Protocadherin Research Articles

Quantitation Analysis of PCDH10 Methylation in Adolescent Idiopathic Scoliosis Using Pyrosequencing Study.

A retrospective and comparative study. To evaluate the difference of DNA methylation in protocadherin10 (PCDH10) genes between adolescent idiopathic scoliosis (AIS) and normal controls, and to assess the association between DNA methylation and the etiology of AIS. The PCDH10 gene showed abnormal expression in AIS. However, the mechanism was still unclear. DNA methylation was an important epigenetic mechanism at the interface between genetics and environmental phenotype, seeming to be a suitable epigenetic mark for the abnormal expression of PCDH10 in AIS. There were 50 AIS patients and 50 healthy controls included in the study. The peripheral blood sample of each participant was taken. The pyrosequencing assay was used to assess the methylation status of PCDH10 promoter and real time PCR (RT-PCR) was used to detect the PCDH10 gene expression. The comparison analysis was performed using independent t test and 2-tailed Pearson coefficients was calculated for the correlation analysis. The average methylation level was 4.32 ± 0.73 in AIS patients and 3.14 ± 0.97 in healthy controls (P < 0.001). The PCDH10 gene expression was 0.23 ± 0.04 in AIS patients and 0.36 ± 0.08 in normal controls (P < 0.0001). Statistically significant linear correlation was found between PCDH10 gene methylation level and Cobb angle of major curve (P < 0.001). Besides, a significant negative correlation between PCDH10 methylation and PCDH10 gene expression was found (P < 0.001). AIS patients were associated with high DNA methylation level and low gene expression of PCDH10 gene rather than normal controls. The high methylation level indicated high Cobb angle of major curves in AIS. The abnormal DNA methylation may widely exist and serve as a potential mechanism for AIS. 3.

Epigenetic Inactivation of Protocadherin 10 by Methylation in Colorectal Cancer

Aberrant promoter methylation of CpG islands of tumor-suppressor genes has been recognized as one of the important tumor markers for cancer detection. The aim of this study was to investigate the promoter methylation status of protocadherin 10 (PCDH10), a tumor suppressor gene, in Iranian colorectal cancer (CRC) patients. Cancerous and the adjacent normal tissues obtained from 38 CRC patients were used to assess the methylation status of PCDH10 with Methylation Specific PCR, in addition, to study the expression level of this gene by quantitative PCR. The relationship between hypermethylation and the demographic characteristics of these patients was analyzed. The promoter methylation level of PCDH10 was statistically different between tumoral and normal tissues in CRC patients. Twenty-seven out of 38 patients showed hypermethylation with a sensitivity of 73% and a specificity of 97%. PCDH10 expression decreased in 15 cases (46%) as 16 cases (50 %) showed overexpression and 1 case (4%) had no changes. Not a significant association was reported between PCDH10 hypermethylation and the clinicopathological characteristics (P&gt;0.05). Our results indicated that PCDH10 methylation has a critical function in CRC, with a nearly elevated sensitivity and a high specificity in the Iranian population, qualify it as a potential candidate biomarker. © 2019 Tehran University of Medical Sciences. All rights reserved. Acta Med Iran 2019;57(8):472-477.

MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma.

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

Serotonin induces Arcadlin in hippocampal neurons

The monoamine hypothesis does not fully explain the delayed onset of recovery after antidepressant treatment or the mechanisms of recovery after electroconvulsive therapy (ECT). The common mechanism that operates both in ECT and monoaminergic treatment presumably involves molecules induced in both of these conditions. A spine density modulator, Arcadlin (Acad), the rat orthologue of human Protocadherin-8 (PCDH8) and of Xenopus and zebrafish Paraxial protocadherin (PAPC), is induced by both electroconvulsive seizure (ECS) and antidepressants; however, its cellular mechanism remains elusive. Here we confirm induction of Arcadlin upon stimulation of an N-methyl-d-aspartate (NMDA) receptor in cultured hippocampal neurons. Stimulation of an NMDA receptor also induced acute (20 min) and delayed (2 h) phosphorylation of the p38 mitogen-activated protein (MAP) kinase; the delayed phosphorylation was not obvious in Acad–/– neurons, suggesting that it depends on Arcadlin induction. Exposure of highly mature cultured hippocampal neurons to 1–10 μM serotonin for 4 h resulted in Arcadlin induction and p38 MAP kinase phosphorylation. Co-application of the NMDA receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (APV) completely blocked Arcadlin induction and p38 MAP kinase phosphorylation. Finally, administration of antidepressant fluoxetine in mice for 16 days induced Arcadlin expression in the hippocampus. Our data indicate that the Arcadlin-p38 MAP kinase pathway is a candidate neural network modulator that is activated in hippocampal neurons under the dual regulation of serotonin and glutamate and, hence, may play a role in antidepressant therapies.

