Overexpression Of HIF-1alpha Research Articles

Abstract 454: The regulation of tumorigenesis by crosstalk between hypoxia and PPARdelta

Abstract Hypoxia, insufficient state of oxygen, is a feature of the tumor environment. Upon tumor growing, inside of tumor mass is hypoxic state. Hypoxia promotes tumor progression, expression of angiogenic mediators and tumor resistance to cancer therapy. Peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors, also play an important role in the regulation of cancer cell growth and angiogenesis. However, it has not been entirely elucidated if hypoxia can regulate PPARdelta activation during tumorigenesis. Therefore, we investigated the relationship between hypoxic stress and PPARdelta activation in tumor cells and the contribution of the crosstalk between these factors to tumor cell growth. Hypoxia (<1% O2) and a hypoxia-mimetic (deferoxamine) increased mRNA levels and protein expression of tumor-promoting mediators such as IL-8, VEGF, and COX-2 in colon cancer cells (HCT116), but not in PPARdelta-knockout HCT116 cells. A synthetic ligand of PPARdelta(GW501516) induced the expression of IL-8, VEGF, and COX-2 in wild-type colon cancer cells but not in PPARdelta-knockout cells. Hypoxia increased both transcriptional activity and mRNA level of PPARdelta in HCT116 cells. In addition, overexpression of HIF-1alpha enhanced transcriptional activity of PPARdelta suggesting that HIF-1alpha regulates the activation of PPARdelta. The proliferation and migration rate of macrophages were enhanced by conditioned media derived from wild-type colon cancer cells but not PPARdelta-knockout cells. These suggest that the expression of IL-8, VEGF, COX-2 induced by hypoxic stress is dependent on PPARdelta activity and that HIF-1alpha and PPARdelta cooperate to promote tumor cell growth. [Supported by a grant from Research Center for Biomolecular Nanotechnology, GIST] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 454.

Down‐regulation of hypoxia inducible factor‐1α: a possible explanation for the protective effects of estrogen on genioglossus fatigue resistance

Patients with obstructive sleep apnea/hypopnea syndrome (OSAHS) often exhibit fatigued or inefficient upper airway dilator muscle activity. It has been shown that estrogen may have some impact on upper airway contractility under normoxic conditions. Chronic intermittent hypoxia (CIH) is a frequent feature of OSAHS, and it may alter muscle susceptibility to oxidative stress, a characteristic of a fatigable nature. Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is responsible for the regulation of oxygen homeostasis under hypoxic conditions. We examined the effects of estrogen on the contractility of the genioglossus by exposing rats to alternating cycles of 6-8% O(2) every 15 s for a total duration of 35 d. The results showed that muscle fatigue resistance was significantly decreased after CIH but was partially reversed after estrogen treatment. Compared with the control group, real-time reverse transcription-polymerase chain reaction and western blotting showed higher levels of HIF-1alpha messenger RNA and protein in the CIH group, but estrogen treatment reduced, in a dose-independent manner, the levels of HIF-1alpha messenger RNA and protein in rats exposed to CIH. We conclude that CIH induced the expression of HIF-1alpha in the genioglossus and altered the physical properties towards a more fatigable phenotype, whereas estrogen inhibited the over-expression of HIF-1alpha, and this may account for the improvement of upper airway muscle endurance in CIH rats.

Hypoxic stress up‐regulates the expression of Toll‐like receptor 4 in macrophages via hypoxia‐inducible factor

Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

Leptin: Clue to poor appetite in oxygen-starved fish

Hypoxia is the most widespread deleterious consequence of eutrophication and has become a major cause of fishery decline. One feature of chronic exposure to hypoxia in fish is inhibition of feeding. In this study, we investigated if the gene that encodes the appetite-suppressing hormone leptin is regulated by hypoxia in zebrafish (Danio rerio). Exposure of adult zebrafish to hypoxic conditions (1+/-0.2 mg O(2) L(-1)) for 4 and 10 days significantly increased leptin-a (zlep-a) mRNA levels in the liver. To evaluate the role of hypoxia-inducible factor 1 (HIF-1) in regulating zlep-a expression, zebrafish embryos were exposed to cobalt chloride (CoCl(2), a HIF-1 inducer) and overexpressed with HIF-1alpha mRNA. Both CoCl(2) treatment and HIF-1alpha overexpression markedly increased zlep-a expression in developing embryos, indicating the possible involvement of HIF-1 in zlep-a regulation. In vivo promoter analysis indicated that zlep-a promoter activity is found in the muscle fibers of zebrafish embryos and enhanced by CoCl(2). This is the first report to show that leptin gene expression in fish is regulated by hypoxia possibly via the involvement of HIF-1.

