The enzyme 15-lipoxygenase-2 (15-LOX-2) utilizes arachidonic acid, a polyunsaturated fatty acid, to synthesize 15(S)-hydroxyeicosatetraenoic acid (HETE). Abundantly expressed in normal prostate epithelium but frequently suppressed in the cancerous tissues, 15-LOX-2 has been suggested as a functional suppressor of prostate cancer, but the mechanism(s) involved remains unknown. To study the functional role of 15-LOX-2 in prostate cancer, we expressed 15-LOX-2 as a fusion protein with GFP in DU145 and PC-3 cells and found that 15-LOX-2 increased cell cycle arrest at G0/G1 phase. When injected into athymic nu/nu mice, prostate cancer cells with 15-LOX-2 expression could still form palpable tumors without significant changes in tumorigenicity. But, the tumors with 15-LOX-2 expression grew significantly slower than those derived from vector controls and were kept dormant for a long period of time. Histological evaluation revealed an increase in cell death in tumors derived from prostate cancer cells with 15-LOX-2 expression, while in vitro cell culture conditions, no such increase in apoptosis was observed. Further studies found that the expression of vascular endothelial growth factor A (VEGF-A) was significantly reduced in prostate cancer cells with 15-LOX-2 expression restored. Our studies suggest that 15-LOX-2 suppresses VEGF gene expression and sustains tumor dormancy in prostate cancer. Loss of 15-LOX-2 functionalities, therefore, represents a key step for prostate cancer cells to exit from dormancy and embark on malignant progression in vivo.
tumor dormancy; angiogenesis; lipoxygenase; prostate cancer; VEGF
15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate and often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we established prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks the arachidonic acid metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67+ cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem/progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia (PIN) or carcinoma and, mechanistically, prostate lobes (especially those of the 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-βgal, p27Kip1 and HP1γ staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.
15-lipoxygenase 2; prostate; hyperplasia; senescence; tumor suppression; stem cells
Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).
RESEARCH DESIGN AND METHODS
Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium–derived factor (PEDF). Liquid chromatography–mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes.
Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE.
12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.
The neuroprotective effect of baicalein is generally attributed to inhibition of
12/15-lipoxygenase (12/15-LOX) and suppression of oxidative stress, but recent
studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α)
through inhibition of prolyl hydrolase 2 (PHD2) and activation of the
phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway. Yet, the
significance and regulation of prosurvival cytokines erythropoietin (Epo) and
vascular endothelial growth factor (VEGF), two transcriptional targets of HIF1α,
in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown.
Here we investigated the causal relationship between the PI3K/Akt signaling
pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary
rat cortical neurons and astrocytes. Our results show that baicalein induced Epo
and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons.
Baicalein also protected neurons against excitotoxicity in a PI3K- and
Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast,
at least a 10-fold higher concentration of baicalein was needed to induce
Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of
neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo
expression requires HIF1α, while PI3K/Akt signaling had little role in
baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct
mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes
for neuroprotection, and provide new insights into the mechanisms and potential
of baicalein in treating brain injury in vivo.
Hypoxia‐inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.
ARPE‐19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme‐linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real‐time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.
HIF‐1α was readily produced by ARPE‐19 cells under hypoxia, but HIF‐2α and HIF‐3α could not be detected even after HIF‐1α silencing. HIF‐1α protein levels showed an increasing trend for the first 24 h while HIF‐1α mRNA levels fluctuated during this time. After 36 h HIF‐1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF‐1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.
