Abstract 3092: FBW7 targets hypoxia inducible factor-1α (HIF-1α) for proteasomal degradation during hypoxia

Abstract Hypoxia Inducible Factor-1α (HIF-1α) mediates expression of genes associated with endothelial cell-mediated angiogenesis and is associated with poor outcomes in a variety of cancers. In normoxia, HIF-1α is ubiquitinated and degraded through interactions with the E3 ubiqutin ligase, von Hippel-Lindau (vHL); however, little is known about the negative regulation of HIF-1α in hypoxia. FBW7, an E3 ubiquitin ligase, has been shown to interact with several transcription factors including those phosphorylated by glycogen synthase kinase 3β (GSK3β). The current study tested the hypothesis that phosphorylation of HIF-1α by GSK3β increases the FBW7-mediated ubiquitination and degradation of HIF-1α, thereby resulting in suppression of the hypoxia-mediated angiogenic response in SKOV-3 ovarian cancer cells. HIF-1α protein and VEGF transcript levels in hypoxia were increased when GSK3β activity was inhibited and were reduced by expression of constitutively active GSK3β (GSK3S9A). Additionally, expression of GSK3S9A increased HIF-1α ubiquitination. Conditioned media from SKOV-3 cells expressing shRNA against GSK3β (shGSK3) had an enhanced effect on endothelial tube formation in a Matrigel matrix, suggesting that GSK3β exerts an inhibitory effect on hypoxia-mediated angiogenesis. Overexpression of the β and γ isoforms of FBW7 decreased HIF-1α stabilization, and HIF-1α interacts with FBW7 by co-immunoprecipitation. Furthermore, knockdown of FBW7 using siRNA resulted in increased HIF-1α levels in hypoxia. These data suggest a new mechanism for negative regulation of HIF-1α during hypoxia that utilizes phosphorylation by GSK3β and interaction with FBW7 leading to ubiquitination and proteasomal degradation. Results of this study better define the signaling pathways necessary for HIF-1α-mediated signaling and may identify new targets that mediate angiogenesis in disease. 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 3092. doi:10.1158/1538-7445.AM2011-3092</jats:p

Impact of Estrogen on Purinergic Signaling in Microvascular Disease

Microvascular ischemia, especially in the heart and kidneys, is associated with inflammation and metabolic perturbation, resulting in cellular dysfunction and end-organ failure. Heightened production of adenosine from extracellular nucleotides released in response to inflammation results in protective effects, inclusive of adaptations to hypoxia, endothelial cell nitric oxide release with the regulation of vascular tone, and inhibition of platelet aggregation. Purinergic signaling is modulated by ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39, which is the dominant factor dictating vascular metabolism of extracellular ATP to adenosine throughout the cardiovascular tissues. Excess levels of extracellular purine metabolites, however, have been associated with metabolic and cardiovascular diseases. Physiological estrogen signaling is anti-inflammatory with vascular protective effects, but pharmacological replacement use in transgender and postmenopausal individuals is associated with thrombosis and other side effects. Crucially, the loss of this important sex hormone following menopause or with gender reassignment is associated with worsened pro-inflammatory states linked to increased oxidative stress, myocardial fibrosis, and, ultimately, diastolic dysfunction, also known as Yentl syndrome. While there is a growing body of knowledge on distinctive purinergic or estrogen signaling and endothelial health, much less is known about the relationships between the two signaling pathways. Continued studies of the interactions between these pathways will allow further insight into future therapeutic targets to improve the cardiovascular health of aging women without imparting deleterious side effects

Regulation of Hypoxic–Adenosinergic Signaling by Estrogen: Implications for Microvascular Injury

Loss of estrogen, as occurs with normal aging, leads to increased inflammation, pathologic angiogenesis, impaired mitochondrial function, and microvascular disease. While the influence of estrogens on purinergic pathways is largely unknown, extracellular adenosine, generated at high levels by CD39 and CD73, is known to be anti-inflammatory in the vasculature. To further define the cellular mechanisms necessary for vascular protection, we investigated how estrogen modulates hypoxic–adenosinergic vascular signaling responses and angiogenesis. Expression of estrogen receptors, purinergic mediators inclusive of adenosine, adenosine deaminase (ADA), and ATP were measured in human endothelial cells. Standard tube formation and wound healing assays were performed to assess angiogenesis in vitro. The impacts on purinergic responses in vivo were modeled using cardiac tissue from ovariectomized mice. CD39 and estrogen receptor alpha (ERα) levels were markedly increased in presence of estradiol (E2). Suppression of ERα resulted in decreased CD39 expression. Expression of ENT1 was decreased in an ER-dependent manner. Extracellular ATP and ADA activity levels decreased following E2 exposure while levels of adenosine increased. Phosphorylation of ERK1/2 increased following E2 treatment and was attenuated by blocking adenosine receptor (AR) and ER activity. Estradiol boosted angiogenesis, while inhibition of estrogen decreased tube formation in vitro. Expression of CD39 and phospho-ERK1/2 decreased in cardiac tissues from ovariectomized mice, whereas ENT1 expression increased with expected decreases in blood adenosine levels. Estradiol-induced upregulation of CD39 substantially increases adenosine availability, while augmenting vascular protective signaling responses. Control of CD39 by ERα follows on transcriptional regulation. These data suggest novel therapeutic avenues to explore in the amelioration of post-menopausal cardiovascular disease, by modulation of adenosinergic mechanisms.</jats:p

