Vascular Endothelial Growth Factor Transcriptional Activation Research Articles

Bach1-Induced Suppression of Angiogenesis Is Dependent on the BTB Domain

The transcription factor BTB and CNC homology 1 (Bach1) impairs angiogenesis after ischemic injury by suppressing Wnt/β-catenin signaling; however, the specific domains responsible for the antiangiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients, and is the early induction gene after ischemic inuced injury. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-ΔBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) were significantly lower in the AdBach1 group, but not in AdBach1-ΔBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-ΔBTB, in human umbilical-vein endothelial cells (HUVECs) was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF transcriptional activity and mRNA expression in HUVECs. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81-89 of the Bach1 BTB domain and the N-terminal CTNNB1 domain of TCF4. Bach1, but not Bach1-ΔBTB, also copreciptated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy. Funding Statement: This work was supported by the Great Program (91639103 to D. Meng), and General Programs (81670450 and 81873469 to D. Meng, 81570081 and 81770083 to S. Li, 81800234 to L. Jiang, 81572713 to X. Zhi, 81873536 to X. Wang) of the National Natural Science Foundation of China, the National Key Research and Development Program of China (2018YFC1313600 to S. Li). Declaration of Interests: The authors state: None. Ethics Approval Statement: HUVECs were isolated (12) from the fresh umbilical cord of normal parturients with informed consent. The procedure was approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College. All experimental protocols were approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23).

Inhibition of tumor growth and angiogenesis of tamoxifen-resistant breast cancer cells by ruxolitinib, a selective JAK2 inhibitor.

Tamoxifen (TAM) is the most widely used treatment for estrogen receptor-positive breast cancer patients. Unfortunately, the majority of these patients exhibit TAM resistance following treatment. We previously reported that proliferation and migration were greater in TAM-resistant MCF-7 (TAMR-MCF-7) cells than in parental MCF-7 cells. Janus kinases (JAKs) are cytosolic tyrosine kinases that transduce signals from plasma membrane cytokines and growth factor receptors. JAK2 selectively phosphorylates signal transducer and activator of transcription (STAT)-3, and the JAK2-STAT3 signaling pathway is known as a crucial signaling pathway for the regulation of cancer progression and metastasis. In the present study, basal phosphorylation of STAT3 was revealed to be greater in TAMR-MCF-7 cells than in control MCF-7 cells. Ruxolitinib, a potent JAK2 inhibitor, was demonstrated to attenuate STAT3 phosphorylation and the proliferation of TAMR-MCF-7 cells. Ruxolitinib also suppressed the enhanced cell migration of TAMR-MCF-7 cells through the inhibition of epithelial mesenchymal transition. Vascular endothelial growth factor (VEGF), a representative target gene of the JAK2-STAT3 pathway, functions as a key regulator of invasion and angiogenesis. Ruxolitinib significantly inhibited VEGF mRNA expression and transcriptional activity. The present study also performed a chick embryo chorioallantoic membrane assay to assess tumor growth and angiogenesis in TAMR-MCF-7 cells. Ruxolitinib reduced tumor weight and the number of blood vessels produced by TAMR-MCF-7 cells in a concentration-dependent manner. These results indicated that JAK2 could be a new therapeutic target for TAM-resistant breast cancer.

Insulin-like growth factor-I induces chemoresistence to docetaxel by inhibiting miR-143 in human prostate cancer.

Elevated levels of insulin-like growth factor-I (IGF-I) are associated with carcinogenesis and cancer progression. However, the molecular mechanisms by which IGF-I promotes prostate cancer development remain to be elucidated. Docetaxel chemotherapy is an important therapeutic strategy in many types of human cancers including prostate cancer. In this study, we showed that IGF-I rendered PC-3 and DU145 cells more resistant to docetaxel treatment. IGF-I treatment decreased miR-143 expression, but increased the expression levels of IGF-I receptor (IGF-IR) and insulin receptor substrate 1 (IRS1), direct targets of miR-143. Overexpression of miR-143 abolished IGF-I-induced chemoresistance to docetaxel treatment, decreased expression levels of IGF-I, IRS1, and vascular endothelial growth factor (VEGF) in prostate cancer cell lines. Furthermore, docetaxel treatment significantly inhibited VEGF transcriptional activation, whereas IGF-I treatment induced VEGF transcriptional activation in a dose-dependent manner. Forced expression of IGF-IR and IRS1 cDNAs without the 3’ UTR regions restored miR-143-inhibited VEGF transcriptional activation. Finally, miR-143 inhibited tumor growth and made cells more sensitive to docetaxel treatment for decreasing tumor growth in vivo. Taken together, our data demonstrates that IGF-I induces docetaxel resistance and upregulates IGF-IR and IRS1 expression through miR-143 downregulation, whereas miR-143 acts as a tumor suppressor by targeting its targets IGF-IR and IRS1.