HIF‐1α appears to be the main isoform of HIF functioning in ARPE‐19 cells. Under hypoxia, HIF‐1α levels are likely self‐regulated by a feedback loop that involves both transcriptional and post‐translational mechanisms. VEGF production by human RPE cells is regulated by HIF‐1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.
diabetic retinopathy; VEGF; erythropoietin; hypoxia‐inducible factor; proline hydroxylase
15-Lipoxygenase-1 (15-LOX-1) is an inducible and highly regulated enzyme in normal human cells that plays a key role in the production of lipid signaling mediators, such as 13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid. 15-LOX-1 significantly contributes to resolution of inflammation and to terminal differentiation of normal cells. 15-LOX-1 is downregulated in human colorectal polyps and cancers. Emerging data support a tumor suppressor role for 15-LOX-1, especially in colon cancer. These data indicate that 15-LOX-1 promotes various antitumorigenic events, including cell differentiation and apoptosis, and inhibits chronic inflammation, angiogenesis, and metastasis. The transcriptional repression of 15-LOX-1 in colon cancer cells is complex and involves multiple mechanisms (e.g., histone methylation, transcriptional repressor binding). Re-expression of 15-LOX-1 in colon cancer cells can function as an important therapeutic mechanism and could be further exploited to develop novel treatment approaches for this common cancer.
15-LOX-1; linoleic acid; apoptosis; colon cancer
This study investigated the role of LOX in promoting invasion and metastasis of epithelial ovarian cancer in a hypoxic environment and its specific signal transduction pathway. Immunohistochemical detection of HIF-1α and LOX protein expression was performed on formalin-fixed paraffin sections of normal ovary, benign ovarian tumors, borderline and malignant epithelial ovarian tumor paraffin sample, using Mann-Whitney U test for independent comparisons and Wilcoxon signed-ranks test for paired comparisons. HIF-1α and LOX were knocked down in epithelial ovarian cancer cells (EOC), and HIF-1α/LOX regulation mechanism and LOX catalytic activity under hypoxia/reoxygenation microenvironment were explored. Cell migration and invasion ability in LOX inhibited HO8910 cells were investigated under hypoxia/reoxygenation conditions, using matrigel cell invasion and migration assays. We found that HIF-1α and LOX are highly expressed in epithelial ovarian cancer tissues, and the expression of both proteins is significantly correlated with the tumor grade, tumor diameter and lymph node metastasis. HIF-1α expression is positively correlated with the expression of LOX. Specifically, the expression of LOX and HIF-1α markedly increases under hypoxic conditions and decreases after reoxygenation. siRNA knockdown of LOX or β-aminoproprionitrile (βAPN), an inhibitor of LOX activity, that attenuates LOX activity, downregulates HIF-1α protein expression and inhibits HO8910 migratory and invasive abilities. LOX catalytic activity is significantly reduced under hypoxic conditions. Moreover, EOC cells display a marked increase in LOX-dependent FAK/AKT activation and cell migration following hypoxia/reoxygenation. Collectively, our study demonstrates that the hypoxia-HIF-1α, LOX-FAK/AKT pathway regulates the migration and invasion of epithelial ovarian cancer cells under hypoxia/reoxygenation conditions, thus, promoting metastasis of ovarian cancer.
lysyl oxidase; ovarian cancer; metastasis; hypoxia; hypoxia-inducible factor; reoxygenation
The von Hippel-Lindau tumor suppressor pVHL regulates the stability of Hypoxia-Inducible Factors (HIF) -1 and –2, oxygen-sensitive basic helix-loop-helix transcription factors, which mediate the hypoxic induction of angiogenic growth factors such as vascular endothelial growth factor (VEGF). Loss of VHL function results in constitutive activation of HIF-1 and HIF-2 and is associated with the development of highly vascularized tumors in multiple organs. We have used a conditional gene targeting approach to investigate the relative contributions of HIF-1 and HIF-2 to VHL-associated vascular tumorigenesis in a mouse model of liver hemangiomas. Here we demonstrate genetically that conditional inactivation of HIF-2α suppressed the development of VHL-associated liver hemangiomas and that angiogenic gene expression in hepatocytes is predominantly regulated by HIF-2 and not by HIF-1. These findings suggest that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors and that pharmacologic targeting of HIF-2 may be an effective strategy for their treatment.
► AAI increases whereas OTA decreases production of proangiogenic VEGF. ► The upregulation of VEGF expression by AAI is caused by induction of SP-1 and HIFs. ► Hypoxia prevents OTA-diminished VEGF production ► The effect of hypoxia on OTA-reduced VEGF is mediated by HIF-2α but not HIF-1α.
