Hypoxia-inducible Factor 1-alpha Inhibitor Research Articles

Impact of hypoxia on glucose metabolism and hypoxia signaling pathways in juvenile horseshoe crabs Tachypleus tridentatus

Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.

HIF-1 inhibitor PX-478 for cancer therapy and other fields

Hypoxia-inducible factor 1-alpha (HIF-1) is a crucial transcription factor for tumor growth and progression. PX-478, a small molecule HIF-1 inhibitor, has been extensively studied for its potential as an anti-cancer agent in preclinical studies. This paper provides an overview of the current status of PX-478 in cancer treatment, with a focus on its application in treating pancreatic ductal adenocarcinoma (PDAC), lung cancer, breast cancer, glioma, and other kinds of cancer. This article discusses the mechanisms of action, pharmacokinetics, and pharmacodynamics of PX-478, as well as the findings of preclinical and clinical studies conducted to date. The evidence suggests that PX-478 has promising potential as a therapeutic agent for various cancer types. However, further research is needed to fully comprehend the compound's potential benefits and limitations and its optimal clinical application.

CXCR4, regulated by HIF1A, promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45+ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.

CircSLC8A1 targets miR-181a-5p/HIF1AN pathway to inhibit the growth, migration and extracellular matrix deposition of human keloid fibroblasts.

Circular RNAs (circRNAs) are identified as important regulators in human diseases, including keloid. The purpose of this study is to reveal the role and molecular mechanism of circSLC8A1 in keloid formation. Expression of circSLC8A1, microRNA (miR)-181a-5p, and hypoxia inducible factor 1 alpha inhibitor (HIF1AN) were detected by quantitative real-time PCR. Protein expression of extracellular matrix (ECM) deposition markers and HIF1AN was detected by western blot analysis. Furthermore, the interaction between miR-181a-5p and circSLC8A1 or HIF1AN was confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Expression of circSLC8A1 was downregulated in keloid tissues and HKFs. Overexpression of circSLC8A1 suppressed HKFs proliferation, migration, ECM deposition, and promoted apoptosis. MiR-181a-5p is targeted by circSLC8A1, and its mimic reversed the effect of circSLC8A1 on the biological function of HKFs. HIF1AN was a target of miR-181a-5p, and it was positively regulated by circSLC8A1. Knockdown of HIF1AN also reversed the negatively regulation of circSLC8A1 on the biological functions of HKFs. Our data showed that circSLC8A1 regulates the miR-181a-5p/HIF1AN axis to restrain HKFs biological functions, confirming that circSLC8A1 might serve as a novel therapeutic target for keloids.

Ubiquitin protein E3 ligase ASB9 suppresses proliferation and promotes apoptosis in human spermatogonial stem cell line by inducing HIF1AN degradation

BackgroundSpermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered.MethodsTo explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence.ResultsThe results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis.ConclusionASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.

