In intestinal epithelial cells, inactivation of APC, a key regulator of the Wnt pathway, activates beta-catenin to initiate tumorigenesis. However, other alterations may be involved in intestinal tumorigenesis. Here we found that RUNX3, a gastric tumor suppressor, forms a ternary complex with beta-catenin/TCF4 and attenuates Wnt signaling activity. A significant fraction of human sporadic colorectal adenomas and Runx3(+/-) mouse intestinal adenomas showed inactivation of RUNX3 without apparent beta-catenin accumulation, indicating that RUNX3 inactivation independently induces intestinal adenomas. In human colon cancers, RUNX3 is frequently inactivated with concomitant beta-catenin accumulation, suggesting that adenomas induced by inactivation of RUNX3 may progress to malignancy. Taken together, these data demonstrate that RUNX3 functions as a tumor suppressor by attenuating Wnt signaling.

Due to a mistake made during manuscript preparation, the Supplemental Data for this article listed incorrect sequences for the primer sets for methylation-specific PCR (MSP). The correct sequences are given below. These corrections do not affect the findings or conclusions of the study.Primer sets used for detection of methylated DNA were 5 0 -ataaagagaaattaggcgc-3 0 and 5 0 -ataaccctcgaaaaacgcg-3 0 (M3), 5 0 -gatgttt gtttaggtcgtagcggtc-3 0 and 5 0 -ccaaactcgaaattcgccgta-3 0 (M2), and 5 0 -tgcgattggttgcgtttcgc-3 0 and 5 0 -cgaaaatacgcataccgcg-3 0 (M1). Primer sets used for detection of unmethylated DNA were 5 0 -ataaagagaaattaggtgt-3 0 and 5 0 -ataaccctcaaaaaacaca-3 0 (M3), 5 0 -tgtttg tttaggttgtagtggttgt-3 0 and 5 0 -cccccaaactcaaaattcaccata-3 0 (M2), and 5 0 -tgtgattggttgtgttttgt-3 0 and 5 0 -caaaaatacacataccaca-3 0 (M1).

The transcription factor RUNX3 functions as a tumor suppressor in the gastrointestinal epithelium, where its loss is an early event in carcinogenesis. While RUNX3 acts concurrently as a mediator of TGF-b signaling and an antagonist of Wnt, the cellular changes that follow its loss and their contribution to tumorigenicity are not fully understood. Here, we report that the loss of Runx3 in gastric epithelial cells results in spontaneous epithelial-mesenchymal transition (EMT). This produces a tumorigenic stem cell-like subpopulation, which remarkably expresses the gastric stem cell marker Lgr5. This phenomenon is due to the compounding effects of the dysregulation of the TGF-b and Wnt pathways. Specifically, Runx32/2 gastric epithelial cells were unexpectedly sensitized for TGF-binduced EMT, during which the resultant induction of Lgr5 was enhanced by an aberrantly activated Wnt pathway. These data demonstrate a protective role for RUNX3 in safeguarding gastric epithelial cells against aberrant growth factor signaling and the resultant cellular plasticity and stemness.

Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT). However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 −/− p53 −/− murine gastric epithelial (GIF-14) cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-β1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-β1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.