Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression

We have previously demonstrated the roles of RhoA, Rac1, and Cdc42 in hypoxia-driven angiogenesis. However, the role of oncogenes in hypoxia signaling is poorly understood. Given the importance of Rho proteins in the hypoxic response, we hypothesized that Rho family members could act as mediators of hypoxic signal transduction. We investigated the cross-talk between hypoxia and oncogene-driven signal transduction pathways and explored the role of Rac1 on hypoxia-induced hypoxia-inducible factor (HIF)-1α and VEGF expression. Since the phosphatidylinositol 3'-kinase (PI3K) pathway is involved in signal transduction of many oncogenes, we explored the role of PI3K on Rac1-mediated expression of HIF-1α and VEGF in hypoxia. We showed that LY-294002, a PI3K inhibitor, suppressed HIF-1α and VEGF induction under hypoxic conditions by up to 50%. Activation of Rac1 resulted in an upregulation of hypoxia-induced HIF-1α expression, which was blocked by LY-294002. These data suggested that Rac1 is an intermediate in the PI3K-mediated induction of HIF-1α. Interestingly, there was a significant downregulation of the tumor suppressor genes p53 and von Hippel-Lindau tumor suppressor (VHL) in cells expressing a constitutively active form of Rac1. Rac1-mediated inhibition of p53 and VHL could therefore be implicated in the upregulation of HIF-1α expression.     

HIF-1alpha and p53: the ODD couple?

Tumor hypoxia activates hypoxia-inducible factor-1 (HIF-1) and induces the accumulation of the tumor suppressor p53. HIF-1 signaling stimulates angiogenesis and mediates cellular adaptation to hypoxia, whereas p53 promotes hypoxia-induced apoptosis. A recent article provides in vitro biophysical evidence supporting a direct interaction between p53 and the oxygen-dependent degradation domain of the HIF-1alpha subunit. The article identifies potential structural parameters required for this interaction and suggests an alternative mechanism by which p53 might impact tumor response to therapy.     

VHL loss of function and its impact on oncogenic signaling networks in clear cell renal cell carcinoma

Loss of von Hippel-Lindau tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma, resulting in the aberrant expression of genes that control cell proliferation, metabolism, invasion and angiogenesis. The molecular mechanisms by which loss of function leads to tumorigenesis are not yet fully defined. The von Hippel-Lindau gene product is part of an ubiquitin ligase complex that targets hypoxia inducible factors for polyubiquitination and proteasomal degradation, linking hypoxia response genes to renal cell carcinoma oncogenesis. Loss von Hippel-Lindau gene function also promotes cell invasiveness in response to hepatocyte growth factor, an important regulator of kidney development and renal homeostasis. Increased cell invasiveness is mediated by another ubiquitin ligase target with relevance to the molecular pathogenesis of renal cell carcinoma: beta-catenin. This discovery and other recent insights into kidney cancer oncogenesis implicate convergent developmental and homeostatic signaling pathways in tumorigenesis, tumor invasiveness and metastasis.     

Incompatible effects of p53 and HDAC inhibition on p21 expression and cell cycle progression

Nutlin-3 selectively activates p53 by inhibiting the interaction of this tumor suppressor with its negative regulator murine double minute 2 (mdm2), while trichostatin A (TSA) is one of the most potent histone deacetylase (HDAC) inhibitors currently available. As both Nutlin-3 and TSA increase the levels of the cell cycle inhibitor p21(cip1/waf1) in cells, we investigated whether a combination of these compounds would further augment p21 levels. Contrary to expectations, we found that short-term exposure to Nutlin-3 and TSA in combination did not have an additive effect on p21 expression. Instead, we observed that activation of p53 prevented the ability of TSA to increase p21 levels. Furthermore, TSA inhibited Nutlin-3-induced expression of p53-dependent mRNAs including P21. This negative effect of TSA on Nutlin-3 was significantly less pronounced in the case of hdm2, another p53 downstream target. Aside from suggesting a model to explain these incompatible effects of Nutlin-3 and TSA, we discuss the implications of our findings in cancer therapy and cell reprogramming.     

