), negative in three cases (luminal A subtype) and weakly expressed in a single case (luminal A subtype); in ER negative category, CLDN3 expression was strong in four cases (one case of Her2/neu subtype and three cases of basal-like subtype), negative in two cases (normal breast-like subtype) and weak in five cases (one case of Her2/neu subtype, one triple-negative subtype, and three basal-like subtype). In Group 2, CLDN3 was overexpressed in 15 cases, histopathologically diagnosed as serous (10 cases), mucinous (two cases), endometrioid (two cases), and mixed carcinomas (one case); a weak expression was noticed in a single case, of the serous subtype; CLDN3 was undetectable in three cases (one serous, one clear cell, and one endometrioid type). Our comparative analysis of CLDN3 profile in breast and ovarian cancer clearly indicates organ specificity.
Loss of the tight junction protein claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast.
Kominsky Scott L,Argani Pedram,Korz Dorian,Evron Ella,Raman Venu,Garrett Elizabeth,Rein Alan,Sauter Guido,Kallioniemi Olli-P,Sukumar Saraswati
Claudins are transmembrane proteins that seal tight junctions, and are critical for maintaining cell-to-cell adhesion in epithelial cell sheets. However, their role in cancer progression remains largely unexplored. Here, we report that Claudin-7 (CLDN-7) expression is lower in invasive ductal carcinomas (IDC) of the breast than in normal breast epithelium, as determined by both RT-PCR (9/10) and Western analysis (6/8). Immunohistochemical (IHC) analysis of ductal carcinoma in situ (DCIS) and IDC showed that the loss of CLDN-7 expression correlated with histological grade in both DCIS (P<0.001, n=38) and IDC (P=0.014, n=31), occurring predominantly in high-grade (Nuclear and Elston grade 3) lesions. Tissue array analysis of 355 IDC cases further confirmed the inverse correlation between CLDN-7 expression and histological grade (P=0.03). This pattern of expression is consistent with the biological function of CLDN-7, as greater discohesion is typically observed in high-grade lesions. In line with this observation, by IHC analysis, CLDN-7 expression was lost in the vast majority (13/17) of cases of lobular carcinoma in situ, which is defined by cellular discohesion. In fact, inducing disassociation of MCF-7 and T47D cells in culture by treating with HGF/scatter factor resulted in a loss of CLDN-7 expression within 24 h. Silencing of CLDN-7 expression correlated with promoter hypermethylation as determined by methylation-specific PCR (MSP) and nucleotide sequencing in breast cancer cell lines (3/3), but not in IDCs (0/5). In summary, these studies provide insight into the potential role of CLDN-7 in the progression and ability of breast cancer cells to disseminate.
Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study.
Tokés Anna-Mária,Kulka Janina,Paku Sándor,Szik Agnes,Páska Csilla,Novák Pál Kaposi,Szilák László,Kiss András,Bögi Krisztina,Schaff Zsuzsa
Breast cancer research : BCR
INTRODUCTION:We compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions. METHODS:Altogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases. RESULTS:CLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue. CONCLUSIONS:The significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.
Identification of Prognostic Candidate Genes in Breast Cancer by Integrated Bioinformatic Analysis.
Wang Charles C N,Li Chia Ying,Cai Jia-Hua,Sheu Phillip C-Y,Tsai Jeffrey J P,Wu Meng-Yu,Li Chia-Jung,Hou Ming-Feng
Journal of clinical medicine
Breast cancer is one of the most common malignancies. However, the molecular mechanisms underlying its pathogenesis remain to be elucidated. The present study aimed to identify the potential prognostic marker genes associated with the progression of breast cancer. Weighted gene coexpression network analysis was used to construct free-scale gene coexpression networks, evaluate the associations between the gene sets and clinical features, and identify candidate biomarkers. The gene expression profiles of GSE48213 were selected from the Gene Expression Omnibus database. RNA-seq data and clinical information on breast cancer from The Cancer Genome Atlas were used for validation. Four modules were identified from the gene coexpression network, one of which was found to be significantly associated with patient survival time. The expression status of 28 genes formed the black module (basal); 18 genes, dark red module (claudin-low); nine genes, brown module (luminal), and seven genes, midnight blue module (nonmalignant). These modules were clustered into two groups according to significant difference in survival time between the groups. Therefore, based on betweenness centrality, we identified and in the nonmalignant module, and in the luminal module, and in the claudin-low module, and and in the basal module as the genes with the highest betweenness, suggesting that they play a central role in information transfer in the network. In the present study, eight candidate biomarkers were identified for further basic and advanced understanding of the molecular pathogenesis of breast cancer by using co-expression network analysis.
Claudins 1, 3, and 4 protein expression in ER negative breast cancer correlates with markers of the basal phenotype.
Blanchard Anne A,Skliris George P,Watson Peter H,Murphy Leigh C,Penner Carla,Tomes Ladislav,Young Tamara L,Leygue Etienne,Myal Yvonne
Virchows Archiv : an international journal of pathology
In the present study we investigated the protein expression of claudins 1, 3, and 4 and their relationship to clinical variables and outcome in a cohort of ER-ve and ER+ve human invasive breast cancers. Immunohistochemical analysis was performed on tissue microarrays representing a total of 412 tumors and interpretable data was derived from 314, 299, and 306 tumors for claudins 1, 3, and 4, respectively. In the ER+ve subset, 5%, 89%, and 52%, and in the ER-ve subset, 39%, 79%, and 79% of tumors stained positively for claudins 1, 3, and 4, respectively (p < 0.0001, p = 0.026, p < 0.0001). Thus, in the two subsets, a significantly higher number of tumors were positive for claudins 3 and 4, compared to claudin 1. In addition, protein expressions of claudins 1 and 4 were significantly higher in those tumors that displayed characteristics of the basal-like subtype of breast cancers (ER-ve, Her-2-ve, EGFR+ve, CK5/6+ve). This study shows a unique pattern of expression for the different claudins in ER-ve and ER+ve tumors. Our data also suggests that increased expression of claudins 1 and 4 was associated with the basal-like subtype of breast cancers, a subtype generally linked to poor outcome.
Expression of tight junction protein claudin-4 in basal-like breast carcinomas.