Brain-derived neurotrophic factor regulates cell motility in human colon cancer.

Brain-derived neurotrophic factor (BDNF) is a potent neurotrophic factor that has been shown to affect cancer cell metastasis and migration. In the present study, we investigated the mechanisms of BDNF-induced cell migration in colon cancer cells. The migratory activities of two colon cancer cell lines, HCT116 and SW480, were found to be increased in the presence of human BDNF. Heme oxygenase-1 (HO)-1 is known to be involved in the development and progression of tumors. However, the molecular mechanisms that underlie HO-1 in the regulation of colon cancer cell migration remain unclear. Expression of HO-1 protein and mRNA increased in response to BDNF stimulation. The BDNF-induced increase in cell migration was antagonized by a HO-1 inhibitor and HO-1 siRNA. Furthermore, the expression of vascular endothelial growth factor (VEGF) also increased in response to BDNF stimulation, as did VEGF mRNA expression and transcriptional activity. The increase in BDNF-induced cancer cell migration was antagonized by a VEGF-neutralizing antibody. Moreover, transfection with HO-1 siRNA effectively reduced the increased VEGF expression induced by BDNF. The BDNF-induced cell migration was regulated by the ERK, p38, and Akt signaling pathways. Furthermore, BDNF-increased HO-1 and VEGF promoter transcriptional activity were inhibited by ERK, p38, and AKT pharmacological inhibitors and dominant-negative mutants in colon cancer cells. These results indicate that BDNF increases the migration of colon cancer cells by regulating VEGF/HO-1 activation through the ERK, p38, and PI3K/Akt signaling pathways. The results of this study may provide a relevant contribution to our understanding of the molecular mechanisms by which BDNF promotes colon cancer cell motility.

DNA-PKcs deficiency inhibits glioblastoma cell-derived angiogenesis after ionizing radiation.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a critical role in non-homologous end-joining repair of DNA double-strand breaks (DSB) induced by ionizing radiation (IR). Little is known, however, regarding the relationship between DNA-PKcs and IR-induced angiogenesis; thus, in this study we aimed to further elucidate this relationship. Our findings revealed that lack of DNA-PKcs expression or activity sensitized glioma cells to radiation due to the defective DNA DSB repairs and inhibition of phosphorylated Akt(Ser473) . Moreover, DNA-PKcs deficiency apparently mitigated IR-induced migration, invasion and tube formation of human microvascular endothelial cell (HMEC-1) in conditioned media derived from irradiated DNA-PKcs mutant M059J glioma cells or M059K glioma cells that have inhibited DNA-PKcs kinase activity due to the specific inhibitor NU7026 or siRNA knockdown. Moreover, IR-elevated vascular endothelial growth factor (VEGF) secretion was abrogated by DNA-PKcs suppression. Supplemental VEGF antibody to irradiated-conditioned media was negated enhanced cell motility with a concomitant decrease in phosphorylation of the FAK(Try925) and Src(Try416) . Furthermore, DNA-PKcs suppression was markedly abrogated in IR-induced transcription factor hypoxia inducible factor-1α (HIF-1α) accumulation, which is related to activation of VEGF transcription. These findings, taken together, demonstrate that depletion of DNA-PKcs in glioblastoma cells at least partly suppressed IR-inflicted migration, invasion, and tube formation of HMEC-1 cells, which may be associated with the reduced HIF-1α level and VEGF secretion. Inhibition of DNA-PKcs may be a promising therapeutic approach to enhance radio-therapeutic efficacy for glioblastoma by hindering its angiogenesis.

