RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAF1/Cip1) expression in cooperation with transforming growth factor {beta}-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 beta1 (TGF-beta1). It is known that TGF-beta1 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-beta-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-beta-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.

FIG. 1.

TGF-β1-dependent induction of p21 expression in SNU16 cells and its requirement for RUNX3. (A) SNU16 cells were stimulated with TGF-β1 (2 ng/ml) for various periods, and their gene expression profile was examined by microarray analysis. Changes in relative expression at the indicated times after TGF-β1 stimulation are presented in graduated color patterns. Red, expression induced by TGF-β1 stimulation; green, expression reduced by TGF-β1 stimulation; black, unchanged expression. (B) SNU16 cells were treated with TGF-β1 (2 ng/ml) for various periods, and the p21 protein levels were analyzed by Western blotting. (C) Suppression of endogenous RUNX3 expression in the SNU16-AS cell line. After reverse transcription (RT), the endogenous RUNX3 mRNA in the SNU16-AS cell line (Endo-RX3) was amplified with RX3-p (5′-CTACGGGACATCCTCTGGCTCC-3′) and RX3-cn (5′-CATCTCTGCCAGCAGCGTGCTG-3′), resulting in an 840-bp fragment. Exogenous antisense RUNX3 mRNA (Exo-AS) was amplified with EF-p (5′-CTTCGCCTCGTGCTTGAGTTGAG-3′) and RX3-p, resulting in a 1,570-bp fragment. The EF-p primer was based on the vector sequence (24), while the RX3-p and RX3-cn primer was based on the RUNX3 cDNA sequence. (D) SNU16 and SNU16-AS cells were cultured for 2 h in the presence or absence of TGF-β1 (2 ng/ml), and p21 expression was analyzed by Northern blotting. The β-actin probe was used as a loading control. (E) The band intensities in panel D were analyzed by densitometry, and the p21 mRNA levels are shown as a graph.

FIG. 2.

Involvement of RUNX3 in TGF-β-dependent p21 induction and cell growth inhibition. (A) SNU16 and SNU16-RX3 cells were treated with the indicated concentration of TGF-β1, and the RUNX3 and p21 expression levels were measured by Northern blotting. The GAPDH probe was used as a loading control. (B) Similarly, SNU16, SNU16-RX3, SNU16-R122C, and SNU16-AS cells were treated with TGF-β1 and the p21 expression levels were measured by Northern blotting. (C) Densitometric analysis of the p21 mRNA band intensities in panel B. (D) SNU16, SNU16-RX3, SNU16-R122C, and SNU16-AS cells were cultured in the presence or absence of 0.03 ng/ml TGF-β1 for 24 h, and the cells were counted. The percentage of growth inhibition of the cells in the presence of TGF-β1 is shown.

FIG. 3.

Effect of IPTG-inducible RUNX3 expression on p21 transcription. (A and B) MKN28-Lac, MKN28-Lac-RX3 (A), and MKN28-Lac-R122C (B) cells were treated with IPTG (5 mM) for the indicated periods, and exogenous RUNX3 and endogenous p21 expression levels were examined by Northern blotting. (C) Densitometric analysis of the p21 mRNA band intensities in panel A. (D) MKN28-Lac-RX3 and MKN28-Lac-R122C cells were cultured in the presence or absence of IPTG (5 mM) for 48 h and counted. The percentage of growth inhibition of the cells is shown.

FIG. 4.

p21 is a direct target of RUNX3. (A) Schematic depiction of the RUNX-binding sites in the p21 promoter (2.3-kb region). (B) SNU16 cells were subjected to ChIP analysis. Their chromatin was immunoprecipitated with the polyclonal anti-RUNX3 antibody, and the p21 promoter region was amplified by PCR. GAPDH was amplified as a negative control. (C) 293 cells were cotransfected with the wild-type or mutant pGL3-p21 promoter construct (50 ng/well in a 24-well plate) and various amounts (0, 0.25, and 0.5 μg) of pCS4-3myc-RX3. Reporter activities were measured by a luminometer.

FIG. 5.

RUNX3 induces p21 expression in cooperation with SMADs. (A and B) MKN28 (A) and 293 (B) cells were cotransfected with pGL3-p21 reporter plasmids and the indicated combination of plasmids. For each transfection, 100 ng/well (24 wells) of each plasmid was used for panel A and 100 ng/well of reporter plasmids was used in all transfections.

FIG. 6.

Comparison of the DNA-binding affinities of RUNX3 and RUNX3-R122C by EMSA. (A) Similar amounts of nuclear extracts obtained from 293 cells transfected with pCS4-myc, pCS4-myc-RUNX3, and pCS4-myc-RX3-R122C were subjected to EMSA using the ³²P-labeled RUNX binding site-bearing double-stranded oligonucleotide RXE as the probe. The m1 and m2 oligonucleotides were used as specific and nonspecific competitors, respectively (see Materials and Methods). The arrows indicate the DNA-RUNX3 and DNA-RUNX3-antibody complexes. (B) EMSA was performed using a fixed amount of probe DNA and increasing amounts of the nuclear extract obtained from 293 cells transfected with pCS4-myc-RUNX3 or pCS4-myc-RX3-R122C. The arrow indicates the DNA-RUNX3 and DNA-RUNX3-R122C complexes. (C) RUNX3 and RUNX3-R122C protein levels in the transfected cells described in panels A and B were determined by using an anti-myc antibody. The arrow indicates RUNX3 and RUNX3-R122C.

FIG. 7.

Interaction between RUNX3 and TGF-β-specific SMADs. (A) 293 cells were transfected with a fixed amount of pCS4-myc-RUNX3, pCS4-myc-RX3-R122C, pCS4-HA-SMAD3, and/or pCS4-HA-SMAD4 as indicated. (Upper panel) The physical interactions between RUNX3 or RX3-R122C and SMADs were examined by immunoprecipitation (IP) analyses using an anti-myc antibody, followed by Western blotting (WB) using the anti-HA antibody. (Middle panel) Expression levels of SMAD3 and SMAD4 as measured by immunoprecipitation with an anti-HA antibody, followed by Western blotting using the same antibody. (Lower panel) Expression levels of RUNX3 and RUNX3-R122C as measured by Western blotting using an anti-myc antibody.

FIG. 8.

RUNX3 and p21 expression patterns in mouse and human gastric epithelium overlap. (A to F). Pyloric areas of stomach tissues were obtained from newborn normal, Runx3^+/−, and Runx3^−/− mutant mice, and the expression levels of Runx3 and p21 were detected by immunohistochemical staining. A to D are adjacent sections. A and B are control staining and HE staining, respectively. Runx3 was detected using the anti-bacterial β-galactosidase antibody since Runx3 was fused with the bacterial β-galactosidase (β-gal) gene and expressed as the Runx3-β-galactosidase fusion protein in Runx3 mutant mice (18). IgG, immunoglobulin G. (G to L). Expression pattern of RUNX3 and p21 in human gastric epithelium. HE staining (G, H), in situ hybridization with RUNX3 (I, J), and in situ hybridization with p21 (K, L) are shown. Arrows and arrowheads indicate normal and cancerous regions, respectively. Enlargement (×200 magnification) of the boxed regions in panels G, I, and K are shown in panels H, J, and L, respectively.