Regulation of Hypoxic&ndash;Adenosinergic Signaling by Estrogen: Implications for Microvascular Injury

Loss of estrogen, as occurs with normal aging, leads to increased inflammation, pathologic angiogenesis, impaired mitochondrial function, and microvascular disease. While the influence of estrogens on purinergic pathways is largely unknown, extracellular adenosine, generated at high levels by CD39 and CD73, is known to be anti-inflammatory in the vasculature. To further define the cellular mechanisms necessary for vascular protection, we investigated how estrogen modulates hypoxic&ndash;adenosinergic vascular signaling responses and angiogenesis. Expression of estrogen receptors, purinergic mediators inclusive of adenosine, adenosine deaminase (ADA), and ATP were measured in human endothelial cells. Standard tube formation and wound healing assays were performed to assess angiogenesis in vitro. The impacts on purinergic responses in vivo were modeled using cardiac tissue from ovariectomized mice. CD39 and estrogen receptor alpha (ER&alpha;) levels were markedly increased in presence of estradiol (E2). Suppression of ER&alpha; resulted in decreased CD39 expression. Expression of ENT1 was decreased in an ER-dependent manner. Extracellular ATP and ADA activity levels decreased following E2 exposure while levels of adenosine increased. Phosphorylation of ERK1/2 increased following E2 treatment and was attenuated by blocking adenosine receptor (AR) and ER activity. Estradiol boosted angiogenesis, while inhibition of estrogen decreased tube formation in vitro. Expression of CD39 and phospho-ERK1/2 decreased in cardiac tissues from ovariectomized mice, whereas ENT1 expression increased with expected decreases in blood adenosine levels. Estradiol-induced upregulation of CD39 substantially increases adenosine availability, while augmenting vascular protective signaling responses. Control of CD39 by ER&alpha; follows on transcriptional regulation. These data suggest novel therapeutic avenues to explore in the amelioration of post-menopausal cardiovascular disease, by modulation of adenosinergic mechanisms

Abstract 4190: Ezrin plays a key role in the regulation of translation in metastatic osteosarcoma

Abstract Our previous studies have demonstrated the association between Ezrin and the metastatic biology of pediatric sarcomas including osteosarcoma (OS) and rhabdomyosarcoma. Mechanistic studies exploring this association have shown that Ezrin expression enhances the survival of metastatic cells upon their arrival to the secondary metastatic site. In order to better understand this role in metastasis, we undertook two non-candidate analyses of Ezrin function including a microarray subtraction of high and low Ezrin expressing cells and a proteomic approach to identify proteins that bind the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis we found Ezrin to be part of a ribonucleoprotein complex and to bind with poly A binding protein 1 (PABA1; PABPC1). Using luciferase reporter-based assays, we have shown that OS cells expressing high levels of Ezrin are able to translate mRNAs containing a stem loop 5′ UTR structure, so called “weakly translated mRNAs,” more efficiently than low Ezrin OS cells. This finding suggests that Ezrin's contribution to the metastatic phenotype may be due in part to enhanced translation of specific mRNAs during metastasis that are normally expressed at low levels outside of the metastatic context. Ongoing studies will now assess the ability of Ezrin to enhance the expression of specific mRNAs in 3-dimensional contexts more relevant to cancer cell growth in vivo. We have developed stable high and low Ezrin osteosarcoma cell lines that express a GFP reporter with or without a complex 5′ UTR stem loop structure in the mRNA transcript and with and without protein destabilizing elements. Initial studies have shown that the addition of the 5′ UTR structure and destabilizing elements progressively limits GFP reporter expression. We plan to confirm that this reduction in expression is due to reduced translation and to assess whether 3-D culture conditions or the in vivo environment enhance expression of these tunable reporters of translation in an Ezrin-dependant manner. We expect these results to provide a novel mechanistic basis to consider how Ezrin may contribute to metastasis. 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 4190. doi:1538-7445.AM2012-4190</jats:p

The Ezrin Metastatic Phenotype Is Associated with the Initiation of Protein Translation

We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis