Cadherin-11 Research Articles

Inhibition of cell adhesion by a cadherin-11 antibody thwarts bone metastasis.

Cadherin-11 (CDH11) is a member of the cadherin superfamily mainly expressed in osteoblasts but not in epithelial cells. However, prostate cancer cells with a propensity for bone metastasis express high levels of cadherin-11 and reduced levels of E-cadherin. Downregulation of cadherin-11 inhibits interaction of prostate cancer cells with osteoblasts in vitro and homing of prostate cancer cells to bone in an animal model of metastasis. These findings indicate that targeting cadherin-11 may prevent prostate cancer bone metastasis. To explore this possibility, a panel of 21 monoclonal antibodies (mAb) was generated against the extracellular (EC) domain of cadherin-11. Two antibodies, mAbs 2C7 and 1A5, inhibited cadherin-11-mediated cell-cell aggregation in vitro using L-cells transfected with cadherin-11. Both antibodies demonstrated specificity to cadherin-11, and neither antibody recognized E-cadherin or N-cadherin on C4-2B or PC3 cells, respectively. Furthermore, mAb 2C7 inhibited cadherin-11-mediated aggregation between the highly metastatic PC3-mm2 cells and MC3T3-E1 osteoblasts. Mechanistically, a series of deletion mutants revealed a unique motif, aa 343-348, in the cadherin-11 EC3 domain that is recognized by mAb 2C7 and that this motif coordinated cell-cell adhesion. Importantly, administration of mAb 2C7 in a prophylactic setting effectively prevented metastasis of PC3-mm2 cells to bone in an in vivo mouse model. These results show that targeting the extracellular domain of cadherin-11 can limit cellular adhesion and metastatic dissemination of prostate cancer cells. Monotherapy using a cadherin-11 antibody is a suitable option for the prevention of bone metastases.

Abstract A045: Cadherin-11, a common therapeutic target in poor prognosis malignancies and rheumatoid arthritis

Abstract Rational: Poor prognosis epithelial-derived cancers often exhibit morphologic and molecular changes characteristic of an epithelial to mesenchymal transition (EMT). EMT markers are predominantly found in tumors with a basal-like phenotype. Increased expression of the mesenchymal cadherins N-cadherin and/or Cadherin-11 (CDH11) and decreased E-cadherin, have been associated with both EMT and tumor progression. Importantly, CDH11 is a therapeutic target in rheumatoid arthritis (RA), an inflammatory disease with properties often compared with cancer. As CDH11 antibody based therapeutics are in clinical trials for RA and we recently showed that the arthritis drug celecoxib has the structural potential to bind CDH11, there is a strong possibility that, if CDH11can be shown to drive malignant progression rather than simply be associated with it, therapeutic options may be rapidly developed. Results: We show that CDH11 is increased early in breast cancer and ductal carcinoma in-situ. CDH11 knockdown and antibodies effective in RA slowed the growth of basal-like breast tumors. CDH11 attenuation decreased proliferation and colony formation of breast, glioblastoma and prostate cancer cells. The arthritis drug celecoxib, which has characteristics consistent with CDH11 binding, an analogue without COX-2 inhibitory activity and other molecules that target CDH11, preferentially affect CDH11 positive cancer cells. Conclusion: Our work demonstrates that cadherin-11 is important for malignant progression, is a therapeutic target in several aggressive tumors and introduces an antibody, novel small molecules and an approved arthritis drug that inhibit its function with potential for rapid clinical translation. Citation Format: Shahin Assefnia, Sivanesan Dakshanamurthy, Jaime M. Guidry Auvil, Constanze Hampel, Panos Anastasiadis, Bhaskar Kallakury, Aykut Uren, David W. Foley, Milton Brown, Lawrence Shapiro, Michael Brenner, David Haigh, Stephen W. Byers. Cadherin-11, a common therapeutic target in poor prognosis malignancies and rheumatoid arthritis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A045.

