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

Abstract

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, CBFb, 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.

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer

“…These data are in agreement with the known tumour suppressor function of members of the Runt family of transcription factors. Thus, Runx2 and Runx3 might act as tumour suppressors in malignant melanoma (Martinez et al, 2005), and Runx3 in breast, gastric, colon, and hepatocellular carcinomas, as well as non-small cell lung cancer (Goel et al, 2004;Sakakura et al, 2005;Lau et al, 2006;Miyagawa et al, 2006;Yanagawa et al, 2007). The reason why the potential tumour suppressor Runx2 is overexpressed in some pancreatic cancer tissues is currently not known, and requires further studies.…”

RUNX3 is multifunctional in carcinogenesis of multiple solid tumors

“…But how does this relate to solid tumors? Inverse correlation between RUNX3 and RUNX2 expression was recently observed in some breast cancer cell lines in which cells with low RUNX3 expression show increased RUNX2 expression (Lau et al, 2006). As all RUNX proteins are closely associated with cancer development, this communication between family members has important implications: dysfunctional RUNX3 would affect RUNX1 and RUNX2 activities on cell proliferation.…”

“…Diverse tumor tissues have been reported to exhibit RUNX3 hypermethylation and inactivation. These include tissues and cell lines that originated from gastric (Li et al, 2002;Oshimo et al, 2004), bladder (Kim et al, 2005;Wolff et al, 2008), colorectal (Ahlquist et al, 2008;Soong et al, 2009;Subramaniam et al, 2009), breast (Lau et al, 2006;Jiang et al, 2008), lung (Sato et al, 2006), pancreatic (Wada et al, 2004), brain cancers (Mueller et al, 2007), and hepatocellular carcinoma . Notably, RUNX3 methylation status is one of the five markers used to classify colorectal tumors associated with very high frequencies of CpG island methylation (CIMP), microsatellite instability, and BRAF mutation (Weisenberger et al, 2006).…”

“…Not only is epigenetic silencing of RUNX3 a frequent occurrence in cancer, cytoplasmic sequestration of RUNX3 was reported in a significant proportion of cancer cases-about 15% of colorectal cancer , 80% of breast cancer (Lau et al, 2006), 38% of gastric cancer , and oral squamous cell carcinomas (Gao et al, 2009). Moreover, RUNX3 cytoplasmic mislocalization is associated with advanced colorectal tumor stages, whereas nuclear RUNX3 expression was correlated with better patient prognosis .…”

Runx3 is required for the differentiation of lung epithelial cells and suppression of lung cancer

“…RUNX3 was also identified as one of the five most informative genes of the CpG island methylator phenotype of colorectal cancer (Weisenberger et al, 2006). In addition to gastric and colon cancer, RUNX3 is frequently inactivated in cancers of the lung, bladder, pancreas, liver, prostate, bile duct, breast, larynx, esophagus, and testicular yolk sac by either DNA hypermethylation or mislocalization of the protein in the cytoplasm (Kato et al, 2003;Goel et al, 2004;Kang et al, 2004;Li et al, 2004;Tozawa et al, 2004;Xiao and Liu, 2004;Ito et al, 2005;Kim et al, 2005b;Lau et al, 2006). Among the lung cancers, RUNX3 promoter hypermethylation is preferentially found in lung adenocarcinomas (Sato et al, 2006).…”