Epithelial Transcription Factor FOXA1 Regulates Prostate Cancer Immune Response

Background: While localized prostate cancer (PCa) can be mitigated by surgery and radiation, metastatic PCa remains a challenge to treat. Androgen deprivation therapies and androgen receptor (AR) pathway inhibitors are mainstay treatments for advanced PCa. Yet, resistance often develops leading to castration-resistant prostate cancer (CRPC). Forkhead Box A1 (FOXA1) is a pioneer transcription factor that plays pivotal roles in regulating AR activity and promoting epithelial differentiation. Studies have shown that FOXA1 is frequently downregulated in CRPC tumors. Congruently, FOXA1 loss is reported to induce aberrant AR signaling, epithelial-mesenchymal transition, and PCa de-differentiation. However, the role of FOXA1 in regulating PCa immune response, an area of much interest recently, has not been reported. CRPC has shown poor response to immune checkpoint inhibitors, due to its immunosuppressive nature. A better understanding of the tumor intrinsic mechanisms regulating PCa tumor immunity will inform the design of better targeted immunotherapeutic approaches. Methods: We performed RNA-seq, ChIP-seq, qPCR, western blot, and ELISA analyses to evaluate how FOXA1 regulates inflammatory response genes. We utilized an in vitro macrophage infiltration transwell assay, in which M2-like macrophages were added to the upper chamber and PCa cells were plated in the lower chamber, to examine how perturbations to PCa cells affect macrophage migration. Finally, we performed bioinformatic analyses of patient datasets to confirm the clinical relevance of FOXA1 repression of inflammatory genes in PCa. Results: Through integration of RNA-seq and ChIP-seq data, we uncovered a novel function of FOXA1 in suppressing inflammatory response pathways. In accordance, patient data analyses revealed that inflammatory response genes were upregulated in FOXA1-low PCa tumors. Mechanistically, we showed that FOXA1 proteins bound an intragenic enhancer of Hypoxia-inducible factor 1-alpha (HIF1A) gene to directly repress its expression, such that FOXA1 loss induced HIF1A upregulation. We further showed that Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) became upregulated upon FOXA1 depletion in a HIF1A-dependent manner. This led to infiltration by immunosuppressive, tumor promoting M2-like macrophages. Inhibiting this HIF1A-CCL2 axis with a HIF1A inhibitor or CCL2 neutralizing antibody blocked macrophage infiltration. Future studies using immunocompetent mouse models are needed to confirm the effect of FOXA1 on macrophage infiltration in vivo and evaluate the preclinical potential of targeting the FOXA1-HIF1A-CCL2 axis in CRPC. Conclusion: This study proposes a novel role for FOXA1 loss in promoting macrophage infiltration via the HIF1A-CCL2 axis. Moreover, our findings suggest that targeting this axis may be a promising approach for the treatment of FOXA1-low CRPC tumors.

Correction to: Investigation of the interaction of hypoxia-inducible factor 1-alpha inhibitor, IDF-11774, with heat shock protein, HSP70, using quantum chemistry calculations

IDF-11774 is an inhibitor of hypoxia-inducible factor-1 (HIF-1) which can bind to heat shock protein (HSP70) and inhibit its chaperon action. To determine the structural, electronic, and thermochemical aspects in interactions between IDF-11774 and its derivatives with active space of HSP70, a density functional theory (DFT) study and quantum theory of atoms in molecule (QTAIM) analysis have been used. The QTAIM method has been utilized to describe the nature of various possible interactions between IDF-11774 and its derivatives with active space of HSP70 in terms of bond critical points. Chemical properties of these compounds were studied using HOMO and LUMO, global hardness, electronic chemical potential, and global electrophilicity power. On the other hand, based on natural bond orbital (NBO) analysis, we have discussed charge transfer between IDF-11774 and its derivatives with active space of HSP70.

MicroRNA-1-3p enhances osteoblast differentiation of MC3T3-E1 cells by interacting with hypoxia-inducible factor 1 α inhibitor (HIF1AN)

Studies have proved that miRNAs participate in the regulation of osteoblast differentiation (OD), and abnormal expression of miRNAs is related with various states of OD. In this study, we investigated the role of miRNA-1-3p in OD using MC3T3-E1 cells. BMP2 is used to induce OD of MC3T3-E1 cells. MiRNA-1-3p mimics or miRNA-1-3p inhibitor was transfected to MC3T3-E1 cells with BMP2. The expression levels of miRNA-1-3p were determined by qRT-PCR. The expression of Runx2, OSX, OPN, and OCN was detected by Western blotting. ALP assay was performed to measure alkaline phosphatase activity. Calcium nodules were evaluated by alizarin red staining. Over-expression of hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) was performed and miRNA-1-3p rescue experiments were carried out. Over-expression of miRNA-1-3p promoted osteogenic differentiations and calcifications, as demonstrated by increased ALP, calcification and osteogenic markers. Knock-down of miRNA-1-3p generated the opposite results. HIF1AN was identified to be directly targeted by miRNA-1-3p. Over-expression of HIF1AN suppressed OD and calcifications, and miRNA-1-3p reversed the effect. Our results demonstrated that miRNA-1-3p could enhance OD of MC3T3-E1 cells through interacting with HIF1AN, which might be employed as therapeutic applications for bone formation and regeneration.