Cadherin-11 Regulates Motility in Normal Cortical Neural Precursors and Glioblastoma

Metastasizing tumor cells undergo a transformation that resembles a process in normal development when non-migratory epithelial cells modulate the expression of cytoskeletal and adhesion proteins to promote cell motility. Here we find a mesenchymal cadherin, Cadherin-11 (CDH11), is increased in cells exiting the ventricular zone (VZ) neuroepithelium during normal cerebral cortical development. When overexpressed in cortical progenitors in vivo, CDH11 causes premature exit from the neuroepithelium and increased cell migration. CDH11 expression is elevated in human brain tumors, correlating with higher tumor grade and decreased patient survival. In glioblastoma, CDH11-expressing tumor cells can be found localized near tumor vasculature. Endothelial cells stimulate TGFβ signaling and CDH11 expression in glioblastoma cells. TGFβ promotes glioblastoma cell motility, and knockdown of CDH11 expression in primary human glioblastoma cells inhibits TGFβ-stimulated migration. Together, these findings show that Cadherin-11 can promote cell migration in neural precursors and glioblastoma cells and suggest that endothelial cells increase tumor aggressiveness by co-opting mechanisms that regulate normal neural development.

The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas.

Effect of ovarian steroids on gene expression related to synapse assembly in serotonin neurons of macaques

Dendritic spines are the elementary structural units of neural plasticity. In a model of hormone replacement therapy (HT), we sought to determine the effect of estradiol (E) and progesterone (P) on gene expression related to synapse assembly in a laser-captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n = 2 animals/treatment), and the results were confirmed for pivotal genes with qRT-PCR on additional laser-captured material (n = 3 animals/treatment). Ovariectomized rhesus macaques were treated with placebo, E, or E + P via Silastic implants for 1 month. The midbrain was obtained, sectioned, and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an arcturus laser dissection microscope (Pixel II). RNA from laser-captured serotonin neurons was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a twofold or greater change in the expression of 63 probe sets in the cell adhesion molecule (CAM) category, and 31 probe sets in the synapse assembly category were similarly altered in E- and E + P-treated animals. qRT-PCR assays showed that E treatment induced a significant increase in ephrin receptor A4 (EPHA4) and in integrin A8 (ITGA8) but not in ephrin receptor B4 (EPHB4) or integrin B8 (ITGB8) expression. E also increased expression of cadherin 11 (CDH11), neuroligin 3 (NLGN3), neurexin 3 (NRXN3), syndecan 2 (SCD2), and neural cell adhesion molecule (NCAM) compared with placebo. Supplemental P treatment suppressed E-induced gene expression. In summary, ovarian steroids target gene expression of adhesion molecules in serotonin neurons that are important for synapse assembly.

Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion‐associated candidate genes

The molecular mechanisms underlying the infiltrative growth of glioblastomas, the most common primary tumours of the central nervous system in adults, are still poorly understood. We aimed to identify and functionally validate novel glioma invasion-associated candidate genes. Microarray-based expression analysis was applied to identify differentially expressed genes in microdissected infiltrating glioma cells in vivo. Promising candidate genes were selected by the invasion-associated gene ontology terms cell adhesion, endocytosis, extracellular matrix and cell migration and validated in vitro by invasion assays and in situ by immunohistochemistry. We identified 180 up-regulated and 61 down-regulated genes (fold change: ≥ 2; P < 0.01) in the infiltration zone relative to more central cell-rich tumour areas of malignant astrocytic gliomas (n = 11). Twenty-seven of these genes matched to invasion-related gene ontology terms. From these, we confirmed the genes encoding cadherin-11 (CDH11), proprotein convertase subtilisin/kexin type 6 (PCSK6) and SH3-domain GRB2-like 3 (SH3GL3) as novel glioma invasion-associated candidate genes, with knockdown of PCSK6 and SH3GL3 inhibiting glioma cell invasion, while inhibition of CDH11 promoted glioma cell invasion in vitro. Immunohistochemistry on glioblastoma tissue sections revealed expression of CDH11 and PCSK6 protein in glioma cells of more central, cell-rich tumour areas, with only weak or absent CDH11 immunoreactivity but consistent PCSK6 staining in infiltrating glioma cells. Using molecular profiling of microdissected primary tumour tissue specimens followed by functional in vitro analysis, we identified and validated CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes that likely contribute to the invasive phenotype of malignant gliomas.