Gene expression profiling in a renal cell carcinoma cell line: dissecting VHL and hypoxia-dependent pathways

The von Hippel-Lindau tumor suppressor, pVHL, is a key player in one of the best characterized hypoxia signaling pathways, the VHL-hypoxia-inducible factor (VHL-HIF) pathway. To better understand the role of VHL in the hypoxia signaling pathways of tumor cells, we used serial analysis of gene expression (SAGE) to investigate hypoxia-regulated gene expression in renal carcinoma cells (786-0), with and without VHL. The gene expression profiles of the cancer cells were compared to SAGE profiles from normal renal proximal tubule cells grown under both normoxia and hypoxia. The data suggest that the role of VHL as a tumor suppressor may be more complex than previously thought. Further, the data reveal that renal carcinoma cells have evolved an alternative hypoxia signaling pathway(s) compared with normal renal cells. These alternative hypoxia pathways demonstrate VHL-dependent and VHL-independent regulation. The genes involved in such pathways include those with potential importance in the physiological and pathological regulation of tumor growth and angiogenesis. Some of the genes identified as showing overexpression in the cancer cells, particularly those encoding secreted or membrane-bound proteins, could be potential biomarkers for tumors or targets for rational therapeutics that are dependent on VHL status.     

Liprin-α4 is a new hypoxia-inducible target gene required for maintenance of cell-cell contacts

Liprin-α1 to liprin-α4 constitute a family of cytoplasmic proteins, which have been found in various multiprotein complexes. For liprin-α1 roles in synapse formation and cell spreading were described but other liprin family members are not well characterized. We show here that liprin-α4 is upregulated in human clear cell renal cell carcinomas (RCC) as compared to normal kidney tissue. Liprin-α4 expression is downregulated by the von Hippel-Lindau tumor suppressor (VHL) and upregulated by hypoxia in RCC cell lines. The liprin-α4 gene promoter is directly activated by binding of the hypoxia-inducible factor 1α (HIF-1α) to HRE consensus binding sites as shown by reporter assays and chromatin immunoprecipitations. RNAi mediated knockdown of liprin-α4 leads to reduced E-cadherin and β-catenin levels at cell junctions and to dissociation of epithelial cell contacts. Our data describe for the first time liprin-α4 as a hypoxia-induced gene potentially involved in cell-cell adhesion.     

The role of p53 deacetylation in p21Waf1 regulation by laminar flow

Laminar flow arrests vascular endothelial cells at the G0/G1 phase with concurrent increase in p53 and p21Waf1. We investigated the molecular mechanism by which laminar flow activates p53 and p21Waf1 in endothelial cells. The application of a laminar flow (12 dyn/cm2) increased the deacetylation at Lys-320 and Lys-373 of p53 and the acetylation at Lys-382 in human umbilical vein endothelial cells. Laminar flow increased the activity of histone deacetylase (HDAC) and the association of p53 with HDAC1. Treating human umbilical vein endothelial cells with trichostatin A (TSA), an HDAC inhibitor, abolished the flow-induced p53 deacetylation at Lys-320 and Lys-373. To investigate the role of the HDAC-deacetylated p53 in the flow activation of p21Waf1, we found that TSA inhibited the activation at both the mRNA and protein levels. Deletion and mutation analyses of the p21Waf1 promoter revealed that flow activated p21Waf1 through p53 and TSA abrogated this p53-dependent activation. The expression plasmid encoding the p53 mutant, with Lys-320 and Lys-373 replaced by Arg, increased the activity of the co-transfected p21Waf1 promoter, which demonstrates that HDAC-deacetylated p53 can transactivate the p21Waf1 gene. The regulation of the p53-p21Waf1 pathway by laminar flow was further supported by observations that flow caused an increase of p21Waf1 level in the wild-type HCT116 (p53+/+) cells but not in the p53-null HCT116 cells.     

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.     