Clinicopathological implications of expressions of hypoxia-related molecules in esophageal superficial squamous cell carcinoma

This study was conducted to clarify whether or not expressions of hypoxia-related molecules would have clinicopathological significance in squamous cell carcinoma (SCC) of the esophagus. Expressions of hypoxia inducible factor-1 alpha (HIF-1alpha), glucose transporter 1 (GLUT-1) and RAC-1 were immunohistochemically analyzed in 96 surgically resected SCCs at pT1b (sm1, 12 cases; sm2, 35 cases; sm3, 49 cases). They were divided into a lymph node metastasis (LNM)-positive group composed of 44 cases and an LNM-negative group composed of 52 cases. Immunohistochemical profiles were estimated based on the staining extent (score: 1+, 2+, 3+) and intensity (score: 1+, 2+, 3+). A significant expression pattern was found in the nucleus for HIF-1alpha, cell membrane for GLUT-1 and cytoplasm for RAC-1. The cases were categorized into a high score group (total score of 4 or more) and a low score group (total score of 3 or less) in each maker, respectively. A comparison made between the LNM-positive group and the LNM-negative group showed that the proportion of cases with a high score was larger in the LNM-positive group than in the LNM-negative group (HIF-1alpha, P = .02; GLUT-1, P = .008; RAC-1, P = .001). Among them, HIF-1alpha was found to be significantly related to the disease-free survival (P = .019) and overall survival (P = .034) as well as LNM (disease-free survival, P = .030; overall survival, P = .030). The multivariate analysis demonstrated that the HIF-1alpha expression would be an independent indicator for prognosis. In the superficial SCCs of the esophagus, GLUT-1 and RAC-1 may be involved in LNM, and HIF-1alpha overexpression is expected to predict an unfavorable clinical outcome.

Angiogenesis Induced by hVEGF165 Gene Controlled by Hypoxic Response Elements in Rabbit Ischemia Myocardium

Hypoxic response element (HRE) offers satisfactory control over expression of hVEGF(165) in cell levels. However, the characteristics of regenerated blood vessels induced by long-term expression of transferred hVEGF(165) under control of HRE in vivo remain unknown. This study aims to investigate the effect of HRE on control of long-term expression of rAAV-delivered hVEGF(165) gene to ischemic myocardium and evaluate characteristics of angiogenesis induced by hVEGF(165) in vivo. Rabbit ischemic heart models were established surgically, rAAV-9HRE-hVEGF(165) was transfected to ischemia hearts subjected to 12 week ischemia. Molecular biological and immunohistochemistry were employed to determine expressions of HIF-1alpha and hVEGF(165). Microvessel densities of CD31(+) and alpha-SMA(+) regenerated vessels were also evaluated. Expressions of both hVEGF(165) mRNA and protein were upregulated following over-expression of endogenous HIF-1alpha early after ischemia, peaked at 4-6 weeks post-MI, declined, and approached pre-ischemia level at the end of 12 weeks of ischemia (P < 0.01). The significantly upregulated CD31 in hVEGF(165)-treated hearts presented from 8 to 12 weeks of ischemia compared with the control (P < 0.01). However, alpha-SMA expression was rapidly downregulated after ischemia and remained lower than the control level by the end of 12 weeks post-MI (P < 0.01). Overexpression of hVEGF(165) controlled by HIF-1alpha-HRE system shows a stably regional angiogenic efficacy in vivo. But, VEGF, as an early angiogenic cytokine, is inadequate for mediating histologically mature vessels in ischemia myocardium.