Aneurysmal Bone Cyst

Aneurysmal bone cyst (ABC) is a primary bone tumor that predominantly affects individuals in the first 2 decades of life. Imaging studies reveal an osteolytic, eccentric, and multicystic lesion. The neoplasm usually behaves in a benign fashion but may recur locally. Intralesional curettage is the treatment of choice, but surgical resection or other therapeutic approaches have been occasionally used. Aneurysmal bone cyst is characterized by the chromosomal translocation t(16;17)(q22;p13), which results in the fusion of the promoter region of the osteoblast cadherin 11 (CDH11) to the entire coding sequence of the ubiquitin protease USP6 in a classic promoter swapping mechanism. The fusion leads to USP6 transcriptional up-regulation and protein overexpression. Several alternate USP6 fusion genes have also been described, and more recent studies have shown that USP6 contributes to the pathogenesis of ABC via (1) activation of metalloproteases that promote osteolysis and matrix degradation and (2) inhibition of proteins involved in osteoblastic maturation. These new discoveries are enhancing our understanding of ABC biology and may lead to the development of novel therapeutic approaches for this tumor.

Cadherin‐11 contributes to pulmonary fibrosis: potential role in TGF‐β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.

HOXC8-Dependent Cadherin 11 Expression Facilitates Breast Cancer Cell Migration through Trio and Rac

HOXC8 expression is upregulated in diverse cancer types, and a high level of HOXC8 is often associated with the aggressive/metastatic phenotypes. We previously reported that the presence of HOXC8 is essential for breast cancer cell migration and metastasis. However, the underlying molecular mechanism of HOXC8 regulation of cell migration is unclear. Here, we demonstrate that the presence of HOXC8 is required for cadherin 11 (CDH11) expression in breast cancer cells and that HOXC8 regulation of cell migration is mediated by CDH11. To understand the role of HOXC8-CDH11 axis in cell migration, we show that depleting either HOXC8 or CDH11 diminishes the formation of actin-based membrane ruffles, an event essential for cell migration. The loss of membrane ruffles in HOXC8- or CDH11-knockdown cells is apparently caused by reduced Rac activity because ectopically expressing active Rac1 restores cytoskeleton reorganization. CDH11 physically interacts with Trio, a Rac GEF. We show that Trio is responsible for the majority of endogenous Rac activity in migratory breast cancer cells. Because knockdown of CDH11 prevents the plasma membrane localization of Trio, our study indicates that CDH11 may play a role in recruiting Trio to the plasma membrane where Trio activates Rac, leading to cell migration. This study reveals a novel HOXC8-CDH11-Trio-Rac signaling axis that contributes significantly to breast cancer cell migration.

In Vitro Approaches to Evaluate Placental Drug Transport by Using Differentiating JEG‐3 Human Choriocarcinoma Cells

