Efficacy of a novel orally active SERD AZD9496 against hormone dependent post-menopausal breast cancer depends on inhibition of cellular aromatase activity.

Treatment of hormone sensitive breast cancer tumors with endocrine therapy such as antiestrogens or aromatase inhibitors has improved the outcome significantly. Studies including our own have shown that downregulation of ERα with pure antiestrogen fulvestrant in combination with aromatase inhibitors may prolong responsiveness of the tumors to endocrine therapy. Fulvestrant has been studied as second line or first line treatment for post-menopausal hormone receptor positive breast cancers as a single agent or in combination with AIs. Studies have also suggested that further escalation of dose may improve benefit. However, dose escalation of fulvestrant, which is administered via intramuscular injection, is difficult due to its poor solubility. To overcome this shortcoming of an injectable drug, a novel orally active antiestrogen, AZD9496 was developed. In addition to being orally active, AZD9496 is designed as a selective ERα downregulator (SERD). In the current study, we compared the effect of AZD9496 and fulvestrant on the growth of MCF-7Ca (human estrogen receptor positive MCF-7 cells stably transfected with human placental aromatase gene) xenografts grown in ovariectomized athymic nude mice. AZD9496 was also compared to fulvestrant in vitro as a single agent or in combination with anastrozole. Our current study shows that AZD9496 is equally effective as fulvestrant at controlling the growth of hormone sensitive human breast cancer tumors. Similar to fulvestrant, AZD9496 inhibits cellular aromatase activity through ERα mediated signaling. However, unlike fulvestrant, combination of AZD9496 with anastrozole did not produce increased tumor inhibition. Our results show that AZD9496 was significantly better at inhibiting cellular aromatase which contributed to its anticancer activity. Next, we measured the effect of AZD9496 on the mouse uterus. Uterine weight of mice treated with AZD9496 was significantly lower than that for mice treated with androstenedione. This reduction in uterine weight was due to AZD9496 mediated inhibition of aromatase activity and not a direct effect on uterine ERα expression. We also observed that anti-cancer efficacy of AZD9496 depended on its ability to inhibit cellular aromatase. These results suggest that AZD9496 may be a better alternative to fulvestrant due to its selectivity for mammary ER and ability to inhibit aromatase in addition of downregulating ERα that can be obtained upon oral administration. As such, AZD9496 may prove to be a better option than fulvestrant for the treatment of hormone sensitive human breast cancer.

Seribantumab, an Anti-ERBB3 Antibody, Delays the Onset of Resistance and Restores Sensitivity to Letrozole in an Estrogen Receptor-Positive Breast Cancer Model.

Heregulin-driven ERBB3 signaling has been implicated as a mechanism of resistance to cytotoxic and antiendocrine therapies in preclinical breast cancer models. In this study, we evaluated the effects of seribantumab (MM-121), a heregulin-blocking anti-ERBB3 monoclonal antibody, alone and in combination with the aromatase inhibitor letrozole, on cell signaling and tumor growth in a preclinical model of postmenopausal estrogen receptor-positive (ER(+)) breast cancer. In vitro, heregulin treatment induced estrogen receptor phosphorylation in MCF-7Ca cells, and long-term letrozole-treated (LTLT-Ca) cells had increased expression and activation levels of EGFR, HER2, and ERBB3. Treatment with seribantumab, but not letrozole, inhibited basal and heregulin-mediated ERBB receptor phosphorylation and downstream effector activation in letrozole-sensitive (MCF-7Ca) and -refractory (LTLT-Ca) cells. Notably, in MCF-7Ca-derived xenograft tumors, cotreatment with seribantumab and letrozole had increased antitumor activity compared with letrozole alone, which was accompanied by downregulated PI3K/MTOR signaling both prior to and after the development of resistance to letrozole. Moreover, the addition of an MTOR inhibitor to this treatment regimen did not improve antitumor activity and was not well tolerated. Our results demonstrate that heregulin-driven ERBB3 signaling mediates resistance to letrozole in a preclinical model of ER(+) breast cancer, suggesting that heregulin-expressing ER(+) breast cancer patients may benefit from the addition of seribantumab to antiendocrine therapy.

Histone deacetylase inhibitor entinostat in combination with a retinoid downregulates HER2 and reduces the tumor initiating cell population in aromatase inhibitor-resistant breast cancer.

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Histone Deacetylase Inhibitor Entinostat Inhibits Tumor-Initiating Cells in Triple-Negative Breast Cancer Cells.