Kulka Janina,Szász Attila Marcell,Németh Zsuzsanna,Madaras Lilla,Schaff Zsuzsa,Molnár István Arthur,Tokés Anna-Mária
Pathology oncology research : POR
Claudins (CLDN) are tight junction proteins which contribute to the paracellular transport and ionic permeability of various epithelia. In recent years they came into focus for their suggested role in carcinogenesis and possible role in cancer therapy. According to our previous studies, in breast tissue CLDN4 is also related to the level of cellular differentiation. Thirty-eight estrogen (ER) and progesterone receptor (PgR) negative, HER2/neu negative, but cytokeratin 5/6 positive basal-like-mainly grade 3-breast carcinomas were compared with twenty-one grade 1, twenty-five grade 2 and twenty grade 3 non-basal-like invasive breast carcinomas, in respect to their CLDN4 expression. The immunohistochemical reactions were evaluated both semiquantitatively and by morphometrical analysis. Statistically significant difference (p = 0.001) was observable regarding CLDN4 expression in the basal-like group as compared to grade 1 and 2 cancers. Further, CLDN4 expression was significantly higher (p = 0.017) in the basal-like compared with the non-basal-like grade 3 carcinomas. Our results suggest that basal-like carcinomas are a subset of breast cancer with high level of CLDN4 protein expression. The finding is in accordance with our former observation that CLDN4 is indeed related to cellular differentiation. This observation may be seen as a further proof that basal-like carcinomas represent a separable group amongst grade 3 breast carcinomas.
Significant elevation of CLDN16 and HAPLN3 gene expression in human breast cancer.
Kuo Shou-Jen,Chien Su-Yu,Lin Che,Chan Szu-Erh,Tsai Hsiu-Ting,Chen Dar-Ren
Cancer development involves the destruction of tight junctions, deprivation of cell polarity, and increased cell mobility. Claudin 16 (CLDN16) is a tight junction protein and plays important roles in the maintenance of cell polarity, cellular arrangement, adhesion, paracellular transport, and ionic permeability of various epithelia. A novel link protein, HAPLN3, functions in hyaluronic acid binding and cell adhesion. Both genes are hypothesized to be related to cancer development and metastasis. The purpose of this study was to estimate the roles of the genes CLDN16 and HAPLN3 in breast cancer. A total of 146 samples were collected from breast cancer tissues and their adjacent normal breast tissues. Reverse transcription and real-time polymerase chain reaction were used to estimate gene expression levels. There were significantly increased gene expression of CLDN16 (p<0.0001) and HAPLN3 (p<0.0001) among breast cancer tissues compared with normal tissues, irrespective of clinical pathological parameters. The absolute increased gene expression level of CLDN16 was significantly negatively correlated with estrogen (r=-0.46; p<0.0001) and progesterone receptor (r=-0.384; p=0.001) staining density. However, a significantly positive correlation (r=0.24; p=0.04) between the absolute increased HAPLN3 gene level and human epidermal receptor 2 staining density was found. There was no significant association between overall survival and the two gene expression levels. The gene up-expression of both CLDN16 and HAPLN3 was suggested to be involved in the development of breast cancer and to be a biomarker and target treatment for breast cancer.
Decreased expression of claudin-1 correlates with recurrence status in breast cancer.
Morohashi Satoko,Kusumi Tomomi,Sato Fuyuki,Odagiri Hiroki,Chiba Hiroki,Yoshihara Shuichi,Hakamada Kenichi,Sasaki Mutsuo,Kijima Hiroshi
International journal of molecular medicine
Claudins (CLDNs) constitute the major transmembrane proteins of tight junctions. It may be hypothesized that changes in or loss of expression of tight junctional proteins such as CLDNs can lead to cellular disorientation and detachment, which is commonly seen in neoplasia. Recent studies have suggested that claudin-1 (CLDN1) plays an important role in invasion and metastasis and claudin-4 (CLDN4) has a particular role in mammary glandular cell differentiation and carcinogenesis. In this study, we examined 83 breast cancer cases and demonstrated immunohistochemical expression patterns of CLDN1/CLDN4 in recurrent and non-recurrent groups. We found significant results between the recurrent and non-recurrent group for expression of CLDN1/CLDN4. The recurrent group (26 cases) showed decreased expression patterns of CLDN1 (p<0.001), compared to the non-recurrent group (57 cases). Decreased expression of CLDN1 (p<0.0001) correlated with short disease-free interval. The lymph node metastasis-positive group showed decreased expression patterns of CLDN1 (p=0.001). However, there was no significance between the recurrent group and non-recurrent group in CLDN4 expression. There was no significance between histological factors and CLDN4 expression. The results indicated that CLDN1 expression correlated with the recurrence status and malignant potential of breast cancer.
Claudin-low breast cancers: clinical, pathological, molecular and prognostic characterization.
Sabatier Renaud,Finetti Pascal,Guille Arnaud,Adelaide José,Chaffanet Max,Viens Patrice,Birnbaum Daniel,Bertucci François
BACKGROUND:The lastly identified claudin-low (CL) subtype of breast cancer (BC) remains poorly described as compared to the other molecular subtypes. We provide a comprehensive characterization of the largest series of CL samples reported so far. METHODS:From a data set of 5447 invasive BC profiled using DNA microarrays, we identified 673 CL samples (12,4%) that we describe comparatively to the other molecular subtypes at several levels: clinicopathological, genomic, transcriptional, survival, and response to chemotherapy. RESULTS:CL samples display profiles different from other subtypes. For example, they differ from basal tumors regarding the hormone receptor status, with a lower frequency of triple negative (TN) tumors (52% vs 76% for basal cases). Like basal tumors, they show high genomic instability with many gains and losses. At the transcriptional level, CL tumors are the most undifferentiated tumors along the mammary epithelial hierarchy. Compared to basal tumors, they show enrichment for epithelial-to-mesenchymal transition markers, immune response genes, and cancer stem cell-like features, and higher activity of estrogen receptor (ER), progesterone receptor (PR), EGFR, SRC and TGFβ pathways, but lower activity of MYC and PI3K pathways. The 5-year disease-free survival of CL cases (67%) and the rate of pathological complete response (pCR) to primary chemotherapy (32%) are close to those of poor-prognosis and good responder subtypes (basal and ERBB2-enriched). However, the prognostic features of CL tumors are closer to those observed in the whole BC series and in the luminal A subtype, including proliferation-related gene expression signatures (GES). Immunity-related GES valuable in basal breast cancers are not significant in CL tumors. By contrast, the GES predictive for pCR in CL tumors resemble more to those of basal and HER2-enriched tumors than to those of luminal A tumors. CONCLUSIONS:Many differences exist between CL and the other subtypes, notably basal. An unexpected finding concerns the relatively high numbers of ER-positive and non-TN tumors within CL subtype, suggesting a larger heterogeneity than in basal and luminal A subtypes.
