Identification of a Survival-independent Metastasis-enhancing Role of Hypoxia-inducible Factor-1α with a Hypoxia-tolerant Tumor Cell Line

During tumor progression, malignant cells must repeatedly survive microenvironmental stress. Hypoxia-inducible factor-1 (HIF-1) signaling has emerged as one major pathway allowing cellular adaptation to stress. Recent findings led to the hypothesis that HIF-1α may enhance the metastatic potential of tumor cells by a survival-independent mechanism. So far it has not been shown that HIF-1α also directly regulates invasive processes during metastasis in addition to conferring a survival advantage to metastasizing tumor cells. In a hypoxia-tolerant tumor cell line (L-CI.5s), which did not rely on HIF-1 signaling for viability in vitro and in vivo, knockdown of Hif-1α reduced invasiveness of the tumor cells in vitro as well as extravasation and secondary infiltration in vivo. Liver metastases associated induction of proinvasive receptor tyrosine kinase Met phosphorylation as well as gelatinolytic activity were Hif-1α-dependent. Indeed, promoter activity of the matrix metalloproteinase-9 (mmp-9) was shown to be Hif-1α-dependent. This study uncovers a new survival-independent biological function of HIF-1α contributing to the efficacy of metastases formation.     

HIF-1-Dependent Induction of Jumonji Domain-Containing Protein (JMJD) 3 under Hypoxic Conditions

Jumonji domain-containing proteins (JMJD) catalyze the oxidative demethylation of a methylated lysine residue of histones by using O₂, α-ketoglutarate, vitamin C, and Fe(II). Several JMJDs are induced by hypoxic stress to compensate their presumed reduction in catalytic activity under hypoxia. In this study, we showed that an H3K27me3 specific histone demethylase, JMJD3 was induced by hypoxia-inducible factor (HIF)-1α/β under hypoxia and that treatment with Clioquinol, a HIF-1α activator, increased JMJD3 expression even under normoxia. Chromatin immunoprecipitation (ChIP) analyses showed that both HIF-1α and its dimerization partner HIF-1β/Arnt occupied the first intron region of the mouse JMJD3 gene, whereas the HIF-1α/β heterodimer bound to the upstream region of the human JMJD3, indicating that human and mouse JMJD3 have hypoxia-responsive regulatory regions in different locations. This study shows that both mouse and human JMJD3 are induced by HIF-1.     

The Role of Hypoxia Inducible Factor-1 Alpha in Bypassing Oncogene-Induced Senescence

Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on Ras^V12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21^CIP1 and p16^INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-Ras^V12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21^CIP1 but not p16^INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21^CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.     

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.     

LncRNA FAM83A-AS1 facilitates tumor proliferation and the migration via the HIF-1α/ glycolysis axis in lung adenocarcinoma

Background: Lung adenocarcinoma (LUAD), the major subtype of lung cancer, is among the leading cause of cancer-related death worldwide. Energy-related metabolic reprogramming metabolism is a hallmark of cancer shared by numerous cancer types, including LUAD. Nevertheless, the functional pathways and molecular mechanism by which FAM83A-AS1 acts in metabolic reprogramming in lung adenocarcinoma have not been fully elucidated.Methods: We used transwell, wound-healing scratch assay, and metabolic assays to explore the effect of FAM83A-AS1 in LUAD cell lines. Western blotting, Co-IP assays, and ubiquitination assays were used to detect the effects of FAM83A-AS1 on HIF-1α expression, degradation, and its binding to VHL. Moreover, an in vivo subcutaneous tumor formation assay was used to detect the effect of FAM83A-AS1 on LUAD.Results: Herein, we identified FAM83A-AS1 as a metabolism-related lncRNA, which was highly correlated with glycolysis, hypoxia, and OXPHOS pathways in LUAD patients using bioinformatics analysis. In addition, we uncovered that FAM83A-AS1 could promote the migration and invasion of LUAD cells, as well as influence the stemness of LUAD cells in vivo and vitro. Moreover, FAM83A-AS1 was shown to promote glycolysis in LUAD cell lines in vitro and in vivo, and was found to influence the expression of genes related to glucose metabolism. Besides, we revealed that FAM83A-AS1 could affect glycolysis by regulating HIF-1α degradation. Finally, we found that FAM83A-AS1 knockdown could inhibit tumor growth and suppress the expression of HIF-1α and glycolysis-related genes in vivo.Conclusion: Our study demonstrates that FAM83A-AS1 contributes to LUAD proliferation and stemness via the HIF-1α/glycolysis axis, making it a potential biomarker and therapeutic target in LUAD patients.     

Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

Purpose

By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs).

Methods

C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45⁺ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition.

Results

DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45⁺ cell infiltration in LGs. Likewise, CD45⁺ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice.

Conclusions

Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.     

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1^R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1''s substrate α-KG. The mutant IDH1^R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC''s treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.     

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis

Background

Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.

Results

Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.

Conclusions

Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.