| Despite a decrease in incidence in recent decades, gastric cancer remains one of the most lethal malignancies and ranks as the fifth most common cause of cancer related death worldwide. Although early stage diagnosis was attempted with endoscopic examination, about 65% of patients with gastric cancer still have regional or distant metastasis at the time of diagnosis. Therefore, the 5-year survival rate of gastric cancer patients with advanced stages still in a very low percentage ranges from 5% to 15%.Invasion, metastasis and relapse after surgery as well as multidrug resistance are all regarded as the contributing reasons for the poor outcome of gastric cancer patients. While, the mechanisms control gastric cancer aggressiveness are not well understood. Since Runt-related transcription factor 2(RUNX2) played an important role in bone-metastasis of tumors and was detected highly expressed in gastric cancer stem cells(GCSCs), this study aims to elucidate the contributing effect of RUNX2 to the tumorigenesis and invasion/metastasis in gastric cancer(GC) and to unveil the underlying mechanism. Immunohistochemistry of large number of gastric cancer samples showed that RUNX2 serves as an important prognositic indicator for gastric cancer patients. A series of in vitro and in orthotopic gastric cancer animal model experiments had been performed to evaluate the function of RUNX2 on aggressiveness and malignancy of gastric cancer. Results indicated that RUNX2 was an essential oncogenic participator in promoting proliferation and tumorigenesis of gastric cancer. Also, RUNX2 was found possessing the migration and invasion abilities of gastric cancer cells in vitro. In an orthotopic gastric cancer model in nude mice, RUNX2 promoted cancer cells local invasion and metastasis to nearby organ of liver. Bioinformatic analysis, Ch IP and luciferase reporter assay were performed to unveil the underlying molecular mechanisms. A binding site of RUNX2 was detected in the promoter region of CXCR4, implying a putative downstream target of RUNX2. CXCR4 knockdown or CXCR4 signal pathway specific inhibitor treatment attenuated RUNX2 mediated the tumorigenicity, invasiveness and liver metastasis of gastric cancer cells. These results demonstrated that RUNX2 servers as an aggressive factor of gastric cancer, and therefore RUNX2/CXCR4 axis could be a potential therapeutic target for gastric cancer patients.Main methods, results and conclusion as follow:1. RUNX2 expression in GCSCs and GC cells.(1) DNA microarray was performed to compare differences of gene expression between spheres and monolayer gastric cancer SGC7901 cell line. Overexpressed RUNX2 was found in sphere-cultured SGC7901 cells, which was cultured in a conditional culture medium and was demonstrated as the GCSCs by our previous work, for 53-fold higher than that in monolayer SGC7901 cells.(2) A bioinformatic analysis was made to evaluate the activation of RUNX2 associated genes based on the DNA microarray data. The Gene Set Enrichment Analysis(GSEA) indicated that RUNX2 gene was significantly activated in tumor sphere with a high enrichment score;(3) Meanwhile, protein level of RUNX2 was also detected with immunofluorescence confocal. Results demonstrated that sphere cultured gastric cancer cells showed an elevation of RUNX2 comparing with monolayer cultured gastric cancer cells;(4) m RNA and protein level of RUNX2 were detected in SGC7901, MGC803 cells and primary gastric cancer cell, XN0422. SGC7901 cell showed lower level of RUNX2 than MGC803 and XN0422 cells.2. RUNX2 expression correlated with clinicopathological features and predicted poor prognosis of patients with gastric cancer.(1) IHC examination in 305 clinical gastric cancer specimens revealed that RUNX2 protein expression is correlated with sorts of clincapathalogical features, such as TNM stage, differentiation status, lymphnode metastasis and depth of invaison. RUNX2 protein was dominantly localized in the nucleus of tumor cells despite a few samples showing both nucleus and cytoplasm location. Diversity of RUXN2 expressions were found in gastric cancer specimens per differentiation status of tumors(P=0.006). A total of 81 cases of patients showed High/Moderate differentiation status, among which 49 cases(49/81, 60.5%) of GC specimens were detected with RUNX2 high expression. Meanwhile, there are 171 cases(171/224, 76.3%) of patients showed RUNX2 high expression among the 224 cases of patients with Low/Poor differentiation status of gastric cancer. RUNX2 was positively correlated with the TNM stages of gastric cancer(P=0.000). In a total of 124 patients with TNM stage I and II gastric cancers, 49 cases(49/124, 39.5%) showed lower expression of RUNX2 and 75 cases(75/124, 60.5%) had higher expressed RUNX2. In contrast, 145 cases(145/181, 80.1%) were detected with RUNX2 High in a total 181 cases of stage III and IV GC patients. RUNX2 high expressing GC cases were often observed in lymph node metastasis(P=0.000) and deep space invasion(P=0.000). Among GC patients with lymph node metastasis, about 79.3% of patients(161/203) were RUNX2 high expression cases. In patients with serosa invasion, over 79.1% of patients(178/225) were detected with RUNX2 high expression. While, the expression of RUNX2 was not correlated with age(P=0.352) and gender(P=0.480); 2). RUNX2 protein was found highly expressed in cancerous tissues rather then in their peritumoral normal