Investigation of the interaction of hypoxia-inducible factor 1-alpha inhibitor, IDF-11774, with heat shock protein, HSP70, using quantum chemistry calculations

Investigation of the interaction of hypoxia-inducible factor 1-alpha inhibitor, IDF-11774, with heat shock protein, HSP70, using quantum chemistry calculations

Down-regulation of microRNA-31 suppresses hepatic fibrosis induced by carbon tetrachloride

MicroRNA-31 (miR-31) is among the most frequently altered microRNAs in human diseases, and altered expression of miR-31 has been detected in a large variety of diseases types. miR-31 could also regulate a variety of cell functions including hepatic fibrosis. Hepatic stellate cells (HSCs) are regarded as the major cell type involved in hepatic fibrosis. Male BALB/c mice (five mice per group aged 6 weeks) received 200 μL of body weight of carbon tetrachloride (10% CCl4) mixed with olive oil intraperitoneally, and the first dose was doubled. To induce hepatic fibrosis, carbon tetrachloride was injected twice a week for 4, 6, 8, and 10 weeks. Control animals were injected with an equal volume of olive oil at the same time intervals. We found that miR-31 expression and fibrosis-related factors in four hepatic fibrosis stages. However, we noted that inhibition of miR-31 was down-regulated fibrosis-related factor expression in F1–F3 stages, but no F4 stage. Thus, we hypothesize that miR-31 may mediate hepatic fibrosis. In this research, we found that inhibition of miR-31 expression significantly inhibited HSC activation. The biological function of miR-31 during HSC activation might be through targeting hypoxia-inducible factor 1-alpha inhibitor (HIF1AN). Inhibition of miR-31 can reduce the transcription factor activity of hypoxia inducible factor 1 (HIF-1) by targeting the biological effects of HIF1AN with the condition of hypoxia. In later hepatic fibrosis could be rescue combining with inhibition of miR-31 and adding heparin-binding EGF-like growth factor (HBEGF).

Circular RNA CDR1as acts as a sponge of miR-135b-5p to suppress ovarian cancer progression.

Background and objectives: Ovarian cancer is the most fatal primary malignancy among gynecological cancers. Circular RNAs (circRNAs) play an important role in the development of various cancers, but the functions of circRNAs in ovarian cancer development have not been studied. We aim to explore the function and mechanism of CDR1as in the progression of ovarian cancer and to provide a new target for the diagnosis and treatment of ovarian cancer.Methods: Ovarian cancer cell proliferation was assessed according to proliferating cell nuclear antigen (PCNA) and Ki67 protein expression levels and MTT and CCK8 assays. The migration ability of cells was detected by scratch-wound assays, and the invasion ability of the cells was determined by Transwell® assays. qRT-PCR and Western blotting were used to verify miRNA and protein expression.Results: CDR1as expression in ovarian tissues was significantly lower in ovarian cancer patients than in patients without ovarian cancer. CDR1as overexpression inhibited the proliferation, invasion and migration of ovarian cancer cells. Silencing CDR1as increased the expression of miR-135b-5p and decreased the expression of hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), thus increasing the proliferation capacity of ovarian cancer cells.Conclusions: CDR1as, acting as a sponge of miR-135b-5p, promotes the expression of HIF1AN and therefore plays a role in tumor inhibition.

A novel HIF1AN substrate KANK3 plays a tumor-suppressive role in hepatocellular carcinoma.

The KN motif and ankyrin repeat domain-containing protein (KANK) family is involved in actin cytoskeleton organization and cell motility. Compared with other KANK members, the biological function of KANK3 is not clear. Here, we identified KANK3 as a new substrate for the oxygen sensor hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), which hydroxylates HIF-1/2α and other ankyrin repeat domain-containing proteins at asparagine residues. An in vitro hydroxylation assay clearly demonstrated asparaginyl hydroxylation of KANK3 by HIF1AN, and mass spectroscopic analysis revealed that KANK3 is hydroxylated at three asparagine residues within the ankyrin repeat domain. Bioinformatics analysis revealed that KANK3 downregulation is correlated with a poor prognosis in several types of cancers, including hepatocellular carcinoma (HCC). In HCC cells, KANK3 knockdown enhanced cell migration and invasion, while its overexpression inhibited these cell behaviors. Interestingly, such effects of KANK3 were not observed under hypoxic conditions, suggesting oxygen-dependent activity of KANK3. Based on these data, we propose that KANK3 acts as a tumor suppressor to control cancer behavior in an oxygen-dependent manner.