2-Oxoglutarate downregulates expression of vascular endothelial growth factor and erythropoietin through decreasing hypoxia-inducible factor-1alpha and inhibits angiogenesis

In oxygenated cells, hypoxia-inducible factor-1 (HIF-1) alpha subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor E3 ligase complex using 2-oxoglutarate as a substrate. We examined the effect of 2-oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The promoter activity of VEGF-luciferase was dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited the HIF-1alpha protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1alpha protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of angiogenesis.     

Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.     

Dual targeting of epigenetic therapy in cancer

Aberrant epigenetic silencing of tumor suppressor genes by promoter DNA hypermethylation and histone deacetylation plays an important role in the pathogenesis of cancer. The potential reversibility of epigenetic abnormalities encouraged the development of pharmacologic inhibitors of DNA methylation and histone deacetylation as anti-cancer therapeutics. (Pre)clinical studies of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have yielded encouraging results, especially against hematologic malignancies. Recently, several studies demonstrated that DNMT and HDAC inhibitors are also potent angiostatic agents, inhibiting (tumor) endothelial cells and angiogenesis in vitro and in vivo. By reactivation of epigenetically silenced tumor suppressor genes with angiogenesis inhibiting properties, DNMT and HDAC inhibitors might indirectly - via their effects on tumor cells - decrease tumor angiogenesis in vivo. However, this does not explain the direct angiostatic effects of these agents, which can be unraveled by gene expression studies and examination of epigenetic promoter modifications in endothelial cells treated with DNMT and HDAC inhibitors. Clearly, the dual targeting of epigenetic therapy on both tumor cells and tumor vasculature makes them attractive combinatorial anti-tumor therapeutics. Here we review the therapeutic potential of DNMT and HDAC inhibitors as anti-cancer drugs, as evaluated in clinical trials, and their angiostatic activities, apart from their inhibitory effects on tumor cells.     

Inhibition of histone deacetylase causes emphysema

In patients with chronic obstructive pulmonary disease (COPD), histone deacetylase (HDAC) expression and activity are reduced in the lung tissue. However, whether HDAC activity controls the maintenance of the lung alveolar septal structures has not been investigated. To explore the consequences of HDAC inhibition and address the question of whether HDAC inhibition causes lung cell apoptosis and emphysema, male Sprague-Dawley rats and human pulmonary microvascular endothelial cells (HPMVEC) were treated with trichostatin A (TSA), a specific inhibitor of HDACs. Chronic TSA treatment increased the alveolar air space area, mean linear intercept, and the number of caspase-3-positive cells in rat lungs. TSA suppressed hypoxia-inducible factor-1α (HIF-1α), VEGF, and lysyl oxidase (LOX) and increased microtubule-associated protein-1 light chain 3 (LC3), p53, and miR34a microRNA expression in both rat lungs and cultured HPMVEC. Gene silencing of HDAC2 using small interfering RNA (siRNA) in cultured HPMVEC resulted in the suppression of HIF-1α, VEGF, and LOX and an increase of p53 expression. These data indicate that HDAC inhibition causes emphysema and that HDAC-dependent mechanisms contribute to the maintenance of the adult lung structure. Our results also suggest that the increase in apoptosis, as a consequence of HDAC inhibition, is associated with decreased VEGF and HIF-1α expression.     

Activation of Peroxisome Proliferator-activated Receptor α (PPARα) Suppresses Hypoxia-inducible Factor-1α (HIF-1α) Signaling in Cancer Cells