Expression patterns of claudins in patients with triple-negative breast cancer are associated with nodal metastasis and worse outcome.
Katayama Ayaka,Handa Tadashi,Komatsu Kei,Togo Maria,Horiguchi Jun,Nishiyama Masahiko,Oyama Tetsunari
Claudins (CLDNs) are key cell adhesion molecules, which compose tight junctions (TJs), and the disruption of TJs is associated with cancer development. Here we immunohistochemically studied expression patterns of CLDNs in 222 primary invasive breast cancers including 68 triple-negative breast cancers (TNBCs), and examined their correlation with epithelial-to-mesenchymal transition (EMT)-related markers, breast cancer stem cell (BCSC) markers, and clinicopathological features including patients' clinical outcome. Tumor margins were classified as three infiltrating growth patterns (expanding, intermediate and infiltrating). For CLDN1, 3, 4, and 7, their expression rates were more frequent in TNBCs than in other subtypes (11.8% vs 0.7%, 26.5% vs 2.0%, 48.5% vs 11.1%, and 32.4% vs 8.7%, respectively; P ≤ 0.001). In 68 TNBCs, we identified high Ki67 labeling index (LI) and the combination of CLDN4 high/CLDN7 low expression as independent predictors of axillary nodal metastasis (P = 0.019; OR, 4.36; 95%CI, 1.28-14.90 and P = 0.007; OR, 5.33; 95%CI, 1.58-17.90). Moreover, the combination of CLDN1 low/CLDN7 low/E-cadherin negative as well as tumor infiltrating patterns were predictors for worse recurrence-free survival by univariate analyses in TNBCs (P = 0.005 and P = 0.011). Our analyses provide further evidence that CLDNs would be valuable prognostic markers in TNBCs.
The expression patterns and correlations of claudin-6, methy-CpG binding protein 2, DNA methyltransferase 1, histone deacetylase 1, acetyl-histone H3 and acetyl-histone H4 and their clinicopathological significance in breast invasive ductal carcinomas.
Xu Xiaoming,Jin Huiying,Liu Yafang,Liu Li,Wu Qiong,Guo Yaxiong,Yu Lina,Liu Zhijing,Zhang Ting,Zhang Xiaowei,Dong Xueyan,Quan Chengshi
BACKGROUND:Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac)). METHODS:We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry. RESULTS:Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression. CONCLUSIONS:Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinoma VIRTUAL SLIDES:The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452.
Claudin 1 in Breast Cancer: New Insights.
Zhou Bowen,Moodie Amanda,Blanchard Anne A A,Leygue Etienne,Myal Yvonne
Journal of clinical medicine
Claudin 1 is a small transmembrane protein responsible for maintaining the barrier function that exists between epithelial cells. A tight junction protein that regulates the paracellular transport of small ions across adjacent cells, claudin 1 maintains cellular polarity and plays a major role in cell-cell communication and epithelial cell homeostasis. Long considered to be a putative tumor suppressor in human breast cancer, new studies suggest a role much more complex. While most invasive breast cancers exhibit a down regulation or absence of claudin 1, some aggressive subtypes that exhibit high claudin 1 levels have now been described. Furthermore, a causal role for claudin 1 in breast cancer progression has recently been demonstrated in some breast cancer cell lines. In this review we highlight new insights into the role of claudin 1 in breast cancer, including its involvement in collective migration and epithelial mesenchymal transition (EMT).
A CLDN1-negative phenotype predicts poor prognosis in triple-negative breast cancer.
Ma Fei,Ding Xiaoyan,Fan Ying,Ying Jianming,Zheng Shan,Lu Ning,Xu Binghe
INTRODUCTION:Triple-negative breast cancer (TNBC) is a heterogeneous disease with no definitive prognostic markers. As a major component of tight junctions, claudins (CLDNs) presumably play an important role in carcinogenesis and progression of breast cancer. This study was aimed at determining the relationship between the expression of CLDNs and the clinical outcomes of TNBCs. MATERIALS AND METHODS:The surgical specimens of primary breast tumors from a consecutive cohort of 173 TNBC patients were retrospectively collected. The membranous expression of CLDN1, CLDN2, CLDN4, and CLDN7 was measured by immunohistochemistry. Then, the associations between CLDN expression, clinicopathological features, and clinical outcomes were assessed. RESULTS:Positive CLDN1, CLDN2, CLDN4, and CLDN7 membrane expression was detected in 44.5%, 54.9%, 76.9%, and 73.4% of the cohort specimens, respectively. A lack of CLDN1 expression was related to only lymph node metastasis (P = 0.014). The rate of CLDN4-positive tumors was significantly increased in tumors of a higher grade (P = 0.003). Importantly, negative CLDN1 expression was associated with worse relapse-free survival (RFS) in both lymph node positive (LN+) and negative (LN-) cases (both P<0.001). Similarly it was also associated with shorter overall survival (OS)(P = 0.003 in LN+ cases; P = 0.018 in LN- cases). In the LN+ subgroup, CLDN2-negative cases had a significantly higher risk of recurrence (P = 0.008). Multivariate analysis revealed that negative CLDN1 expression was an independent prognostic factor for high risk of both recurrence and death (HR 5.529, 95% CI 2.664-11.475, P<0.001; HR 3.459, 95% CI 1.555-7.696, P = 0.002). However, neither CLDN4 nor CLDN7 expression was associated with survival. CONCLUSION:In TNBC, the CLDN1-negative phenotype predicts a high risk of recurrence and death. The absence of CLDN1 expression is strongly suggested to be an independent adverse prognostic factor in this heterogeneous subtype of breast cancer.
[Claudins as prognostic factors of breast cancer].