Prognostic relevance of hypoxia inducible factor-1α expression in patients with melanoma

Overexpression of hypoxia inducible factor (HIF)-1alpha has been found in several human cancers and is thought to correlate with aggressive disease and poor response. A retrospective analysis was carried out on 89 patients with primary cutaneous melanoma. HIF-1alpha expression was assessed by immunohistochemistry in formalin-fixed, paraffin wax-embedded tumour sections. Overall survival (OS) and disease-free survival (DFS) were determined using univariate and multivariate analyses. Of the 89 patients, 78 (87.6%) expressed HIF-1alpha, and the remaining 11 patients (12.4%) did not. HIF-1alpha expression correlated with age (P = 0.002), but not with the main predictive factors in melanoma. Survival analysis disclosed no difference between the groups for OS and DFS. In multivariate analysis, only Breslow Index and ulceration were significantly associated with poor OS. Our results indicate that HIF-1alpha overexpression is present in most primary melanomas, but is not associated with clinicopathological variables, patient prognosis or survival.

Regulation of HIF-1α and VEGF by miR-20b Tunes Tumor Cells to Adapt to the Alteration of Oxygen Concentration

The regulation of HIF-1α is considered to be realized by pVHL-mediated ubiquitin-26S proteasome pathway at a post-transcriptional level. The discovery of a class of small noncoding RNAs, called microRNAs, implies alternative mechanism of regulation of HIF-1α. Here, we show that miR-20b plays an important role in fine-tuning the adaptation of tumor cells to oxygen concentration. The inhibition of miR-20b increased the protein levels of HIF-1α and VEGF in normoxic tumor cells; the increase of miR-20b in hypoxic tumor cells, nevertheless, decreased the protein levels of HIF-1α and VEGF. By using luciferase reporter vector system, we confirmed that miR-20b directly targeted the 3′UTR of Hif1a and Vegfa. On the other hand, the forced overexpression of HIF-1α in normoxic tumor cells downregulated miR-20b expression. However, HIF-1α knockdown in hypoxic tumor cells caused the increase of miR-20b. The differential expression of miR-20b has important biological significance in tumor cells, either enhancing the growth or favoring the survival of tumor cells upon the oxygen supply. Thus, we identify a novel molecular regulation mechanism through which miR-20b regulates HIF-1α and VEGF and is regulated by HIF-1α so to keep tumor cells adapting to different oxygen concentrations.

Construction of recombinant adenovirus vector containing a modified gene that codes for human hypoxia-inducible factor-1α without oxygen-dependent degradation domain

The expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in heart allografts is an important mechanism in response to ischemia/reperfusion (I/R) injury which represents the single major non-immunologic factor implicated in pathogenesis of chronic graft dysfunction (CGD). Adenoviral mediated overexpression of HIF-1alpha is a useful way to investigate the molecular mechanisms of I/R injury and the cardiac function during heart transplantation. The oxygen-dependent degradation (ODD) domain of HIF-1alpha can lead to degradation of the HIF-1alpha protein in normoxia. This will be an obstacle to steady expression of HIF-1alpha in heart allograft after transduction. In this study, we obtained the coding sequence of HIF-1alpha without ODD domain (HIF-1alphaDeltaODD) through a PCR-based method, and then generated the HIF-1alphaDeltaODD-expressing adenovirus. In normoxia, adenoviral mediated expression of HIF-1alphaDeltaODD shows constitutive activity in human cardiomyocytes, and can up-regulate heme oxygenase (HO)-1 mRNA levels significantly compared with the group transduced with HIF-1alpha-expressing adenovirus. The constructed HIF-1alphaDeltaODD-expressing adenovirus can be used to transduce allografts in animal studies to investigate the mechanism of CGD and provide a useful model to study the regulation mechanisms of genes regulated by HIF-1alpha alone.