Human choriocarcinoma cells have been used as models for studying transcellular drug transport through placental trophoblasts. However, these models allow the transport of low-molecular-weight drugs through intercellular gap junctions. This study aimed at investigating the differentiation patterns of JEG-3 choriocarcinoma cells under different culture conditions and establishing the appropriate model of in vitro syncytiotrophoblast drug transport. Paracellular permeability was estimated by measuring the transepithelial electrical resistance (TEER) across JEG-3 cell layers. The mRNA expression levels of non-expressed in choriocarcinoma clone 1 (NECC1) and breast cancer resistance protein (BCRP), and those of E-cadherin (ECAD) and cadherin-11 (CDH11), which are adherens junction-associated proteins related to fusogenic ability of syncytiotrophoblasts differentiated from cytotrophoblasts, protein expression levels were considered as the differentiation signals. The highest TEER values were obtained in the JEG-3 cells cultured in the Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (1:1) mixed medium (CS-C(®) ; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan). By comparing the TEER values and the differentiation signals, the authors identified at least five JEG-3 cell-differentiation patterns. The differentiation pattern of JEG-3 cultured in CS-C resembled the syncytiotrophoblast-like differentiation signal characterizations in vivo. In conclusion, the syncytiotrophoblast-like models of differentiating JEG-3 cells cultured in CS-C might be appropriate for evaluating drug transport across the placental trophoblast.

OB‐cadherin cloning and expression in a model of wound repair in horses

Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast-to-myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB-cadherin expression. To clone equine cadherin-11 (CDH11) cDNA and to study its spatiotemporal expression profile during the repair of body and limb wounds, thereby contributing to a better understanding of the repair process. Cloning was by a PCR technique. Expression was studied in intact skin and in 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal CDH11 gene expression was determined by RT-PCR while OB-cadherin protein expression was mapped immunohistochemically. Equine CDH11 is a highly conserved gene and protein. mRNA was not expressed in equine skin whereas the wound repair process was characterised by a significantly higher expression in the thorax than in limb samples. mRNA expression pattern was paralleled by protein data as confirmed by immunohistochemistry. The data suggest that deficient OB-cadherin expression in the first phases of wound repair contributes to the excessive proliferative response seen in horse limb wounds. Future studies should verify the quantitative, temporal expression of this protein in order to provide the basis for targeted therapies that might prevent the development of EGT in horse wound repair.

Expressão de caderinas e fator repressor em tumor de pacientes com câncer de mama na presença ou ausência de células malignas em medula óssea

OBJETIVO: Acredita-se que o perfil gênico do tumor é diferente daquele do tecido normal. Além disso, é possível que o tumor adquira características que lhe conferem potencial de gerar metástases em sítios específicos. A expressão tumoral de Caderinas 3 e 11 (envolvidas em adesão e migração celular), e de CBX3 (repressor transcricional) pode estar relacionada a maior agressividade e potencial invasivo em pacientes com câncer de mama (CM). Nosso objetivo é comparar a expressão de Caderina 3, Caderina 11 e CBX3 em tumor e tecido mamário normal adjacente e correlacionar a expressão tumoral destes genes com colonização da medula óssea (MO) por células tumorais. MÉTODOS: Amostras de tumor, tecido normal peritumoral e MO foram obtidas de 30 pacientes com CM estadio clínico I-III. A expressão de Caderina 3, Caderina 11 e CBX3 foi avaliada por RT-PCR em tempo real. A presença de células malignas em MO foi determinada por detecção da expressão de citoqueratina 19 por Nested-RT-PCR. RESULTADOS: Dezessete pacientes (56,7%) apresentaram células malignas em MO. Houve maior expressão de Caderina 3 e Caderina 11, mas não de CBX3 em tumor em relação ao tecido normal. Não encontramos associação entre expressão tumoral de Caderina 3, Caderina11 e CBX3 e a presença de células malignas em MO. CONCLUSÃO: A hiperexpressão de Caderina 3 e 11 em tumores em relação ao tecido normal sugere que estes transcritos possa mestar relacionados à carcinogênese. A expressão tumoral de Caderina 3, Caderina 11 e CBX3 não parece associada com a disseminação para a MO.

