Simultaneous bilateral breast carcinoma: Histopathological characteristics and CD44/catenin-cadherin profile

AIMS: Family history of breast carcinoma, multicentric tumor foci in one breast, and in situ lobular carcinoma increase the risk of bilateral breast cancer (BBC), synchronous or metachronous. Synchronous tumors are designated as simultaneous breast carcinoma if they appear at the same time. The CD44 family and cadherin/catenin immunophenotype of this group of BBCs has not yet been evaluated. The aim of this study was to compare clinicopathological characteristics and immunohistochemical profiles of simultaneous BBC and corresponding lymph node metastases in eight patients. METHODS AND RESULTS: In toto 15 primary and 9 metastatic tumors were evaluated. The expression of CD44 variant isoforms, beta-catenin, E, P and N-cadherin were evaluated by immunohistochemistry. Rare types of breast carcinoma were frequent in this group of patients. There were 6 pleomorphic lobular, 5 invasive ductal of usual type, 3 atypical medullary carcinomas, 2 mucinous and one invasive micropapillary carcinoma. The expression CD44v6 was most frequent, followed by CD44v3-10, CD44v5, and CD44v3. CD44v4 was generally not expressed. E-cadherin was expressed in 80% primary tumors, 40% expressed N-cadherin, and 66% expressed P-cadherin. CONCLUSIONS: Generally, simultaneous carcinomas had different morphology and different immunophenotype. Each primary tumor was more similar to its corresponding metastatic tumor than to the contralateral primary tumor.     

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

Epithelial cadherin (E‐cadherin) is a 120 kDa cell–cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as β‐catenin. Loss of E‐cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down‐regulation of E‐cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH‐6 and IBH‐4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E‐cadherin and related proteins in IBH‐6 and IBH‐4 cell lines was evaluated. In IBH‐6 and IBH‐4 cell extracts, only an 89 kDa E‐cadherin form (Ecad89) was detected, which is truncated at the C‐terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E‐cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH‐6 and IBH‐4 cells showed an intracellular scattered E‐cadherin localization. β‐catenin accompanied E‐cadherin localization, and actin stress fibers were identified in both cell types. E‐cadherin mRNA levels were remarkably low in IBH‐6 and IBH‐4 cells. The E‐cadherin mRNA and genomic sequence encoding exons 14–16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up‐regulation of Twist mRNA was found in both cell lines. In conclusion, IBH‐6 and IBH‐4 breast cancer cells show down‐regulation of E‐cadherin expression with aberrant protein localization, and up‐regulation of Twist; these features can be related to their invasive/metastatic characteristics. J. Cell. Physiol. 222: 596–605, 2010. © 2009 Wiley‐Liss, Inc.     

E-cadherin和CD44V6在食管上皮癌变过程及癌组织中的表达

研究不同类型的食管上皮增生和癌组织的 E- cadherin (E- cad)和 CD44 V6的表达 ,并探讨其与食管癌发生和发展的关系。应用免疫组织化学 SABC法 ,观察 10例正常、 3例消化性溃疡、 2 5例单纯性增生、 15例不典型增生的食管粘膜上皮 ,5例食管原位癌与 5 4例浸润癌组织中的 E- cad和 CD44 V6蛋白的表达情况。结果显示正常食管鳞状上皮和高分化肿瘤细胞膜和细胞浆 E- cad和 CD44 V6染色 ,非典型增生、低分化肿瘤细胞两种蛋白抗体表达减弱或呈阴性。E- cad和 CD44 V6的表达与癌组织的组织学分级、类型和淋巴结转移有关 (P<0 .0 1,P<0 .0 5 ) ,与癌组织的浸润深度无关 (P>0 .0 5 )。提示E- cad和 CD44 V6表达减弱是癌组织低分化和高度恶性的生物学标志 ,但其与淋巴结转移的关系有待进一步研究     

宫颈癌中骨桥蛋白和CD44v6的表达及临床意义

目的探讨骨桥蛋白(OPN)和CD44v6在宫颈浸润癌中的表达及其意义。方法应用免疫组化SP法检测OPN和CD44v6在10例慢性宫颈炎、30例宫颈上皮内瘤样变及50例宫颈浸润癌组织中的表达。结果OPN在以上组织中的阳性表达率分别为10.00%(1/10)、36.67%和60.00%,慢性宫颈炎与宫颈浸润癌之间的表达差异有显著性(P<0.01);CD44v6阳性表达率分别为10.00%(1/10)、43.33%和68.00%,慢性宫颈炎与宫颈浸润癌之间的表达差异有显著性(P<0.01)。宫颈浸润癌组织中OPN和CD44v6表达均与患者年龄、肿瘤病理分级、组织学类型无关(P>0.05),而与淋巴结转移有关(P<0.05)。OPN与CD44v6的表达之间有显著正相关性(r=0.829,P<0.01)。结论OPN、CD44v6可能参与了宫颈癌的发生、发展和转移过程,联合检测它们的表达可作为判断宫颈癌的预后和术后复发的评估指标。     

