The human cadherin 11 is a pro-apoptotic tumor suppressor modulating cell stemness through Wnt/β-catenin signaling and silenced in common carcinomas

Abstract

Genetic alterations of 16q21-q22, the locus of a 6-cadherin cluster, are frequently involved in multiple tumors, suggesting the presence of critical tumor suppressor genes (TSGs). Using 1 Mb array comparative genomic hybridization (aCGH), we refined a small hemizygous deletion (~1 Mb) at 16q21-22.1, which contains a single gene Cadherin-11 (CDH11, OB-cadherin). CDH11 was broadly expressed in human normal adult and fetal tissues, while its silencing and promoter CpG methylation were frequently detected in tumor cell lines, but not in immortalized normal epithelial cells. Aberrant methylation was also frequently detected in multiple primary tumors. CDH11 silencing could be reversed by pharmacologic or genetic demethylation, indicating an epigenetic mechanism. Ectopic expression of CDH11 strongly suppressed tumorigenecity and induced tumor cell apoptosis. Moreover, CDH11 was found to inhibit Wnt/β-catenin and AKT/Rho A signaling, as well as actin stress fiber formation, thus further inhibiting tumor cell migration and invasion. CDH11 also inhibited epithelial-to-mesenchymal transition and downregulated stem cell markers. Thus, our work identifies CDH11 as a functional tumor suppressor and an important antagonist of Wnt/β-catenin and AKT/Rho A signaling, with frequent epigenetic inactivation in common carcinomas.

Figure 1

(a) Representative 1 Mb aCGH result showing a small hemizygous deletion including the CDH11 locus in NPC cell lines. Cytoband of 16q is shown. Normalized log2 signal intensity ratios from −1 to 1 are plotted. Each dark blue-colored dot represents a single BAC clone. Two BAC clones closest to the CDH11 locus (RP11-467L24 and RP11-229O3) are labeled with red dots and red rectangle frames. The CDH11 locus is shown in lower panel as in Ensemble Human Contig view (http://www.ensemble.org/). (b) CDH11 is broadly expressed in human normal adult tissues and fetal tissues, with GAPDH as a control. Sk.M., skeleton muscle; B.M., bone marrow; L.N., lymph node.

Figure 2

(a) Schematic structure of the CDH11 CGI. Exon 1 (indicated with a black rectangle), CpG sites (short vertical lines), MSP sites, and BGS region analyzed are indicated. (b) CDH11 is frequently silenced and methylated in multiple carcinoma cell lines but expressed and unmethylated in immortalized but non-transformed epithelial cell lines (underlined). ESCC, esophageal carcinoma; GsCa, gastric carcinoma; CRC, colorectal cancer;M, methylated; U, unmethylated. (c) Validation of the specificity of MSP system for CDH11. No product was obtained for unbisulfited DNA. (d) Pharmacologic demethylation with Aza alone or combined with trichostatin A (A+T), or genetic demethylation in DKO cell line restored CDH11 expression in methylated/silenced tumor cell lines.

Figure 3

Detailed BGS analyses of CDH11 promoter methylation. (a) NPC cell lines and immortalized normal cell line NP69. (b) Summarized percentage methylation of CDH11 in multiple carcinoma cell lines. (c) Pharmacologic and genetic demethylation of CDH11. The CDH11 transcription start site is shown as bent arrows. Circles, CpG sites analyzed; row of circles, an individual promoter allele that was cloned, randomly selected, and sequenced; filled circle, methylated CpG site; open circle, unmethylated CpG site.

Figure 4

(a–c) Representative MSP analysis of CDH11 methylation in primary carcinomas and normal tissues. Ca, carcinoma; ESCC, esophageal carcinoma; CRC, colorectal cancer; GsCa, gastric carcinoma; N, paired tumor-adjacent normal tissues; T, tumor; M, methylated; U, unmethylated.

Figure 5

Subcellular localization and functional analyses of CDH11. (a) Confocal microscopy assay showed that CDH11 (red) is clearly expressed on the cell membrane in CDH11-transfected HONE1 cells using mouse anti-CDH11 monoclonal antibody, with double immunostaining for E-cadherin (green) as a control. DAPI counterstaining (blue) was used to visualize DNA. (b) CDH11 and E-cadherin protein expression detected in human normal tissues of brain, testis, lung and trachea by western blot. *Indicates truncated variant band. (c) CDH11 inhibits tumor cell growth. Representative colony formation assays. Quantitative analyses of colony numbers are shown in the right as values of mean±s.d., *P<0.05, **P<0.01. (d) CDH11 induces tumor cell apoptosis. TUNEL assays of CDH11 and vector-expressing tumor cells. *P<0.05. (e) Western blot showing upregulation of cleaved caspase 9, 7, 3 and cleaved poly (ADP-ribose) polymerase (PARP) in CDH11-expressing tumor cells.

Figure 6

(a) Inhibition of TcF activity and CCND1, c-Myc and MMP7 promoter reporter activities in CDH11-expressing tumor cells. *P<0.05. (b) Ectopic expression of CDH11 disrupted Wnt/β-catenin signaling pathway. Western blot was performed using antibodies against total β-catenin, active β-catenin, phospho-β-catenin (Ser552), phospho-GSK3β, CCND1, c-Myc and MMP7. α-Tubulin was used as a control. (c) CDH11 sequestered nuclear β-catenin to the cytoplasm. Endogenous β-catenin localization was visualized in CDH11- and vector-transfected HONE1 cells by indirect immunofluorescence. Original magnification, × 400.

Figure 7

(a) Impaired actin stress fiber organization in CDH11-expressing HONE1 cells. Original magnification, × 400. (b) Expression of several key regulators of actin stress fiber reorganization in CDH11- and vector-transfected cells. Western blot analysis was done using antibodies against phospho-Akt, phospho-JNK, phospho-Rho A and total Rho A. (c) Wound-healing assay of tumor cells transfected with either vector or CDH11. Pictures were taken at 0, 12, 24 or 40 h. Right panel: width of remaining open wound measured in relation to time 0 h separation. (d) CDH11 inhibited the invasive activity of tumor cells. Original magnification, × 400. *P<0.05.

Figure 8

(a) Morphology changes of HONE1 cells transfected with CDH11 or empty vector by phase-contrast microscopy. Original magnification, × 400. (b) Indirect immunofluorescence detecting the expression of E-cadherin and Vimentin in CDH11- or vector-transfected HONE1 cells. (c) Western blot showing the expression of E-cadherin and Vimentin in CDH11- or vector-transfected cells. α-Tubulin was used as a loading control. (d) Downregulation of representative stem cell markers in CDH11-transfected tumor cells. *Indicates significantly downregulated bands.