Abstract 467: Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells

Abstract Recent studies have shown the crucial role of miRNAs in regulating cancer initiation, progression and metastatic process via influencing gene expression patterns. However, the distinct mechanism by which miRNA regulates tumorigenesis remains as an area requiring detailed research investigations. In our earlier studies, we have demonstrated a positive correlation between MDM2 expression and hypoxia inducible factor -1alpha (HIF-1α) levels in both normoxic and hypoxic conditions using prostate cancer cell lines. Subsequently, we postulated that the upregulation of HIF-1α is most likely regulated by MDM2 in cancers cells that are positive for this gene expression. Hence, to confirm our hypothesis, we utilized miScript miRNA PCR Array analysis (Qiagen, USA) and compared the gene expression profile of the hypoxia signaling pathway in LNCaP (prostate cancer cells) and LNCaP-MST (MDM2 transfected prostate cancer cells) under normoxic conditions. This study was expected to outline how MDM2 impacts the differential expression of miRNAs and leading to enhancement of tumor angiogenesis via increase in HIF-1α or other genes of the hypoxia signaling pathway. Our preliminary data indicate that hsamiR99a5p, hsamiR125b5p, hsamiR1385p, hsamiR199b5p and hsalet7c5p are significantly upregulated in LNCaP-MST cells compared to LNCaP. In addition, some of the miRNAs such as hsalet7d5p, hsalet7e5p, hsalet7f5p, hsalet7g5p and hsalet7i5p are significantly downregulated in the same cells. Among the upregulated miRNAs, hsamiR125b5p emerged as an important gene that is known to downregulate the protein levels of hypoxia inducible factor 1alpha inhibitor (HIF1AN or FIH), which can normally inhibit the transcriptional activity of HIF-1α in both normoxic and hypoxic conditions. Therefore, overexpression of hsamiR125b5p seems to be associated with the increase in HIF-1α level. From the aforementioned findings, it is confirmed that the MDM2 overexpression is impacting miRNA levels such as hsamiR125b5p that are involved in hypoxia related signaling pathway. Thus, our results offer a convincing evidence that MDM2 can promote angiogenesis and increase metastatic potential of cancers through altering the miRNA expression levels. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Saad Ebrahim Alobid, Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Appu Rathinavelu. Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 467. doi:10.1158/1538-7445.AM2017-467

Cobalt Chloride Enhances the Stemness of Human Dental Pulp Cells

IntroductionHypoxia is a factor in controlling stem cell stemness. We investigated if cobalt chloride (CoCl2), a chemical agent that mimics hypoxia in vitro, affected human dental pulp cell (hDPC) stemness by examining cell proliferation, stem cell marker expression, and osteogenic differentiation. MethodshDPCs were cultured with or without 25 or 50 μmol/L CoCl2. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine cell proliferation. The number of STRO-1+ cells was determined by flow cytometry. The messenger RNA expression of the stem cell markers REX1, OCT4, SOX2, and NANOG and the osteogenic-associated genes ALP, COLI, and RUNX2 were evaluated using reverse transcription polymerase chain reaction or real-time polymerase chain reaction. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and mineralization assays. ResultsAlthough 25 and 50 μmol/L CoCl2 suppressed hDPC proliferation, 50 μmol/L CoCl2 increased the number of STRO-1+ cells. Moreover, CoCl2 dose dependently induced stem cell marker expression. Additionally, CoCl2 treatment suppressed osteogenic-associated gene expression, ALP activity, and calcium deposition. The addition of apigenin, a hypoxia-inducible factor 1-alpha inhibitor, reversed the inhibitory effect of CoCl2 on ALP activity. ConclusionsThis study indicated that CoCl2 may enhance hDPC stemness.

