Frequent inactivation of RUNX3 by promoter hypermethylation and protein mislocalization in oral squamous cell carcinomas

Abstract

Purpose: RUNX3 is a functionally important component in transforming growth factor-beta (TGF-beta) mediated signaling pathway. Epigenetic silencing expression of RUNX3, as well as aberrant cytoplasmic retention of RUNX3 protein are causally involved in gastric carcinogenesis. Here, we examined the expression of RUNX3 gene and protein in oral squamous cell carcinomas (OSCCs) and analyzed the methylation status of RUNX3 promoter region.

Methods: About 10 normal oral mucosa and 30 OSCCs were collected to examine RUNX3 expression by RT-PCR analysis and immunohistochemistry assay using anti-RUNX3 monoclonal antibody R3-6E9. Methylation-specific PCR was carried out on the same specimens to analyze the methylation status of RUNX3 promoter. In addition, the stored paraffin-embedded specimens, including 40 oral leucoplakia (OLK) and 120 OSCCs, were examined by immunohistochemistry assay.

Results: RUNX3 gene and protein were underexpressed in OSCCs due to promoter hypermethylation. Protein mislocalization occurred frequently. Both downregulation of RUNX3 protein expression (P = 0.001) and protein mislocalization (P = 0.001) were correlated with the differentiation grades in OSCCs.

Conclusions: RUNX3 plays an important role in oral carcinogenesis. It may be a useful diagnostic marker and a potential therapeutic target for OSCC.

Fig. 1

Expression levels of RUNX3 gene in OSCC specimens by RT-PCR analysis. N normal tissues, T tumor tissues. GAPDH is used as a positive control for RNA quality and loading

Fig. 2

a Analysis of methylation status in normal tissue by MSP. N the normal tissue. All the samples demonstrate only unmethylated DNA PCR products. Lane M amplified products with primes recognizing the methylated sequence (221 bp), Lane U amplified products with primers recognizing the unmethylated sequence (221 bp). The gastric cell line, MKN74, was used as a positive control (P); the peripheral lymphocytes (L) were used as a negative control. b Analysis of methylation in OSCCs and the matched adjacent normal tissues by MSP. T tumor tissue, N the matched adjacent normal tissue; c sequence analysis of methylated-specific PCR products for RUNX3 promoter region. WT the wild-type RUNX3 gene sequence, U the sequence of PCR products amplified with the primers recognizing the unmethylated sequence (shown here from nucleotides 65057–65113). The CpG sites were underlined. In the products the unmethylated cytosine residues were changed to thymidine. M, the sequence of PCR products amplified with primers recognizing the methlylated sequence (shown here from nucleotides 65043 to 65098). In the products the unmethylated cytosine residues were changed to thymidine, while the methylated cytosine residues were unchanged

Fig. 3

Immunodetection of RUNX3 protein in normal oral mucosa, OLKs and OSCCs with the specific monoclonal antibody R3-6E9. The normal oral mucosa (a), OLK without dysplasia (b), OLK with mild dysplasia (c), OLK with moderate dysplasia (d), OLK with Severe dysplasia (e), well differentiated OSCC (f), moderately differentiated OSCC (g), and poorly differentiated OSCC (h) are showed in the figure. The boxed regions (×40) are enlarged on the right (×400)

Fig. 4

Immunoreactivity patterns of RUNX3 protein in normal oral mucosa and OSCCs with the specific monoclonal antibody R3-6E9. a Expression of RUNX3 in normal oral mucosa, b loss of RUNX3 expression in OSCC, c cytoplasmic mislocalization of RUNX3 expression in OSCC, d, nuclear expression of RUNX3 in OSCC, the boxed regions (×100) are enlarged on the right (×400). e, n, t, the expression of RUNX3 in the tumor tissues and the adjacent normal tissues, n the adjacent normal tissues, t the tumor tissues. The boxed regions (×40) are enlarged on the right (×400)

Fig. 5

The frequency of downregulation of RUNX3 expression in the different differentiation grades in oral carcinogenesis. Normal tissues 0%, OLK without dysplasia 0%, OLK with mild dysplasia 0%, OLK with moderate dysplasia 0%, OLK with severe dysplasia and carcinoma in situ 20%, Well differentiated OSCC 24.6%, Moderately differentiated OSCC 62.2%, Poorly differentiated OSCC 90.5%