Paternal DNA Methylation May Be Associated With Gestational Age at Birth.

Background:How epigenetic modifications of DNA are associated with gestational age at birth is not fully understood. We investigated potential effects of differential paternal DNA methylation (DNAm) on offspring gestational age at birth by conducting an epigenome-wide search for cytosine-phosphate-guanine (CpG) sites.Methods:Study participants in this study consist of male cohort members or partners of the F1-generation of the Isle of Wight Birth Cohort (IoWBC). DNAm levels in peripheral blood from F1-fathers (n = 92) collected around pregnancy of their spouses were analyzed using the Illumina 450K array. A 5-step statistical analysis was performed. First, a training-testing screening approach was applied to select CpG sites that are potentially associated with gestational age at birth. Second, functional enrichment analysis was employed to identify biological processes. Third, by centralizing on biologically informative genes, Cox proportional hazards models were used to assess the hazard ratios of individual paternal CpGs on gestational age adjusting for confounders. Fourth, to assess the validity of our results, we compared our CpG-gestational age correlations within a Born into Life Study in Sweden (n = 15). Finally, we investigated the correlation between the detected CpGs and differential gene expression in F2 cord blood in the IoWBC.Results:Analysis of DNAm of fathers collected around their partner’s pregnancy identified 216 CpG sites significantly associated with gestational age at birth. Functional enrichment pathways analyses of the annotated genes revealed 2 biological pathways significantly related to cell-cell membrane adhesion molecules. Differential methylation of 9 cell membrane adhesion pathway-related CpGs were significantly associated with gestational age at birth after adjustment for confounders. The replication sample showed correlation coefficients of 2 pathway-related CpGs with gestational age at birth within 95% confidence intervals of correlation coefficients in IoWBC. Finally, CpG sites of protocadherin (PCDH) gene clusters were associated with gene expression of PCDH in F2 cord blood.Conclusions:Our findings suggest that differential paternal DNAm may affect gestational age at birth through cell-cell membrane adhesion molecules. The results are novel but require future replication in a larger cohort.

PCDH17 increases the sensitivity of colorectal cancer to 5-fluorouracil treatment by inducing apoptosis and autophagic cell death

5-Fluorouracil (5-FU) is known as a first-line chemotherapeutic agent against colorectal cancer (CRC), but drug resistance occurs frequently and significantly limits its clinical success. Our previous study showed that the protocadherin 17 (PCDH17) gene was frequently methylated and functioned as a tumor suppressor in CRC. However, the relationship between PCDH17 and 5-FU resistance in CRC remains unclear. Here, we revealed that PCDH17 was more highly expressed in 5-FU-sensitive CRC tissues than in 5-FU-resistant CRC tissues, and high expression of PCDH17 was correlated with high BECN1 expression. Moreover, this expression profile contributed to superior prognosis and increased survival in CRC patients. Restoring PCDH17 expression augmented the 5-FU sensitivity of CRC in vitro and in vivo by promoting apoptosis and autophagic cell death. Furthermore, autophagy played a dominant role in PCDH17-induced cell death, as an autophagy inhibitor blocked cell death to a greater extent than the pancaspase inhibitor Z-VAD-FMK. PCDH17 inhibition by siRNA decreased the autophagy response and 5-FU sensitivity. Mechanistically, we showed that c-Jun NH2-terminal kinase (JNK) activation was a key determinant in PCDH17-induced autophagy. The compound SP600125, an inhibitor of JNK, suppressed autophagy and 5-FU-induced cell death in PCDH17-reexpressing CRC cells. Taken together, our findings suggest for the first time that PCDH17 increases the sensitivity of CRC to 5-FU treatment by inducing apoptosis and JNK-dependent autophagic cell death. PCDH17 may be a potential prognostic marker for predicting 5-FU sensitivity in CRC patients.