Effects of resveratrol and methoxyamine on the radiosensitivity of iododeoxyuridine in U87MG glioblastoma cell line.

The purpose of this study was to evaluate the combination effect of resveratrol and methoxyamine on radiosensitivity of iododeoxyuridine in spheroid culture of U87MG glioblastoma cell line using colony formation and alkaline comet assays. Spheroids on day-20 with 350 µm diameters were treated with 20 µM resveratrol and/or 6 mM methoxyamine and/or 1 µM iododeoxyuridine for one volume doubling time (67 h), and then irradiated with 2 Gy gamma-radiation ((60)Co) in different groups. After treatment, viability of the cells, colony forming ability and DNA damages were obtained by blue dye exclusion, colony formation and alkaline comet assay, respectively. Our results showed that methoxyamine and resveratrol could significantly reduce colony number and induce the DNA damages of glioblastoma spheroid cells treated with iododeoxyuridine in combination with gamma-rays. Therefore, methoxyamine as base excision repair inhibitor and resveratrol as hypoxia inducible factor 1-alpha inhibitor in combination with iododeoxyuridine as radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Vascular endothelial growth factor (VEGF) regulation by hypoxia inducible factor-1 alpha (HIF1A) starts and peaks during endometrial breakdown, not repair, in a mouse menstrual-like model.

How is vascular endothelial growth factor (VEGF) expression regulated by hypoxia inducible factor 1 alpha (HIF1A) during menstruation? After progesterone (P4) withdrawal, HIF1A was activated and it directly up-regulated VEGF mRNA expression and this regulation was the highest during endometrium breakdown in the mouse menstrual-like model. VEGF, an important angiogenic factor, is known to be essential for endometrial repair, particularly in angiogenesis and re-epithelialization. However, its upstream regulation has not been fully clarified. HIF1 is the first transcription factor response to hypoxia and is closely associated with angiogenesis; it is also an upstream regulator of VEGF mRNA. We investigated the changes in the expression of HIF1A and VEGF after P4 withdrawal and after HIF1A inhibition. The total number of mice used was 62. The treatment duration in the mouse menstrual-like model was 8 days. The mouse menstrual-like model and mouse and human decidual endometrial stromal cells were established to mimic menstruation. Protein and mRNA expressions of HIF1A and VEGF were investigated by immunohistochemistry, Western blot and quantitative PCR. The direct interaction between HIF1A and the Vegf promoter was also investigated by chromatin immunoprecipitation. HIF1A inhibition in vivo and in vitro was achieved by administration of an HIF1A inhibitor and by siRNA knockdown, respectively. HIF1A was translocated to the nucleus from 8 to 16 h after P4 withdrawal, while VEGF mRNA expression was the highest at 12 h. HIF1A directly bound to Vegf promoter during endometrial breakdown, which peaked at 12 h. HIF1A inhibition suppressed VEGF mRNA and protein expression in the mouse menstrual-like model and decidualized stromal cells. Inhibition of HIF1A also suppressed endometrial breakdown. Although HIF1A regulation of VEGF mRNA was confirmed in the mouse menstrual-like model and decidual endometrium stromal cells, the functional regulation of VEGF protein was not further determined. Here, we report that the functional regulation of VEGF was complicate in menstruation. We also report that HIF1A plays a key role in endometrial breakdown. The National Nature Science Foundation of China (No. 30901608), the National Basic Research Program of China (2010CB530403) and the National Science and Technology Support Program (No. 2012BAI32B05). The authors have no conflicts of interest to disclose. This study is not a clinical trial.