Mortality following breast cancer diagnosis is mainly due to the development of distant metastasis. To escape from the primary site, tumor cells undergo the epithelial-to-mesenchymal transition (EMT), which helps them acquire a more motile and invasive phenotype. In our previous study, we showed that class I selective HDAC inhibitor entinostat reverses the EMT phenotype through reversal of epigenetic repression of E-cadherin. Recent evidence suggests that a subset of cells within a breast tumor may drive the metastatic outgrowth following escape from the primary site. These cells, termed tumor-initiating cells (TIC), represent a great threat to overall prognosis. They are critical in terms of drug resistance and tumor initiation at metastatic sites. Acquisition of EMT traits has also been shown to impart TIC phenotype to the cells, making EMT a "dual-threat" for prognosis. In the current study, we show that entinostat treatment can reduce the percentage of TIC cells from triple-negative breast cancer (TNBC) cells. Entinostat treatment was able to reduce the CD44(high)/CD24(low) cell population, ALDH-1 activity, as well as protein and mRNA expression of known TIC markers such as Bmi-1, Nanog, and Oct-4. Next, we inoculated MDA-MB-231 cells transfected with firefly luciferase (231/Luc) in mammary fat pad of NSG mice. The mice were then treated with entinostat (2.5 mg/kg/d), and tumor development and formation of metastasis were assessed by bioluminescence imaging. Treatment with entinostat significantly reduced tumor formation at the primary site as well as lung metastasis. As such, entinostat may help prevent development of distant metastasis.

Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer.

INTRODUCTION: Although aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2. METHODS: In vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance. RESULTS: Basal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP. CONCLUSIONS: One of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.

HDAC inhibitor entinostat restores responsiveness of letrozole-resistant MCF-7Ca xenografts to aromatase inhibitors through modulation of Her-2.

We previously showed that in innately resistant tumors, silencing of the estrogen receptor (ER) could be reversed by treatment with a histone deacetylase (HDAC) inhibitor, entinostat. Tumors were then responsive to aromatase inhibitor (AI) letrozole. Here, we investigated whether ER in the acquired letrozole-resistant tumors could be restored with entinostat. Ovariectomized athymic mice were inoculated with MCF-7Ca cells, supplemented with androstenedione (Δ(4)A), the aromatizable substrate. When the tumors reached about 300 mm(3), the mice were treated with letrozole. After initial response to letrozole, the tumors eventually became resistant (doubled their initial volume). The mice then were grouped to receive letrozole, exemestane (250 µg/d), entinostat (50 µg/d), or the combination of entinostat with letrozole or exemestane for 26 weeks. The growth rates of tumors of mice treated with the combination of entinostat with letrozole or exemestane were significantly slower than with the single agent (P < 0.05). Analysis of the letrozole-resistant tumors showed entinostat increased ERα expression and aromatase activity but downregulated Her-2, p-Her-2, p-MAPK, and p-Akt. However, the mechanism of action of entinostat in reversing acquired resistance did not involve epigenetic silencing but rather included posttranslational as well as transcriptional modulation of Her-2. Entinostat treatment reduced the association of the Her-2 protein with HSP-90, possibly by reducing the stability of Her-2 protein. In addition, entinostat also reduced Her-2 mRNA levels and its stability. Our results suggest that the HDAC inhibitor may reverse letrozole resistance in cells and tumors by modulating Her-2 expression and activity.

Zoledronic acid reverses the epithelial-mesenchymal transition and inhibits self-renewal of breast cancer cells through inactivation of NF-κB.

Zoledronic acid, a third-generation bisphosphonate, has been shown to reduce cell migration, invasion, and metastasis. However, the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT), a cellular process essential to the metastatic cascade, remain unclear. Therefore, the effects of zoledronic acid on EMT, using triple-negative breast cancer (TNBC) cells as a model system, were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers, N-cadherin, Twist, and Snail, and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability, induced cell-cycle arrest, and decreased the proliferative capacity of TNBC, suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype, the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins, BMI-1 and Oct-4, and both prevented and eliminated mammosphere formation. To understand the mechanism of these results, the effect of zoledronic acid on established EMT regulator NF-κB was investigated. Zoledronic acid inhibited phosphorylation of RelA, the active subunit of NF-κB, at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter, providing a direct link between inactivation of NF-κB signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased, correlating modulation of EMT to decreased self-renewal. On the basis of these results, it is proposed that through inactivation of NF-κB, zoledronic acid reverses EMT, which leads to a decrease in self-renewal.