Clustered protocadherins methylation alterations in cancer

BackgroundClustered protocadherins (PCDHs) map in tandem at human chromosome 5q31 and comprise three multi-genes clusters: α-, β- and γ-PCDH. The expression of this cluster consists of a complex mechanism involving DNA hub formation through DNA-CCTC binding factor (CTCF) interaction. Methylation alterations can affect this interaction, leading to transcriptional dysregulation. In cancer, clustered PCDHs undergo a mechanism of long-range epigenetic silencing by hypermethylation.ResultsIn this study, we detected frequent methylation alterations at CpG islands associated to these clustered PCDHs in all the solid tumours analysed (colorectal, gastric and biliary tract cancers, pilocytic astrocytoma), but not hematologic neoplasms such as chronic lymphocytic leukemia. Importantly, several altered CpG islands were associated with CTCF binding sites. Interestingly, our analysis revealed a hypomethylation event in pilocytic astrocytoma, suggesting that in neuronal tissue, where PCDHs are highly expressed, these genes become hypomethylated in this type of cancer. On the other hand, in tissues where PCDHs are lowly expressed, these CpG islands are targeted by DNA methylation. In fact, PCDH-associated CpG islands resulted hypermethylated in gastrointestinal tumours.ConclusionsOur study highlighted a strong alteration of the clustered PCDHs methylation pattern in the analysed solid cancers and suggested these methylation aberrations in the CpG islands associated with PCDH genes as powerful diagnostic biomarkers.

PCDH10 inhibits invasion of lymphoma cells by regulating β-catenin.

The aim of this study was to investigate the effect of protocadherin 10 (PCDH10) on the invasive potential of lymphoma cells by regulating matrix metalloproteinase-7 (MMP7) and MMP9 via targeting β-catenin. The mRNA and protein expressions of PCDH10, β-catenin, MMP7 and MMP9 in lymphoma cell lines were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Raji cells with low expression of PCDH10 were transiently transfected with pCMV5-HA-PCDH10 or pcDNA5-His-β-catenin. Meanwhile, HUT-78 cells with high expression of PCDH10 were transfected with PCDH10-shRNA or β-catenin-shRNA. Subsequently, the expression levels of β-catenin, MMP7 and MMP9 in transfected lymphoma cells were determined as well. In addition, the regulatory effects of PCDH10 on the invasive potential of lymphoma cells were explored by transwell assay. PCDH10 expression was negatively correlated with the expressions of β-catenin, MMP7 and MMP9 in several lymphoma cell lines. Transfection of HA-PCDH10 in human-derived malignant B lymphoma cell line Raji markedly down-regulated the protein levels of β-catenin, MMP7 and MMP9. However, the mRNA level of β-catenin was not influenced by PCDH10. Interference with PCDH10 in HUT-78 cells with high expression of PCDH10 significantly increased the protein expressions of β-catenin, MMP7, and MMP9. However, no significant changes were observed in the mRNA expression of β-catenin. In addition, knockdown of β-catenin in cells with high expression of PCDH10 remarkably down-regulated the expression levels of MMP7 and MMP9. PCDH10 overexpression in lymphoma cells downregulates β-catenin expression, as well as inhibits the expressions of MMP7 and MMP9, eventually inhibiting the invasive potential of lymphoma cells.

Semaphorin 3A (SEMA3A), protocadherin 9 (PCdh9), and S100 calcium binding protein A3 (S100A3) as potential biomarkers of carcinogenesis and chemoresistance of different neoplasms, including ovarian cancer - review of literature.