Szász Marcell A
Different expression of claudins and E-cadherin has been described in the pathogenesis and progression of breast cancer. Changes in the expression of these junctional molecules have also been described as being of prominent importance in other cancers as well. Thus, we aimed at exploring the potential prognostic relevance of these cell junctional molecules in breast carcinoma cases. Expression of claudin-1, -3, -4, -5, -7, -8, -10, -15, -18 and E-cadherin at mRNA level was evaluated in correlation with survival in publicly available datasets containing expression measurements of 1809 breast cancer patients. Breast cancer tissues of 636 patients were evaluated with tissue microarray technique and immunohistochemical method for claudin-1, -2, -3, -4, -5, -7 and E-cadherin protein expression. In 96 cases lymph node metastases were also subjects of the study. Claudin expression bears prognostic information in itself. Based on bioinformatic data analysis, the meta-gene of claudin-3, -4, -7 and E-cadherin has proved the most powerful in predicting survival. An immunohistochemical protein profile consisting of claudin-2, -4 and E-cadherin was able to predict outcome in the most effective manner in the training set. Combining the overlapping members of the distinct methods resulted in the CC index (consisting of claudin-4 and E-cadherin, a.k.a. CURIO), which was able to accurately predict relapse-free survival in the validation cohort (p=0.029) in a more efficient way than its components. Cox regression analysis including clinicopathological variables and the average CC score showed that in univariate analysis most of them were prognostic but most of them lost independent prognostic value in multivariate analysis except for the CC index, the subtypes defined by immunoprofiling and vascular invasion. On the other hand, the CC index was able to further refine prognosis splitting good vs. poor prognosis patients into two clusters in these subgroups. Evaluation of lymph node metastases has shown that decreased expression of claudin-1 and elevated expression of claudin-4 can predict worse prognosis in breast cancers spreading to the regional lymph nodes. The defined claudin-cadherin index provides additional prognostic information besides the routinely utilized diagnostic approaches and factors. The level of expression of certain claudins can be of prognostic significance in regional lymph node metastases.
Claudin expression in high-grade invasive ductal carcinoma of the breast: correlation with the molecular subtype.
Lu Shaolei,Singh Kamaljeet,Mangray Shamlal,Tavares Rose,Noble Lelia,Resnick Murray B,Yakirevich Evgeny
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Claudin proteins are a major component of the tight junctions. Dysregulation of claudin protein expression has been described in a number of malignancies. Gene expression profiling has stratified breast cancers into distinct molecular subtypes: luminal, HER2 positive (HER2+), and basal-like. Recently, a novel claudin-low molecular subtype has been described. In this study, we correlated the expression patterns of claudins with the molecular subtypes of breast cancer. On the basis of immunohistochemical expression, 226 grade 3 invasive ductal carcinomas were stratified into 65 luminal (estrogen receptor positive (ER+)), 65 HER2+, 86 basal-like, including 14 metaplastic carcinomas (ER-, HER2-, CK5/6, and/or epidermal growth factor receptor positive), and 10 unclassified. Tissue microarrays were analyzed for the expression of claudins 1, 3, 4, 7, and 8 by immunohistochemistry and scored semiquantitatively. High levels of expression were detected in 17% of all cases for claudin 1, 32% claudin 3, 41% claudin 4, 44% claudin 7, and 40% claudin 8. Luminal cancers exhibited increased claudins 7 and 8; basal-like tumors demonstrated increased expression of claudins 1 and 4. Low expression of all five claudins was detected in 30 of 226 cases (13%) and this group was designated 'claudin-low'. The majority of the claudin-low subgroup were basal-like cancers (23 of 30, 77%). In contrast, only 1 of 30 (3%) claudin-low tumors was of the luminal phenotype and 6 of 30 cases (20%) were HER2+ (P<0.001). Within the basal-like subgroup, 64% of the metaplastic and 19% of the non-metaplastic tumors were claudin-low. The claudin-low group was strongly associated with disease recurrence (P=0.0093). In conclusion, this study is the first to examine comprehensively the differential expression of claudins 1, 3, 4, 7, and 8 in the molecular subtypes of high-grade breast cancer. Claudin-low subtype is a frequent phenomenon in metaplastic and basal-like breast cancer and appears to be a strong predictor of disease recurrence.
Prognostic significance of claudin expression changes in breast cancer with regional lymph node metastasis.
Szasz A M,Tokes A M,Micsinai M,Krenacs T,Jakab Cs,Lukacs L,Nemeth Zs,Baranyai Zs,Dede K,Madaras L,Kulka J
Clinical & experimental metastasis
Adherent and tight junction molecules have been described to contribute to carcinogenesis and tumor progression. Additionally, the group of claudin-low tumors have recently been identified as a molecular subgroup of breast carcinoma. In our study, we examined the expression pattern of claudins, beta-catenin and E-cadherin in invasive ductal (IDCs) and lobular (ILCs) carcinomas and their corresponding lymph node metastases (LNMs). Tissue microarrays of 97 breast samples (60 invasive ductal carcinomas, 37 invasive lobular carcinomas) and their corresponding LNMs have been analyzed immunohistochemically for claudin-1, -2, -3, -4, -5, -7, beta-catenin and E-cadherin expression. The stained slides were digitalized with a slide scanner and the reactions were evaluated semiquantitatively. When compared to LNMs, in the IDC group beta-catenin and claudin-2, -3, -4 and -7 protein expression showed different pattern while claudin-1, -2, -3, -4 and -7 were differently expressed in the ILC group. Lymph node metastases developed a notable increase of claudin-5 expression in both groups. Decrease or loss of claudin-1 and expression of claudin-4 in lymph node metastases correlated with reduced disease-free survival in our patients. According to our observations, the expression of epithelial junctional molecules, especially claudins, is different in primary breast carcinomas compared to their lymph node metastases as demonstrated by immunohistochemistry. Loss of claudin junctional molecules might contribute to tumor progression, and certain claudin expression pattern might be of prognostic relevance.
Increased claudin-4 expression is associated with poor prognosis and high tumour grade in breast cancer.
