CDH3 Gene Research Articles

Identification of oxidative stress-induced gene expression profiles in cavernosal endothelial cells.

The aim of the present study was to explore the regulation status of genes in oxidative stress (OS)‑induced endothelial dysfunction and to elucidate the mechanism of action of OS‑associated genes, which induce cavernosal endothelial dysfunction in erectile dysfunction (ED). OS was established in purified cavernosal endothelial cells (CECs) using xanthine/xanthine oxidase and the differentially expressed OS‑associated genes were analyzed using gene microarrays. In addition, an ED rat model was established through bilateral internal iliac artery ligation with hyperlipidemia and was verified by an intracavernosal pressure test. The selected OS‑associated genes were validated in the CECs and ED rat model using reverse transcription‑quantitative polymerase chain reaction. Student's t‑test and one‑way analysis of variance were performed using SBC analysis system. Gene microarray analysis revealed that 13090 (31.92%) genes were expressed in the control group, whereas 12039 (29.35%) genes were expressed in the treated group. The cut‑off value for differential expression was set at 2.0 fold‑change and 2480 genes were found to be differentially expressed compared with the control group. Of these cells, 1454 were upregulated and 1026 were downregulated. Cluster analysis identified relevant cell signaling pathways that were hypothesized to be significant in OS‑associated endothelial dysfunction, including the cytokine‑cytokine receptor interactions, nitrogen metabolism, coagulation cascades and cell adherens. Cxcl12, Tgfbr1, Asns, Bdkrb1 and Cdh3 genes showed a corresponding variation in the CECs and ED rat model compared with the results of the gene microarray analysis. In conclusion, in the present study, the network of differentially expressed genes and OS‑associated signaling pathways identified using gene microarray analysis were validated in the CECs and ED rat model. The results indicated that OS may lead to endothelial dysfunction through certain cell signaling pathways, inducing ED. However, further functional verification is required in order to elucidate the underlying mechanisms of OS‑associated cell signaling pathways in ED.

Resolving the genetic heterogeneity of prelingual hearing loss within one family: Performance comparison and application of two targeted next generation sequencing approaches

Here, we report an unconventional Chinese pedigree consisting of three branches all segregating prelingual hearing loss (HL) with unclear inheritance pattern. After identifying the cause of one branch as maternally inherited aminoglycoside-induced HL, targeted next generation sequencing (NGS) was applied to identify the genetic causes for the other two branches. One affected subject from each branch was subject to targeted NGS whose genomic DNA was enriched either by whole-exome capture (Agilent SureSelect All Exon 50 Mb) or by candidate genes capture (Agilent SureSelect custom kit). By NGS analysis, we identified that patients from Branch A were compound heterozygous for p.E1006K and p.D1663V in the CDH23 (DFNB12) gene; and patients from Branch B were homozygous for IVS7-2A>G in the SLC26A4 (DFNB4) gene. Both CDH23 mutations altered conserved calcium binding sites of the extracellular cadherin domains. The co-occurrence of three different genetic causes in this family was exceedingly rare but fully compatible with the mutation spectrum of HL. Our study has also raised several technical and analytical issues when applying the NGS technique to genetic testing.

Identification of CDH23 mutations in Korean families with hearing loss by whole-exome sequencing.