Ovarian cancer is the fifth leading cause of cancer-related deaths in women. Its high mortality rate results from lack of adequate and sensitive methods allowing for the detection of the early stages of the disease, as well as low efficiency of the treatment, caused by the cytotoxic drug resistance of cancer cells. Unfortunately, tumours are able to develop new pathways and protective mechanisms that allow them to survive toxic conditions of chemotherapy. Therefore, intensive search for new genes and proteins involved in resistance to cytotoxic drugs is still needed, especially from a clinical point of view. The article presents an overview of the available literature on the role of semaphorin 3A (SEMA3A), protocadherin 9 (PCDH9), and S100 calcium binding protein A3 (S100A3) in carcinogenesis and chemoresistance of various tumors including ovarian cancer. As it turns out, the role of described genes/proteins is not limited only to their native biological activity but they function also as an oncogenic or suppressor factors in the tumor development. Moreover, they can also play an important role in development of drug resistance, as it was shown in ovarian cancer cell lines.

Reduced protocadherin17 expression in leukemia stem cells: the clinical and biological effect in acute myeloid leukemia

BackgroundLeukemia stem cell (LSC)-enriched genes have been shown to be highly prognostic in acute myeloid leukemia (AML). However, the prognostic value of tumor suppressor genes (TSGs) that are repressed early in LSC remains largely unknown.MethodsWe compared the public available expression/methylation profiling data of LSCs with that of hematopoietic stem cells (HSCs), in order to identify potential tumor suppressor genes in LSC. The prognostic relevance of PCDH17 was analyzed on a cohort of 173 AML patients from The Cancer Genome Atlas (TCGA), and further validated in three independent cohorts (n = 339).ResultsWe identified protocadherin17 (PCDH17) and demonstrated that it was significantly down-regulated and hypermethylated in LSCs compared with HSCs. Our analyses of primary AML patient samples also confirmed these deregulations. Clinically, low PCDH17 expression was associated with female sex (P = 0.01), higher WBC (P < 0.0001), higher percentages of blasts in bone marrow (BM) and peripheral blood (PB) (P = 0.04 and < 0.001, respectively), presence of FLT3-internal tandem duplications (P = 0.002), mutated NPM1 (P = 0.02), and wild-type TP53 (P = 0.005). Moreover, low PCDH17 expression predicted worse overall survival (OS) in four independent cohorts as well as in the molecularly defined subgroups of AML patients. In multivariable analyses, low PCDH17 expression retained independent prognostic value for OS. Biologically, PCDH17 expression-associated gene signatures were characterized by deregulations of EMT- and Wnt pathway-related genes.ConclusionsPCDH17 gene was silenced by DNA methylation in AML. Low PCDH17 expression is associated with distinct clinical and biological features and improves risk stratification in patients with AML.

In silico Identification and Expression of Protocadherin Gene Family in Octopus vulgaris.

Connecting millions of neurons to create a functional neural circuit is a daunting challenge. Vertebrates developed a molecular system at the cell membrane to allow neurons to recognize each other by distinguishing self from non-self through homophilic protocadherin interactions. In mammals, the protocadherin gene family counts about 50 different genes. By hetero-multimerization, protocadherins are capable of generating an impressive number of molecular interfaces. Surprisingly, in the California two-spot octopus, Octopus bimaculoides, an invertebrate belonging to the Phylum Mollusca, over 160 protocadherins (PCDHs) have been identified. Here we briefly discuss the role of PCDHs in neural wiring and conduct a comparative study of the protocadherin gene family in two closely related octopus species, Octopus vulgaris and O. bimaculoides. A first glance at the expression patterns of protocadherins in O. vulgaris is also provided. Finally, we comment on PCDH evolution in the light of invertebrate nervous system plasticity.

Methylation and Expression of Nonclustered Protocadherins Encoding Genes and Risk of Precancerous Gastric Lesions in a High-Risk Population.