Lanigan Fiona,McKiernan Eadaoin,Brennan Donal J,Hegarty Shauna,Millikan Robert C,McBryan Jean,Jirstrom Karin,Landberg Goran,Martin Finian,Duffy Michael J,Gallagher William M
International journal of cancer
The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin-4, a tight junction protein, is overexpressed in breast tumour cells compared to normal epithelial cells, which generally corresponds to a loss in polarity. The aim of this study was to investigate the distribution and potential clinical value of claudin-4 in breast cancer, and to evaluate its usefulness as a prognostic and predictive biomarker. Expression of claudin-4 was initially examined by Western blot analysis in a cohort of 88 breast tumours, and was found to correlate positively with tumour grade and negatively with ER. Claudin-4 expression was then evaluated by immunohistochemistry in a larger cohort of 299 tumours represented on a tissue microarray. Claudin-4 expression correlated positively with tumour grade and Her2, and negatively with ER. High claudin-4 expression was also associated with worse breast cancer-specific survival (p = 0.003), recurrence-free survival (p = 0.025) and overall survival (p = 0.034). Multivariate analysis revealed that claudin-4 independently predicted survival in the entire cohort (HR 1.95; 95%CI 1.01-3.79; p = 0.047) and in the ER positive subgroup treated with adjuvant tamoxifen (HR 4.34; 95%CI 1.14-16.53; p = 0.032). This relationship between increased claudin-4 expression and adverse outcome was validated at the mRNA level in a DNA microarray dataset of 295 breast tumours. We conclude that high levels of claudin-4 protein are associated with adverse outcome in breast cancer patients, including the subgroup of patients treated with adjuvant tamoxifen.
Prognostic significance of CD24 and claudin-7 immunoexpression in ductal invasive breast cancer.
Bernardi M A,Logullo A F,Pasini F S,Nonogaki S,Blumke C,Soares F A,Brentani M M
This study aimed to identify the CD24 and CD44 immunophenotypes within invasive ductal breast carcinoma (IDC) subgroups defined by immunohistochesmistry markers and to determine its influence on prognosis as well as its association with the expression of Ki-67, cytokeratins (CK5 and CK18) and claudin-7. Immunohistochemical expression of CD44 and CD24 alone or in combination was investigated in 95 IDC cases arranged in a tissue microarray (TMA). The association with subgroups defined as luminal A and B; HER2 rich and triple negative, or with the other markers and prognosis was analyzed. CD44+/CD24- and CD44-/CD24+ were respectively present in 8.4% and 16.8% of the tumors, a lack of both proteins was detected in 6.3%, while CD44+/CD24+ was observed in 45.3% of the tumors. Although there was no significant correlation between subgroups and different phenotypes, the CD44+/CD24- phenotype was more common in the basal subgroups but absent in HER2 tumors, whereas luminal tumors are enriched in CD44-/CD24+ and CD44+/CD24+ cells. The frequency of CD44+/CD24- or CD44-/CD24+ was not associated with clinical characteristics or biological markers. There was also no significant association of these phenotypes with the event free (DFS) and overall survival (OS). Single CD44+ was evident in 57.9% of the tumors and was marginally associated to grading and not to any other tumor characteristics as well as OS and DFS. CD24+ was positive in 74.7% of the tumors, showing a significant association with estrogen receptor, progesterone receptor and Ki-67 and a marginal association with CK18 and claudin-7. Expression of claudin-7 and Ki-67 did not associate with the cancer subgroups, while a positive association between CK18 and the luminal subgroups was found (P=0.03). CK5, CK18 and Ki-67 expression had no influence in OS or DFS. Single CD24+ (P=0.07) and claudin-7 positivity (P=0.05) were associated with reduced time of recurrence, suggesting a contribution of these markers to aggressiveness of breast cancer.
Claudin-3 and claudin-4: distinct prognostic significance in triple-negative and luminal breast cancer.
Kolokytha Panagiota,Yiannou Petros,Keramopoulos Dimitris,Kolokythas Argyrios,Nonni Afroditi,Patsouris Efstratios,Pavlakis Kitty
Applied immunohistochemistry & molecular morphology : AIMM
INTRODUCTION:To investigate the immunohistochemical expression of claudin-1, claudin-3, and claudin-4 in triple-negative breast carcinomas and compare it with several clinicopathologic parameters as well as their expression in luminal cancers. MATERIALS AND METHODS:A total of 128 cases of breast carcinoma were included in the study. For all these cases, immunohistochemistry for estrogen and progesterone receptors, Ki-67, and Her2 had already been performed, whereas Her2 2+ cases had been further characterized as positive or negative for Her2 amplification with the chromogenic in situ hybridization technique. Seventy-six tumors were triple negative. The remaining 52 were luminal cancers. All tumors were evaluated for the expression of claudin-1, claudin-3, and claudin-4. RESULTS:In the triple-negative group, the positive expression of claudin-3 and claudin-4 was related to unfavorable and favorable prognostic factors, respectively. Claudin-1 was not related to any parameter under evaluation. In the luminal cancer group, claudin-4 positivity was related to a shorter disease-free survival, whereas the inverse was observed for claudin-3. Moreover, all 3 claudins increased with increase of the grade and Ki-67 value in the luminal cancers. CONCLUSION:A distinct prognostic significance in the expression of claudin-3 and mostly of claudin-4 between triple-negative and luminal breast carcinomas was identified. Specifically, in triple-negative carcinomas, claudin-4 positivity could probably be considered as a biomarker of favorable prognosis, whereas in luminal cancers with claudin-4-positive expression, the administration of targeted therapy should eventually be part of the patients' management in the near future.
Identification of a claudin-4 and E-cadherin score to predict prognosis in breast cancer.
Szasz Attila M,Nemeth Zsuzsanna,Gyorffy Balazs,Micsinai Mariann,Krenacs Tibor,Baranyai Zsolt,Harsanyi Laszlo,Kiss Andras,Schaff Zsuzsa,Tokes Anna-Maria,Kulka Janina
The elevated expression of claudins (CLDN) and E-cadherin (CDH-1) was found to correlate with poor prognostic features. Our aim was to perform a comprehensive analysis to assess their potential to predict prognosis in breast cancer. The expression of CLDN-1, -3-5, -7, -8, -10, -15, -18, and E-cadherin at the mRNA level was evaluated in correlation with survival in datasets containing expression measurements of 1809 breast cancer patients. The breast cancer tissues of 197 patients were evaluated with tissue microarray technique and immunohistochemical method for CLDN-1-5, -7, and E-cadherin protein expression. An additional validation set of 387 patients was used to test the accuracy of the resulting prognostic score. Based on the bioinformatic screening of publicly-available datasets, the metagene of CLDN-3, -4, -7, and E-cadherin was shown to have the most powerful predictive power in the survival analyses. An immunohistochemical protein profile consisting of CLDN-2, -4, and E-cadherin was able to predict outcome in the most effective manner in the training set. Combining the overlapping members of the above two methods resulted in the claudin-4 and E-cadherin score (CURIO), which was able to accurately predict relapse-free survival in the validation cohort (P = 0.029). The multivariate analysis, including clinicopathological variables and the CURIO, showed that the latter kept its predictive power (P = 0.040). Furthermore, the CURIO was able to further refine prognosis, separating good versus poor prognosis subgroups in luminal A, luminal B, and triple-negative breast cancer intrinsic subtypes. In breast cancer, the CURIO provides additional prognostic information besides the routinely utilized diagnostic approaches and factors.
