CDCP1 is a novel stem cell marker that is expressed in several types of cancer. The mechanisms by which CDCP1 expression is regulated, and the clinical implications of this marker, have not been clarified. In this report, we examine the epigenetic regulation of CDCP1 expression in cell lines and clinical samples from patients with breast cancer. Many CpG sequences were localized around the transcription initiation site of CDCP1. These CpG motifs were found to be poorly methylated in cell lines with high levels of CDCP1 expression and heavily methylated in cell lines with low levels of CDCP1 expression. The in vitro methylation of CpG sites decreased CDCP1 promoter activity, and the addition of a demethylating reagent restored activity. In 25 breast cancer samples, an inverse correlation was noted between the CDCP1 expression level and the proportion of methylated to non-methylated CpG sites. Tumours with high-level CDCP1 expression showed higher levels of proliferation, as revealed by immunohistochemical detection of the MIB-1 antigen, than tumours with low-level CDCP1 expression. These findings indicate that the expression of CDCP1 is regulated by methylation of its promoter region in tumours. CDCP1 expression may prove to be useful in the further characterization of cancers.
"DNA methylation is an important mechanism for gene transcriptional silencing. CpG hypermethylation in DNA promoter regions is responsible for gene silencing –. DNA methylation status was regulated by DNMT, which has de novo methylation activity. "
[Show abstract][Hide abstract] ABSTRACT: Cardiac progenitor cells (CPCs) have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+)CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF), are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α) could enhance the expression of c-kit. However, the mechanism is unknown.
CPCs were isolated from adult mouse hearts, c-kit(+) and c-kit(-) CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+)CPCs, made c-kit(-)CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+) and c-kit(-) CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT) mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(-) CPCs.
SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+)CPCs and make c-kit(-)CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT activity, as well as the subsequent demethylation of the c-kit gene.
PLoS ONE 07/2013; 8(7):e69134. DOI:10.1371/journal.pone.0069134 · 3.23 Impact Factor
"CDCP1 gene regulation has been investigated in various cancer cell lines. PC3 cells show abundant expression of CDCP1 protein and a low frequency of methylation in transcription-initiation sites . We found that both HMW and LMW forms of CDCP1 are expressed in PC3 cells (Figure 2). "
[Show abstract][Hide abstract] ABSTRACT: KAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.
We used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.
We demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.
These novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.
BMC Cancer 03/2012; 12(1):81. DOI:10.1186/1471-2407-12-81 · 3.36 Impact Factor
"The Trask/CDCP1 gene is flanked by the markers D3S1029 and D3S1478 (from the NCBI genome map) referenced in the study by Wistuba et al, identifying it as a gene with loss of heterozygosity in a large fraction of lung cancers, but not normal or pre-neoplastic bronchial epithelium (Wistuba et al 2000). Further consistent with a potential tumor suppressing role is the evidence showing that its expression is reduced or silenced by promoter methylation in some cancer cell lines (Ikeda et al 2006). In this study we find that the expression of Trask relative to the normal epithelium is reduced or lost in some cancers of the breast or colon. "
[Show abstract][Hide abstract] ABSTRACT: Trask/CDCP1 is a transmembrane glycoprotein widely expressed in epithelial tissues whose functions are just beginning to be understood, but include a role as an anti-adhesive effector of Src kinases. Early studies looking at RNA transcript levels seemed to suggest overexpression in some cancers, but immunostaining studies are now providing more accurate analyses of its expression. In an immuno-histochemical survey of human cancer specimens, we find that Trask expression is retained, reduced or sometimes lost in some tumors compared with their normal epithelial tissue counterparts. A survey of human cancer cell lines also show a similar wide variation in the expression of Trask, including some cell types with the loss of Trask expression, and additional cell types that have lost the physiological detachment-induced phosphorylation of Trask. Three experimental models were established to interrogate the role of Trask in tumor progression, including two gain-of-function models with tet-inducible expression of Trask in tumor cells lacking Trask expression, and one loss-of-function model to suppress Trask expression in tumor cells with abundant Trask expression. The induction of Trask expression and phosphorylation in MCF-7 cells and in 3T3v-src cells was associated with a reduction in tumor metastases while the shRNA-induced knockdown of Trask in L3.6pl cancer cells was associated with increased tumor metastases. The results from these three models are consistent with a tumor-suppressing role for Trask. These data identify Trask as one of several potential candidates for functionally relevant tumor suppressors on the 3p21.3 region of the genome frequently lost in human cancers.
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