Hypoxia-inducible factor-1α suppresses the expression of macrophage scavenger receptor 1

Macrophages are distributed in all peripheral tissues and play a critical role in the first line of the innate immune defenses against bacterial infection by phagocytosis of bacterial pathogens through the macrophage scavenger receptor 1 (MSR1). Within tissues, the partial pressure of oxygen (pO2) decreases depending on the distance of cells from the closest O2-supplying blood vessel. However, it is not clear how the expression of MSR1 in macrophages is regulated by low pO2. On the other hand, hypoxia-inducible factor (HIF)-1alpha is well known to control hypoxic responses through regulation of hypoxia-inducible genes. Therefore, we investigated the effects of hypoxia and HIF-1alpha on MSR1 expression and function in the macrophage cell line RAW264. Exposure to 1% O2 or treatment with the hypoxia-mimetic agent cobalt chloride (CoCl2) significantly suppressed the expression of MSR1 mRNA, accompanied by a markedly increase in levels of nuclear HIF-1alpha protein. The overexpression of HIF-1alpha in RAW264 cells suppressed the expression of MSR1 mRNA and protein, transcriptional activity of the MSR1 gene, and phagocytic capacity against the Gram-positive bacteria Listeria monocytogenes. The suppression of MSR1 mRNA by hypoxia or CoCl2 was inhibited by YC-1, an inhibitor of HIF-1alpha, or by the depletion of HIF-1alpha expression by small interference RNA. These results indicate that hypoxia transcriptionally suppresses MSR1 expression through HIF-1alpha.

Recepteur d'Origine Nantais Tyrosine Kinase Is a Direct Target of Hypoxia-inducible Factor-1α-mediated Invasion of Breast Carcinoma Cells

Hypoxia-inducible factor-1alpha (HIF-1alpha) overexpression was shown to be associated with invasion and metastasis of tumors and tumor cell lines. The identification of molecular targets that contribute to HIF-1alpha-mediated invasion is under intensive investigation. We have analyzed the role of recepteur d'origine nantais (RON), a tyrosine kinase receptor for macrophage-stimulating protein (MSP) that plays a role in breast cancer cell invasion as one of the molecular targets of HIF-1alpha. Analysis of a panel of breast cancer cell lines indicated a correlation between HIF-1alpha and RON expression. Treatment of HIF-1alpha- and RON-positive breast cancer cells with HIF-1alpha inhibitor, echinomycin, led to the inhibition of HIF-1alpha activity and RON expression. We have identified HIF-1alpha binding site on the RON promoter. Chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter confirmed the binding of HIF-1alpha to RON promoter. HIF-1alpha inhibitor-, echinomycin-, or short hairpin RNA-mediated selective knockdown of HIF-1alpha or HIF-1alpha target RON tyrosine kinase abrogated RON gene expression, and the RON ligand macrophage-stimulating protein mediated invasion of breast cancer cells. Consequently, the data presented herein demonstrated RON as a novel molecular target of HIF-1alpha and suggest a potential therapeutic role for HIF-1alpha or RON tyrosine kinase inhibitors in the blockade of RON tyrosine kinase-mediated invasion of carcinoma cells.

BCL-xL Is a Target Gene Regulated by Hypoxia-inducible Factor-1α

The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) plays pivotal roles in physiology and pathophysiology. Constitutive or hypoxia-induced HIF-1alpha overexpression is observed in many types of cancers including prostate adenocarcinoma, in which it is associated with resistance to apoptosis and therapeutic agents. BCL-xL, a hypoxia-responsive, anti-apoptotic protein of the Bcl-2 family, is also overexpressed in prostate carcinoma and many other cancers. Despite this connection, whether BCL-xL expression is directly regulated by HIF-1alpha is not known. We used prostate cancer PC-3 cell with constitutive high HIF-1alpha level as a model to address this important question. We first generated prostate cancer PC-3 cells in which HIF-1alpha was stably knocked-down (HIF-KD) by using small interference RNA. BCL-xL was dramatically decreased in HIF-KD PC-3 cells, in parallel with sensitization to apoptosis with caspase-3 activation as well as decreased cell proliferation. We then demonstrated that HIF-1alpha directly regulated BCL-xL transcription by binding to a hypoxia-responsive element in the BCL-xL promoter (-865 to -847) by reporter gene assay, chromatin immunoprecipitation, and electrophoretic mobility shift and supershift assays. HIF-1alpha-dependent BCL-xL overexpression may be an important mechanism by which HIF-1alpha protects prostate cancer cells from apoptosis and leads to treatment resistance.

