The role of Pcdh10 in neurological disease and cancer.

BACKGROUND: Protocadherin 10 (PCDH 10), a member of the superfamily of protocadherins, is a Ca2+-dependent homophilic cell-cell adhesion molecule expressed on the surface of cell membranes. Protocadherin 10 plays a critical role in the central nervous system including in cell adhesion, formation and maintenance of neural circuits and synapses, regulation of actin assembly, cognitive function and tumor suppression. Additionally, Pcdh10 can serve as a non-invasive diagnostic and prognostic indicator for various cancers. METHODS: This paper collects and reviews relevant literature in Pubmed. CONCLUSION: This review describes the latest research understanding the role of Pcdh10 in neurological disease and human cancer, highlighting the importance of scrutinizing its properties for the development of targeted therapies and identifying a need for further research to explore Pcdh10 functions in other pathways, cell types and human pathologies.

Innovative mouse models for the tumor suppressor activity of Protocadherin-10 isoforms.

BACKGROUND: Nonclustered mouse protocadherin genes (Pcdh) encode proteins with a typical single ectodomain and a cytoplasmic domain with conserved motifs completely different from those of classic cadherins. Alternative splice isoforms differ in the size of these cytoplasmic domains. In view of the compelling evidence for gene silencing of protocadherins in human tumors, we started investigations on Pcdh functions in mouse cancer models. METHODS: For Pcdh10, we generated two mouse lines: one with floxed exon 1, leading to complete Pcdh10 ablation upon Cre action, and one with floxed exons 2 and 3, leading to ablation of only the long isoforms of Pcdh10. In a mouse medulloblastoma model, we used GFAP-Cre action to locally ablate Pcdh10 in combination with Trp53 and Rb1 ablation. From auricular tumors, that also arose, we obtained tumor-derived cell lines, which were analyzed for malignancy in vitro and in vivo. By lentiviral transduction, we re-expressed Pcdh10 cDNAs. RNA-Seq analyses were performed on these cell families. RESULTS: Surprisingly, not only medulloblastomas were generated in our model but also tumors of tagged auricles (pinnae). For both tumor types, ablation of either all or only long isoforms of Pcdh10 aggravated the disease. We argued that the perichondrial stem cell compartment is at the origin of the pinnal tumors. Immunohistochemical analysis of these tumors revealed different subtypes. We obtained several pinnal-tumor derived (PTD) cell lines and analyzed these for anchorage-independent growth, invasion into collagen matrices, tumorigenicity in athymic mice. Re-expression of either the short or a long isoform of Pcdh10 in two PTD lines counteracted malignancy in all assays. RNA-Seq analyses of these two PTD lines and their respective Pcdh10-rescued cell lines allowed to identify many interesting differentially expressed genes, which were largely different in the two cell families. CONCLUSIONS: A new mouse model was generated allowing for the first time to examine the remarkable tumor suppression activity of protocadherin-10 in vivo. Despite lacking several conserved motifs, the short isoform of Pcdh10 was fully active as tumor suppressor. Our model contributes to scrutinizing the complex molecular mechanisms of tumor initiation and progression upon PCDH10 silencing in many human cancers.

Circular RNA PCDH10 regulates the tumorigenesis of pancreatic cancer through the miR-338-3p/hTERT axis.

Protocadherin-10 (PCDH10) was previously identified as a pancreatic cancer (PC) suppressor by reducing telomerase activity through binding with human telomerase reverse transcriptase (hTERT). However, we did not observe any effects of PCDH10 on hTERT mRNA or protein expression. Our research found that the PCDH10 gene could be transcribed into linear mRNA or circular RNA, and FUS could bind to the introns flanking the circularized exons, inducing the PCDH10 linear mRNA to shift to circPCDH10 in PC cells. Knockdown of circPCDH10 significantly inhibited PC progression. Mechanistically, circPCDH10 acted as a sponge of miR-338-3p, which could negatively regulate hTERT expression in PC cells. The inhibitory effects of circPCDH10 knockdown on PC cells could be notably reversed by miR-338-3p inhibition and ectopic expression of hTERT. Overall, we propose that the increased FUS expression in PC cells made circPCDH10 the preferred product of the PCDH10 gene, and circPCDH10 might promote PC progression through upregulation of hTERT expression by targeting miR-338-3p.