Aristolochic acid I (AAI) and ochratoxin A (OTA) cause chronic kidney diseases. Recently, the contribution of hypoxic injuries and angiogenic disturbances to nephropathies has been suggested, but underlying mechanisms have not been fully clarified yet.
In porcine kidney epithelial cell line, LLC-PK1 cells, treatment with non-toxic doses of AAI increased whereas with OTA decreased production of vascular endothelial growth factor (VEGF), the angiogenic factor with well-defined functions in kidney. Moreover, the activity of transcription factors regulating VEGF expression was differentially affected by examined compounds. Activity of hypoxia inducible factors (HIFs) and SP-1 was increased by AAI but diminished by OTA. Interestingly, AP-1 activity was inhibited while NFκB was not influenced by both toxins. Mithramycin A, a SP-1 inhibitor, as well as chetomin, an inhibitor of HIFs, reversed AAI-induced up-regulation of VEGF synthesis, indicating the importance of SP-1 and HIFs in this effect. Additionally, adenoviral overexpression of HIF-2α but not HIF-1α prevented OTA-diminished VEGF production suggesting the protective effect of this isoform towards the consequences exerted by OTA.
These observations provide new insight into complex impact of AAI and OTA on angiogenic gene regulation. Additionally, it adds to our understanding of hypoxia influence on nephropathies pathology.
AA, aristolochic acid; AAI, aristolochic acid I; AAII, aristolochic acid II; AA-ATN, aristolochic acid-induced acute tubular necrosis; AAN, aristolochic acid-induced nephropathy; AdGFP, adenoviral vectors containing GFP cDNA; AdHIF-1,-2α, adenoviral vectors containing HIF-1,-2α cDNA; β-gal, β-galactosidase; BEN, Balkan endemic nephropathy; CKDs, chronic kidney diseases; EMT, epithelial to mesenchymal cell transformation; GFP, green fluorescent protein; HIF, hypoxia inducible factor; HRE, hypoxia responsive element; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; LLC-PK1, porcine kidney epithelial cell line; IARC, The International Agency for Research on Cancer; OTA, ochratoxin A; ROS, reactive oxygen species; RT, room temperature; TGFβ, transforming growth factor β; VEGF, vascular endothelial growth factor; Nephropathy; Kidney diseases; Vascular endothelial growth factor; Angiogenesis; Hypoxia; LLC-PK1
Growing tumors are hypoxic and respond to microenvironmental stress through increased expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor, resulting in an adaptive switch to glycolytic metabolism, angiogenic signaling, survival, and metastasis. HIF-1α expression is associated with tumor resistance to cytotoxic therapy and inferior patient outcomes. Pancreatic cancer is the most hypoxic of all solid tumors and remains refractory to current chemoradiotherapy. We have seen nuclear HIF-1α in 88% of human pancreatic ductal carcinoma but in only 16% of normal pancreas. Stroma adjacent to the pancreatic ductal carcinoma also showed HIF-1α in 43% of cases. We investigated the novel selective HIF-1α inhibitor PX-478 on in vitro and in vivo radiation response of human pancreatic cancer models. Inhibition of HIF-1α by PX-478 increased cell killing by radiation. In mice with Panc-1, CF-PAC-1, or SU.86.86 pancreatic xenografts, concurrent administration of PX-478 potentiated the antitumor effects of fractionated radiation, with or without combined treatment with 5-fluorouracil or gemcitabine. Alternative sequencing of PX-478 with fractionated radiotherapy suggests optimal radiosensitization with concurrent or neoadjuvant administration of drug. Early tumor responses to combined PX-478/radiation treatment could be rapidly and repeatedly quantified by vascular imaging biomarkers. Dual-tracer dynamic contrast enhanced-magnetic resonance imaging and ultrasound imaging discriminated response to combined treatment prior to detection of differences in anatomic tumor size at 10 days posttreatment. Therefore, PX-478 is a mechanistically appealing and potentially clinically relevant enhancer of pancreatic cancer radiosensitivity, inhibiting tumor and stromal HIF-1 proangiogenic signaling and reducing the innate radiation resistance of hypoxic tumor cells.
Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined that betulinic acid (BA), a triterpene from the bark of white birch, had the inhibitory effects on hypoxia-mediated activation of STAT3 in androgen independent human prostate cancer PC-3 cells.
BA inhibited the protein expression and the transcriptional activities of hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. Consistently, BA blocked hypoxia-induced phosphorylation, DNA binding activity and nuclear accumulation of STAT3. In addition, BA significantly reduced cellular and secreted levels of vascular endothelial growth factor (VEGF), a critical angiogenic factor and a target gene of STAT3 induced under hypoxia. Furthermore, BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxic PC-3 cells, implying anti-angiogenic activity of BA under hypoxic condition. Of note, chromatin immunoprecipitation (ChiP) assay revealed that BA inhibited binding of HIF-1α and STAT3 to VEGF promoter. Furthermore, silencing STAT3 using siRNA transfection effectively enhanced the reduced VEGF production induced by BA treatment under hypoxia.
Taken together, our results suggest that BA has anti-angiogenic activity by disturbing the binding of HIF-1α and STAT3 to the VEGF promoter in hypoxic PC-3 cells.
Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor involved in the adaptation of mammals to hypoxia and plays a crucial role in cancer angiogenesis. Recent evidence suggests a leading role for HIF-1 in various inflammatory and infectious diseases. Here we describe the role of HIF-1 in Staphylococcus aureus infections by investigating the HIF-1-dependent host cell response. For this purpose, transcriptional profiling of HIF-1α-deficient HepG2 and control cells, both infected with Staphylococcus aureus, was performed. Four hours after infection, the expression of 190 genes, 24 of which were regulated via HIF-1, was influenced. LOX (encoding lysyl oxidase) was one of the upregulated genes with a potential impact on the course of S. aureus infection. LOX is an amine oxidase required for biosynthetic cross-linking of extracellular matrix components. LOX was upregulated in vitro in different cell cultures infected with S. aureus and also in vivo, in kidney abscesses of mice intravenously infected with S. aureus and in clinical skin samples from patients with S. aureus infections. Inhibition of LOX by β-aminopropionitrile (BAPN) did not affect the bacterial load in kidneys or blood but significantly influenced abscess morphology and collagenization. Our data provide evidence for a crucial role of HIF-1-regulated LOX in abscess formation.
The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.
Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase.
5-Lipoxygenase; Prostate cancer; Apoptosis; PKC-epsilon
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
Intratumoral hypoxia, a frequent finding in metastatic cancer, results in the activation of the hypoxia-inducible factors (HIFs). HIFs are implicated in many steps of breast cancer metastasis, including metastatic niche formation through induction of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins, enzymes that remodel collagen at the metastatic site and recruit bone marrow-derived cells (BMDCs) to the metastatic niche. We investigated the effect of two chemically and mechanistically distinct HIF inhibitors, digoxin and acriflavine, on breast cancer metastatic niche formation. Both drugs blocked the expression of LOX and LOXL proteins, collagen cross-linking, CD11b+ BMDC recruitment, and lung metastasis in an orthotopic breast cancer model. Patients with HIF-1α-overexpressing breast cancers are at increased risk of metastasis and mortality and our results suggest that such patients may benefit from aggressive therapy that includes a HIF inhibitor.