Hypoxic stress‐induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine-elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr-1 and Sp1 contribute by stimulating the gene encoding the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla-derived PC12 cells. Hypoxia (5% O(2)) also activates hypoxia inducible factor (HIF) 1alpha, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1alpha over-expression also elevate PNMT promoter-driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1alpha over-expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1alpha inducers CoCl(2) or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1alpha expression construct show markedly higher levels of Egr-1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr-1 and Sp1 binding sites in the proximal -893 bp of PNMT promoter precludes HIF1alpha stimulation while a potential hypoxia response element (-282 bp) in the promoter shows weak HIF1alpha affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1alpha induction of Egr-1 and Sp1 rather than by co-activation by Egr-1, Sp1 and HIF1alpha. In addition, the rise in HIF1alpha protein leading to Egr-1 and Sp1 stimulation of PNMT appears to include HIF1alpha gene activation rather than simply prevention of HIF1alpha proteolytic degradation.

Estrogen and progestin regulate HIF-1α expression in ovarian cancer cell lines via the activation of Akt signaling transduction pathway

Our previous study revealed that estrogen regulates nm23-H1 expression thus promoting cell migration-invasion via activating PIK3/Akt pathway. In this study, we explored the effect of hormone on hypoxia-inducible factor-1 (HIF-1alpha), a key factor in cancer invasion and metastasis, via activation of Akt signaling transduction pathway. We treated two ovarian cancer cell lines ES-2 and SKOV3 with 17beta-estradiol, methoxyprogesterone acetate (MPA) only, or hormone combined with and Akt, MAPK pathway inhibitor, or transefected with siRNA targeting Akt sequenced with hormone. Expression of HIF-1alpha was measured by Western blotting. We observed the effect of hormone on nm23-H1 expression after the cells were transfected by siRNA targeting HIF-1alpha or treated with CoCl2 to induce HIF-1alpha overexpression. The 17beta-estradiol increased HIF-1alpha expression in ovarian cancer cells, and this upregulatory effect was abrogated by Akt inhibitor LY294002 (P<0.05) and Akt siRNA interference (P<0.05), but not affected by MAPK inhibitor PD980059 (P>0.05). MPA had the opposite effect. Nm23-H1 protein expression in ES-2 and SKOV3 cells were decreased after treatment with 17beta-estradiol (P<0.05), whereas MPA had the opposite effect. The effect was attenuated by HIF-1alpha siRNA (P<0.05) and enhanced by HIF-1alpha overexpression after CoCl2 treatment (P<0.05). Our data suggest that estrogen and progestin regulate HIF-1alpha expression via Akt signaling pathway, affecting nm23-H1 expression in influencing cell metastasis.

Increase in gene dosage is a mechanism of HIF‐1α constitutive expression in head and neck squamous cell carcinomas

The HIF-1alpha protein plays a key role in the cellular response to hypoxia via transcriptional regulation of genes involved in erythropoiesis, angiogenesis, and metabolism. Overexpression of HIF-1alpha is commonly found in solid tumors in significant association with increased patient mortality and resistance to therapy. The predominant mode of HIF-1alpha regulation by hypoxia occurs at the level of protein stability. In addition to hypoxia, HIF-1alpha protein stability and synthesis is regulated by nonhypoxic signals such as inactivation of tumor suppressors and activation of oncogenes. Here, we show that an increase in gene dosage may contribute to HIF-1alpha mRNA and protein overexpression in a nonhypoxic environment in head and neck squamous cell carcinomas (HNSCC). Increased HIF-1alpha gene dosage was found in one out of five HNSCC-derived cell lines and three out of 27 HNSCC primary tumors. Significantly, increased gene dosage in those samples was associated with high HIF-1alpha mRNA and protein levels. Normoxic overexpression of HIF-1alpha protein in HNSCC-derived cell lines was also paralleled by higher expression levels of HIF-1alpha target genes. Array CGH analysis confirmed the copy number increase of HIF-1alpha gene and revealed that the gene is contained within a region of amplification at 14q23-q24.2 both in the cell line and primary tumors. In addition, FISH analysis revealed the presence of 11-13 copies on a tetraploid background in SCC2 cells. These data suggest that increased HIF-1alpha gene dosage is a mechanism of HIF-1alpha protein overexpression in HNSCC that possibly prepares the cells for a higher activity in an intratumoral hypoxic environment.