BackgroundPatient genetic heterogeneity renders it difficult to discover disease-cause genes. Whole-exome sequencing is a powerful new strategy that can be used to this end. The purpose of the present study was to identify a hitherto unknown mutation causing autosomal recessive nonsyndromic hearing loss (ARNSHL) in Korean families.MethodsWe performed whole-exome sequencing in 16 individuals from 13 unrelated small families with ARNSHL. After filtering out population-specific polymorphisms, we focused on known deafness genes. Pathogenic effects of the detected mutations on protein structure or function were predicted via in silico analysis.ResultsWe identified compound heterozygous CDH23 mutations in hearing-loss genes of two families. These include two previously reported pathological mutations, p.Pro240Leu and p.Glu1595Lys, as well as one novel mutation, p.Asn342Ser. The p.Pro240Leu mutation was found in both families. We also identified 26 non-synonymous variants in CDH23 coding exons from 16 hearing-loss patients and 30 Korean exomes.ConclusionThe present study is the first to show that CDH23 mutations cause hearing loss in Koreans. Although the precise contribution made by such mutations needs to be determined using a larger patient cohort, our data indicate that mutations in the CDH23 gene are one of the most important causes of non-syndromic hearing loss in East Asians. Further exome sequencing will identify common mutations or polymorphisms and contribute to the molecular diagnosis of, and development of new therapies for, hereditary hearing loss.

HOXA9 promotes homotypic and heterotypic cell interactions that facilitate ovarian cancer dissemination via its induction of P-cadherin.

BackgroundEpithelial ovarian cancer (EOC) is a lethal disease that frequently involves the peritoneal cavity. Dissemination of EOC is a multi-step process in which exfoliated tumor cells survive in the peritoneal fluid as multi-cellular aggregates and then form invasive implants on peritoneal surfaces. The mechanisms that control this process are poorly understood. We previously identified that high expression of the developmental patterning gene HOXA9 is associated with poor survival in EOC patients. In this study, we investigated the significance and mechanisms of HOXA9 in controlling aggregation and implantation of floating EOC cells.MethodsHOXA9 was inhibited by shRNAs or expressed in EOC cells that were propagated in suspension cultures and in the peritoneal cavity of mice. Cell death was assayed by flow cytometry and ELISA. Cell aggregation, attachment and migration were evaluated by microscopy, transwell chamber assays and histopathologic analysis. DNA-binding of HOXA9 and its effect on expression of the cell adhesion molecule P-cadherin were assayed by chromatin immunoprecipitation, quantitative RT-PCR and Western blot. HOXA9 and P-cadherin expression was evaluated in publicly available datasets of EOC clinical specimens.ResultsWe identified that HOXA9 promotes aggregation and inhibits anoikis in floating EOC cells in vitro and in xenograft models. HOXA9 also stimulated the ability of EOC cells to attach to peritoneal cells and to migrate. HOXA9 bound the promoter of the CDH3 gene that encodes P-cadherin, induced CDH3 expression in EOC cells, and was associated with increased CDH3 expression in clinical specimens of EOC. Inhibiting P-cadherin in EOC cells that expressed HOXA9 abrogated the stimulatory effects of HOXA9 on cell aggregation, implantation and migration. Conversely, these stimulatory effects of HOXA9 were restored when P-cadherin was reconstituted in EOC cells in which HOXA9 was inhibited.ConclusionThese findings indicate that HOXA9 contributes to poor outcomes in EOC in part by promoting intraperitoneal dissemination via its induction of P-cadherin.

A new Atp2b2 deafwaddler allele, dfwi5, interacts strongly with Cdh23 and other auditory modifiers

Tight regulation of calcium (Ca2+) concentrations in the stereocilia bundles of auditory hair cells of the inner ear is critical to normal auditory transduction. The plasma membrane Ca2+ ATPase 2 (PMCA2), encoded by the Atp2b2 gene, is the primary mechanism for clearance of Ca2+ from auditory stereocilia, keeping intracellular levels low, and also contributes to maintaining adequate levels of extracellular Ca2+ in the endolymph. This study characterizes a novel null Atp2b2 allele, dfwi5, by examining cochlear anatomy, vestibular function and auditory physiology in mutant mice. Loss of auditory function in PMCA2 mutants can be attributed to dysregulation of intracellular Ca2+ inside the stereocilia bundles. However, extracellular Ca2+ ions surrounding the stereocilia are also required for rigidity of cadherin 23, a component of the stereocilia tip-link encoded by the Cdh23 gene. This study further resolves the interaction between Atp2b2 and Cdh23 in a gene dosage and frequency-dependent manner, and finds that low frequencies are significantly affected by the interaction. In +/dfwi5 mice, one mutant copy of Cdh23 is sufficient to cause broad frequency hearing impairment. Additionally, we report another modifying interaction with Atp2b2 on auditory sensitivity, possibly caused by an unidentified hearing loss gene in mice.