Minimal 16q Genomic Loss Implicates Cadherin-11 in Retinoblastoma

Abstract Retinoblastoma is initiated by loss of both RB1 alleles. Previous studies have shown that retinoblastoma tumors also show further genomic gains and losses. We now define a 2.62 Mbp minimal region of genomic loss of chromosome 16q22, which is likely to contain tumor suppressor gene(s), in 76 retinoblastoma tumors, using loss of heterozygosity (30 of 76 tumors) and quantitative multiplex PCR (71 of 76 tumors). The sequence-tagged site WI-5835 within intron 2 of the cadherin-11 (CDH11) gene showed the highest frequency of loss (54%, 22 of 41 samples tested). A second hotspot for loss (39%, 9 of 23 samples tested) was detected within intron 2 of the cadherin-13 (CDH13) gene. Furthermore, deletion of the exons of CDH11 and/or WI-5835 was shown by quantitative multiplex PCR in 17 of 30 (57%) of previously untested tumors. Immunoblot analyses revealed that 91% (20 of 22) retinoblastoma exhibited either a complete loss or a decrease of the intact form of CDH11 and 8 of 13 showed a prevalent band suggestive of the variant form. Copy number of WI-5835 for these samples correlated with CDH11 protein expression. CDH11 staining was evident in the inner nuclear layer in early mouse retinal development and in small transgenic murine SV40 large T antigen–induced retinoblastoma tumors, but advanced tumors frequently showed loss of CDH11 expression by reverse transcription-PCR, suggestive of a role for CDH11 in tumor progression or metastasis. CDH13 protein and mRNA were consistently expressed in all human and murine retinoblastoma compared with normal adult human retina. Our analyses implicate CDH11, but not CDH13, as a potential tumor suppressor gene in retinoblastoma.

Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways

Cadherins, a family of cell-cell adhesion molecules, provide recognition signals that are important for cell sorting and aggregation during tissue development. This study was performed to determine whether distinct cadherin repertoires define tissue-specific lineages during differentiation of immature C3H10T1/2 and C2C12 mesenchymal cells. Both cell lines expressed mRNA for N-cadherin (N-cad), cadherin-11 (C11), and R-cadherin (R-cad). After induction of osteogenesis by recombinant human BMP-2 (rhBMP-2) treatment, steady state N-cad mRNA slightly increased in C3H10T1/2 cells. Likewise, the abundance of C11 mRNA increased in both cell lines, although the changes were more remarkable in C2C12 cells. By contrast, R-cad expression was almost shut off by rhBMP-2. The immature but committed osteoblastic MC3T3-E1 cells exhibited only minor changes in N-cad and C11 mRNA abundance after rhBMP-2 treatment. Whereas adipogenic differentiation was associated with a net decrease of N-cad and C11 expression in C3H10T1/2 cells, induction of myogenesis in C2C12 cells resulted in up-regulation of N-cad, while R-cad mRNA became undetectable in either case. Similarly, the adipocytic 3T3-L1 cells expressed very low levels of all cadherins when fully differentiated. Therefore, the repertoire of cadherins present in undifferentiated mesenchymal cells undergoes distinct changes during transition to mature cell phenotypes. Although neither N-cad nor C11 represent strict tissue-specific markers, the relative abundance of these mesenchymal cadherins defines lineage-specific signatures, perhaps providing recognition signals for aggregation and differentiation of committed precursors.

Human Osteoblasts Express a Repertoire of Cadherins, Which Are Critical for BMP-2–Induced Osteogenic Differentiation

Direct cell-cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription-polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS-2 and MG-63, expressed mRNA for cadherin-11 (C11) and N-cadherin (N-cad). HOBs and BMCs also expressed low levels of cadherin-4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein-2 (BMP-2) treatment of either BMCs or HOBs. Likewise, N-cad mRNA did not change during BMP-2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down-regulated by BMP-2 treatment of normal cells. Both C11 and C4 were localized to sites of cell-cell contact in both HOBs and BMCs, colocalized with beta-catenin, and bands corresponding to cadherins were coimmunoprecipitated by a beta-catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N-cad partially inhibited Ca2+-dependent cell-cell adhesion and completely prevented BMP-2-induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin-mediated cell-to-cell adhesion is critical for normal human osteoblast differentiation.