Effect of selumetinib on the growth of anastrozole-resistant tumors.

Despite significant improvement in the treatment outcome of hormone responsive postmenopausal breast cancer, some patients eventually acquire resistance to aromatase inhibitors (AIs). Using our MCF-7Ca xenograft model, we observed that although AIs such as anastrozole initially inhibit tumor growth effectively, tumors eventually began to grow. Our previous data show that anastrozole-resistant tumors upregulate growth factor receptor pathways as they adapt to grow in the low estrogen environment. Therefore, in the current study, we investigated the effect of inhibiting the growth factor receptor pathways with a MEK-1/2 inhibitor selumetinib (AZD6244, ARRY-142866). We treated the mice with anastrozole-resistant tumors with selumetinib alone or in combination with anastrozole. MCF-7Ca cells were inoculated sc into ovariectomized athymic nude mice supplemented throughout the experiment with androstenedione (100 µg/day), the substrate for aromatase conversion to estrogen. Once the tumors reached a measurable size (~300 mm(3)), the mice were treated with anastrozole (200 µg/day), supplemented with androstenedione (Δ(4)A). The tumors in the anastrozole group doubled in volume after 6 weeks, at which time the animals were regrouped to receive the following treatments: (i) anastrozole, (ii) anastrozole withdrawal (Δ(4)A alone), (iii) selumetinib (25 mg/kg/d, bid, po), and (iv) selumetinib + anastrozole, (n = 10 mice/group). The treatments were given for 6 weeks (till week 12) and then the mice were euthanized, the tumors were collected and analyzed. The tumors of mice treated with selumetinib + anastrozole had significantly lower growth rates than those treated with single agents (p = 0.008). Western blot analysis of the tumors showed that treatment with anastrozole resulted in upregulation of proteins in the growth factor receptor cascade such as p-mTOR, pAkt, pMEK, and pMAPK. This was accompanied by downregulation of ERα protein, consistent with previous findings. The treatment of mice with selumetinib resulted in downregulation of activated MAPK, along with p-mTOR, which likely resulted in upregulation of ERα. Our results suggest that inhibition of the growth factor receptor pathway with selumetinib can reverse anastrozole resistance.

The importance of HER2 signaling in the tumor-initiating cell population in aromatase inhibitor-resistant breast cancer.

Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless, a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways, particularly human epidermal growth factor receptor 2 (EGFR2/HER2), have been implicated in AI resistance. However, the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers, such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells, as well as from in vivo xenograft tumors, indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall, these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP, HER2, EGFR/HER1, and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance.

Inhibition of oxygen-induced hypoxia-inducible factor-1alpha degradation unmasks estradiol induction of vascular endothelial growth factor expression in ECC-1 cancer cells in vitro.

Estradiol (E(2)) rapidly and strongly induces vascular endothelial growth factor (VEGF) transcription in uterine endometrial epithelial cells in vivo. We have shown that this is mediated by both the estrogen receptor-alpha and hypoxia-inducible factor (HIF)-1alpha. By contrast, E(2) induces little or no VEGF expression in cultured breast or endometrial cancer cells, which lack HIF-1alpha due to the abnormally high concentration of oxygen ( approximately 20%) to which they are exposed. To test the hypothesis that restoring HIF-1alpha in cultured cells would restore the ability of E(2) to induce VEGF expression, we treated human endometrial cancer cells (ECC-1) with cobalt chloride (CoCl(2);100 microm), which prevents oxygen-induced HIF-1alpha degradation. HIF-1alpha was absent in untreated ECC-1 cells but detectable by 4 h after treatment with CoCl(2) alone, as was a significant increase in VEGF mRNA. E(2) plus CoCl(2) induced detectable HIF-1alpha expression at 2 h and an even higher level than that induced by CoCl(2) alone at 4 h; this HIF-1alpha was localized in the nuclei. This was accompanied by increasing VEGF expression, with the increase at 4 h severalfold higher than that induced by CoCl(2) alone and was concurrent with recruitment of both HIF-1alpha and estrogen receptor-alpha to the VEGF promoter. These results confirm that HIF-1alpha plays an essential role in E(2)-induced expression of VEGF. Through the induction of increased microvascular permeability and the consequent exudation of plasma growth factors, VEGF in turn may play an essential role in cancer cell proliferation in vivo.

Estrogen rapidly activates the PI3K/AKT pathway and hypoxia-inducible factor 1 and induces vascular endothelial growth factor A expression in luminal epithelial cells of the rat uterus.