Abstract 1014: Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies

Abstract Traditional anticancer therapies are limited in their efficacy because they often lead to drug-resistant tumors and display toxicity to normal cells. Control of angiogenesis represents one of the key goals in cancer therapy, as exemplified by targeting the VEGF pathway with bevacizumab.Chronic hypoxia, a hallmark of many solid tumors is responsible for the elevated levels of VEGF and other pro-proliferative factors. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, and pro-proliferative mechanisms that promote tumor growth. The specific hypothesis behind the proposed research is that angiogenesis could be effectively controlled by down-regulation of hypoxia-inducible gene expression via a disruption of key transcription factor-coactivator interactions with designed molecules. The team at BioCycive and its collaborators at USC are currently developing such compounds that block hypoxia-inducible transcription of key genes. We have designed and synthesized a novel dimeric epidithiodiketopiperazine, ETP2, which targets pleiotropic coactivator p300 disrupts HIF1α-p300 complex in vitro and effectively down-regulates hypoxia-inducible signaling and gene expression. ETP 2 effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of breast and lung cancer cell lines. We have shown excellent antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. A significant decrease in the vascularization of these tumors upon treatment with ETP 2 was also noticed, which is consistent with the proposed mechanism of inhibition of vascularization via down-regulation of hypoxia-inducible signaling.Our efficacy studies with renal carcinoma cells lines resulted in similar findings, where we successfully reached the primary endpoint of efficacy (reduction in tumor volume of >50%) and secondary endpoint of survival. Nude mice engrafted with 786-O (renal cell carcinoma) were treated with ETPs at 1 mg/kg given IV every three days for 45 days. All tumors were imaged at the end of the study and harvested for histopathology studies. MTD studies clearly indicated that ETP 2 was non-toxic within the range of injected doses, with an average MTD of 21.1 mg/kg. Our preliminary PK studies show that the ETP 2 is a lipophilic molecule, with a plasma half-life of 3.5-5 days. Our results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance. Citation Format: Ramin Dubey, Ivan Grishagin, Usha Nagavarapu, Chenera Balan, Shalabh Gupta, Bogdan Z. Olenyuk. Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2014-1014

Abstract 4170: MicroRNA-135b influences androgen and estrogen receptor protein levels and regulates proliferation of hormone-receptor positive prostate and breast cancer cells.

Abstract MicroRNAs (miRNAs) regulate genes and impact on a wide range of cellular signaling pathways and biological processes in both physiological and pathological states. To understand their role in prostate and breast cancer, we have carried out systematic functional high-throughput miRNA-overexpression studies in cell lines, and assessed the effect of miRNAs on androgen receptor (AR) and estrogen receptor (ER) protein levels and on cell proliferation. We have observed that overexpression of miR-135b affected the growth of cells where the endogenous expression of miR-135b was low. Furthermore, the cells with low miR-135b expression mostly had a higher expression of AR and ER in prostate respective breast cancer cells. We identified that miR-135b is able to target AR and regulate the AR protein level in prostate cancer cells. Our aim was thus to further explore the function and significance of miR-135b in prostate and breast cancer. We found that miR-135b inhibited the growth of AR-positive prostate cancer (LNCaP, 22Rv1 and VCaP) and ER-positive breast cancer (MCF-7 and BT-474) cells, whereas it did not have any effect on the AR- and ER-negative cells. Here, we demonstrate that miR-135b decreases the ER protein level in breast cancer cells and that miR-135b expression inversely correlates with ER protein and mRNA as well as AR mRNA levels in 101 clinical breast cancer samples and with AR protein level in 47 clinical prostate cancer samples. In order to characterize novel target genes and to understand the mechanisms for the observed effect, we conducted gene expression analyses and identified 65 genes that were deregulated by miR-135b at three different time points (12 h, 24 h and 36 h). We discovered that for almost 50% of the genes, the expression significantly (p≤0.05) positively or negatively correlated with the expression of miR-135b in clinical breast cancer samples, suggesting that they may be relevant. For 14 genes we observed a significant inverse correlation with miR-135b in the clinical material. Hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) showed an inverse correlation and is predicted to be targeted by miR-135b by TargetScan 6.2. Our results indicate that miR-135b influences the protein level of HIF1AN. This could imply that miR-135b is involved in the regulation of oxygen homeostasis, and subsequently angiogenesis and glycolytic metabolism, in breast and prostate cancer tumors by negatively regulating the inhibitor of HIF1α that plays an important role in cancer. Citation Format: Anna Aakula, Suvi-Katri Leivonen, Pekka Kohonen, Petteri Hintsanen, Tero Aittokallio, Yvonne Ceder, Anne-Lise Børresen-Dale, Merja Perälä, Päivi Östling, Olli Kallioniemi. MicroRNA-135b influences androgen and estrogen receptor protein levels and regulates proliferation of hormone-receptor positive prostate and breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2013-4170