HOTAIR Induces Methylation of PCDH10, a Tumor Suppressor Gene, by Regulating DNMT1 and Sponging with miR-148b in Gastric Adenocarcinoma.

PURPOSE: HOX transcript antisense intergenic RNA (HOTAIR), as a long non-coding RNA, has been reported to regulate carcinogenesis by epigenetic mechanism in various cancers. Protocadherin 10 (PCDH10) is one of the well-known tumor suppressor genes, and is frequently methylated in gastric cancers (GC). We aimed to investigate the detailed pathway of how HOTAIR contributes to the target gene in gastric carcinogenesis. MATERIALS AND METHODS: We investigated the mechanism of HOTAIR on carcinogenesis and metastasis of GC. Methylation-specific PCR was performed to identify the interaction between HOTAIR and PCDH10. In addition, we investigated the interaction between miR-148b and HOTAIR by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: The expression of HOTAIR was significantly upregulated in GC tissues (p<0.05) and GC cell lines (p<0.01), while PCDH10 was downregulated in GC tissues (p<0.05). The knockdown of HOTAIR (si-HOTAIR1 and 2) significantly upregulated the mRNA/protein expression of PCDH10 and reduced the methylation of PCDH10 compared to the control in MKN 28 and MKN 74. Si-HOTAIR1 and 2 significantly reduced DNA methyltransferase 1 (DNMT1) expression, and overexpression of HOTAIR increased DNMT1 expression. In RIP, we found that miR-148b interacted with HOTAIR. Si-HOTAIRs increased miR-148b expression, and miR-148b mimic inversely reduced HOTAIR expression. Si-HOTAIRs and miR-148b mimic reduced DNMT1 expression and increased PCDH10 expression compared to the control. CONCLUSION: This study demonstrated that HOTAIR interacts with miR-148b and DNMT1, eventually leading to PCDH10 methylation, which contributes to the progression of GC. Our findings provide a better understanding for detailed pathway of HOTAIR in epigenetic mechanism of GC.

PCDH10 exerts tumor-suppressor functions through modulation of EGFR/AKT axis in colorectal cancer.

Protocadherin 10 (PCDH10) is identified as a tumor suppressor in multiple cancers. The molecular mechanisms that mediate the functions of PCDH10 have yet to be fully elucidated. Here, we demonstrated that ectopic expression of PCDH10 in colorectal cancer (CRC) cells induced cell cycle retardation and increased apoptosis through regulation of the p53/p21/Rb axis and Bcl-2 expression. Overexpression of PCDH10 reversed the epithelial-mesenchymal transition (EMT) process with morphological changes and EMT marker alterations. Mechanistic study revealed that PCDH10 inhibited AKT/GSK3ß signaling pathway which in turn reduced ß-catenin activity and thus attenuated Snail and Twist1 expression. Furthermore, PCDH10 inhibited the stemness of CRC cells, including spheroid formation and stem cell markers. A proteomics approach revealed that PCDH10 could interact with EGFR, which was further verified by co-immunoprecipitation. Moreover, restoration of PCDH10 expression reduced EGFR phosphorylation. Accordingly, our work proposes a novel pathway by which PCDH10 directly engages in the negative regulation of EGFR/AKT/ß-catenin signaling pathway, resulting in tumor suppression.

Evaluation of Tumor Regulatory Genes and Apoptotic Pathways in The Cytotoxic Effect of Cytochalasin H on Malignant Human Glioma Cell Line (U87MG).