Hypoxia-inducible factor 1; Lysyl oxidase; Bone marrow derived cells; Breast cancer metastasis; Metastatic niche; HIF inhibitor
Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). Under hypoxia, HIFs regulate a variety of pro-angiogenic and pro-glycolysis pathways. In solid cancers, regions of hypoxia are commonly present throughout the tissue because of the chaotic vascular architecture and regions of necrosis. In these regions, the hypoxic state fluctuates in a spatial and temporal manner. Transient hypoxic cycling causes an increase in the activity of the HIF proteins above what is typical for non-pathologic tissue. The extent of hypoxia strongly correlates to poor patient survival, therapeutic resistance and an aggressive tumour phenotype, but the full contribution of hypoxia and the HIFs to tumour biology is an area of active investigation. Recent reports link resistance to conventional therapies and the metastatic potential to a stem-like tumour population, termed cancer stem cells (CSCs). We and others have shown that within brain tumours CSCs reside in two niches, a perivascular location and the surrounding necrotic tissue. Restricted oxygen conditions increase the CSC fraction and promote acquisition of a stem-like state. Cancer stem cells are critically dependant on the HIFs for survival, self-renewal, and tumour growth. These observations and those from normal stem cell biology provide a new mechanistic explanation for the contribution of hypoxia to malignancy. Further, the presence of hypoxia in tumours may present challenges for therapy because of the promotion of CSC phenotypes even upon successful killing of CSCs. The current experimental evidence suggests that CSCs are plastic cell states governed by microenvironmental conditions, such as hypoxia, that may be critical for the development of new therapies targeted to disrupt the microenvironment.
HIF; hypoxia; cancer stem cell
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator composed of HIF-1α and HIF-1β subunits. Several dozen HIF-1 targets are known, including the gene encoding vascular endothelial growth factor (VEGF). Under hypoxic conditions, HIF-1α expression increases as a result of decreased ubiquitination and degradation. The tumor suppressors VHL (von Hippel-Lindau protein) and p53 target HIF-1α for ubiquitination such that their inactivation in tumor cells increases the half-life of HIF-1α. Increased phosphatidylinositol 3-kinase (PI3K) and AKT or decreased PTEN activity in prostate cancer cells also increases HIF-1α expression by an undefined mechanism. In breast cancer, increased activity of the HER2 (also known as neu) receptor tyrosine kinase is associated with increased tumor grade, chemotherapy resistance, and decreased patient survival. HER2 has also been implicated as an inducer of VEGF expression. Here we demonstrate that HER2 signaling induced by overexpression in mouse 3T3 cells or heregulin stimulation of human MCF-7 breast cancer cells results in increased HIF-1α protein and VEGF mRNA expression that is dependent upon activity of PI3K, AKT (also known as protein kinase B), and the downstream kinase FRAP (FKBP-rapamycin-associated protein). In contrast to other inducers of HIF-1 expression, heregulin stimulation does not affect the half-life of HIF-1α but instead stimulates HIF-1α synthesis in a rapamycin-dependent manner. The 5′-untranslated region of HIF-1α mRNA directs heregulin-inducible expression of a heterologous protein. These data provide a molecular basis for VEGF induction and tumor angiogenesis by heregulin-HER2 signaling and establish a novel mechanism for the regulation of HIF-1α expression.
The hypoxia inducible transcription factors (HIFs) control many mediators of vascular response, including both angiogenic factors and small molecules such as nitric oxide (NO). In studying how endothelial HIF response itself affects metastasis, we found that loss of HIF-1α in endothelial cells reduces NO synthesis, retards tumor cell migration through endothelial layers, and restricts tumor cell metastasis, and that loss of HIF-2α has in each case the opposite effect. This results from differential regulation of NO homeostasis that in turn regulates vascular endothelial growth factor expression in an NO-dependent feedback loop. These opposing roles for the two HIF factors indicate that both they and endothelial cells regulate metastasis as malignancy progresses.
► Endothelial cell hypoxic response is a critical determinant of metastatic success ► HIF isoforms differentially regulate NO homeostasis in endothelium ► iNOS is essential for hypoxic NO production in endothelial cells ► HIF isoforms in endothelium differentially promote and hinder metastasis
One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.
colon cancer; gastrin; intracellular signaling; VEGF
The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.
A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.
Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.
Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.