Activation of peroxisome proliferator-activated receptor α (PPARα) has been demonstrated to inhibit tumor growth and angiogenesis, yet the mechanisms behind these actions remain to be characterized. In this study, we examined the effects of PPARα activation on the hypoxia-inducible factor-1α (HIF-1α) signaling pathway in human breast (MCF-7) and ovarian (A2780) cancer cells under hypoxia. Incubation of cancer cells under 1% oxygen for 16 h significantly induced HIF-1α expression and activity as assayed by Western blotting and reporter gene analysis. Treatment of the cells with PPARα agonists, but not a PPARγ agonist, prior to hypoxia diminished hypoxia-induced HIF-1α expression and activity, and addition of a PPARα antagonist attenuated the suppression of HIF-1α signaling. Activation of PPARα attenuated hypoxia-induced HA-tagged HIF-1α protein expression without affecting the HA-tagged HIF-1α mutant protein level, indicating that PPARα activation promotes HIF-1α degradation in these cells. This was further confirmed using proteasome inhibitors, which reversed PPARα-mediated suppression of HIF-1α expression under hypoxia. Using the co-immunoprecipitation technique, we found that activation of PPARα enhances the binding of HIF-1α to von Hippel-Lindau tumor suppressor (pVHL), a protein known to mediate HIF-1α degradation through the ubiquitin-proteasome pathway. Following PPARα-mediated suppression of HIF-1α signaling, VEGF secretion from the cancer cells was significantly reduced, and tube formation by endothelial cells was dramatically impaired. Taken together, these findings demonstrate for the first time that activation of PPARα suppresses hypoxia-induced HIF-1α signaling in cancer cells, providing novel insight into the anticancer properties of PPARα agonists.     

Trichostatin A,a histone deacetylase inhibitor suppresses NADPH Oxidase 4‐Derived Redox Signalling and Angiogenesis

Histone deacetylase (HDAC) inhibitors are known to suppress abnormal development of blood vessels. Angiogenic activity in endothelial cells depends upon NADPH oxidase 4 (Nox4)‐dependent redox signalling. We set out to study whether the HDAC inhibitor trichostatin A (TSA) affects Nox4 expression and angiogenesis. Nox4 expression was measured by real time PCR and Western blot analysis in endothelial cells. Hydrogen peroxide (H₂O₂) was measured by amplex^® red assay in endothelial cells. Nox4 was knocked down by Nox4 shRNA. In vitro angiogenic activities such migration and tubulogenesis were assessed using wound healing and Matrigel assays, respectively. In vivo angiogenic activity was assessed using subcutaneous sponge assay in C57Bl/6 and Nox4‐deficient mice. Trichostatin A reduced Nox4 expression in a time‐ and concentration‐dependent manner. Both TSA and Nox4 silencing decreased Nox4 protein and H₂O₂. Mechanistically, TSA reduced expression of Nox4 via ubiquitination of p300‐ histone acetyltransferase (p300‐HAT). Thus, blocking of the ubiquitination pathway using an inhibitor of ubiquitin‐activating enzyme E1 (PYR‐41) prevented TSA inhibition of Nox4 expression. Trichostatin A also reduced migration and tube formation, and these effects were not observed in Nox4‐deficient endothelial cells. Finally, transforming growth factor beta1 (TGFβ1) enhanced angiogenesis in sponge model in C57BL/6 mice. This response to TGFβ1 was substantially reduced in Nox4‐deficient mice. Similarly intraperitoneal infusion of TSA (1 mg/kg) also suppressed TGFβ1‐induced angiogenesis in C57BL/6 mice. Trichostatin A reduces Nox4 expression and angiogenesis via inhibition of the p300‐HAT‐dependent pathway. This mechanism might be exploited to prevent aberrant angiogenesis in diabetic retinopathy, complicated vascular tumours and malformations.     

Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells

Hypoxia-induced angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Rho family small GTPases are involved in this process, and Racl, a prominent member of the Rho family, may be critical in regulating hypoxia-induced gene activation of several angiogenesis factors and tumor suppressors. To fur-ther define Racl function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock down gene expression of Racl in gastric cancer cell line AGS that expresses a high level of Racl.Both the mRNA and protein levels of Racl in the AGS cells were decreased dramatically after transfection with a Racl-specific siRNA vector. When the conditioned medium derived from the Racl downregulated AGS cells was applied to the human endothelial cells. it could significantly inhibit the cell proliferation. Further study proved that, VEGF and HIF-la, two angiogenesis promoting factors, were found to be downregulated whereas p53 and VHL, which are tumor suppressors and angiogenesis inhibitors. were upregulated in the Racl siRNA transfected cells. Our results suggest that Racl may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting the Racl GTPase.