CCAAT/Enhancer Binding Protein β (C/EBPβ) Isoforms as Transcriptional Regulators of the Pro-Invasive CDH3/P-Cadherin Gene in Human Breast Cancer Cells

P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ) transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

Compound Heterozygosity of the Functionally Null <i>Cdh23<sup>v-ngt</sup></i> and Hypomorphic <i>Cdh23<sup>ahl</sup></i> Alleles Leads to Early-onset Progressive Hearing Loss in Mice

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23ahl allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23v-ngt) null allele with mice carrying the hypomorphic Cdh23ahl allele on the C57BL/6J background, and we then analyzed the animals’ balance and hearing phenotypes. Although the Cdh23v-ngt/ahl compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23ahl/ahl homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23v-ngt/ahl mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23v-ngt/ahl mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.

Prevalence and Clinical Features of Hearing Loss Patients with CDH23 Mutations: A Large Cohort Study

Screening for gene mutations in CDH23, which has many exons, has lagged even though it is likely to be an important cause for hearing loss patients. To assess the importance of CDH23 mutations in non-syndromic hearing loss, two-step screening was applied and clinical characteristics of the patients with CDH23 mutations were examined in this study. As a first screening, we performed Sanger sequencing using 304 probands compatible with recessive inheritance to find the pathologic mutations. Twenty-six possible mutations were detected to be pathologic in the first screening. For the second screening, using the probes for these 26 mutations, a large cohort of probands (n = 1396) was screened using Taqman amplification-based mutation analysis followed by Sanger sequencing. The hearing loss in a total of 52 families (10 homozygous, 13 compound heterogygous, and 29 heterozygous) was found to be caused by the CDH23 mutations. The majority of the patients showed congenital, high frequency involved, progressive hearing loss. Interestingly, some particular mutations cause late onset moderate hearing loss. The present study is the first to demonstrate the prevalence of CDH23 mutations among non-syndromic hearing loss patients and indicated that mutations of the CDH23 gene are an important cause of non-syndromic hearing loss.

Riedel's thyroiditis - a case report with genes' expression studies

BackgroundGenetic background of Riedel's thyroiditis remains unknown. Herein, we describe our results of studies on genes expression levels in Riedel's thyroiditis.Case report and genetic findingsWe report the case of 48-year old woman with Riedel's thyroiditis who has presented unusual course of disease with non-specific cervical discomfort, though as with no pain and/or no compression symptoms. After surgery, thyroid specimens were quantitatively evaluated, regarding PIK3CA, PIK3CD, PIK3CG, Tg, TGFB1, THRB, COL1, CDKN1C, CDH3 and CACNA2D2 genes expression levels, by real-time PCR in the ABI PRISM® 7500 Sequence Detection System. Out of 10 above genes, in 2 cases the expression was higher than in respective Controls of unchanged thyroid tissue. In the remaining 8 cases, expression in question became comparable or lower as in Controls.DiscussionThe association between increased expression levels of PIK3CA and CDH3 genes and Riedel's thyroiditis is not well-defined. However, the increased expression of PIK3CA and CDH3 genes in our case report and in previous studies of other authors on various malignancies may suggest possible molecular relation between Riedel's thyroiditis and certain neoplastic processes, the relation of which requires further genetic evaluation. It is to be stressed that gene expression studies in Riedel's thyroiditis are difficult to perform, mainly due to fibrosis, resulting in scarce thyroid specimens and - in consequence - small amount of genetic material.

