RUNX3 Suppresses Gastric Epithelial Cell Growth by Inducing p21^WAF1/Cip1 Expression in Cooperation with Transforming Growth Factor β-Activated SMAD

Abstract

RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor ␤1 (TGF-␤1). It is known that TGF-␤1 induces cell growth arrest by activating CDKN1A (p21 WAF1/Cip1 ), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-␤-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-␤-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.The RUNX family of transcription factors plays pivotal roles in normal development and neoplasias. In mammals, the RUNX family genes consist of RUNX1/AML1, RUNX2, and RUNX3 (see reference 37 for the nomenclature). All RUNX family members share the central Runt domain, which is well conserved and recognizes a specific DNA sequence, but each has relatively divergent N-and C-terminal regions. RUNX1 and RUNX2 are required for hematopoiesis and osteogenesis, respectively, and are genetically altered in leukemia and bone disease (15,20,26,27,29). In contrast, RUNX3 is involved in neurogenesis (10, 17) and thymopoiesis (36, 41) and functions as a tumor suppressor of gastric cancer (7,18). About 60% of primary gastric cancer specimens do not express RUNX3 due to a combination of hemizygous deletion and DNA hypermethylation of the RUNX3 promoter region (7,13,18,28).

RUNX3 is multifunctional in carcinogenesis of multiple solid tumors

“…Earlier shown to bind directly to TGF-b signaling effector SMAD3 for synergistic induction of Immunoglobulin germline C a promoter (Shi and Stavnezer, 1998; Hanai et al, 1999) during Immunoglogulin A class switching, RUNX3 is now also known to have a function in TGF-b-induced growth inhibition. After stimulation by TGF-b cytokines, RUNX3 cooperates with SMAD proteins to directly upregulate transcription of the proapoptotic gene Bim1 and increase expression of the cyclin-dependent kinase inhibitor p21 WAF1 in gastric cancer cells (Chi et al, 2005;Yano et al, 2006). Moreover, the ability of RUNX3 to target SMADs to distinct nuclear foci on stimulation by TGF-b further strengthens the case for RUNX3 as a regulator of the TGF-b signaling pathway (Zaidi et al, 2002).…”

“…This seems to stem from multiple defects in its protein properties. The R122C mutant has reduced affinity for DNA with the consensus RUNX-binding site; it is also less efficient in binding SMADs (Chi et al, 2005). The combination of these defects results in impaired participation in TGF-b signaling, as seen by reduced activation of p21 WAF1 promoter during TGF-b stimulation (Chi et al, 2005).…”

Claudin-1 Has Tumor Suppressive Activity and Is a Direct Target of RUNX3 in Gastric Epithelial Cells

“…RUNX3 has been shown to contribute to tumor suppressor activity as a component of the TGF- tumor suppressor pathway 9 through the attenuation of cell growth with a CDK inhibitor 13 , induction of apoptosis 15 , and inhibition of angiogenesis 16 and metastasis 31 in gastric cancers. Here, we showed that RUNX3 also exerts its tumor functions as a tumor suppressor under the TGF--signalling pathway 9 .…”

KLF4, p21 and context-dependent opposing forces in cancer

“…For example, RUNX3 (a transcription factor of the Runt family) has been shown to be required for TGFβ-induced CDKN1A expression 108 and cell-cycle arrest 109 , whereas Notch signalling has been shown to be required for TGFβ-induced epithelial to mesenchymal transition 110 . Interestingly, KLF4 is also linked to these pathways.…”