GDNF increases cell motility in human colon cancer through VEGF–VEGFR1 interaction

Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor, has been shown to affect cancer cell metastasis and invasion. However, the molecular mechanisms underlying GDNF-induced colon cancer cell migration remain unclear. GDNF is found to be positively correlated with malignancy in human colon cancer patients. The migratory activities of two human colon cancer cell lines, HCT116 and SW480, were found to be enhanced in the presence of human GDNF. The expression of vascular endothelial growth factor (VEGF) was also increased in response to GDNF stimulation, along with VEGF mRNA expression and transcriptional activity. The enhancement of GDNF-induced cancer cell migration was antagonized by a VEGF-neutralizing antibody. Our results also showed that the expression of VEGF receptor 1 (VEGFR1) was increased in response to GDNF stimulation, whereas GDNF-induced cancer cell migration was reduced by a VEGFR inhibitor. The GDNF-induced VEGF expression was regulated by the p38 and PI3K/Akt signaling pathways. Treatment with GDNF increased nuclear hypoxia-inducible factor 1 α (HIF1α) accumulation and its transcriptional activity in a time-dependent manner. Moreover, GDNF increased hypoxia responsive element (HRE)-containing VEGF promoter transcriptional activity but not that of the HRE-deletion VEGF promoter construct. Inhibition of HIF1α by a pharmacological inhibitor or dominant-negative mutant reduced the GDNF-induced migratory activity in human colon cancer cells. These results indicate that GDNF enhances the migration of colon cancer cells by increasing VEGF-VEGFR interaction, which is mainly regulated by the p38, PI3K/Akt, and HIF1α signaling pathways.

Abstract 599: Regulation of hypoxia-inducible factor stability by peptidyl prolyl isomerase Pin1

Abstract Pin1 is an isomerase that binds to phosphorylated Ser/Thr-Pro motifs and regulates a subset of phosphoproteins through isomerization of the phosphorylated bonds. Pin1 has been reported to play an important role in differentiation, cell proliferation and immune responses. Interestingly, Pin1 is overexpressed in many types of cancer tissues including breast, prostate, lung and colon. However, molecular mechanism underlying its oncogenic functions has not been fully elucidated. Here, we report that Pin1 directly or indirectly interacts with hypoxia-inducible factor (HIF) -1α and -2α in HCT116 cells. Pin1 can bind both HIF-1α and -2α in vitro. We also found that Pin1 interacted more strongly with HIF-2α than with HIF-1α at the endogenous level. Pin1 binding may cause stabilization of HIF-1α and HIF-2α protein, given that their levels were significantly increased under hypoxic condition. Pin1 knockdown decreased the expression of HIF-1α and HIF-2α proteins and vascular endothelial growth factor (VEGF) mRNA under hypoxic conditions. On the other hand, overexpression of Pin1 resulted in an increase in HIF-1α protein levels in hypoxic cells, and enhanced hypoxia-induced VEGF transcriptional activation. These results suggest that Pin1 interplays with HIF-1α and -2α, and is a potential chemopreventive and therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 599. doi:1538-7445.AM2012-599

Myc‐associated zinc finger protein (MAZ) is regulated by miR‐125b and mediates VEGF‐induced angiogenesis in glioblastoma

In patients with glioblastomas, vascular endothelial growth factor (VEGF) is a key mediator of tumor-associated angiogenesis. Glioblastomas are notorious for their capacity to induce neovascularization, driving continued tumor growth. Here we report that miR-125b is down-regulated in glioblastoma-associated endothelial cells, resulting in increased expression of its target, myc-associated zinc finger protein (MAZ), a transcription factor that regulates VEGF. The down-regulation of miR-125b was also observed on exposure of endothelial cells to glioblastoma-conditioned medium or VEGF, resulting in increased MAZ expression. Further analysis revealed that inhibition of MAZ accumulation by miR-125b, or by MAZ-specific shRNAs, attenuated primary human brain endothelial cell migration and tubule formation in vitro, phenomena considered to mimick angiogenic processes in vitro. Moreover, MAZ expression was elevated in brain blood vessels of glioblastoma patients. Altogether these results demonstrate a functional feed-forward loop in glioblastoma-related angiogenesis, in which VEGF inhibits the expression of miR-125b, resulting in increased expression of MAZ, which in its turn causes transcriptional activation of VEGF. This loop is functionally impeded by the VEGF receptor inhibitor vandetanib, and our results may contribute to the further development of inhibitors of tumor-angiogenesis.