gastric tissues. Seventy two persant(220/305) GC tissues was detected a high level expression of RUNX2 as compared to 21.6%(66/305) in their adjacent gastric tissues(P=0.00);(3) Highly expressed RUNX2 in gastric cancers was significantly associated with increased mortality(P=0.00). Patients with RUNX2 low expression GC had a much longer lifetime after surgery, as compared with the patients with RUNX2 high expressing GCs;(4) COX’s proportion hazard regression analysis indicated that RUNX2 overexpression(HR 3.454, 95%CI 1.408-8.230, P=0.007), together with depth of invasion(HR 1.620, 95%CI 1.022-2.567, P=0.040) and lymphatic metastasis(HR 1.539, 95%CI 1.089-2.591, P=0.045), contributed to the poor prognosis of gastric cancer patients, indicating that RUNX2 level could be a prediction factor for prognosis of gastric cancer. RUNX2 expression was not correlated with T stage(HR 1.293, 95%CI 0.846-1.975, P=0.235) and differentiation status(HR 0.704, 95%CI 0.488-1.016, P=0.061).3. RUNX2 promotes tumorigenesis and invasion/metastasis in vivo and in vitro.(1) RUNX2 promotes tumorigenesis of GC cells in nude mice. In order to detect the xenografts formation ability, subcutaneously injection in nude mice was performed. Size and weight of xenograft tumors formed were carefully observed, recorded and calculated. Xenografts formed by SGC7901 cells with ectpoic expression of RUNX2 was much bigger in size and heavier in weight, as compared with the wildtype or mock SGC7901 cells;(2) RUNX2 stimulates invasion and metastasis of gastric cancer in vitro and in vivo. Cell scratching assay was performed to evaluate the migration ability of gastric cancer cells. Results demonstrated that migration ability of ectpoic RUNX2 expressed SGC7901 cells were dramatically enhanced compared to that of the wildtype or mock SGC7901 cells. While, MGC803 and XN0422 cells with higher RUNX2 level showed longer migrated distance and robust migration ability than MGC803-sh RUNX2 and XN0422-sh RUNX2 cells. Cell invasion assay was used to investigate the tumor invasion ability. Results indicated that overexpression of RUNX2 significantly enhanced the invasion and metastasis ability of GC cells. While, silencing of RUNX2 impaired this important function of cancer cells. An orthotopic gastric cancer animal model was established in order to evaluate the invasion and metastasis ability of gastric cancer cells in vivo. Results indicated that RUNX2 motivates the invasive potential of orthotopic tumors in the stamoch and promotes metastasis in nearby organ, such as liver.4. RUNX2 could directly transactivate CXCR4.(1) Among the invasion related genes, CXCR4 ranked the top score by transcription factor bioinformatic analysis(Score=11.462). CXCR4 showed the most significant changes between groups of MGC803-Mock vs MGC803-sh RUNX2 and XN0422-Mock vs XN0422-sh RUNX2 cells. Real-time PCR test was used to sort bioinformatically predicted downstream genes of RUNX2 from a series of aggression associated genes, such as MMPs, VEGFs, EMT associated genes and CXCR4. MMPs and VEGFs were reported to be the downstream target in many types of cancers, such as osteosarcoma, breast cancer, and prostate cancer. CXCR4 was the only commonly down-regulated gene among all the candidates in both MGC803 and XN0422 groups;(2) Ch IP analysis was performed to examine the interaction between RUNX2 and the promoter of CXCR4. A 277 bp fragment in promoter of CXCR4 was identified as RUNX2 binding region;(3) Bioinformatic analysis suggested a 15 bp putative RUNX2 binding site in this region. This binding site was confirmed by Luciferase reporter assay and EMSA when comparing wildtype with mutated promoter fragment of CXCR4;(4). Meanwhile, we also constructed several reporter vectors with fragment delete mutations. Luciferase reporter assay indicated that the first 50 bp of this 277 bp fragment might be the binding site, where the predicted 15 bp region was located right within this first 50 bp region. These data indicated that a 15 bp region was responsible for the binding of RUNX2 on the promoter region of CXCR4;(5). WB detection of RUNX2 and CXCR4 protein showed postive correlationship between RUNX2 and CXCR4 in GC specimens.5. CXCR4 was an important downstream target of RUNX2 in promoting gastric cancer malignancy.(1). Inhibition of CXCR4 signaling in RUNX2 elevated gastric cancer SGC7901-ex RUNX2 dramatically impaired the chemotaxis effect indicated by chemotaxis assay. Inhibition of CXCR4 signal pathway was made by lentivirus infection to stablely silence CXCR4 or adding a CXCR4 signaling specific inhibitor, AMD3100, to SGC7901 cells;(2). Cell invasion assay indicated impaired invasive ability of SGC7901 cells after inhibition of CXCR4 signaling.(3). Furthermore, in vivo experiments in orthotopic gastric cancer model in nude mice indicated that the invasiveness of orthotopic tumors in stamoch and disseminational metastasis in liver were impaired after CXCR4 signal pathway was interrupted in SGC7901 cells. These data indicated that RUNX2 acts as a nuclear transcription factor that transactivates CXCR4 involved in malignancy and aggressiveness in gastric cancer.In conclusion, RUNX2 plays an important role in promoting the invasion and metastasis of GC by transcriptionally up-regulating CXCR4. RUNX2 thus serves as a prognostic indicator and a potential therapeutic target for GC. |
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