Claudin 1 expression in basal-like breast cancer is related to patient age.
Blanchard Anne A,Ma Xiuli,Dueck Kevin J,Penner Carla,Cooper Steven C,Mulhall Drew,Murphy Leigh C,Leygue Etienne,Myal Yvonne
BACKGROUND:Defects in tight junctions, gate-keepers of the integrity of the epidermal barrier function, are known to contribute to cancer development. As such, enhancing our understanding of how the expression of proteins involved in these junctions is regulated in cancer, remains a priority. Although the expression of one of these proteins, claudin 1, is down regulated in most invasive human breast cancers (HBC), we have recently shown that high levels of claudin 1, characterized tumors belonging to the very aggressive basal-like breast cancer (BLBC) subtype. In these tumors, the claudin 1 protein, usually localized in the cell membrane, is often mislocalized to the cytoplasm. METHODS:To examine the clinical relevance of this observation, we have generated and analyzed an invasive HBC tissue microarray consisting of 151 breast tumor samples; 79 of which presented a basal-like phenotype (i.e. ER-ve, PR-ve HER2-ve, CK5/6 or EGFR+ve). We also interrogated the outcome of claudin 1 knockdown in a human BLBC cell line, BT-20. RESULTS:Immunohistochemical analysis of this patient cohort revealed a significant association between high claudin 1 expression and BLBCs in women 55 years of age and older. Interestingly, no significant association was found between claudin 1 and nodal involvement, tumor grade or tumor size. Regression analysis however, showed a significant positive association between claudin 1 and claudin 4, even though claudin 4 did not significantly correlate with patient age. Claudin 1 knockdown in BT-20 cells resulted in decreased cell migration. It also significantly altered the expression of several genes involved in epithelial-mesenchymal-transition (EMT); in particular, SERPINE 1 (PAI1) and SSP1 (osteopontin), known to inhibit EMT and cancer cell migration. Conversely, genes known to maintain EMT through their interaction, SNAIL2, TCF4 and FOXC2 were significantly down regulated. CONCLUSIONS:The association of high claudin 1 protein levels observed in tumors derived from older women with BLBC, suggests that claudin 1 has the potential to serve as a marker which can identify a specific subgroup of patients within the BLBC subtype and thus, further contribute to the characterization of these ill-defined breast cancers. More importantly, our studies strongly suggest that claudin 1 directly participates in promoting breast cancer progression, possibly through the alteration of expression of EMT genes.
Claudin-4 Expression Is Associated With Disease-Free Survival in Breast Carcinoma-in-Situ: Mean Follow-up of 8.2 Years.
Duarte Giuliano M,Almeida Natalie Rios,Tocchet Fernando,Espinola Juliana,Barreto Carolina Teixeira de Resende,Pinto Glauce Aparecida,Soares Fernando Augusto,Marshall Priscila,Russano de Paiva Silva Geisilene
Clinical breast cancer
INTRODUCTION:Claudins are tight junctions associated with breast cancer prognosis. The claudin-low intrinsic subtype of invasive carcinoma is associated with high-grade carcinoma, low junction molecule expression, and worse response to chemotherapy. However, it is not known whether the expression of claudins may provide clues as to carcinoma-in-situ (CIS) prognosis. The aim of this study was evaluate claudin-4 expression in CIS and its association with disease-free survival and histologic type of local recurrence (in situ or invasive). METHODS:A tissue microarray block, constructed from 137 pure CIS paraffin blocks, was submitted to immunohistochemical staining for claudin-4, β-catenin, E-cadherin, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and Ki-67. A claudin-4 score categorized samples as claudin-4-low or -high. Clinical and treatment data were obtained from medical records. RESULTS:Claudin-4 expression was evaluated in 86 samples; 88.4% were high and 11.6% low. Mean follow-up was 98.4 months, and the local recurrence rate was 10.4%. There was a significant difference in disease-free survival between claudin-4-high and -low (4.9 and 1.9 years, respectively, P = .02); however, there was no difference between them in histologic type of recurrence (invasive or in situ) (P = .44). CONCLUSION:In our samples, high claudin-4 expression in CIS was more frequent than low expression. Claudin-4-low expression had a worse prognosis in CIS (inferior disease-free survival), but it was similar to high claudin-4 in histologic type of local recurrence.
Prognostic significance of Claudin 12 in estrogen receptor-negative breast cancer.
Iravani Omid,Yip George Wai-Cheong,Thike Aye Aye,Chua Pei Jou,Jane Scully Olivia,Tan Puay-Hoon,Bay Boon-Huat
Journal of clinical pathology
AIMS:Altered expression of the Claudin (CLDN) superfamily of tight junction proteins has been reported in breast cancer. The aim of this study was to examine the immunohistochemical expression of CLDN 12 and its prognostic significance in breast cancer tissues. METHODS:Immunohistochemical expression of CLDN 12 was performed on tissue microarrays consisting of 232 cases of breast carcinoma and correlated with clinicopathological features as well as survival of the patients with breast cancer. RESULTS:For the estrogen receptor (ER)-negative subgroup of patients with breast cancer, CLDN 12 expression was shown to be an independent predictor of poor overall survival (HR=2.345; p=0.020) and disease-free survival (HR=2.177; p=0.026) but not for the ER-positive tumours. CONCLUSIONS:The findings suggest that CLDN 12 expression could be clinically useful for predicting the survival of the ER-negative subgroup of patients with breast cancer.