OBJECTIVE: The aim of current study was to provide a proof-of-concept on the mechanism of PLAU and PCDH10 gene expressions and caspases-3, -8, and -9 activities in the apoptotic pathway after treatment of malignant human glioma cell line (U87MG) with cytochalasin H. MATERIALS AND METHODS: In the present experimental study, we have examined cytochalasin H cytotoxic activities as a new therapeutic agent on U87MG cells in vitro for the first time. The cells were cultured and treated with 10-5-10-9 M of cytochalasin H for 24, 48 and 72 hours. The assessment of cell viability was carried out by (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay at 578 nm. The data are the average of three independent tests. mRNA expression changes of PLAU and PCDH10 were then evaluated by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The fluorometric of caspases-3, -8, and -9 activities were carried out. The morphology changes in the U87MG cells were observed by fluorescence microscope. RESULTS: MTT assay showed that cytochalasin H (10-5 M) inhibited the U87MG cancer cells proliferation after 48 hours. Analysis of qRT-PCR showed that the PLAU expression was significantly decreased in comparison with the control (P<0.05). The expression of PCDH10 also showed a significant increase when compared to the control (P<0.001). Fluorescence microscope indicated morphological changes due to apoptosis in U87MG cancer cells, after treatment with cytochalasin H (10-5 M, 48 hours). The fluorometric evaluation of caspase-3, -8, and -9 activities showed no significant difference between the caspases and the control group. CONCLUSION: This study shows the effect of caspase-independent pathways of the programmed cell death on the U87MG cancer cell line under cytochalasin H treatment. Further studies are needed to explore the exact mechanism.

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.

Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration.

Melanocytes derive from neural crest cells, which are a highly migratory population of cells that play an important role in pigmentation of the skin and epidermal appendages. In most vertebrates, melanocyte precursor cells migrate solely along the dorsolateral pathway to populate the skin. However, zebrafish melanocyte precursors also migrate along the ventromedial pathway, in route to the yolk, where they interact with other neural crest derivative populations. Here, we demonstrate the requirement for zebrafish paralogs pcdh10a and pcdh10b in zebrafish melanocyte precursor migration. pcdh10a and pcdh10b are expressed in a subset of melanocyte precursor and somatic cells respectively, and knockdown and TALEN mediated gene disruption of pcdh10a results in aberrant migration of melanocyte precursors resulting in fully melanized melanocytes that differentiate precociously in the ventromedial pathway. Live cell imaging analysis demonstrates that loss of pchd10a results in a reduction of directed cell migration of melanocyte precursors, caused by both increased adhesion and a loss of cell-cell contact with other migratory neural crest cells. Also, we determined that the paralog pcdh10b is upregulated and can compensate for the genetic loss of pcdh10a. Disruption of pcdh10b alone by CRISPR mutagenesis results in somite defects, while the loss of both paralogs results in enhanced migratory melanocyte precursor phenotype and embryonic lethality. These results reveal a novel role for pcdh10a and pcdh10b in zebrafish melanocyte precursor migration and suggest that pcdh10 paralogs potentially interact for proper transient migration along the ventromedial pathway.

Spatial rearrangement of Purkinje cell subsets forms the transverse and longitudinal compartmentalization in the mouse embryonic cerebellum.

Transversely oriented lobules and longitudinally arrayed stripes of Purkinje cell subsets subdivide the cerebellar cortex into multiple compartments that are involved in diverse functions. In the mammalian cerebellum, anterior, and posterior lobules, which are involved in somatosensorimotor function, show an alternation of aldolase C (zebrin II) -positive and -negative stripes, whereas the central lobules (lobules VIb-VII and crus I), which are implicated in nonmotor functions, show a laterally expanded arrangement solely of aldolase C-positive stripes. To understand the developmental process of this compartmental pattern, we identified groups of Purkinje cell subsets in the entire mouse cerebellum at embryonic day (E) 14.5 by staining Purkinje cell subset markers. We then tracked four major domains of Protocadherin 10 (Pcdh10)-positive Purkinje cell subsets (medial, dorsal, central, and mid-lateral subsets), which were clearly demarcated during E14.5-17.5. These domains of Purkinje cell subsets shifted predominantly in the longitudinal direction to be positioned in the anterior and posterior lobules. However, a particular portion of the medial and mid-lateral domains, and the whole of the central domain shift in the lateral direction to be positioned in the central lobules. The results indicate that while the longitudinal shift of domains of Purkinje cell subsets forms the longitudinally striped compartments in the anterior and posterior cerebellum, the lateral shift of particular domains of Purkinje cell subsets underlies the laterally expanded arrangement of stripes in central lobules. Thus, the rearrangement of Purkinje cell subsets in the embryonic cerebellum is critically related to the compartmental organization in the mammalian cerebellum.