A mutation in the cdh23 gene causes age-related hearing loss in Cdh23nmf308/nmf308 mice

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23nmf308/nmf308 mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23nmf308/nmf308 mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23nmf308/nmf308 mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23nmf308/nmf308 mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5–10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23nmf308/nmf308 mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23nmf308/nmf308 mouse is an excellent animal model for investigating the mechanisms involved in human AHL.

Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice

Inbred strain variants of the Cdh23 gene have been shown to influence the onset and progression of age-related hearing loss (AHL) in mice. In linkage backcrosses, the recessive Cdh23 allele (ahl) of the C57BL/6J strain, when homozygous, confers increased susceptibility to AHL, while the dominant allele (Ahl+) of the CBA/CaJ strain confers resistance. To determine the isolated effects of these alleles on different strain backgrounds, we produced the reciprocal congenic strains B6.CBACa-Cdh23Ahl+ and CBACa.B6-Cdh23ahl and tested 15-30 mice from each for hearing loss progression. ABR thresholds for 8 kHz, 16 kHz, and 32 kHz pure-tone stimuli were measured at 3, 6, 9, 12, 15 and 18 months of age and compared with age-matched mice of the C57BL/6J and CBA/CaJ parental strains. Mice of the C57BL/6N strain, which is the source of embryonic stem cells for the large International Knockout Mouse Consortium, were also tested for comparisons with C57BL/6J mice. Mice of the C57BL/6J and C57BL/6N strains exhibited identical hearing loss profiles: their 32 kHz ABR thresholds were significantly higher than those of CBA/CaJ and congenic strain mice by 6 months of age, and their 16 kHz thresholds were significantly higher by 12 months. Thresholds of the CBA/CaJ, the B6.CBACa-Cdh23Ahl+, and the CBACa.B6-Cdh23ahl strain mice differed little from one another and only slightly increased throughout the 18-month test period. Hearing loss, which corresponded well with cochlear hair cell loss, was most profound in the C57BL/6J and C57BL/6NJ strains. These results indicate that the CBA/CaJ-derived Cdh23Ahl+ allele dramatically lessens hearing loss and hair cell death in an otherwise C57BL/6J genetic background, but that the C57BL/6J-derived Cdh23ahl allele has little effect on hearing loss in an otherwise CBA/CaJ background. We conclude that although Cdh23ahl homozygosity is necessary, it is not by itself sufficient to account for the accelerated hearing loss of C57BL/6J mice.

An ENU-Induced Mutation of Cdh23 Causes Congenital Hearing Loss, but No Vestibular Dysfunction, in Mice

Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea-induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca(2+)-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23(V2360E/V2360E) mutation alters the structural conformation of the protein and affects Ca(2+)-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12.

Abstract 3414: EMT-associated genes are altered in giant cell tumor of bone

Abstract The giant cell tumor of bone (GCT) is a benign bone-destructive neoplasm, with uncertain biological behavior, with features such as a large number of multinucleated giant cells and aggressive behavior. The local recurrence of GCT is often observed, especially in the first three years after treatment, giving a rate of recurrence ranging from 20% to 50% of cases. Further aggravating the recurrence, is the fact that after a relapse, patients often also presents metastasis in other organs, mainly in the lungs. In this study we investigate the expressions of epithelial and mesenchymal (EMT) markers (ADAM23, CDH1, CDH3, MMP14, p16, TIMP2 and VIM) in GCTs by quantitative qPCR. The expression of the MMP14, TIMP2 and VIM genes increased in more than 96% samples and ADAM23, CDH1, CDH3 and p16 genes had expression reduced in more than 91% of GCTs samples. Since ADAM23, CDH1, CDH3 and p16 have CpG islands in their promoter regions and low expression in tumor tissue, their methylation patterns were analyzed by MSP-PCR. For ADAM23 and p16 genes were detected a high frequency of hypermethylation. The lower expression of ADAM23 and p16 are directly related to the recurrence and metastasis in GCTs. The genes MMP14, TIMP2 and VIM are related to angiogenesis, degradation of extracellular matrix and cell migration and presented with increased expression in GCT, also confirmed by immunohistochemistry. These observations suggest that dysregulation of these genes may be a key factor responsible for the initiation and perpetuation of EMT in GCT, leading to cellular migration and malignant transformation, contributing to development of metastases and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3414. doi:10.1158/1538-7445.AM2011-3414

Digenic inheritance of an autosomal recessive hypotrichosis in two consanguineous pedigrees

Hypotrichosis is a human hereditary hair loss disorder in which affected individuals show sparse to complete absence of hair on scalp and/or on different body parts. To date, at least eight isolated autosomal recessive and dominant forms of hypotrichosis loci have been mapped on different human chromosomes, and the corresponding genes have been identified. Detailed clinical and molecular studies were undertaken of the hereditary hypotrichosis observed in the two consanguineous families (A and B) presented here. Human genome scan, using >500 highly polymorphic microsatellite markers, identified equal evidence of linkage of the hypotrichosis phenotype on chromosomes 12q21.2-q22 and 16q21-q23.1 in both the families. The novel hypotrichosis locus on chromosome 12q21.2-q22 spans 16.3 cM (17.62 Mb), flanked by markers D12S326 and D12S101. At this locus, maximum multipoint logarithm of the odds ratio (LOD) scores of 3.68 and 3.31 were obtained in families A and B, respectively. The second hypotrichosis locus on chromosome 16q21-q23.1, identified in the two families, spans 5.58 cM (8.28 Mb) and is flanked by markers D16S3031 and D16S512. Maximum multipoint LOD scores of 3.17 and 3.31 were obtained with markers mapped at this locus in families A and B, respectively. DNA sequence analysis of six candidate genes (PLEKHG7, SLC6A15, VEZT, DUSP6, KERA and KITLG), located in the linkage interval on chromosome 12q21.2-q22, failed to detect potential sequence variants in the affected individuals of the two families. However, DNA sequence analysis of CDH3 gene, located on chromosome 16q21-q23.1, detected a single base pair homozygous insertion (c.1024_1025insG and p.342insGfsX345) in exon 9 in family A and deletion of four base pair (c.1859_1862delCTCT and p.620delSfsX629) in exon 13 in family B. We described for the first time digenic inheritance of an autosomal recessive hypotrichosis phenotype in two unlinked loci on chromosomes 12q21.2-q22 and 16q21-q23.1 in two unrelated consanguineous Pakistani families.

CDH3/P-cadherin overexpression in breast carcinomas: its regulatory mechanisms, the role in cell invasion, and the association with cancer stem cell properties