Hypoxia Inducible Factor (HIF) is a master heterodimeric transcriptional regulator of oxygen (O2) homeostasis critical to proper angiogenic responses. Due to the distinctive coexpression of HIF-1α and HIF-2α subunits in endothelial cells, our goal was to examine the genetic elimination of HIF transcriptional activity in response to physiological hypoxic conditions by using a genetic model in which the required HIF-β subunit (ARNT, Aryl hydrocarbon Receptor Nuclear Translocator) to HIF transcriptional responses was depleted. Endothelial cells (ECs) and aortic explants were isolated from ArntloxP/loxP mice and infected with Adenovirus -Cre/GFP or control -GFP. We observed that moderate levels of 2.5% O2 promoted vessel sprouting, growth, and branching in control aortic ring assays while growth from Adenovirus -Cre infected explants was compromised. Primary Adenovirus -Cre infected EC cultures featured adverse migration and tube formation phenotypes. Primary pulmonary or cardiac ARNT-deleted ECs also failed to proliferate and survive in response to 8 or 2.5% O2 and hydrogen peroxide treatment. Our data demonstrates that ARNT promotes EC migration and vessel outgrowth and indispensible for the proliferation and preservation of ECs in response to the physiological environmental cue of hypoxia. Thus, these results demonstrate that ARNT plays a critical intrinsic role in ECs and support a critical role for the collaboration of HIF-1 and HIF-2 transcriptional activity in these cells.
Angiogenesis; ARNT; HIF; physiological hypoxia; endothelium
Genetic and biochemical evidence suggests a functional link between human 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3) in normal differentiation of the epidermis; LOX-derived fatty acid hydroperoxide is isomerized by the atypical eLOX3 into a specific epoxyalcohol that is a potential mediator in the pathway. Mouse epidermis expresses a different complement of LOX enzymes, and therefore this metabolic linkage could differ. To test this concept, we compared the substrate specificities of recombinant mouse and human eLOX3 toward sixteen hydroperoxy stereoisomers of arachidonic and linoleic acids. Both enzymes metabolized R-hydroperoxides 2–3 times faster than the corresponding S enantiomers. Whereas 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) is the best substrate for human eLOX3 (2.4 sec−1; at 30 µM substrate), mouse eLOX3 shows the highest turnover with 8R-HPETE (2.9 sec−1) followed by 8S-HPETE (1.3 sec−1). Novel product structures were characterized from reactions of mouse eLOX3 with 5S-, 8R-, and 8S-HPETEs. 8S-HPETE is converted specifically to a single epoxyalcohol, identified as 10R-hydroxy-8S,9S-epoxyeicosa-5Z,11Z,14Z-trienoic acid. The substrate preference of mouse eLOX3 and the unique occurrence of an 8S-LOX enzyme in mouse skin point to a potential LOX pathway for the production of epoxyalcohol in murine epidermal differentiation.
hepoxilin; epoxyalcohol; HPETE; fatty acid hydroperoxide; epidermis; differentiation; ichthyosis
β-arrestins 1 and 2 are multifunctional adaptor proteins originally discovered for their role in desensitizing seven-transmembrane receptor signaling via the heterotrimeric guanine nucleotide binding proteins. Recently identified roles of β-arrestins include regulation of cancer cell chemotaxis and proliferation. Herein we report that β-arrestin1 expression regulates breast tumor colonization in nude mice and cancer cell viability during hypoxia. β-arrestin1 robustly interacts with nuclear hypoxia-induced factor 1α (HIF-1α) that is stabilized during hypoxia and potentiates HIF-1-dependent transcription of the angiogenic factor VEGF-A. Increased expression of β-arrestin1 in human breast cancer (infiltrating ductal carcinoma or IDC and metastatic IDC) correlates with increased levels of VEGF-A. While the anti-angiogenic drug thalidomide inhibits HIF-1-dependent VEGF transcription in breast carcinoma cells, it does not prevent HIF-1α stabilization, but leads to aberrant localization of HIF-1α to the perinuclear compartments and surprisingly stimulates nuclear export of β-arrestin1. Additionally, imatinib mesylate that inhibits release of VEGF induces nuclear export of β-arrestin1-HIF-1α complexes. Our findings suggest that β-arrestin1 regulates nuclear signaling during hypoxia to promote survival of breast cancer cells via VEGF signaling and that drugs that induce its translocation from the nucleus to the cytoplasm could be useful in anti-angiogenic and breast cancer therapies.
Arrestin; HIF-1; VEGF; hypoxia