Epigenetic silencing of protocadherin 10 in colorectal cancer.

Colorectal cancer (CRC) is one of the most common types of malignant tumor in the world and occurs through a multi-step process resulting from the accumulation of genetic and epigenetic alterations of the genome. Although the molecular mechanisms of the pathogenesis of CRC remain unclear, the inactivation of tumor suppressor genes (TSGs) through promoter methylation serves an important role. Aberrant methylation is a well-defined marker of CRC. At present, the epigenetic silencing of protocadherin 10 (PCDH10) has been identified as an important TSG with key roles in colorectal carcinogenesis, invasion and metastasis as a frequent and early event. Advances in gene methylation detection in tumor tissues and body fluids have led to the development of non-invasive screening methods for CRC. The present study aimed to review the epigenetic alteration of PCDH10 in CRC development, and the potential of PCDH10 to be a non-invasive biomarker for CRC.

Prognostic signature of protocadherin 10 methylation in curatively resected pathological stage I non-small-cell lung cancer.

Although curative resection is the current treatment of choice for localized non-small-cell lung cancer (NSCLC), patients show a wide spectrum of survival even after complete resection of pathological stage I NSCLC. Thus, identifying molecular biomarkers that help to accurately select patients at high risk of relapse is an important key to improving the treatment strategy. The purpose of this study was to evaluate the prognostic signature of protocadherin 10 (PCDH10) promoter methylation in curatively resected pathological stage I NSCLC. Using methylation-specific polymerase chain reaction assays, methylation of PCDH10 promoter was assessed in cancer tissues of 109 patients who underwent curative resection of pathological stage I NSCLC. Associations between PCDH10 methylation status and disease outcome was analyzed. PCDH10 promoter methylation was detected in 46/109 patients (42.2%). Patients with methylated PCDH10 showed significantly worse recurrence-free, overall, and disease-specific survival compared with those without methylation (P < 0.0001, P = 0.0004, P = 0.0002, respectively). Multivariate Cox proportional hazard regression analysis revealed that adjusted hazard ratios of methylated PCDH10 were 5.159 for recurrence-free, 1.817 for overall, and 5.478 for disease-specific survival (P = 0.0005, P = 0.1475, P = 0.0109, respectively). The pattern of recurrence was not significantly different between patients with and without PCDH10 methylation (P = 0.5074). PCDH10 methylation is a potential biomarker that predicts a poor prognosis after curative resection of pathological stage I NSCLC. Assessment of PCDH10 methylation status might assist in patient stratification for determining an appropriate adjuvant treatment and follow-up strategy.

Gene methylation in gastric cancer.

Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. Over 70% of new cases and deaths occur in developing countries. In the early years of the molecular biology revolution, cancer research mainly focuses on genetic alterations, including gastric cancer. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs, and microRNAs. Aberrant DNA methylation in the promoter regions of gene, which leads to inactivation of tumor suppressor and other cancer-related genes in cancer cells, is the most well-defined epigenetic hallmark in gastric cancer. The advantages of gene methylation as a target for detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as serum and gastric washes have led to many studies of application in gastric cancer. This review focuses on the most common and important phenomenon of epigenetics, DNA methylation, in gastric cancer and illustrates the impact epigenetics has had on this field.