Protocadherin PCDH10, involved in tumor progression, is a frequent and early target of promoter hypermethylation in cervical cancer

Abstract

Cervical cancer (CC) is the second most common cancer in women. Currently, no tractable molecular-based therapeutic targets exist for patients with invasive CC and no predictive markers of risk assessment for progression of precancerous lesions are identified. New molecular insights into CC pathogenesis are urgently needed. Towards this goal, we first determined the copy number alterations of chromosome 4 and then examined the role of PCDH10 mapped to 4q28 as a candidate tumor suppressor gene. We identified monosomy 4 in 47% of 81 invasive CC studied by SNP array and found that 91% of 130 invasive CC harboring methylation in the promoter region of the PCDH10 gene. We then showed that aberrant promoter hypermethylation of PCDH10 is associated with downregulation of gene expression and cell lines exposed to demethylating agent resulted in profound reactivated gene expression. We also showed that the promoter methylation in the PCDH10 gene occurs at an earliest identifiable stage of low-grade squamous intraepithelial lesion. Our studies demonstrate that inactivation of PCDH10 may be a critical event in CC progression and form a potentially useful therapeutic target for CC.

Figure 1

Identification of chromosome 4 copy number alterations in invasive CC by 250K NspI SNP array. Each vertical column represents a sample with genomic regions representing from pter (top) to qter (bottom). Prefix “T” indicates primary tumor; “Cl” indicates cell line. The blue-red scale bar (-2 to +2) at the bottom represents the copy number changes relative to mean across the samples. The intensities of blue and red indicate relative decrease and increase in copy numbers, respectively. G-banded ideogram of chromosome 4 is shown on the extreme right. All tumors that exhibited chromosome 4 losses are shown from largest to smallest region of losses. Inferred copy number view of MS-751 cell line showing 4p monosomy from normal (2N) (red line) is shown on right.

Figure 2

Analysis of PCDH10 methylation in cervical cancer cell lines and primary tumors. A. MSP analysis in cervical cancer cell lines. U, unmethylated; M, methylated. B. Bisulphite MSP cloning and sequencing of the PCDH10 gene. T, tumor; Cl, cell line. CpG sites examined are numbered sequentially as shown above. Filled circles indicate methylated CpG sites, semi-filled circles indicate partial/heterogeneous methylation, and empty circles indicate unmethylated CpGs. Methylation status by MSP is shown on the right (in bracket), M, methylated; U, unmethylated.

Figure 3

Real-time quantitative PCR analysis of PCDH10 gene expression compared to control gene. A. Expression plot showing PCDH10 (brace) and GAPDH (bracket) gene expression in primary invasive cervical cancer. B. Expression plot showing PCDH10 (brace) and GAPDH (bracket) gene expression in cervical cancer cell lines. Note higher expression of PCDH10 in unmethylated cell line C-33A. C and D. Expression plots showing levels of expression of control gene GAPDH (C) and PCDH10 (D) in cell lines after exposure to azacytidine. Note no difference in control gene in untreated and azacytidine-treated cell lines, while PCDH10 gene expression is reactivated in treated cell lines. Arrow and arrowhead indicate high basal as well as after Azacytidine-exposure, respectively, of PCDH10 expression levels in an unmethylated cell line C-33A.

Figure 4

Analysis of PCDH10 methylation in cervical precancerous lesions. A. MSP analysis. B. Bisulphite MSP cloning and sequencing of the PCDH10 gene. U, unmethylated; M, methylated. LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions. CpG sites examined are numbered sequentially as shown above in panel B. Filled circles indicate methylated CpG sites and empty circles indicate unmethylated CpGs. Pap smear numbers indicating methylation status by MSP are shown on right.