The relation between hypoxia‐inducible factor (HIF)‐1alpha expression with p53 expression and outcome in surgically treated supraglottic laryngeal cancer

The purpose of this study was to examine whether a relationship exists between HIF-1alpha expression and the pro-apoptotic protein p53 in supraglottic laryngeal squamous cell carcinomas (SCCs), which could provide information concerning patient prognosis. The study population was composed of 106 previously untreated men with SCC of the supraglottic larynx. All the patients underwent surgical resection of the tumor and bilateral neck dissection. Immunohistochemical analysis of HIF-1alpha and p53 protein expression was performed in relation with clinicopathological parameters and prognosis. HIF-1alpha nuclear expression was detected in 71% of primary carcinomas and 55% of the paired lymph node metastases. There was a significant positive correlation between HIF-1alpha and T-classification but no associations were observed with other clinicopathological variables and with prognosis. There was no correlation between the expression of HIF-1alpha and p53. HIF-1alpha overexpression in combination with p53 immunostaining was not associated with disease recurrence or survival. The data suggest that HIF-1alpha expression does not have a prognostic value in surgically treated supraglottic laryngeal SCC, and that immunohistochemical determination of p53 does not allow improving the clinical significance of HIF-1alpha.

Induction of trefoil factor (TFF)1, TFF2 and TFF3 by hypoxia is mediated by hypoxia inducible factor‐1: implications for gastric mucosal healing

Mucosal microcirculation is compromised during gastric damage induced by non-steroidal anti-inflammatory drugs, such as aspirin. Consequently, oxygen supply to epithelial cells is decreased. The trefoil factor (TFF) peptides are involved in mechanisms of defence and repair in the gastrointestinal tract but their regulation at sites of gastric injury is unknown. Hypoxia and expression of TFF genes and peptides were measured in the damaged stomach of aspirin-treated rats. In a human gastric cell line (AGS cells), the effects of hypoxia and of hypoxia inducible factor (HIF)-1 (through transient transfection of HIF-1alpha siRNA or over-expression of HIF-1alpha) on TFF gene expression were evaluated. Hypoxyprobe immunostaining, up-regulation of TFF2 (1.9-fold) and TFF3 (1.8-fold) and a non-significant increase of TFF1 (1.5-fold) mRNA were observed in the damaged stomach of aspirin-treated rats, compared with control animals. Hypoxia (3% O(2), 16 h) induced mRNA for TFF1 (5.8-fold), TTF2 (9.1-fold) and TFF3 (9.3-fold) in AGS cells, an effect mediated by HIF-1, as transient transfection of HIF-1alpha siRNA reduced the effects of hypoxia. Over-expression of HIF-1alpha by transfection in non-hypoxic epithelial cells produced a similar pattern of TFF induction to that observed with hypoxia and transactivated a TFF1 reporter construct. Hypoxia inducible factor-1 mediated the induction of TFF gene expression by hypoxia in gastric epithelial cells. Low oxygen levels and up-regulation of TFF gene expression in the damaged stomach of aspirin-treated rats suggest that hypoxia induced expression of TFF genes at sites of gastric injury.

Mechanism of Cancer Cell Adaptation to Metabolic Stress: Proteomics Identification of a Novel Thyroid Hormone-mediated Gastric Carcinogenic Signaling Pathway

Gastric cancer is the second most common cancer worldwide and has a poor prognosis. To determine the mechanism of adaptation to metabolic stress in cancer cells, we used gastric cancer as a model system to reveal the potential signaling pathways involved. Two-dimensional polyacrylamide gel electrophoresis coupled with ESI-Q-TOF MS/MS analysis was used to identify differentially expressed proteins between gastric tumor tissues and the corresponding noncancerous tissues. In total, 107 spots with significant alteration (+/-over 2-fold, p < 0.05) were positively identified by MS/MS analysis. Altered expression of representative proteins was validated by RT-PCR and Western blotting. Cluster analysis of the changed proteins revealed an interesting group of metabolic proteins, which suggested accumulation of triiodothyronine (T(3); the major functional component of thyroid hormone) and overexpression of hypoxia-induced factor (HIF) in gastric carcinoma. These observations were further confirmed by electrochemiluminescence immunoassay and immunohistochemistry. T(3)-induced expression of HIF1-alpha and vascular endothelial growth factor was further verified using a gastric cancer cell line and in vivo mouse model. Because the early accumulation of HIF1-alpha was found to be independent of de novo transcription, we also found that the cytosolic cascade phosphatidylinositol 3-kinase/Akt pathway sensitive to T(3) stimulus was involved. Furthermore we demonstrated that T(3)-induced overexpression of HIF1-alpha was mediated by fumarate accumulation and could be enhanced by fumarate hydratase inactivation but inhibited by 2-oxoglutarate. These results provide evidence for alteration of metabolic proteins and dysfunction of thyroid hormone regulation in gastric tumors, and a novel thyroid hormone-mediated tumorigenic signaling pathway is proposed. Our findings are considered a significant step toward a better understanding of adaptations to metabolic stress in gastric carcinogenesis.

