In this study, we focused on the critical role of the histone H3K4 demethylase RBP2 in angiogenesis of gastric cancer, especially the direct regulation of VEGF by RBP2. First, we determined the association of RBP2 expression, VEGF expression and MVD status in human gastric cancer tissue species. Second, we detected the direct regulation of VEGF by RBP2 both in human gastric cancer cell lines and RBP2-targeted mutant mice, which showed that H3K4 demethylation by RBP2 expression was important for VEGF expression and MVD status. RBP2 bound to the promoter of VEGF directly and activated the promoter of VEGF for regulation at the transcriptional level. Third, VEGF expression induced by RBP2 overexpression activated tumorigenesis and angiogenic potential in human gastric cancer cells and animal models, whereas silencing of RBP2 expression had the reverse effect. Therefore, abnormal RBP2 expression and VEGF activation might explain the poor prognosis with gastric cancer and contribute directly to gastric tumor angiogenesis and aggressive gastric cancer biology.
Angiogenesis is a critical process in the invasion, growth and metastasis of most solid tumors . Angiogenesis is complex and involves a large number of molecules. VEGF, fibroblast growth factor, angiopoietin, Notch, transforming growth factor β, Hedgehog and WNT signaling cascades orchestrate angiogenesis through the direct or indirect regulation of quiescence, migration and the proliferation of endothelial cells [19–23]. VEGF has critical roles in tumor angiogenesis . Small-molecule compounds and human/humanized monoclonal antibodies interrupting VEGF signaling have been developed as anti-angiogenic therapeutics for cancer [25, 26]. Epigenetic regulation in angiogenesis is a new pathway in carcinogenesis and metastasis. Lysine acetylation and cytosine methylation are important transcriptional regulators of angiogenic genes in endothelial cells. Lysine acetylation and cytosine methylation inhibitors idiosyncratically tune the transcriptome and affect expression of key modulators of angiogenesis such as VEGF and endothelial nitric oxide synthase .
We have found that RBP2, a new histone H3K4 demethylase, is involved in carcinogenesis by escaping cell senescene and inhibiting CDKIs [15, 16]. In this research, we first found RBP2 activation associated with VEGF expression and tumorgenesis and angiogenesis in human gastric cancer: RBP2 expression was positively associated with VEGF mRNA and protein expression and with MVD as identified by CD31 and CD34 antibody staining and cell proliferation by Ki67 overexpression, which suggests a link between RBP2 activation and VEGF overexpression. RBP2 overexpression significantly affected VEGF expression in gastric cancer cells via demethylation of H3K4. In RBP2-targeted mutant mice with H3K4 demethylation of RBP2 silenced and H3K4 trimethylation enhanced, the expression of VEGF and the aniogenic phenotype were also inhibited. As well, RBP2 expression was involved in VEGF promoter activity in gastric cancer cells. Specifically, we identified one potential RBP2 binding site on the VEGF promoter. Mutation of the site profoundly attenuated but did not completely eliminate RBP2-mediated transactivation of the VEGF promoter. Therefore, we confirmed by ChIP assay that this binding site was functional, which showed active recruitment of RBP2 to the binding site.
H3-K4 methylation is associated with transcription activation [28–30]. As a histone demethylase specific for di- and tri-methylated H3-K4, RBP2 acts as a transcriptional repressor by inhibiting H3-K4 methylation at its target promoters [31, 32]. However, we found that VEGF was a direct target of RBP2. Not as the suppression of CDKIs promoters by RBP2 in gastric cancer, RBP2 induced the activation of VEGF promoter by directly binding to the CCGCCC DNA motif . Another important H3K4 demethylase, LSD1, which is overexpressed in numerous cancers, was found involved in inducing VEGF expression in prostate cancer . LSD1 was also identified to act through its demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with the CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation . In addition, RBP2 is present in a number of chromatin-remodeling complexes [36, 37]. Polycomb-repressive complex 2 recruits RBP2 to its target genes via physical interaction . This situation may occur with the promoter of the VEGF gene. However, we need more evidence to determine whether RBP2 affects the VEGF promoter methylation status, which is involved in the regulation of VEGF expression  in human gastric cancer cells.
We detected the critical role of RBP2 in angiogenesis by the biological effect in human cancer cells and animal models. With RBP2 siRNA, RBP2 expression was silenced and VEGF expression inhibited. The clone formation of human gastric cancer cells was also suppressed in this process, which was partially reversed by the overexpression of VEGF in RBP2 siRNA-transfected cells. We found RBP2 involved in gastric carcinogenesis by the regulation of VEGF. In nude mice, tumors from transfected gastric cancer cells stably expressing RBP2 shRNA were smaller, with lower VEGF expression, and less MVD and cell proliferation than control cells. Recent genome-wide analyses of mouse embryonic stem cells and human leukemic cell lines revealed hundreds of RBP2 target genes and many of them implicated in development, proliferation and differentiation controls [32, 38, 39]. RBP2 was identified as a key molecule in drug tolerance of cancer cells and maintaining cancer stem cells [40, 41]. Our new findings that RBP2 is critical in constitutive and inducible VEGF expression might suggest its clinical implication in gastric tumor angiogenesis and progression.
Surgery plays a central role in the overall management of operable gastric cancer. Histone deacetylase inhibitors, based on epigenetic development, are being used clinically , and histone demethylase inhibitors are being addressed in clinical trials . Our study identified a novel molecular mechanism for RBP2 and provides better understanding of the molecular basis for angiogenetic signaling pathways, which might aid in the design of effective therapeutic modalities to control gastric cancer growth and metastasis.