1. Introduction
1.3 Protocadherin 10 (PCDH10)
Protocadherins are members of a non-classic subfamily of calcium-dependent cell–cell adhesion molecules in the cadherin superfamily. Subfamily members of protocadherins contain similar extracellular domain structure, a transmembrane domain but a different cytoplasmic tail.11 Protocadherin 10 (PCDH10/OL-PCDH /KIAA1400) is the human orthologue of mouse Pcdh10. It is located at chromosome 4q28.3. There are two transcript variants of PCDH10, namely PCDH10 variant 1 (PCDH10 V1) and PCDH10 variant 2 (PCDH10 V2). The first exon of PCDH10 V1 encodes the extracellular domain which contains six ectodomain repeats (extracellular repeat domains, EC domains), as well as the transmembrane domain and the cytoplasmic domain, while the remaining exons encode an extension for the cytoplasmic tail. On the other hand, transcript of PCDH10 V2 ends in the first intron, resulting a single exon gene that encodes the three domains stated above.11
PCDH10 is mainly expressed in the olfactory bulb, most parts of the limbic system (hence the name OL-PCDH) and in the cerebellum, suggesting its function in cerebellum development.11 Recent analysis of PCDH10 knockout mice demonstrated that the striatal neurons could not extend their axons normally in the brain. Since
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PCDH10 was highly concentrated in the axons’ growth zones, it suggests that PCDH10
may play a role in sustaining the normal migration of striatal axons.12 13 PCDH10 is also found to be expressed in other organs such as heart, kidney, lung, trachea and colon as revealed by reverse transcriptase polymerase chain reaction (RT-PCR). 14 15
PCDH10 was also reported to induce homophilic cell-cell adhesion ability.16
However, the cell-cell adhesion ability was weaker than that of the classical cadherins, suggesting that protocadherins may be involved in other functions such as signal transduction besides physical linking of cells.
Recently, regulatory elements in PCDH10 gene promoter was further identified and characterized by Li et al. using human prostate cancer cells. It was shown that the region between nucleotides -144 and -99 was essential to the promoter activity, and within this region, a CAAT box and a GT box were found to be important to the promoter regulation.17
1.3.1 Recent studies of PCDH10
In a study by Miyamoto et al. in 2005, CpG islands in the 5’ promoter region of PCDH10 was found to be aberrantly methylated in breast cancer cell lines and in
42.8% primary breast cancer tested. On the contrary, there is no CpG islands hypermethylation found in normal human mammary epithelial cells.18 In 2006, Ying et
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al. identified PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal
and multiple other carcinomas with frequent methylation. Moreover, transcriptional silencing of PCDH10 could be reversed by pharmacologic demethylation drug, suggesting PCDH10 may be regulated by epigenetic modification. In the same study, ectopic expression of PCDH10 in several carcinoma cell lines (nasopharyngeal carcinoma cell line CNE-1 and the esophageal carcinoma cell line EC109) strongly suppressed tumor cell growth, migration, invasion and colony formation ability.15 In 2007, the same research group identified the frequent methylation and silencing of PCDH10 in lymphoma cell lines as well as in primary tumors but not in normal
peripheral blood mononuclear cells (PBMCs) or lymph nodes, suggesting methylation of PCDH10 may also involve in lymphomagenesis.19 Methylation of PCDH10 in gastric cancer cell lines and in 81.7% primary gastric tumors tested was reported by Yu et al. in 2009,20 supporting its role as a tumor suppressor in various carcinomas.
Monosomy 4 and hypermethylation of promoter region of PCDH10 were also reported by Narayan et al.21 and Wang et al.22 in invasive cervical cancer. However, loss of heterozygosity using microsatellite markers (D4S2423) adjacent to PCDH10 was not observed.22 Recently, Cheung et al. reported the first application of methylated DNA immunoprecipitation-chip to detect epigentically regulated regions in human testicular cancer. In this study, one of the genes identified was PCDH10 and its promoter region
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was found to be hypermethylated in testicular embryonal carcinoma cell line NT2.23 On the other hand, Yu et al. utilized high-resolution melting analysis to detect the methylation status of PCDH10 in gastric, colorectal and pancreatic cancers and reported that the methylation level of PCDH10 was significantly higher in tumor tissues than in normal tissues in all three type of cancers, supporting its role as a tumor suppressor.24
1.3.2 PCDH10 and apoptosis
In Yu et al. study in 2009, PCDH10 re-expression in gastric cancer cell line MKN45 was found to induce apoptosis and reduce cell proliferation. cDNA array analysis of MKN45 stably transfected with PCDH10 revealed that several genes involved in apoptosis, proliferation, and invasion/metastasis pathways were modulated by PCDH10.20
Another study by Ding et al. in 2009 reported that downregulation of human brain expressed X-linked 1 (hBex1) inhibited apoptosis induced by imatinib in Bcr/Abl+ leukemic cells. Further investigations pointed out that hBex1 is associated with PCDH10, and silencing of PCDH10 also attenuated apoptosis induced by imatinib in
hBex1 transfected cells, suggesting that PCDH10 may participate in the apoptosis of cancer cells.25
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1.3.3 Deletion of chromosome 4q28 and cancer
High loss of heterozygosity (LOH) frequency in 4q28 region was first reported in primary hepatocellular carcinoma cases by Hammond et al. in 1999, indicating important tumor suppressors within or in vicinity of this region.26 In 2004, Knösel et al.
used comparative genomic hybridization (CGH) to screen CRCs for chromosomal aberrations that were associated with metastatic phenotype. Deletions were prominent at 4q27-4q28.27 In addition, loss in copy number at 4q28.3 in pancreatic adenocarcinoma,28 a 4.5-megabase deletion at 4q28 in prostate cancer cells29 and chromosomal loss at 4q28-32 in breast cancer were also reported.30
1.3.4 Studies of PCDH10 at our lab
Previous studies of LOH at chromosome 4q25-4q28.3 identified 3 regions with high frequency of LOH; PCDH10 is located within one of these regions. Epigenetic studies of PCDH10 in CRC cell lines and patient tumor samples revealed that 5’ promoter region of PCDH10 was highly methylated when compared to the paired normal tissue.
Treatment of five CRC cell lines with demethylation drug 5-aza-2’-deoxycytidine also restored RNA expression of PCDH10, implying that PCDH10 was regulated by epigenetic modification. Investigation of PCDH10 V1 RNA expression in CRC
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primary samples also indicated a decreased expression level in tumor than in normal tissue. However, expression pattern of PCDH10 V2 was inconsistent; though predisposed to co-express with PCDH10 V1 when the expression level of it was high.
Functional assays using PCDH10 V1 single stable expression clones in CRC cell line HCT116 showed that both proliferation and colony formation ability of cells decreased, but no similar observations were found in PCDH10 V2 single stable expression clones.
Moreover, PCDH10 V1 and PCDH10 V2 were found to co-localize in cell membrane.
In conclusion, PCDH10 may be a putative tumor suppressor gene associated with colorectal carcinogenesis.
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