Dataset Information

ZEB1 Upregulates VEGF Expression and Stimulates Angiogenesis in Breast Cancer.

ABSTRACT: Although zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a key factor in the regulation of breast cancer differentiation and metastasis, its potential role in modulating tumor angiogenesis has not been fully examined. Here, we present the novel finding that conditioned medium derived from ZEB1-expressing MDA-MB-231 cells significantly increased the capillary tube formation of human umbilical vein endothelial cells (HUVECs), whereas ZEB1 knockdown by RNA interference had the opposite effect. ZEB1 caused marked upregulation of the expression of vascular endothelial growth factor A (VEGFA) at both mRNA and protein levels. Pre-incubation of HUVECs with anti-VEGFA neutralized antibody attenuated ZEB1-mediated tube formation of HUVECs. In breast cancer tissues, expression of ZEB1 was positively correlated with those of VEGFA and CD31. At the molecular level, ZEB1 activated VEGFA transcription by increasing SP1 recruitment to its promoter, which was mediated via the activation of PI3K and p38 pathways. Using a nude mouse xenograft model, we demonstrated that elevated expression of ZEB1 promotes in vivo tumorigenesis and angiogenesis in breast cancer. Collectively, we found that ZEB1-expressing breast cancer cells increase VEGFA production and thus stimulate tumor growth and angiogenesis via a paracrine mechanism.

SUBMITTER: Liu L

PROVIDER: S-EPMC4755590 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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ZEB1 Upregulates VEGF Expression and Stimulates Angiogenesis in Breast Cancer.

Liu Lingjia L Tong Qi Q Liu Shuo S Cui Jianlin J Zhang Quansheng Q Sun Wei W Yang Shuang S

PloS one 20160216 2

Although zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a key factor in the regulation of breast cancer differentiation and metastasis, its potential role in modulating tumor angiogenesis has not been fully examined. Here, we present the novel finding that conditioned medium derived from ZEB1-expressing MDA-MB-231 cells significantly increased the capillary tube formation of human umbilical vein endothelial cells (HUVECs), whereas ZEB1 knockdown by RNA interference had the op ...[more]

PMID: 26882471

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Project description:BackgroundSpalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown.MethodsAnalyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression.ResultsSALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM.ConclusionThese findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway.

Project description:Cancer-associated fibroblasts (CAFs), a predominant component of the tumor microenvironment, contribute to aggressive angiogenesis progression. In clinical practice, traditional anti-angiogenic therapy, mainly anti-VEGF, provides extremely limited beneficial effects to breast cancer. Here, we reveal that FOS-like 2 (FOSL2), a transcription factor in breast CAFs, plays a critical role in VEGF-independent angiogenesis in stromal fibroblasts. Methods: FOSL2 and Wnt5a expression was assessed by qRT-PCR, western blotting and immunohistochemistry in primary and immortalized CAFs and clinical samples. FOSL2- or Wnt5a-silenced CAFs and FOSL2-overexpressing NFs were established to explore their proangiogenic effects. Invasion, tubule formation, three-dimensional sprouting assays, and orthotopic xenografts were conducted as angiogenesis experiments. FZD5/NF-κB/ERK signaling activation was evaluated by western blotting after blocking VEGF/VEGFR with an anti-VEGF antibody and axitinib. Dual luciferase reporter assays and chromatin immunoprecipitation were performed to test the role of FOSL2 in regulating Wnt5a expression, and Wnt5a in the serum of the patients was measured to assess its clinical diagnostic value for breast cancer patients. Results: Enhanced FOSL2 in breast CAFs was significantly associated with angiogenesis and clinical progression in patients. The supernatant from CAFs highly expressing FOSL2 strongly promoted tube formation and sprouting of human umbilical vein endothelial cells (HUVECs) in a VEGF-independent manner and angiogenesis as well as tumor growth in vivo. Mechanistically, the enhanced FOSL2 in CAFs was regulated by estrogen/cAMP/PKA signaling. Wnt5a, a direct target of FOSL2, specifically activated FZD5/NF-κB/ERK signaling in HUVECs to promote VEGF-independent angiogenesis. In addition, a high level of Wnt5a was commonly detected in the serum of breast cancer patients and closely correlated with microvessel density in breast tumor tissues, suggesting a promising clinical value of Wnt5a for breast cancer diagnostics. Conclusion: FOSL2/Wnt5a signaling plays an essential role in breast cancer angiogenesis in a VEGF-independent manner, and targeting the FOSL2/Wnt5a signaling axis in CAFs may offer a potential option for antiangiogenesis therapy.

Dataset Information

ZEB1 Upregulates VEGF Expression and Stimulates Angiogenesis in Breast Cancer.

Publications

ZEB1 Upregulates VEGF Expression and Stimulates Angiogenesis in Breast Cancer.

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