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RUNX3 Is Frequently Inactivated by Dual Mechanisms of Protein Mislocalization and Promoter Hypermethylation in Breast Cancer

¹ Oncology Research Institute and ² Department of Pathology, National University of Singapore; ³ Bioinformatics Institute; ⁴ Institute of Molecular and Cell Biology, Singapore, Singapore; ⁵ H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida; and ⁶ Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Requests for reprints: Saraswati Sukumar, Oncology Research Institute. Phone: 410-614-2479; Fax: 410-614-4073; E-mail: saras{at}jhmi.edu or Yoshiaki Ito, Oncology Research Institute, Clinical Research Centre Level 5, National University of Singapore, 10 Medical Drive, Singapore 117597. Phone: 65-6586-9646; Fax: 65-6779-1117; E-mail: itoy{at}imcb.a-star.edu.sg.

A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBF-ß, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment. (Cancer Res 2006; 66(13): 6512-20)

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