Nonclustered protocadherins (PCDH) family is a group of cell-cell adhesion molecules. We have found differentially methylated genes in the nonclustered PCDHs family associated with Helicobacter pylori (H. pylori) infection in prior genome-wide methylation analysis. To further investigate the methylation and expression of nonclustered PCDHs encoding genes in H. pylori--related gastric carcinogenesis process, four candidate genes including PCDH7, PCDH10, PCDH17, and PCDH20 were selected, which were reported to be tumor suppressors for digestive cancers. A total of 747 participants with a spectrum of gastric lesions were enrolled from a high-risk population of gastric cancer. Promoter methylation levels of four genes were significantly higher in H. pylori-positive subjects than the negative group (all P < 0.001). Elevated methylation levels of PCDH10 and PCDH17 were observed with the increasing severity of gastric lesions (both P trend < 0.001). In the protein expression analysis, PCDH17 expression was inversely associated with gastric lesions; the OR [95% confidence interval (CI)] was 0.49 (0.26-0.95) for chronic atrophic gastritis (CAG), 0.31 (0.15-0.63) for intestinal metaplasia, and 0.38 (0.19-0.75) for indefinite dysplasia and dysplasia, compared with superficial gastritis. In addition, PCDH10 expression was significantly lower in CAG (OR, 0.40; 95% CI, 0.24-0.68). The inverse association between methylation and protein expression of PCDH10 and PCDH17 was further supported when we explored the methylation and mRNA expression in The Cancer Genome Atlas database (all P < 0.001). Our study found elevated promoter methylation and decreased expression of PCDH10 and PCDH17 in advanced gastric lesions, suggesting that elevated PCDH10 and PCDH17 methylation may be an early event in gastric carcinogenesis. Cancer Prev Res; 11(11); 717-26. ©2018 AACR.

Estrogen receptor‑α is a determinant of protocadherin‑8 expression in breast cancer cells

Protocadherin‑8 (PCDH8) is a member of the protocadherin subfamily within the cadherin superfamily of cell adhesion molecules. PCDH8 is silenced in a variety of solid tumors including those of the breast, and believed to be a tumor suppressor. Here we show that PCDH8 is suppressed by estrogen in MCF-7 and T47D breast cancer cells. The suppression is dependent on estrogen receptor‑α (ERα) expression. Knockdown of ERα alone induces the expression of PCDH8. The inverse correlation between PCDH8 and ERα expression is also apparent from the analysis of PCDH8 expression in breast tumors from the TCGA-BRCA dataset. Since estrogen produces mitogenic effects in ERα positive breast cancer cells, estrogen-mediated suppression of PCDH8 is consistent with its purported tumor suppressor function. Functional studies on PCDH8 in breast cancer cells may yield valuable insights into the mechanisms of estrogen-mediated epithelial-mesenchymal transition.

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions.

Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein 'bait'. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs was analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

Aberrant methylation of PCDH17 gene in high-grade serous ovarian carcinoma.

Aberrant DNA methylation of protocadherins (PCDHs) has been associated with development and progression of various types of cancer. It could represent possible direction in the search for critically needed tumor biomarkers for ovarian cancer. To investigate methylation of δ2 group of non-clustered PCDHs in high-grade serous ovarian carcinoma (HGSOC) tissue in comparison with control tissue. We used next-generation sequencing for detecting regions with the most altered methylation. For further confirmation of discovered alterations we used methylation-sensitive high-resolution melting analysis. PCDH17 methylation was detected in almost 70% of HGSOC patients without any methylation in the group of control samples and was found both in the late stage tumors as well as in the early stage ones. Other selected PCDHs did not show any relevant changes in methylation. Subsequent gene expression analysis of PCDH17 revealed decreased expression in all of the tumor samples in comparison to the control ones. Statistically significant negative correlation was found between methylation and levels of expression suggesting potentially methylation-based silencing. Methylation of PCDH17 could play an important role in development and progression of HGSOC and has potential to become a target in the search for new clinical biomarkers.

Aberrant promoter methylation reduced the expression of protocadherin 17 in nasopharyngeal cancer.

This study aimed to explore the underlying mechanism of protocadherin 17 (PCDH17) downregulated in nasopharyngeal cancer (NPC). NPC tumor and adjacent tissue samples were collected from NPC patients who received therapy in the Chinese PLA General Hospital. Meanwhile, a normal epithelial cell lines NP96 and 6 NPC and cell lines C666-1, CEN1, CEN2, HNE1, HEN2, and HONE1 were prepared. Then, the expression level of PCDH17 and the methylation level of PCDH17 promoter in both tissues samples and cell lines were determined using the PCR method. Moreover, PCDH17 was overexpressed in CNE2 and HONE1 using Lipo2000. Following this, the proliferation and apoptosis of CNE2 and HONE1 were assessed using MTT and flow cytometry. The expression of PCDH17 was significantly downregulated in NPC tissues compared with the adjacent tissues as well as in the NPC cell lines compared with the normal NP96 cells. Overexpressed PCDH17 could significantly inhibit the proliferation of CNE2 and HONE1 cells but obviously promote the apoptosis of these two cell lines. Aberrant hypermethylation in the promoter might be the explanation of PCDH17 downregulated in PCDH17 and promoted the development of NPC.