We have previously shown that 17beta-estradiol (E(2)) increases vascular endothelial growth factor A (Vegfa) gene expression in the rat uterus, resulting in increased microvascular permeability, and that this involves the simultaneous recruitment of hypoxia-inducible factor 1 (HIF1) and estrogen receptor alpha (ESR1) to the Vegfa gene promoter. Both events require the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. However, those studies were carried out using whole uterine tissue, and while most evidence indicates that the likely site of E(2)-induced Vegfa expression is luminal epithelial (LE) cells, other studies have identified stromal cells as the site of that expression. To address this question, the pathway regulating Vegfa expression was reexamined using LE cells rapidly isolated after E(2) treatment. In addition, we further characterized the nature of the receptor through which E(2) triggers the signaling events that lead to Vegfa expression using the specific ESR1 antagonist ICI 182,780. In agreement with previous results in the whole uterus, E(2) stimulated Vegfa mRNA expression in LE cells, peaking at 1 h (4- to 14-fold) and returning to basal levels by 4 h. Treatment with E(2) also increased phosphorylation of AKT in LE cells, as well as of the downstream mediators FRAP1 (mTOR), GSK3B, and MDM2. The alpha subunit of HIF1 (HIF1A) was present in LE cells before E(2) treatment, was unchanged 1 h after E(2), but was >2-fold higher by 4 h. Chromatin immunoprecipitation analysis showed that HIF1A was recruited to the Vegfa promoter by 1 h and was absent again by 4 h. The E(2) activation of the PI3K/AKT pathway, HIF1A recruitment to the Vegfa promoter, and Vegfa expression were all blocked by ICI 182,780. In summary, the rapid E(2)-induced signaling events that lead to the expression of Vegfa observed previously using the whole uterus occur in LE cells and appear to be initiated via a membrane form of ESR1.

Estrogen-induced activation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor expression, and edema in the uterus are mediated by the phosphatidylinositol 3-kinase/Akt pathway.

Vascular endothelial growth factor (VEGF) plays an essential role in normal uterine physiology and function as well as endometrial cancer and other uterine disorders. Recently we showed that estrogen regulation of VEGF expression in the rat uterus involves rapid recruitment of both estrogen receptor (ER)-alpha and hypoxia-inducible factor (HIF)-1alpha to the VEGF promoter. Estrogen is known to stimulate both the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways, which have been linked to the activation of both of these transcription factors. Therefore, the involvement of these pathways in estrogen-induced VEGF expression was investigated. Inhibitors of the MAPK (U0126) or PI3K pathways (wortmannin or LY294002) were administered ip to immature female rats 1 h before 17beta-estradiol (E(2)) treatment. E(2) activation of both pathways occurred and was completely inhibited by the appropriate antagonist. Only PI3K inhibitors, however, blocked E(2) stimulation of VEGF mRNA expression and E(2)-induced uterine edema. In vivo chromatin immunoprecipitation analysis showed that this was associated with a failure of both HIF-1alpha and ERalpha to bind to the VEGF promoter. To determine whether inhibiting the PI3K pathway affected ERalpha induction of other estrogen target genes, the expression of creatine kinase B and progesterone receptor A/B was also examined. The expression of each was also inhibited by wortmannin, as was ERalpha binding to the creatine kinase B promoter. In conclusion, although estrogen activates both the MAPK and PI3K pathways in the rat uterus, activation of HIF-1alpha and ERalpha, and therefore regulation of VEGF gene expression is dependent only on the PI3K/Akt pathway. Furthermore, activation of the PI3K pathway appears to be a common requirement for the expression of estrogen-induced genes. These findings not only shed light on estrogen action in normal target tissues but also have important implications for cancer biology because excessive PI3K, HIF-1alpha, and VEGF activity are common in estrogen-dependent tumors.

New insight into the transcriptional regulation of vascular endothelial growth factor expression in the endometrium by estrogen and relaxin.