Methylation of the claudin 1 promoter is associated with loss of expression in estrogen receptor positive breast cancer.
Di Cello Francescopaolo,Cope Leslie,Li Huili,Jeschke Jana,Wang Wei,Baylin Stephen B,Zahnow Cynthia A
Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA). Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+) breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.
Upregulation of Claudin-4, CAIX and GLUT-1 in distant breast cancer metastases.
Jiwa Laura S,van Diest Paul J,Hoefnagel Laurien D,Wesseling Jelle,Wesseling Pieter, ,Moelans Cathy B
BACKGROUND:Several studies have shown that the immunophenotype of distant breast cancer metastases may differ significantly from that of the primary tumor, especially with regard to differences in the level of hormone receptor protein expression, a process known as receptor conversion. This study aimed to compare expression levels of several membrane proteins between primary breast tumors and their corresponding distant metastases in view of their potential applicability for molecular imaging and drug targeting. METHODS:Expression of Claudin-4, EGFR, CAIX, GLUT-1 and IGF1R was assessed by immunohistochemistry on tissue microarrays composed of 97 paired primary breast tumors and their distant (non-bone) metastases. RESULTS:In both the primary cancers and the metastases, Claudin-4 was most frequently expressed, followed by GLUT-1, CAIX and EGFR.From primary breast cancers to their distant metastases there was positive to negative conversion, e.g. protein expression in the primary tumor with no expression in its paired metastasis, in 6%, 19%, 12%, 38%, and 0% for Claudin-4 (n.s), GLUT-1 (n.s), CAIX (n.s), EGFR (n.s) and IGF1R (n.s) respectively. Negative to positive conversion was seen in 65%, 47%, 43%, 9% and 0% of cases for Claudin-4 (p = 0.049), GLUT-1 (p = 0.024), CAIX (p = 0.002), EGFR (n.s.) and IGF1R (n.s.) respectively. Negative to positive conversion of Claudin-4 in the metastasis was significantly associated with tumor size (p = 0.015), negative to positive conversion of EGFR with negative PR status (p = 0.046) and high MAI (p = 0.047) and GLUT-1 negative to positive conversion with (neo)adjuvant chemotherapy (p = 0.039) and time to metastasis formation (p = 0.034). CAIX and GLUT-1 expression in the primary tumor were significantly associated with high MAI (p = 0.008 and p = 0.038 respectively). CONCLUSION:Claudin-4 is frequently expressed in primary breast cancers but especially in their metastases and is thereby an attractive membrane bound molecular imaging and drug target. Conversion in expression of the studied proteins from the primary tumor to metastases was fairly frequent, except for IGF1R, implying that the expression status of metastases cannot always be reliably predicted from the primary tumor, thereby necessitating biopsy for reliable assessment.
Clinicopathologic relevance of claudin 5 expression in breast cancer.
Sugimoto Hitoshi,Nagahara Makoto,Bae Yuan,Nakagawa Tsuyoshi,Ishikawa Toshiaki,Sato Takanobu,Uetake Hiroyuki,Eishi Yoshinobu,Sugihara Kenichi
American journal of clinical pathology
OBJECTIVES:Claudins are major adhesion molecules in tight junctions and are strongly expressed in various cancers. We thus investigated the expression of claudin 5, a member of the claudin family, in breast cancer. METHODS:A total of 193 patients with breast cancer were identified based on their pathologic diagnosis. The expression of each claudin 5 was analyzed in the tumor by immunohistochemical staining. Parametric correlations were done between claudin 5 expression and the clinicopathologic findings. RESULTS:Claudin 5 expression in patients with recurrent breast cancer was statistically significantly higher (P = .004). In addition, analysis of the correlation with other clinicopathologic factors showed statistically significant differences with respect to lymphatic invasion (P = .014), venous invasion (P = .048), estrogen receptor status (P = .002), and human epidermal growth factor 2 status (P = .007). Multivariate analysis revealed that claudin 5 expression was an independent predictive factor in the recurrence for relapse-free survival (RFS) (P = .020). Kaplan-Meier analysis showed that the RFS rate was significantly lower in the high claudin 5 expression group (P = .001). CONCLUSIONS:Patients with breast cancer with high claudin 5 expression had a significantly lower RFS rate. Our findings suggest that claudin 5 may be useful as a new biomarker of a risk factor.
High-level cytoplasmic claudin 3 expression is an independent predictor of poor survival in triple-negative breast cancer.
Jääskeläinen Anniina,Soini Ylermi,Jukkola-Vuorinen Arja,Auvinen Päivi,Haapasaari Kirsi-Maria,Karihtala Peeter
BACKGROUND:The subtype of claudin-low breast cancer can be reliably determined only by gene-expression profiling. Attempts have been made to develop immunohistochemical surrogates, which nearly always focus on membranous claudin expression. METHODS:We assessed the immunohistochemical expression of both membranous and cytoplasmic claudins 3, 4 and 7 in a series of 197 non-metastatic breast cancers, enriched with triple-negative breast cancers (TNBCs; 60%). The expression of epithelial-to-mesenchymal transition-regulating transcription factors Sip1, Zeb1 and vimentin had previously been determined in the same material. RESULTS:In multivariate analysis, strong cytoplasmic claudin 3 expression was associated with poor relapse-free survival (RFS), disease-free survival, distant disease-free survival, breast cancer-specific survival and overall survival among TNBC patients (for RFS, RR 5.202, 95% CI 1.210-22.369, p = 0.027, vs. T-class, RR 0.663, 95% CI 0.168-2.623, p = 0.558, and N-class, RR 3.940, 95% CI 0.933-16.631, p = 0.062). Cytoplasmic claudin 3 expression was also associated with strong nuclear Sip1 expression (p = 0.000053), TNBC phenotype (p = 0.012) and within them, non-basal-like phenotype (p = 0.026). Cytoplasmic claudin 7 was associated with dismal RFS (RR 6.328, 95% CI 1.401-28.593, p = 0.016, vs. T-class, RR 0.692, 95% CI 0.242-1.982, p = 0.493, and N-class, RR 2.981, 95% CI 1.1016-8.749, p = 0.047). Low cytoplasmic expression of claudins 3, 4 and 7 together also predicted poor RFS (RR 6.070, 95% CI 1.347-27.363, p = 0.019, vs. T-class, RR 0.677, 95% CI 0.237-1.934, p = 0.467, and N-class, RR 3.167, 95% CI 1.079-9.290, p = 0.036). CONCLUSIONS:Immunohistochemical expression levels of cytoplasmic claudins 3 and 7 appear to be novel prognostic factors in TNBC.
Claudin 4 expression in triple-negative breast cancer: correlation with androgen receptors and Ki-67 expression.
Abd-Elazeem Mona A,Abd-Elazeem Marwa A
Annals of diagnostic pathology
Breast cancer is the most common malignancy in women and the leading cause of cancer mortality worldwide. Triple-negative breast cancer (TNBC) is an important phenotype of breast cancer that accounts for a relatively small number of breast cancer cases but still represent a focus of increasing interest at the clinical, biological, and epidemiological level. Claudins are the major component of the tight junction, and only a few studies have addressed the role of claudins in breast cancer, especially TNBC. Androgen receptors (ARs), as members of the nuclear receptor superfamily, are known to be involved in a complex network of signaling pathways that collectively regulate cell proliferation. However, roles of AR in breast cancer development and progression have not been very clearly understood. The proliferation marker Ki-67 has been confirmed as an independent predictive and prognostic factor in early breast cancer. The aims of this study are to identify the clinicopathologic associations and prognostic value of claudin 4 expression in TNBC and to correlate claudin 4 expression with AR status and Ki-67 expression. Paraffin blocks obtained from 56 female patients with triple-negative primary invasive ductal breast carcinomas were analyzed for claudin 4, AR, and Ki-67 immunohistochemical expression. High levels of claudin 4 expression were detected in 66.1% of TNBC cases. There was a significant positive correlation with age, tumor size, grade, nodal status, metastasis, and Ki-67 expression (all P < .05) and negative correlation with AR status (P < .001). Androgen receptor showed positivity in 29 cases (51.78%). There was a statistical negative correlation with the all the studied clinicopathologic parameters, claudin 4 and Ki-67 expression. High claudin 4 expression, negative AR expression, and high Ki-67 index would provide a strong prognostic power to differentiate the patients with worse outcome among TNBC patients. Moreover, target treatment for TNBC cells expressing claudin 4 or AR enriched would be valuable for future therapies.
Claudin-Low Breast Cancer; Clinical & Pathological Characteristics.
Dias Kay,Dvorkin-Gheva Anna,Hallett Robin M,Wu Ying,Hassell John,Pond Gregory R,Levine Mark,Whelan Tim,Bane Anita L
Claudin-low breast cancer is a molecular type of breast cancer originally identified by gene expression profiling and reportedly associated with poor survival. Claudin-low tumors have been recognised to preferentially display a triple-negative phenotype, however only a minority of triple-negative breast cancers are claudin-low. We sought to identify an immunohistochemical profile for claudin-low tumors that could facilitate their identification in formalin fixed paraffin embedded tumor material. First, an in silico collection of ~1600 human breast cancer expression profiles was assembled and all claudin-low tumors identified. Second, genes differentially expressed between claudin-low tumors and all other molecular subtypes of breast cancer were identified. Third, a number of these top differentially expressed genes were tested using immunohistochemistry for expression in a diverse panel of breast cancer cell lines to determine their specificity for claudin-low tumors. Finally, the immunohistochemical panel found to be most characteristic of claudin-low tumors was examined in a cohort of 942 formalin fixed paraffin embedded human breast cancers with >10 years clinical follow-up to evaluate the clinico-pathologic and survival characteristics of this tumor subtype. Using this approach we determined that claudin-low breast cancer is typically negative for ER, PR, HER2, claudin 3, claudin 4, claudin 7 and E-cadherin. Claudin-low tumors identified with this immunohistochemical panel, were associated with young age of onset, higher tumor grade, larger tumor size, extensive lymphocytic infiltrate and a circumscribed tumor margin. Patients with claudin-low tumors had a worse overall survival when compared to patients with luminal A type breast cancer. Interestingly, claudin-low tumors were associated with a low local recurrence rate following breast conserving therapy. In conclusion, a limited panel of antibodies can facilitate the identification of claudin-low tumors. Furthermore, claudin-low tumors identified in this manner display similar clinical, pathologic and survival characteristics to claudin-low tumors identified from fresh frozen tumor material using gene expression profiling.
Claudin-2 is an independent negative prognostic factor in breast cancer and specifically predicts early liver recurrences.
Kimbung Siker,Kovács Anikó,Bendahl Pär-Ola,Malmström Per,Fernö Mårten,Hatschek Thomas,Hedenfalk Ingrid
BACKGROUND:Predicting any future metastatic site of early-stage breast cancer is important as it significantly influences the prognosis of advanced disease. This study aimed at investigating the potential of claudin-2, over-expressed in breast cancer liver metastases, as a biomarker for predicting liver metastatic propensity in primary breast cancer. METHODS:Claudin-2 expression was analyzed in two independent cohorts. Cohort 1 included 304 women with metastatic breast cancer diagnosed between 2002 and 2007, while cohort 2 included 237 premenopausal women with early-stage node-negative breast cancer diagnosed between 1991 and 1994. Global transcriptional profiling of fine-needle aspirates from metastases was performed, followed by immunohistochemical analyses in archival primary tumor tissue. Associations between claudin-2 expression and relapse site were assessed by univariable and multivariable Cox regression models including conventional prognostic factors. Two-sided statistical tests were used. RESULTS:CLDN2 was significantly up-regulated (P < 0.001) in liver metastases compared to other metastatic sites. Claudin-2 protein was more frequently expressed in primary tumors from patients who subsequently developed liver metastases (P = 0.02) and high expression was associated with a shorter metastasis-free interval (cohort 1, HR = 1.4, 95% CI = 1.0-1.9; cohort 2, HR = 2.2, 95% CI = 1.3-3.5). Specifically, a significantly shorter interval between primary tumor diagnosis and liver-specific recurrence was observed among patients with high levels of claudin-2 expression in the primary tumor (cohort 1, HR = 2.3, 95% CI = 1.3-3.9). CONCLUSION:These results suggest a novel role for claudin-2 as a prognostic biomarker with the ability to predict not only the likelihood of a breast cancer recurrence, but more interestingly, the liver metastatic potential of the primary tumor.