CD44v6: a target for antibody-based cancer therapy

The human CD44 gene encodes type 1 transmembrane glycoproteins involved in cell-cell and cell-matrix interactions. The structural heterogeneity of the gene products is caused primarily by alternative splicing of at least 10 out of 20 exons. Certain CD44 variant isoforms, in particular those containing CD44 variant domain 6 (CD44v6), have been implicated in tumourigenesis, tumour cell invasion and metastasis. Here we will give an overview of immunohistochemically determined CD44v6 expression in human malignancies (primary epithelial and nonepithelial tumours as well as metastases) and normal tissues, and review several examples of the clinical use of CD44v6-specific antibodies. In nonmalignant tissues, CD44v6 expression is essentially restricted to a subset of epithelia. Intense and homogeneous expression of CD44v6 was reported for the majority of squamous cell carcinomas and a proportion of adenocarcinomas of differing origin, but was rarely seen in nonepithelial tumours. This expression pattern has made CD44v6 an attractive target for antibody-guided therapy of various types of epithelium-derived cancers.Abbreviations CD44 type 1 transmembrane glycoprotein, cell surface receptor for hyaluronate - CD44s (CD44H) standard form of CD44 - CD44v6 splice variant exon 6 of CD44 - CTC common toxicity criteria - 2F10, VFF4, VFF7, VFF18 (BIWA 1), U36, V6B3, HB-256, Var 3.1 monoclonal antibodies targeting the CD44v6 antigen - SCC squamous cell carcinoma     

DDR1 regulates the stabilization of cell surface E‐cadherin and E‐cadherin‐mediated cell aggregation

The stabilization of cell surface E‐cadherin is important for the maintenance of apical junction complexes and epithelial polarity. Previously, we reported that discoidin domain receptor 1 (DDR1) forms a complex with E‐cadherin at adhesive contacts; however, the regulatory role of DDR1 in the stabilization of cell surface E‐cadherin and E‐cadherin‐mediated cell behaviors remained undefined. To gain insight into these questions, we utilized two stable clones depleted for DDR1 via the small interfering RNA (siRNA) technique, and we over‐expressed DDR1 in MDCK cells. We performed Western blotting, immunofluorescence analysis, flow cytometry, and cell aggregation studies to investigate the effect of DDR1 on cell surface E‐cadherin. The results showed that both DDR1/2 and E‐cadherin use their extracellular domains to form DDR/E‐cadherin complexes. Neither the depletion nor the over‐expression of DDR1 changed the expression level of E‐cadherin in MDCK cells. Collagen disrupted the formation of E‐cadherin complexes and caused E‐cadherin to accumulate in the cytoplasm; however, over‐expression of DDR1 stabilized E‐cadherin on the cell surface and decreased its cytoplasmic accumulation. Furthermore, independently of collagen stimulation, the depletion of DDR1 resulted in a decrease in the level of cell surface E‐cadherin, which consequently caused its cytoplasmic accumulation and decreased E‐cadherin‐mediated cell aggregation. These results indicate that DDR1 can increase the stability of cell surface E‐cadherin and promote MDCK cell aggregation, which may be mediated through the formation of DDR1/E‐cadherin complexes. Overall, these findings have implications for the physiological roles of DDR1 in association with the maintenance of both the adhesion junction and epithelial polarity. J. Cell. Physiol. 224: 387–397, 2010. © 2010 Wiley‐Liss, Inc.     

H‐Cadherin expression reduces invasion of malignant melanoma

Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell–cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E‐cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H‐cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H‐cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H‐cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re‐expression of H‐cadherin decreases migration and invasion capacity, as well as anchorage‐independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re‐express H‐cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down‐regulation of H‐cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

CD44s and CD44v6 expression in head and neck epithelia

Background

CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.

Methods

Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.

Results

In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.

Conclusion

CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.     

Inhibition of cell proliferation by CD44: Akt is inactivated and EGR‐1 is down‐regulated

Objective: CD44 is a transmembrane glycoprotein and can facilitate signal transduction by serving as a platform for molecular recruitment and assembly. A number of studies have suggested that CD44 can either positively or negatively regulate cell proliferation. The purpose of this study was to investigate how CD44 can inhibit cell proliferation. Materials and methods: We engineered E6.1 Jurkat cells to express CD44. Importantly, these cells lack endogenous CD44 expression. Molecular pathways involved with cell proliferation were studied using RT²‐PCR array, siRNA, Western blotting and by employing pharmacological inhibitors of ERK1/2, p38 and the PI3K/Akt pathways. Results: We found that CD44 expression significantly inhibited cell proliferation and down‐regulated EGR‐1 expression and EGR‐1 targets cyclin D1 and cyclin D2. Transfection of control E6.1 Jurkat cells with EGR‐1 siRNA also inhibited cell proliferation, confirming its role. Disruption of the PI3K/Akt pathway with pharmacological inhibitors reduced both EGR‐1 expression and cell proliferation, recapitulating the properties of CD44 expressing cells. Akt was hypophosphorylated in cells expressing CD44 showing its potential role in negatively regulating Akt activation. Strikingly, constitutively active Akt rescued the proliferation defect showing requirement for active Akt, in our system. Conclusion: Our results suggest a novel pathway by which CD44 inactivates Akt, down‐regulates EGR‐1 expression and inhibits cell proliferation.     

