Epigenetic Silencing Of NKD2, A Major Component Of Wnt Signaling, Promotes Breast Cancer Growth

Background:Breast cancer is the most common malignancy in women worldwide, and it is the second leading cause of cancer-related death among women. The incidence of breast cancer is much lower in China than in Western countries. The geographical variation indicates that environmental factors may play a significant role in the risk of breast cancer development. Epigenetic changes are strongly influenced by environmental factors. Aberrant epigenetic changes have been frequently reported in human breast cancer and are associated with development of breast cancer. The naked cuticle (NKD) family includes Drosophila naked cuticle and its two vertebrate orthologs, naked cuticle homolog 1 (NKD1) and 2 (NKD2). NKD1 is located in chromosome 16q12.1, and NKD2 is located in chromosome 5p15.3. Loss of heterozygosity has been frequently found in these regions in different types of tumors, including breast cancer. The promoter region of NKD2 is hypermethylated in glioblastoma cells. In metastic human and mouse osteosarcoma cells, overexpression of NKD2 significantly reduces cell proliferation, migration and invasion ability in vitro and in vivo.Aims:To study the epigenetic regulation and function of NKD2 in breast cancer and to explore the effect of NKD2 in Wnt signaling.Methods:Six breast cancer cell lines (MCF-7, ZR75-1, MDA-MB-468, MDA-MB-231, T47D and BT474) and 68 cases of primary human breast cancer were studied using methylation specific PCR, immunohistochemistry, semi RT-PCR. Cell proliferation assay, colony formation experiment and flow cytometry techniques were employed to analyse the function of NKD2 in breast cancer. The effect of NKD2 in Wnt signaling is studied by dual-Luciferase reporter assay, western blot and SiRNA techeniques. Mouse xenograft model was employed to further analyze the function of NKD2 in breast cancer.Results:Loss of NKD2 expression and complete methylation were found in MDA-MB-231 and MDA-MB-468 cells. Our results revealed that MCF7, ZR75-1, BT474 and T47D cells expressed NKD2 and its promoter was partial methylated. NKD2 was methylated in 51.4%(35/68) of human primary breast cancer samples. NKD2 methylation was significantly associated with reduction of NKD2 expression, and tumor stage (p<0.05). NKD2 suppressed breast cancer cell proliferation both in vitro and in vivo. NKD2 induced G0/1 arrest and inhibited Wnt signaling in breast cancer cells.Conclusion:NKD2 is frequently methylated in human breast cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses breast cancer proliferation by inhibiting WNT signaling.