C-Myc- Dependent Stabilization of Hif-1alpha in MM: Therapeutic Implications

Bone marrow (BM) angiogenesis has been associated with multiple myeloma (MM) progression. Here we demonstrate that oncogenic c-Myc, also prominently associated with adverse prognosis in MM, regulates Hif-1α promoter activity, Hif-1alpha protein levels, and consequently the expression of pro-angiogenic target genes such as VEGF in MM cells. As observed with c-Myc knockdown, anti-MM agents with known anti-angiogenic activity including bortezomib, lenalidomide, and enzastaurin also downregulate Hif-1alpha and VEGF. Moreover, tyrophostin adaphostin induces similar effects, associated with anti-angiogenic activity, in a zebrafish model. The correlation between c-Myc, Hif-1alpha and tumor angiogenesis was further confirmed in a xenograft mouse model of human MM. Mechanistically, our in vitro data show that oncogenic c-Myc stabilizes caspase-8, and thereby protects Hif-1alpha against caspase-8- dependent degradation and attenuation of VEGF secretion. These sequelae also contribute to enhanced VEGF secretion triggered by MM cell-bone marrow stromal cell binding. Indeed, conditioned media from adaphostin- treated co-cultures inhibit endothelial cell growth and tubule formation. In addition, HIF1alpha gene demonstrated significant increased expression (p=0.02, Mann Whitney test) in tumor cells versus plasma cells derived from healthy donors. Importantly, when tested on a dataset of 559 patients, a significant link between HIF1alpha overexpression and poor prognosis was found (p=0,0069). These data further point to the possible relevance of HIF1alpha in the progression of a subset of MM patients. In summary, our data provide strong evidence of a key role of Hif-1alpha in MM angiogenesis and the therapeutic potential of specific Hif-1alpha and c-Myc inhibitors. Moreover they demonstrate additional biologic sequelae of novel MM therapies.

Hypoxia-inducible factor 1 alpha expression increases during colorectal carcinogenesis and tumor progression

BackgroundHypoxia-inducible factor 1 alpha (HIF-1α) is involved in processes promoting carcinogenesis of many tumors. However, its role in the development of colorectal cancer is unknown. To investigate the significance of HIF-1α during colorectal carcinogenesis and progression we examined its expression in precursor lesions constituting the conventional and serrated pathways, as well as in non-metastatic and metastatic adenocarcinomas.MethodsImmunohistochemistry and Western blot is used to analyse HIF-1α expression in normal colonic mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well as in non-metastatic and metastatic colorectal adenocarcinomas. Eight colorectal carcinoma cell lines are tested for their HIF-1α inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry.ResultsIn normal mucosa, HPP and TA-LGD HIF-1α was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1α were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1α overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1α expression was strongly accumulated in perinecrotic regions. In these cases HIF-1α activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1α was also seen in periiflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1α expression and nuclear translocation.ConclusionWe conclude that HIF-1α expression occurs in early stages of colorectal carcinogenesis and achieves a maximum in the invasive stage independent of the metastatic status. Perinecrotic activation of HIF-1α in invasive tumors underlines a dual role of HIF-1α by regulating both pro-survival and pro-death processes. HIF-1α up-regulation in response to LPS-mediated stimulation and periinflammatory expression in invasive carcinomas suggest its involvement in inflammatory events. These patterns of HIF-1α inducibility could contribute indirectly to the acquisition of a metastatic phenotype.