Down-regulated of PCDH10 predicts poor prognosis in hepatocellular carcinoma patients.

Protocadherin10 (PCDH10), a member of the nonclustered protocadherin family, functions as a tumor suppressor in many cancers. The aim of this study was to evaluate the expression level and prognostic value of PCDH10 in hepatocellular carcinoma (HCC) patients.Quantitative real-time polymerase chain reaction was used to analyze the expression level of PCDH10 in HCC tissues and adjacent nontumor tissues. The association of PCDH10 expression with clinicopathological features of patients was evaluated by chi-squared test. Overall survival was estimated using the Kaplan–Meier method. Besides, the patient prognosis was also evaluated by Cox regression analysis.PCDH10 expression was significantly lower in HCC tissues than that in adjacent nontumor tissues (P = .000). Kaplan–Meier curves showed that patients with lower PCDH10 expression had a worse overall survival. Moreover, PCDH10 expression level was associated tumor size (P = .005), tumor node metastasis stage (P = .002), smoking status (P = .000), and drinking status (P = .005). Multivariate analysis showed that the expression of PCDH10 (P = .000; hazard ratio = 4.784; 95% confidence interval: 2.550–8.977) was an independently associated with poor overall survival rates, as well as smoking status and drinking status.Our findings indicated that the decreased expression of PCDH10 was closely associated with poor prognosis of HCC patients. It might be considered as a valuable biomarker for HCC.

Aberrant promoter methylation of PCDH10 as a potential diagnostic and prognostic biomarker for patients with breast cancer.

Protocadherin-10 (PCDH10) is a tumor suppressor gene. Its expression level is downregulated by promoter methylation in certain types of human tumors. The aim of the present study was to examine the expression level and promoter methylation status of PCDH10 in breast cancer cells and to evaluate the association of PCDH10 methylation and tumor progression and prognosis. MethyLight was used to detect the methylation status of PCDH10 in breast cancer tissues and healthy breast tissues. Reverse transcription-quantitative polymerase chain reaction was used to assess the mRNA expression level of PCDH10, as well as to evaluate the association between PCDH10 methylation and clinicopathological features, along with patients' overall survival (OS). PCDH10 5′-C-phosphate-G-3′ (CpG) methylated sites were identified in tumor tissues and matched healthy tissues (n=392). Tumor tissues and matched healthy tissues exhibited identifiable PCR results, with PCDH10 gene promoter methylation identified in ductal carcinoma in situ (66%), invasive ductal carcinoma (82%), invasive ductal carcinoma with lymph node metastasis (85.32%) and hereditary breast cancer tissues (72.37%). PCDH10 mRNA expression was significantly decreased in breast cancer tissues compared with healthy breast tissues (P=0.032). PCDH10 methylation was associated with tumor size (P=0.004), but not associated with other clinical factors. Survival analysis revealed that the patients exhibiting methylated-PCDH10 had significantly poorer OS times than patients exhibiting unmethylated-PCDH10 (P<0.0001). Receiver operating characteristic analysis indicated a sensitivity of 75%, a specificity of 62.5%, and an area under the curve of 0.682 for PCDH10. Additionally, the results of the present study indicated that PCDH10 methylation status may be a useful diagnostic and prognostic evaluation biomarker for breast cancer. The results suggested that PCDH10 methylation is a common occurrence in primary breast cancer and is associated with poor survival rates among patients with breast cancer.