Heat shock protein 72 associated with CD44v6 in human colonic adenocarcinoma

CD44v6 is a splice variant of CD44 (CD44v), probably promoting cancer cell adherence to vascular endothelium and base membranes and enhancing the invasion and metastasis of colonic carcinomas. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to be overexpressed in epithelial carcinoma cells. There may be a possible association between the expression of HSP72 and CD44v6 during the growth and progression of colonic carcinoma cells. The aim of the study was to investigate the interaction between heat shock protein 72 and CD44v6 in human colonic carcinomas. The localization of HSP72 and CD44v6 in human colonic carcinomas was determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and CD44v6 in colonic carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. Our results revealed that colonic carcinoma synchronously co-expressed higher levels of HSP72 and CD44v6 than that in adjacent normal colonic tissues. HSP72 and CD44v6 were mainly immunolocalized in the cytoplasm, and also immunolabelled on the cell membrane. Based on immunoprecipitation and Western immunoblots, we found that HSP72 was associated with CD44v6 precursor fragments in human colonic carcinoma cells. The interaction between HSP72 and CD44v6 in human colonic carcinoma cells may contribute to study the pathogenesis and immunotherapy of colonic carcinoma.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

E‐cadherin determines Caveolin‐1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin‐1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E‐cadherin in CAV1‐dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E‐cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co‐expression of E‐cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav‐1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E‐cadherin expression in B16F10 (E‐cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co‐expression of CAV1 and E‐cadherin in B16F10 (cav‐1/E‐cad) cells abolishes tumor formation, lung metastasis, increased Rac‐1 activity, and cell migration observed with B16F10 (cav‐1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac‐1 activation in these cells.     

Proper E‐cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer

We reported that Disabled‐2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E‐cadherin. Dab2 depletion in Caco‐2 cells led to E‐cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium‐induced colitis increased Dab2/E‐cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E‐cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E‐cadherin delocalization. Epithelial Dab2 decrement preceded that of E‐cadherin. We suggest that Dab2, by inhibiting E‐cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.     

大肠癌组织及癌旁组织中c—myc，COX-2和CD44v6基因表达量差异的检测

目的：通过检测大肠癌组织和癌旁组织中c-myc,COX-2以及CD44v6的表达水平,探讨这三种基因在大肠癌发生和发展中的意义。方法：应用实时荧光定量PCR技术检测了10例大肠癌组织和相应癌旁组织中c-myc,COX-2以及CD44v6基因表达水平的差异,并探讨了各基因在癌纽织中的表达水平与大肠癌临床病理指标之间的关系。结果：c-myc,COX-2以及CD44v6在大肠癌组织和癌旁组织中的表达均有非常显著性差异（P〈0．01）;癌组织中COX-2和CD44v6的表达与淋巴结转移、分化程度及Dukes分期有关（P〈0．05）。结论：c—myc,COX-2和CD44v6的异常表达均与大肠癌密切相关,三者从不同方面对大肠癌的发生和发展起到了重要作用,可作为早期诊断和预后的参考指标。     

FAM3B promotes progression of oesophageal carcinoma via regulating the AKT–MDM2–p53 signalling axis and the epithelial‐mesenchymal transition

FAM3B has been suggested to play important roles in the progression of many cancers, such as gastric, oral, colon and prostate cancer. However, little is known about the role of FAM3B in human esophageal squamous cell carcinoma (ESCC). In the present study, we found that FAM3B expression was higher in ESCC tissues than in adjacent normal tissues. Using quantitative real‐time polymerase chain reaction, we found similar results in cell lines. FAM3B expression was significantly related to T/TNM stage. Importantly, Kaplan–Meier analysis revealed that a high expression level of FAM3B predicted a poor outcome for ESCC patients. Overexpression of FAM3B inhibits ESCC cell death, increases oesophageal tumour growth in xenografted nude mice, and promotes ESCC cell migration and invasion. Further studies confirmed that FAM3B regulates the AKT–MDM2–p53 pathway and two core epithelial‐to‐mesenchymal transition process markers, Snail and E‐cadherin. Our results provide new insights into the role of FAM3B in the progression of ESCC and suggest that FAM3B may be a promising molecular target and diagnostic marker for ESCC.