One of the most basic characteristics of cancer cells is the loss of cell-cell adhesion and acquirement of invasive properties. During cancer progression, the regulation and expression of cell-cell adhesion molecules (like cadherins) play a pivotal role. In most invasive tumors, E-cadherin is downregulated and N-cadherin is de novo expressed (known as epithelial-to-mesenchymal transition). However, in some invasive carcinomas, E-cadherin expression is maintained, with the concomitant de novo expression of another cadherin – P-cadherin. Their co-expression occurs frequently in breast carcinomas, which show a worst patient prognosis when compared with patients harboring tumors with loss of E-cadherin as single event. In the last ten years, our group has been mainly interested in understanding the role of P-cadherin overexpression in cancer cells, as well as the gene regulatory mechanisms behind its aberrant expression. P-cadherin has been extensively studied concerning its function and prognostic value in breast cancer. Its overexpression has been identified in 30% of invasive carcinomas, being highly associated with proliferative lesions of high histological grade and decreased patient survival. Recently, we showed that P-cadherin overexpression confers an invasive capacity to breast cancer cells, inducing the secretion of MMPs, which are responsible by the cleavage of its extracellular domain, giving rise to a P-cadherin soluble form. We proved that this fragment is a pro-invasive factor, which needs to be inhibited to render cancer cells non-invasive. Still, we identified the intracellular signaling pathway that regulates and activates the P-cadherin (CDH3) gene promoter, inducing P-cadherin overexpression in breast cancer cells. We found that an antiestrogen is able to increase CDH3 promoter activity, as well as to induce activating histone epigenetic modifications at putative C/EBPβ binding sites in the CDH3 gene promoter. We showed, for the first time, that C/EBPβ is able to regulate P-cadherin overexpression. Furthermore, our recent data supports the idea that P-cadherin has a role in cancer stem cell biology. We found that breast cancer cell lines, presenting the highest levels of P-cadherin, show the highest expression of the stem cell markers CD49f, CD44 and CD24, as well as ALDH1 activity. Importantly, we reproduced these results in primary tumors, where we found that P-cadherin overexpression occurs in breast carcinomas with a cancer stem cell phenotype (CD44+CD24-/low) and ALDH1 expression. These results are now being experimentally addressed by mammosphere, 2D and 3D culture in vitro assays, to be then confirmed in vivo. In conclusion, our results open new avenues in breast cancer treatment, since P-cadherin is likely to be a good therapeutic target for invasive carcinomas overexpressing this protein. Scientifically, our studies will help to understand better the pathogenesis of breast cancer and other cancer models involving cadherin’s alterations.

Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70-80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs

We report a novel mutation (erlong, erl) of the cadherin 23 (Cdh23) gene in a mouse model for DFNB12 characterized by progressive hearing loss beginning from post-natal day 27 (P27). Genetic and sequencing analysis revealed a 208T>C transition causing an amino acid substitution (70S-P). Caspase expression was up-regulated in mutant inner ears. Hearing was preserved (up to 35-dB improvement) in pan-caspase inhibitor Z-VAD-FMK-treated mutants compared to untreated mutants (P < 0.05). Outer hair cell (OHC) loss in the cochleae of Z-VAD-FMK-treated mutants was significantly reduced compared to those of untreated mice. Thus, the erl mutation can lead to hearing loss through apoptosis. This is the first genetic mouse model of hearing loss shown to respond to otoprotective drug therapy. The short interval from initial hearing loss to deafness (P27-P90) makes this model ideal for screening and validating otoprotective drugs.

712 ICI 182,780 induces p-cadherin overexpression in breast cancer cells through chromatin remodelling at the promoter level: role of C/EBPbeta in CDH3 gene activation

SMAD signaling is essential for follicular development. The distribution of activated (phosphorylated) SMADs during folliculogenesis has not been described in detail. The present results indicate that oocytes from preantral and antral follicles contain the mRNA for the receptor regulated Smads (-1, -2, -3, -5 and -9), Smad4 (co-SMAD), and Smad6 and Smad7 (inhibitory SMADs). Levels of Smad5, Smad6 and Smad7 mRNA decreased in fully-grown oocytes compared to growing oocytes. Immunostaining for pSMAD1/5/9, pSMAD2 and pSMAD3 was evident in oocytes from primordial, primary, secondary and antral follicles. We also observed substantial staining for pSMADs in intact fully-grown oocytes from antral follicles. In granulosa cells, immunostaining for both pSMAD2/3 and pSMAD1/5/9 was apparent in preantral granulosa cells from primordial to secondary follicles. In antral follicles, immunostaining for pSMADs became more intense in the cumulus cells surrounding the oocyte. Immunostaining for pSMAD2 and pSMAD1/5/9 was also apparent in the epithelium lining of the oviduct. Clearly, oocytes, preantral granulosa cells, cumulus cells and the epithelium of the oviduct are major targets of SMAD-mediated pathways in female reproductive tissues. The finding that pSMAD pathways are active in oocytes raises new questions regarding the role of TGF-beta superfamily members in directly promoting oocyte development. The extent to which defects in pSMAD signaling in oocytes or the oviduct contribute to infertility in humans or animals remains an open question.