Increased uterine capillary permeability, which can be induced by both estrogen and relaxin, is required for endometrial growth and implantation. This effect is mediated in both cases by estrogen receptors (ERs), via stimulation of vascular endothelial growth factor (VEGF) expression. The sites on the VEGF promoter through which induction occurs, however, are completely unclear. We have used the technique of chromatin immunoprecipitation in vivo to localize the site of ER action and identify other transcription factors that are involved. We have found that ERa associates with Sp1/Sp3 at a GC-rich region of the promoter. More interesting, however, is the observation that estrogen also induces rapid, transient binding of hypoxia-inducible factor 1 (HIF-1), which mediates VEGF transcription in response to hypoxia, to the promoter. The estrogen-induced HIF-1 binding closely matches the estrogen-induced pattern of VEGF expression in the uterus, suggesting that HIF-1 is involved in that induction, and probably that of many other genes as well (HIF-1 is now known to regulate the expression of more than 40 genes). It is likely that studies now under way will also link relaxin-induced VEGF expression to HIF-1. This is based on the similarities in the effects of the two hormones on VEGF expression and on their shared ability to activate the PI3K and MAPK pathways, both of which can activate HIF-1.

Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor alpha and hypoxia-inducible factor 1 to the vascular endothelial growth factor promoter.

Vascular endothelial growth factor (VEGF) plays a pivotal role in the regulation of microvascular permeability and angiogenesis, processes essential for normal endometrial growth and implantation. Estrogen [17beta-estradiol (E2)], via its receptor (ER alpha), rapidly stimulates VEGF expression in the uterus at the transcriptional level. The VEGF gene promoter, however, lacks a consensus estrogen response element (ERE), and attempts to identify the site through which E2 induces VEGF expression have yielded contradictory results. To address this question, we modified the chromatin immunoprecipitation method to identify transcription factors that interact with the VEGF promoter in the rat uterus in response to E2. Chromatin immunoprecipitation showed that both Sp1 and Sp3 were associated with a proximal, GC-rich region of the promoter before E2 treatment. E2 induced an increase in Sp1 binding and the recruitment of ER alpha, and the coactivator p300 to this region. The association of ER alpha persisted, however, after VEGF mRNA levels had declined again (at 4 h), indicating that other factors might be involved in that expression. Western analysis showed that both the alpha- and beta-subunits of the transcription factor hypoxia-inducible factor 1 (HIF-1), which regulates VEGF expression in response to hypoxia and several hormones and growth factors, were present in the uterus. Furthermore, E2 rapidly induced their recruitment to the -944 to -611 bp region of the VEGF promoter, which contains the hypoxia response element to which HIF-1 binds. This binding was transient, matching the pattern of E2-induced VEGF expression. These results indicate that HIF-1 is an important mediator of E2-induced VEGF expression in the uterus. In addition, E2 also induced a later increase in HIF-1alpha mRNA and protein expression in the uterus, suggesting that it may be required for longer term effects of E2 on the uterus as well. In contrast to the uterus, HEC1A cells cultured in 95% air-5% CO2 (and therefore 20% O2) contained no HIF-1alpha, consistent with the inability of E2 to stimulate the expression of VEGF by these and other cell types in vitro.

Pulmonary vascular permeability and ischemic injury in gelsolin-deficient mice.

Gelsolin is a potent actin filament regulatory protein that controls cytoskeletal assembly and disassembly. Because cellular gelsolin deficiency leads to pronounced actin stress fiber formation and defective chemotaxis, and similar cytoskeletal remodeling results in endothelial barrier dysfunction, we hypothesized that gelsolin deficient mice would exhibit increased vascular permeability. To test this hypothesis, we compared baseline lung lavage (BAL) protein concentration, wet/dry weight ratio, and osmotic reflection coefficient for albumin (sigma alb) in gelsolin-deficient (gsn-/-) and C57BL/6 (wild-type) mice. In addition, we assessed lung permeability in response to ischemia by evaluating BAL protein concentration after 4, 8, or 24 h of left pulmonary arterial (LPA) occlusion, and lung wet/dry weight ratio and histology after 24 h of LPA occlusion, in gsn-/- and wild-type animals, as compared with control and sham-operated mice. Baseline measurements revealed that BAL protein concentration was 18-fold higher in gsn-/- than in wild-type mice, whereas sigma alb averaged 0.62 + 0.15 in wild-type, as compared with 0.31 + 0.05 in gsn-/- animals, indicating that gelsolin deficiency caused increased pulmonary vascular permeability. Ischemia increased lung permeability (BAL protein and lung wet/dry weight) in both wild-type and gsn-/- mice. However, whereas the fold-increase in BAL protein concentration was less in gsn-/- mice (2- to 4-fold) as compared with wild-type (22- to 34-fold), the duration of ischemia-induced permeability changes was prolonged. Lung wet/dry weight and gross histology following ischemia were comparable in wild-type and gsn-/- animals. These data suggest that gelsolin significantly contributes to maintenance of vascular barrier function in the lung.