Acacetin inhibits VEGF expression, tumor angiogenesis and growth through AKT/HIF-1α pathway

Acacetin (5,7-dihydroxy-4′-methoxyflavone) is a flavone compound, some of which have anti-cancerous effects. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and tumor growth. In this study, we found that acacetin decreased the steady level of VEGF mRNA level and inhibited VEGF transcriptional activation. To further determine the potential mechanism of acacetin in inhibiting VEGF expression, we showed that acacetin inhibited HIF-1α expression and AKT activation. Over-expression of HIF-1α or AKT restored acacetin-decreasing VEGF transcriptional activation, indicating that AKT and HIF-1 are the essential downstream targets of acacetin for inhibiting VEGF expression in the cells. Moreover, acacetin significantly inhibited ovarian cancer cell-induced angiogenesis and tumor growth in vivo through inhibiting HIF-1α and VEGF expression. Acacetin did not change HIF-1α mRNA level, but inhibited HIF-1α protein level through increasing its degradation and decreasing its stability. These results indicate that acacetin may be a useful natural compound for ovarian cancer prevention and treatment.

Calcineurin Inhibitor-Induced and Ras-Mediated Overexpression of VEGF in Renal Cancer Cells Involves mTOR through the Regulation of PRAS40

Malignancy is a major problem in patients treated with immunosuppressive agents. We have demonstrated that treatment with calcineurin inhibitors (CNIs) can induce the activation of proto-oncogenic Ras, and may promote a rapid progression of human renal cancer through the overexpression of vascular endothelial growth factor (VEGF). Interestingly, we found that CNI-induced VEGF overexpression and cancer cell proliferation was inhibited by rapamycin treatment, indicating potential involvement of the mammalian target of rapamycin (mTOR) pathway in this tumorigenic process. Here, we examined the role of mTOR pathway in mediating CNI- and Ras-induced overexpression of VEGF in human renal cancer cells (786-0 and Caki-1). We found that the knockdown of raptor (using siRNA) significantly decreased CNI-induced VEGF promoter activity as observed by promoter-luciferase assay, suggesting the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. It is known that mTOR becomes activated following phosphorylation of its negative regulator PRAS40, which is a part of mTORC1. We observed that CNI treatment and activation of H-Ras (through transfection of an active H-Ras plasmid) markedly increased the phosphorylation of PRAS40, and the transfection of cells using a dominant-negative plasmid of Ras, significantly decreased PRAS40 phosphorylation. Protein kinase C (PKC)-ζ and PKC-δ, which are critical intermediary signaling molecules for CNI-induced tumorigenic pathway, formed complex with PRAS40; and we found that the CNI treatment increased the complex formation between PRAS40 and PKC, particularly (PKC)-ζ. Inhibition of PKC activity using pharmacological inhibitor markedly decreased H-Ras-induced phosphorylation of PRAS40. The overexpression of PRAS40 in renal cancer cells significantly down-regulated CNI- and H-Ras-induced VEGF transcriptional activation. Finally, it was observed that CNI treatment increased the expression of phosho-PRAS40 in renal tumor tissues in vivo. Together, the phosphorylation of PRAS40 is critical for the activation of mTOR in CNI-induced VEGF overexpression and renal cancer progression.

Abstract 16: Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells

Abstract In addition to act as an antiapoptotic protein, bcl-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia inducible factor 1 (HIF-1) -mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogates the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2011-16

Increased Akt-mTOR Signaling in Lung Epithelium Is Associated with Respiratory Distress Syndrome in Mice

Pregnancy in women with diabetes is associated with a higher risk of perinatal complications. In particular, infants of diabetic mothers frequently suffer from respiratory distress syndrome (RDS), which is a leading cause of death in preterm infants and is considered to be primarily due to hyperinsulinemia in infants in response to maternal hyperglycemia. To elucidate the mechanism of how insulin signaling induces RDS, bronchoalveolar epithelium-specific Akt1 transgenic (TG) mice were generated. Akt1 overexpression in fetal lung epithelium resulted in RDS in preterm infants born by Caesarean section at embryonic day 18.5 (E18.5). The expression levels of hypoxia-inducible factor 2α (HIF-2α) and its target vascular endothelial growth factor (VEGF) were downregulated in the lung of Akt1 TG mice. Inhibition of the Akt-mammalian target of rapamycin (mTOR) signaling axis by rapamycin restored the expression of VEGF and improved the lung pathology of Akt1 TG pups. Rapamycin also attenuated the RDS phenotype in wild-type mice delivered preterm at E17.5. In cultured lung epithelial cells, insulin reduced VEGF expression and transcriptional activity of HIF-2 on VEGF promoter in an mTOR-dependent manner. Thus, aberrant activation of the Akt-mTOR pathway in lung epithelium plays a causal role in the pathogenesis of infant RDS, presumably through downregulation of HIF-2-dependent VEGF expression in the lung.

Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells

In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2.

Mechanisms of Platelet-Activating Factor-induced Enhancement of VEGF Expression

It has been previously reported that platelet-activating factor (PAF) induces the expression of vascular endothelial growth factor (VEGF) via the downregulation of p53 activity. In this study, we attempted to characterize the mechanism by which p53 activity negatively regulates PAF-induced VEGF expression. PAF increased luciferase activity as well as VEGF mRNA expression in human non-small cell lung cancer cell line H1299 transfected with VEGF luciferase reporter plasmid (VEGF-Luc). Cotransfection of the cells with wt p53, but not mutant p53, effected a blockage of PAF-induced VEGF mRNA expression. The ChIP assay revealed that p53 did not bind to the VEGF promoter. Transfection of Egr-1 or Sp-1 expression vector increased VEGF luciferase activity in VEGF-Luc-transfected cells, and this was inhibited by transfection with wt p53. The results of the Immunoprecipitation and immunoblot analysis showed that p53 binds to Egr-1 and Sp-1. Additionally, our electrophoretic mobility shift assay demonstrated that PAF induced the mobilization of Egr-1 and Sp-1 to the nucleus, and this activity was inhibited by transfection with wt p53. These data indicate that PAF inhibits protein complexes between p53 and Egr-1/Sp-1 via the downregulation of p53 levels, thus increasing the free form levels of Egr-1 and Sp-1, ultimately resulting in the transcriptional activation of VEGF.

Abstract A51: Acacetin in inhibiting tumor growth and angiogenesis

Abstract Background: Acacetin (5,7-dihydroxy-4′-methoxyflavone) is a member of the flavonoid compound familys, some of which have anti-cancerous effects. Angiogenesis is a key process during carcinogenesis and tumor growth; however, but the effect and the mechanisms of acacetin on angiogenesis and tumor growth remain to be elucidated. Methods: In order to determine the effect of acacetin on the expression of vascular endothelial growth factor (VEGF) and HIF-1α expression, we tested VEGF expression by reporter assay and HIF-1α expression by Western blotting and RT-PCR assay. To test whether overexpression of active form of AKT increases HIF-1 expression, transient transfection was performed. Tumor angiogenesis and tumor formation in vivo were examined by chicken chorioallantoic membrane (CAM). Results: Acacetin decreased the steady level of VEGF mRNA under the normoxia and hypoxia conditions while inhibiting, and inhibited VEGF transcriptional activation. To further determine the potential mechanism of acacetin in inhibiting VEGF expression, we showed demonstrated that acacetin inhibited HIF-1α expression and AKT activation. Overexpression of HIF-1α or AKT abolished acacetin-decreasing VEGF transcriptional activation, indicating that AKT and HIF-1 are the essential downstream targets of acacetin for inhibiting VEGF expression in the cells. Moreover, acacetin significantly inhibited ovarian cancer cells-induced angiogenesis and tumor growth in vivo through inhibiting HIF-1α and VEGF expression. Acacetin inhibited HIF-1α protein expression through increasing its degradation and decreasing its stability, but did not alter HIF-1α mRNA levels. Conclusion: These results indicate that acacetin inhibits tumor agnigenensis and growth by blocking the AKT/HIF-1/VEGF pathway. It may be a useful natural compound for cancer prevention and treatment. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A51.

Opposite functions of HIF-α isoforms in VEGF induction by TGF-β1 under non-hypoxic conditions

Transforming growth factor (TGF)-β1 has biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in the late stages promoting tumor angiogenesis and metastasis. We demonstrate here that tumor-producing TGF-β1 induces vascular endothelial growth factor (VEGF) in prostate cancer cells, and hypoxia-inducible factor (HIF)-1α and HIF-2α has opposite functions in TGF-β1 regulation of VEGF expression under non-hypoxic conditions. The promoter response of VEGF to TGF-β1 was upregulated by the transfection of HIF-2α or siHIF-1α but downregulated by HIF-1α and siHIF-2α. Both HIF-1α and HIF-2α were induced by TGF-β1 at mRNA and protein levels, however, their nuclear translocation was differentially regulated by TGF-β1, suggesting its association with their opposite effects. VEGF induction by TGF-β1 occurred in a Smad3-dependent manner, and the Smad-binding element 2 (SBE2, -992 to -986) and hypoxia response element (-975 to -968) in the VEGF promoter were required for the promoter response to TGF-β1. Smad3 cooperated with HIF-2α in TGF-β1 activation of VEGF transcription and Smad3 binding to the SBE2 site was greatly impaired by knockdown of HIF-2α expression. Moreover, the VEGF promoter response to TGF-β1 was synergistically elevated by co-transfection of Smad3 and HIF-2α but attenuated by HIF-1α in a dose-dependent manner. Additionally, TGF-β1 was found to increase the stability of VEGF transcript by facilitating the cytoplasmic translocation of a RNA-stabilizing factor HuR. Collectively, our data show that tumor-producing TGF-β1 induces VEGF at the both transcription and post-transcriptional levels through multiple routes including Smad3, HIF-2α and HuR. This study thus suggests that autocrine TGF-β1 production may contribute to tumor angiogenesis via HIF-2α signaling under non-hypoxic conditions, providing a selective growth advantage for prostate tumor cells.

Inhibition of vascular endothelial growth factor expression by Chinese medicine of Hedyotis diffusa Willd herbal compounds

The Hedyotis diffusa Willd herbal compounds (HDWHCs) are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic efficacy in China. However, the underlying mechanisms remain to be elucidated. In this study, we used freeze-dried powder of the water extracts of HDWHCs to investigate the potential mechanisms of HDWHCs in cancer treatment. HDWHCs treatment significantly inhibited vascular endothelial growth factor (VEGF) mRNA levels and VEGF transcriptional activation in cancer cells. HDWHCs also had a remarkable inhibitory effect on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha). Forced expression of HIF-1α restored VEGF transcriptional activation inhibited by HDWHCs, indicating that HDWHCs suppressed VEGF expression through decreasing HIF-1alpha expression. Moreover, HDWHCs inhibited cyclooxygenase-2 (COX-2) expression, and overexpression of HIF-1alpha restored HDWHCs’ inhibitory effect on COX-2 at transcriptional level. These findings may provide better understanding of HDWHCs’ anti-cancer mechanism in cancer treatment.

Abstract 1247: Transcriptional regulation of VEGF by WT1 and AR in prostate cancer

Abstract Identifying molecular mechanisms that influence prostate cancer progression is central to understanding how this disease develops. Angiogenesis is key for cancer development and growth as tumors require new blood vessels for nutrients and oxygen. Vascular endothelial growth factor (VEGF) is the most important signaling protein involved in angiogenesis and is up-regulated by oncogene expression among other things. Previous studies have shown that the zinc finger transcription factor WT1 (Wilms tumor 1) transcriptionally regulated VEGF in multiple cell lines. Additionally, WT1 activation of VEGF required an intact zinc finger domain and was mediated by DNA binding. Potential WT1 binding sites were identified in the VEGF promoter using in silico approaches and shown to bind WT1 protein using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Luciferase reporter assays showed increased activation of the VEGF promoter by over-expression of WT1. Additional factors known to induce VEGF expression are growth factors, hypoxia and hormones. How androgen regulates VEGF transcription is unknown, but we have identified two potential androgen receptor (AR) binding sites within 2kb of the transcriptional start site and demonstrated reporter activation by R1881 treatment of transfected LNCaP cells. This study examined the effect of mutating selected WT1 and AR binding sites in the VEGF promoter to determine whether they were responsible for activation of VEGF transcription. Mutating these binding sites by site-directed mutagenesis significantly decreased activation of the VEGF promoter by WT1 and the androgen analog R1881 in LNCaP cells. As predicted, treatment with the anti-androgen casodex similarly eliminated hormone response of the VEGF promoter, suggesting that androgens are functioning via androgen signaling. This was confirmed in vivo by both western blot analysis of VEGF protein and quantitative real-time PCR measurement of VEGF mRNA in cells treated with casodex. This is important because androgen signaling is vital to normal prostate development and malignant prostate growth. Overall these results show that both WT1 and AR transcriptionally regulate VEGF expression in prostate cancer cells and these factors directly bind and activate the promoter in vivo. Understanding how angiogenesis is regulated will lead to improved therapy and prevention of prostate cancer progression. This work was supported by NIHR15CA11360 (GF), Sigma Xi GIAR220034 (KE), and GSSKSU (KE) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1247.

Impaired Function of Hematopoietic Stem Cells with Abrogated Hypoxia Induced Expression of Vascular Endothelial Growth Factor

Hypoxia has been suggested to be a characteristic of the bone marrow (BM) microenvironment where hematopoietic stem cells (HSC) reside. The major molecular response to hypoxia is the activation of hypoxia inducible factors (HIF). These transcription factors activate gene expression by binding to hypoxia response elements (HRE) in the promoter region of target genes, such as vascular endothelial growth factor (VEGF). It has been demonstrated that VEGF is indispensable for HSC survival in the adult and that VEGF mediated HSC survival is cell intrinsic. We are therefore investigating the role of hypoxia-regulated VEGF expression in HSC survival. For our studies we use the transgenic mouse model VEGFδ/δ. In this model, the HRE in the VEGF promoter is mutated which prevents HIF binding. Thus, hypoxia-induced VEGF transcriptional activation is abrogated while basal VEGF expression remains unaffected. Steady state hematopoiesis was not affected by the mutation since distribution between the B-cell, T-cell and myeloid lineages in both peripheral blood and BM of mice homozygous for the mutation was comparable to wild type animals. Furthermore, we could not detect any differences in the frequency of lineage-, Sca1+, c-kit+, CD34- cells in the BM between VEGFδ/δ and wild type mice. However, when assessing HSC functional properties in transplantation assays, BM from mice homozygous for the mutation was demonstrated to have dramatically reduced HSC function. Only around 60 % of lethally irradiated recipients that were transplanted with VEGFδ/δ BM could be rescued while recipients receiving wild type BM survived as expected. In addition, during early hematopoietic reconstitution, a skewing towards myeloid differentiation at the expense of B-cell differentiation could be observed in peripheral blood of recipients reconstituted with VEGFδ/δ BM. Moreover, in a competitive repopulation assay, VEGFδ/δ BM was shown to have a severe competitive disadvantage compared to wild type BM. Our findings indicate an important role for hypoxia related VEGF expression in reconstituting HSCs and suggest that low oxygen levels might be an important characteristic of the HSC niche.

Overexpression of Vascular Endothelial Growth Factor and the Development of Post-Transplantation Cancer

Cancer is an increasing and major problem after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors. We propose that the effect of calcineurin inhibitors on the expression of vascular endothelial growth factor (VEGF) leads to an angiogenic milieu that favors tumor growth. Here, we used 786-0 human renal cancer cells to investigate the effect of cyclosporine (CsA) on VEGF expression. Using a full-length VEGF promoter-luciferase construct, we found that CsA markedly induced VEGF transcriptional activation through the protein kinase C (PKC) signaling pathway, specifically involving PKC zeta and PKC delta isoforms. Moreover, CsA promoted the association of PKC zeta and PKC delta with the transcription factor Sp1 as observed by immunoprecipitation assays. Using promoter deletion constructs, we found that CsA-mediated VEGF transcription was primarily Sp1 dependent. Furthermore, CsA-induced and PKC-Sp1-mediated VEGF transcriptional activation was partially inhibited by von Hippel-Lindau protein. CsA also promoted the progression of human renal tumors in vivo, wherein VEGF is overexpressed. Finally, to evaluate the in vivo significance of CsA-induced VEGF overexpression in terms of post-transplantation tumor development, we injected CT26 murine carcinoma cells (known to form angiogenic tumors) into mice with fully MHC mismatched cardiac transplants. We observed that therapeutic doses of CsA increased tumor size and VEGF mRNA expression and also enhanced tumor angiogenesis. However, coadministration of a blocking anti-VEGF antibody inhibited this CsA-mediated tumor growth. Collectively, these findings define PKC-mediated VEGF transcriptional activation as a key component in the progression of CsA-induced post-transplantation cancer.