Abstract 4981: ICI182,780 induces P-cadherin upregulation in breast cancer cells through histone modifications at the promoter level: The role of C/EBPbeta in CDH3 gene activation

Abstract P-cadherin is a classical cadherin which has been extensively studied concerning its functions and its prognostic value in breast cancer. Moderate to intense expression of P-cadherin has been identified in 30% - 50% of invasive ductal carcinomas of the breast, being reported to be strongly associated with proliferative and high histological grade, decreased patient survival and as a marker of the aggressive behaviour of basal-like and HER-2 tumour subtypes. In vitro, P-cadherin overexpression is also able to induce increased cell invasion and migration. However, only a few works have been published in order to decipher the regulatory mechanisms behind P-cadherin overexpression in breast cancer. P-cadherin is up-regulated by the antiestrogen ICI 182,780, suggesting that the lack of ERalpha signalling may be responsible for the aberrant P-cadherin expression in breast cancer, but the mechanisms underlying this ICI-induced aggressive behaviour are completely unknown. The aim of this study was to determine whether the induction of the CDH3 gene is a direct effect of ICI 182,780 at CDH3 promoter level or would require the prior induction of other genes/proteins or epigenetic mechanisms. We found that ICI 182,780 increases CDH3 promoter activity as well as induces an epigenetic mechanism that lead to activating histone modifications at putative C/EBPbeta binding sites in at the CDH3 promoter. We also demonstrate that this transcription factor is able to directly activate P-cadherin transcription in breast cancer cells and that these two proteins are highly associated in human breast carcinomas, being both significantly related with high grade and proliferative invasive breast carcinomas. This study brings forward the characterization of putative intracellular pathways and epigenetic mechanisms that are able to induce P-cadherin overexpression in breast cancer cells and, in general, contribute to clarify the ability of ICI 182,780 to induce expression of signal transduction genes normally repressed by oestrogen/ER signalling. In addition, we showed for the first time that the transcription factor C/EBPbeta is able to regulate P-cadherin overexpression in breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4981.

ICI 182,780 induces P-cadherin overexpression in breast cancer cells through chromatin remodelling at the promoter level: a role for C/EBP in CDH3 gene activation

CDH3/P-cadherin is a classical cadherin. Overexpression of which has been associated with proliferative lesions of high histological grade, decreased cell polarity and poor survival of patients with breast cancer. In vitro studies showed that it can be up-regulated by ICI 182,780, suggesting that the lack of ERalpha signalling is responsible for the aberrant P-cadherin overexpression and for its role in inducing breast cancer cell invasion and migration. However, the mechanism by which ER-signalling inhibition leads to P-cadherin expression is still unknown. The aim of this study was to explore the molecular mechanism linking the ERalpha-signalling and P-cadherin-regulated expression in breast cancer cell lines. This study showed that ICI 182,780 is able to increase CDH3 promoter activity, inducing high levels of the active chromatin mark H3 lysine 4 dimethylation. We also observed, for the first time, that the transcription factor C/EBPbeta is able to up-regulate CDH3 promoter activity in breast cancer cells. Moreover, we showed that the expression of P-cadherin and C/EBPbeta are highly associated in human breast carcinomas and linked with a worse prognosis of breast cancer patients. This study demonstrates the existence of an epigenetic regulation by which ICI 182,780 up-regulates P-cadherin expression in MCF-7/AZ breast cancer cells through chromatin remodelling at CDH3 promoter, bringing forward the growing evidence that ERalpha signalling-abrogation by anti-oestrogens is able to induce the expression of ERalpha-repressed genes which, in the appropriate cell biology context, may contribute to a breast cancer cell invasion phenotype.CDH3 GenBank accession no. NT_010498.