The limited correlation between the apoptotic index and tumor volume may be due (at least in part) to the possibility that tumor growth in this model is influenced mainly by proliferative activity rather than apoptosis

The limited correlation between the apoptotic index and tumor volume may be due (at least in part) to the possibility that tumor growth in this model is influenced mainly by proliferative activity rather than apoptosis. Open in a separate window Figure 5 ZEB2 promotes tumor angiogenesis and CTC survival(A) SNU-398 stable cells (1 107 cells/mouse) were injected subcutaneously into the flanks of nude mice (= 8) as described in the Materials and Methods. between ZEB2 and Sp1. Clinical data showed that ZEB2 expression was positively associated with Sp1 expression, and that the expression of both of these factors had prognostic significance for predicting survival in cancer patients. This study suggests that invasion is usually linked to cancer cell survival and angiogenesis by ZEB2 during cancer progression, and increases our understanding of the pathways via which EMT-inducing transcription factors regulate the complex process of metastasis. 0.05. siSCR, scrambled siRNA. In addition, real-time qPCR analysis showed that VEGF was downregulated by knockdown of ZEB2 (Physique ?(Figure1D)1D) and upregulated by ZEB2 overexpression (see below). To explore whether suppression of ZEB2 reduces VEGF promoter activity, SNU-398 cells were transiently co-transfected with siRNA specific to ZEB2 and a reporter plasmid driven by the VEGF promoter (?2361/+298). Knockdown of ZEB2 significantly reduced VEGF promoter activity by 32% (Physique ?(Figure1E).1E). Survivin and cyclin D1 mRNA expression was also reduced by knockdown of ZEB2 (Physique ?(Figure1D1D). ZEB2 induces transcription of VEGF, cyclin D1, and survivin in an Sp1-dependent manner We then explored whether Sp1 is usually involved in ZEB2-mediated VEGF transcription. A reporter assay showed that ZEB2 significantly upregulated VEGF promoter (?2361/+298 and ?267/+50 regions) activity in SW480 (Physique ?(Figure2A)2A) and HEK293E (Supplementary Figure 1A) cells. Three Sp1-binding sites and two Egr-1-binding sites are present in the ?85/?50 region and are reported to be involved in VEGF transcription [17, 18]. Open in a separate window Physique 2 ZEB2 induces transcription of VEGF, cyclin D1, and survivin CD34 in an Sp1-dependent manner(A) SW480 cells were co-transfected with a ZEB2 expression vector and VEGF promoter (?2361/+298 and ?267/+50) reporter constructs for 48 h. Firefly luciferase activity representing VEGF promoter activity was measured after 48 h and normalized to Renilla luciferase activity to measure the transfection efficiency. (B) Mutation analysis of LY 255283 Sp1 sites and Egr-1 sites in the VEGF promoter (?85/+50). Reporter constructs made up of Sp1 site or Egr-1 site mutations were used in the reporter assay with SW480 cells. Values represent mean standard deviation. * 0.05. (C, E, F, G) SW480 cells were co-transfected with the ZEB2 expression vector and Sp1-specific siRNA for 48 h. (C) Real-time qPCR analysis to determine the effect of Sp1-specific siRNA on VEGF mRNA induction LY 255283 by ZEB2 in SW480 cells. (D) Reporter assay to determine the effect of mutant ZEB2 lacking the Smad-binding domain name on VEGF promoter activity. (E, F) Real-time qPCR analysis of the mRNA levels of cyclin D1 (E) and survivin (F) in SW480 cells. Values represent mean standard deviation. * 0.05 compared with empty vector + control siRNA; 0.05 compared with ZEB2 + control siRNA. (G) Transfected cells were lysed for immunoblot analysis. An anti-myc antibody was used to detect myc-tagged ZEB2. Densitometry quantification was performed around the immunoblots, using GAPDH as a loading control. We analyzed the functional involvement of the Sp1 sites in the ?85/?50 region by performing reporter assays using mutated VEGF promoter constructs. Mutation of the Sp1 sites resulted in a drastic decrease in ZEB2-induced activation of the VEGF promoter in SW480 (Physique ?(Figure2B)2B) and HEK293E (Supplementary Figure 1B) cells, indicating the functional significance of the proximal Sp1 sites for the effects of ZEB2. Of note, mutation of the Sp1 sites also dramatically decreased basal VEGF promoter activity, which is usually consistent with previous reports [17], suggesting the possible involvement of these sites in basal VEGF promoter activity. By contrast, mutation of the Egr-1 sites did not dramatically change ZEB2-induced VEGF promoter activity, although it partially reduced basal VEGF promoter activity (Physique ?(Physique2B2B and Supplementary Physique 1B). We also explored whether Sp1 is required for ZEB2-induced VEGF transcription. Real-time qPCR analysis showed that ZEB2-mediated transcription of VEGF was diminished in SW480 cells following knockdown of Sp1 by siRNA (Physique ?(Figure2C).2C). In addition, a reporter assay exhibited that mutant ZEB2 lacking the Smad-binding domain name (amino acid residues 437C487) activated VEGF promoter to a similar extent as wild-type ZEB2 in SW480 cells (Physique ?(Figure2D),2D), suggesting that ZEB2 upregulated VEGF expression in a Smad-independent, but Sp1-dependent, manner. We also explored the function of Sp1 in ZEB2-mediated cyclin D1 and survivin expression. Real-time qPCR analysis showed that ZEB2-mediated transcription of cyclin D1 (Physique ?(Figure2E)2E) and survivin (Figure ?(Physique2F)2F) was reduced in SW480 cells following knockdown of Sp1 by siRNA. Immunoblot analysis showed that Sp1 was required for ZEB2-induced survivin and cyclin D1 expression (Physique ?(Figure2G).2G). Together, these results suggest that ZEB2 induces VEGF, cyclin D1, and survivin in an Sp1-dependent LY 255283 manner. ZEB2 promotes HUVEC proliferation through upregulation of VEGF VEGF is usually a well-known.