Transcription factor RUNX2 up-regulates chemokine receptor CXCR4 to promote invasive and metastatic potentials of human gastric cancer

Abstract

Runt-related transcription factor 2 (RUNX2) is a regulator of embryogenesis and development, but has also been implicated in the progression of certain human cancer. This study aimed to elucidate the role of RUNX2 in the invasive and metastatic potentials of human gastric cancer (GC) and the underlying mechanisms. We found that the levels of RUNX2 expression in gastric cancer tissues were correlated with the differentiation degrees, invasion depth and lymph node metastasis. COX regression analysis indicated that RUNX2 was an independent prognostic indicator for GC patients. RUNX2 significantly increased the migration and invasion ability of GC cells in vitro and enhanced the invasion and metastatic potential of GC cells in an orthotopic GC model of nude mice. Mechanistically, RUNX2 directly bound to the promoter region of the gene coding for the chemokine receptor CXCR4 to enhance its transcription. CXCR4 knockdown or treatment with AMD3100, a CXCR4 inhibitor, attenuated RUNX2-promoted invasion and metastasis. These results demonstrate that RUNX2 promotes the invasion and metastasis of human GC by transcriptionally up-regulating the chemokine receptor CXCR4. Therefore, the RUNX2-CXCR4 axis is a potential therapeutic target for GC.

Keywords: CXCR4; RUNX2; gastric cancer; invasion; metastasis.

Figure 1. The expression of RUNX2 in human GC specimens is correlated with the outcome of GC patients

(A) RUNX2 is not or only weakly expressed in normal gastric tissue as detected by IHC staining. (B and C) RUNX2 expression in GC tissues is correlated with different stages of differentiation and depth of tumor invasion. Arrows indicate RUNX2 positive GC cells. (D) Positive staining of RUNX2 in GC metastatic foci of lymph node. (E) Kaplan-Meier Overall survival curves indicate that patients with RUNX2^High staining have shorter life time after surgery than patients with RUNX2^Low tumors (RUNX2^High, n = 220 and RUNX2^Low, n = 85). Scale bar = 50 μm.

Figure 2. RUNX2 promotes the migration and invasion of GC cells in vitro

(A) The expression level of RUNX2 in SGC7901 cells is lower than that in MGC803 and XN0422 cells at mRNA and protein levels detected by qPCR and WB. (B) The cell scratching assay shows that RUNX2-overexpressing SGC7901 cells migrate at longer distance than control cells. (C) After silencing RUNX2, the migrated distance of MGC803 and XN0422 cells was significantly shortened as compared with mock cells. (D) RUNX2-overexpressing SGC7901 cells showed higher invasiveness than control SGC7901 cells as analyzed in invasion assay. (E) RUNX2-knockdown in both MGC803 and XN0422 cells impaired their invasion ability. **P < 0.01, Student's t test.

Figure 3. RUNX2 enhances GC cell invasion and metastasis in orthotopic transplantation mouse model

(A) GC cells genetically engineered to overexpress or silence RUNX2 were implanted into the stomach serosa of nude mice (n = 5 for each group). The invasion and metastasis of transplanted tumors were examined after eight weeks. Representative images of orthotopic xenograft tumor sections show enhanced invasion abilitiy of tumors formed by RUNX2-overexpressing SGC7901 cells, as compared to SGC7901-Control cells. Black dotted line indicates the submucosa of the stomach. (B) Representative images show that RUNX2-knockdown in MGC803 and XN0422 cells impairs the invasiveness of xenografts. Black arrow shows tumor cell invasion into the mucosa. (C) Representative images showing liver metastasis of tumors formed by RUNX2-overexpressing SGC7901 cells as compared to SGC7901-Control cells. Empty triangle shows liver metastatic foci. (D) Representative images show that RUNX2-knockdown in MGC803 and XN0422 cells impairs their metastatic potential. Empty triangle shows liver metastatic foci. (E) Overall survival curves show that mice implanted with RUNX2-overexpressing SGC7901 cells have a shorter lifespan than mice implanted with control SGC7901 cells (n = 5 for each group). (F) Mice implanted with RUNX2-knockdown MGC803 cells and XN0422 primary cells show better outcome than their counterparts (n = 5 for each group).

Figure 4. CXCR4 is a candidate RUNX2-targeting gene

(A) Among invasion-related candidate RUNX2-targeting genes predicted by bioinformatic analysis, CXCR4 gene was markedly reduced by RUNX2 knockdown in both MGC803 cells (left) and XN0422 cells (right) as detected by qPCR. The expression level of genes was presented as relative fold increased normalized with GAPDH. *P < 0.05, **P < 0.01, Student's t test. (B) Western Blot shows increased CXCR4 in RUNX2-overexpressing SGC7901 cells but decreased CXCR4 in RUNX2-silenced MGC803 and XN0422 cells.

Figure 5. RUNX2 binds to with CXCR4 promoter

(A) ChIP shows a candidate binding region (3F to 4R, 277 bp) of RUNX2 in CXCR4 promoter. (B) Luciferase activity assay shows that the predicted site (−1046 to −1032), but not mutants, is bound by RUNX2 in CXCR4 promoter. (C) Direct binding of RUNX2 to CXCR4 promoter region determined by EMSA. Lane 1 and 4 for nuclear extract binding reaction; lane 2 for negative control; lane 3 for competition test; lane 5 to 8 for supershift. **P < 0.01, Student's t test.

Figure 6. CXCR4 mediates RUNX2-promoted invasiveness and metastasis of GC cells

(A) Chemotaxis of GC cells in response to the CXCR4 ligand SDF-1a (CXCL12, 10 nM/L). (B) Silencing CXCR4 or treatment with AMD3100 (50 ng/mL) attenuates the invasion ability of RUNX2-overexpressing SGC7901 cells. (C) Representative images of xenograft tumor section show that tumors formed by SGC7901-exRUNX2-Mock cells, but not SGC7901-exRUNX2-shCXCR4 cells, invade into the submucosa (black dotted line). (D) Representative images show that AMD3100 treatment reduced the invasion ability of RUNX2-overexpressing SGC7901 cells (black dotted line). Mice (n = 5 for each group) were intra-peritoneally injected with AMD3100 (7.5 mg/kg) every 3 days and the tumors were harvested after GC cell orthotopic implantation for 8 weeks. Scale bar = 50 μm. (E and F) Overall survival curves show that mice bearing tumors formed by SGC7901-exRUNX2 cells with CXCR4 knockdown or AMD3100 treatment have a long survival than mice implanted with mock or PBS treated SGC7901-exRUNX2 cells (n = 5 for each group). **P < 0.01, Student's t test.