RUNX3 drives metastasis in gastric cancer cells. A, Subcutaneous xenograft tumors were obtained by inoculation of 1 × 106 HGC-27 control and RUNX3 KO cells, respectively. Typical images are shown (n = 5). B, The weight of tumors was quantified; ****, P < 0.0001 by a two-tailed Student t test. C, Liver metastasis model by splenic inoculation of 1 × 106 HGC-27 cells. Representative images of tumors in liver metastasis in control, RUNX3 KO, and RUNX3 KO rescued with reintroduction of RUNX3 (RUNX3 OE) cells are shown (n = 5, respectively). D, Percentage of the metastatic tumor area in the liver tissue was measured using Image J and graphed (mean + SD); **, P < 0.01; *, P < 0.05 by a two-tailed Student t test. E, Representative images of tumor formation in spleen by inoculation of 1 × 106 HGC-27 cells in control and RUNX3 KO are shown (n = 5, respectively). F, The weight of tumors in spleen was quantified. G, Kaplan–Meier plots in liver metastasis models present overall survival for mice after inoculation of control and RUNX3 KO of HGC-27 cells; *, P < 0.05 by a two-tailed Student t test. H, Orthotopic transplantation model of 1 × 106 HGC-27 cells. Representative images of tumors in stomach (1 in blue, circled by dot line), peritoneal invasion (2, circled by dot line), and liver metastasis (3, indicated by an arrow head) in control, and also tumors in stomach (circled by dot line) in RUNX3 KO cells are shown (n = 5).
Funding
NUS | Cancer Science Institute of Singapore, National University of Singapore (CSI)
MOH | National Medical Research Council (NMRC)
Japan Agency for Medical Research and Development (AMED)
Chiba University (千葉大学)
ARTICLE ABSTRACT
Gastric cancer metastasis is a major cause of mortality worldwide. Inhibition of RUNX3 in gastric cancer cell lines reduced migration, invasion, and anchorage-independent growth in vitro. Following splenic inoculation, CRISPR-mediated RUNX3-knockout HGC-27 cells show suppression of xenograft growth and liver metastasis. We interrogated the potential of RUNX3 as a metastasis driver in gastric cancer by profiling its target genes. Transcriptomic analysis revealed strong involvement of RUNX3 in the regulation of multiple developmental pathways, consistent with the notion that Runt domain transcription factor (RUNX) family genes are master regulators of development. RUNX3 promoted “cell migration” and “extracellular matrix” programs, which are necessary for metastasis. Of note, we found pro-metastatic genes WNT5A, CD44, and VIM among the top differentially expressed genes in RUNX3 knockout versus control cells. Chromatin immunoprecipitation sequencing and HiChIP analyses revealed that RUNX3 bound to the enhancers and promoters of these genes, suggesting that they are under direct transcriptional control by RUNX3. We show that RUNX3 promoted metastasis in part through its upregulation of WNT5A to promote migration, invasion, and anchorage-independent growth in various malignancies. Our study therefore reveals the RUNX3-WNT5A axis as a key targetable mechanism for gastric cancer metastasis.
Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.