FDG-PET imaging for the evaluation of antiglioma agents in a rat model

The increasing development of novel anticancer agents demands parallel advances in the methods used to rapidly assess their therapeutic efficacy (TE) in the preclinical phase. We evaluated the ability of small-animal PET, using the (18)F-fluoro-deoxy-D-glucose (FDG) radiotracer, to predict the TE of a number of anticancer agents in the rat C6 glioma model following 3 days of treatment. Semi-quantitative measurements of changes in FDG uptake during the course of treatment (standardized uptake value response [SUV(r)]) were found to be significantly lower in tumors treated with the hypoxia-inducible factor-1alpha inhibitor YC-1 (15 mg/kg) than in tumors in the control group. No significant SUV(r) change was observed following a similar 3-day regimen with the proapoptotic agent NS1619 (20 microg/kg), the combination of YC-1 and NS1619, or the alkylating agent temozolomide (7.5 mg/kg). Quantitative immunohistochemical studies demonstrated significantly lower levels of glucose transporter-1 (GLUT-1) expression in the YC-1-treated tumors, thereby correlating with the low SUV(r) observed in this group. The ability of SUV(r) to predict gold-standard outcomes of TE was further validated as YC-1-treated tumors had decreased volumes compared to control tumors. As such, we successfully demonstrated the ability of FDG-PET to rapidly determine the TE of novel agents for the treatment of glioma in the preclinical phase of evaluation.

ARC-111 inhibits hypoxia-mediated hypoxia-inducible factor-1α accumulation

ARC-111, a small-molecule topoisomerase I inhibitor, is a potent cytotoxic drug against multiple human cancer cell lines under normoxic conditions (Li et al., Cancer Res 2003; 63:8400-8407). In this study, we explore the potential of ARC-111 as a hypoxia-inducible factor-1alpha inhibitor under hypoxic conditions. The transcription factor, hypoxia-inducible factor-1alpha, is an essential regulator of tumorigenesis and an attractive molecular target for cancer therapy. We demonstrate that ARC-111 specifically inhibits hypoxia-induced accumulation of hypoxia-inducible factor-1alpha, but not other short half-life proteins in multiple human cancer cell lines. ARC-111 inhibits hypoxia-inducible factor-1alpha protein synthesis specifically and does not inhibit protein synthesis globally. We demonstrate that inhibition of hypoxia-inducible factor-1alpha accumulation by ARC-111 is independent of proteasomal degradation. In addition, we demonstrate using topoisomerase I-resistant cell lines that topoisomerase I is required for ARC-111-mediated hypoxia-inducible factor-1alpha inhibition. Experiments performed with nocodazole indicate that ARC-111 inhibits hypoxia-inducible factor-1alpha accumulation in a cell-cycle-independent manner. Analysis of AKT and mammalian target of rapamycin phosphorylation reveals that ARC-111 does not exhibit inhibitory effect on the phosphatidylinositol-3-kinase AKT mammalian target of rapamycin signaling pathway. It has been previously shown that topotecan, a topoisomerase I inhibitor, can also modulate hypoxia-induced hypoxia-inducible factor-1alpha accumulation (Rapisarda et al., Cancer Res 2003; 64:1475-1482). In addition to inhibiting hypoxia-induced accumulation of hypoxia-inducible factor-1alpha, ARC-111 exhibits antiproliferative effects against multiple human cancer cell lines. We demonstrate that topoisomerase I is required for the antiproliferative effects of ARC-111. Antiproliferative effects of ARC-111, however, are oxygen-independent, which is distinguishable from inhibition of hypoxia-inducible factor-1alpha accumulation by ARC-111, which is only observed under hypoxia. The results indicate that inhibiting hypoxia-inducible factor-1alpha accumulation and exhibiting antiproliferation of ARC-111 are through distinct mechanisms of action, which reinforce the potential anticancer effect of ARC-111 on hypoxic tumors.