Abstract 1079: PCDH7 is overexpressed in advanced prostate cancer and modulates aggressive phenotype in prostate cancer cells

Abstract Introduction: Most of prostate cancer (PCa) deaths are a result of distant metastasis and due to emergence of castrate resistant PCa (CRPC). Protocadherins (PCDHs) are members of the cadherin superfamily that regulate cell adhesion. Their extracellular domains contain cadherin-like repeats, but they differ significantly from classical cadherins with respect to their unique cytoplasmic domains that lack the conserved motifs for binding β-catenin and p120-catenin. PCDH7, a cadherin superfamily transmembrane protein that is known to function in cell-cell recognition and adhesion, is reported to be overexpressed in breast cancer and non-small cell lung cancer and promoted tumor metastasis. The role of PCDH7 in PCa is investigated for the first time in the present investigation. Materials and Methods: PCa cells- LNCaP, C4-2b, DU145, PC3, 22Rv1 and RWPE1 were used in this study. PCDH7 protein and mRNA levels were checked by Western Blotting and quantitative real time PCR respectively. Immunohistochemistry was done to check expression in TRAMP mice FFPE prostate tissue sections and human prostate tissues. Publically available data set (Trento 2016) was used to analyze PCDH7 expression in NEPC patients. PCDH7 lentiviral knock down was performed using PCDH7 shRNA in PC3 cells. Cell migration and invasion were done using IncuCyte® Scratch Wound Cell Migration and Invasion System. Colony formation was assessed by staining with 0.4% crystal violet after 3 weeks of cell seeding. Results: PCDH7 mRNA and protein is overexpressed in CRPC cells (C4-2b, 22Rv1, DU145 and PC3 cells as compared to castrations sensitive LNCaP cells as well as normal prostate RWPE1 cells. We observed highest levels for PCDH7 in enzalutamide refractory 22Rv1 cells. We also observed a significant increased expression of PCDH7 during tumor progression (30wk vs 12wk) in prostate tissues of TRAMP mice. Our results also show that PCDH7 is amplified in 9% of PCa patients and overexpressed in 43% patients. Immunohistochemical analysis of PCDH7 revealed high expression of PCDH7 in human prostate cancer tissues. EGF treatment enhanced ERK and AKT activities in time-dependent manner and the phosphorylation remain sustained even at late time points in PCDH7 positive PC3 cells as compared to decline in phosphorylation PCDH7 negative RWPE1 cells. shRNA-mediated knockdown of PCDH7 reduced ERK and AKT activities. Knockdown of PCDH7 also resulted in decreased cell migration, reduced cell invasion, and decreased colony formation. Collectively, these data suggest PCDH7 plays a crucial role in advanced PCa. Conclusion: Our results are the first direct demonstration of PCDH7 expression in PCa. Our exciting observations reveal a critical role for PCDH7 in prostate cancer progression. These results suggest that PCDH7 may be an attractive target for therapeutic intervention in PCa, in general, and CRPC in particular. Citation Format: Gauri Shishodia, Prakash Srinivasan Shanmugam, Sweaty Koul, Hari Koul. PCDH7 is overexpressed in advanced prostate cancer and modulates aggressive phenotype in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1079.

Expression of protocadherin8: Function as a tumor suppressor in hypopharyngeal carcinoma.

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the expression of PCDH8 and its clinicopathological significance in hypopharyngeal carcinoma. We also examined the possible inactivation mechanism of PCDH8. A total of 80 pairs of hypopharyngeal carcinoma tumor tissues and non-tumor tissues were investigated to examine the immunohistochemical expression of PCDH8. Prognostic value and clinicopathological significance of PCDH8 expression were examined by Kaplan-Meier and log-rank test and Cox's ones. Ten pairs of tumor tissues and non-tumor tissues were analyzed by RT-PCR and 4 pairs by Western blot respectively. Promoter methylation of PCDH8 was observed in 14 pairs of tumor tissues and non-tumor tissues by methylation-specific PCR (MSP). The expression of PCDH8 in tumor tissues was depressed immunohistochemically when compared with non-tumor tissues and was significantly lower in the advanced pathological stage. Meanwhile, the expression of PCDH8 served as an independent prognostic risk factor for overall survival of hypopharyngeal carcinoma. The mRNA and protein levels of PCDH8 in tumor tissues was also down-regulated than in non-tumor tissues. Moreover, aberrant promoter methylation of PCDH8 occurred frequently in tumor tissues, rather than in non-tumor tissues. For the first time, our study demonstrates the tumor-suppressive function of PCDH8 and its epigenetic inactivation by promoter methylation in hypopharyngeal carcinoma. It suggests that PCDH8 may serve as a useful prognostic biomarker and a potential therapeutic target for hypopharyngeal carcinoma patients.