Infection with high-risk human papillomavirus (HPV) has been linked to several human cancers, the most prominent of which is cervical cancer. The integration of the viral genome into the host genome is one of the manners in which the viral oncogenes E6 and E7 achieve persistent expression. The most well-studied cellular targets of the viral oncogenes E6 and E7 are p53 and pRb, respectively. However, recent research has demonstrated the ability of these two viral factors to target many more cellular factors, including proteins which regulate epigenetic marks and splicing changes in the cell. These have the ability to exert a global change, which eventually culminates to uncontrolled proliferation and carcinogenesis.

The Runt‐related transcription factors (RUNX) belong to an ancient family of metazoan genes involved in developmental processes. Through multiple protein‐interacting partners, RUNX proteins have been implicated in diverse signaling pathways and cellular processes. The frequent inactivation of RUNX genes in cancer indicates crucial roles for RUNX in tumor suppression. This review discusses the abilities of RUNX proteins, in particular RUNX3, to integrate oncogenic signals or environmental cues and to initiate appropriate tumor suppressive responses.

Little is known about the mechanisms of gastric carcinogenesis, partly because it has been a challenge to identify characterize gastric stem cells. Runx genes regulate development and their products are transcription factors associated with cancer development. A Runx1 enhancer element, eR1, is a marker of hematopoietic stem cells. We studied expression from eR1 in the stomach and the roles of gastric stem cells in gastric carcinogenesis in transgenic mice. We used in situ hybridization and immunofluorescence analyses to study expression of Runx1 in gastric tissues from C57BL/6 (control) mice. We then created mice that expressed enhanced green fluorescent protein (EGFP) or CreERT2 under the control of eR1 (eR1-CreERT2;Rosa-Lox-Stop-Lox [LSL]-tdTomato, eR1-CreERT2;Rosa-LSL-EYFP mice). Gastric tissues were collected and lineage-tracing experiments were performed. Gastric organoids were cultured from eR1-CreERT2(5-2);Rosa-LSL-tdTomato mice and immunofluorescence analyses were performed. We investigated the effects of expressing oncogenic mutations in stem cells under control of eR1 using eR1-CreERT2;LSL-KrasG12D/+ mice; gastric tissues were collected and analyzed by histology and immunofluorescence. Most proliferation occurred in the isthmus; 86% of proliferating cells were RUNX1-positive and 76% were MUC5AC-positive. In eR1-EGFP mice, EGFP signals were detected mainly in the upper part of the gastric unit, and 83% of EGFP-positive cells were located in the isthmus/pit region. We found that eR1 marked undifferentiated stem cells in the isthmus and a smaller number of terminally differentiated chief cells at the base. eR1 also marked cells in the pyloric gland in the antrum. Lineage-tracing experiments demonstrated that stem cells in the isthmus and antrum continuously gave rise to mature cells to maintain the gastric unit. eR1-positive cells in the isthmus and pyloric gland generated organoid cultures in vitro. In eR1-CreERT2;LSL-Kras G12D/+ mice, MUC5AC-positive cells rapidly differentiated from stem cells in the isthmus, resulting in distinct metaplastic lesions similar to that observed in human gastric atrophy. Using lineage-tracing experiments in mice, we found that a Runx1 enhancer element, eR1, promotes its expression in the isthmus stem cells of stomach corpus as well as pyloric gland in the antrum. We were able to use eR1 to express oncogenic mutations in gastric stem cells, proving a new model for studies of gastric carcinogenesis.

The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFβ, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers.

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-β 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-β and Wnt pathways. Specifically, Runx3−/−p53−/− gastric epithelial cells were unexpectedly sensitized for TGF-β-induced 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.