HIF-1α Binding Research Articles

Deficiency of Rbpj Leads to Defective Stress-Induced Hematopoietic Stem Cell Functions and Hif Mediated Activation of Non-canonical Notch Signaling Pathways.

Deregulated notch signaling has been associated with human pathobiology. However, functions of notch pathways in hematopoiesis remain incompletely understood. Here, we ablated canonical notch pathways, through genetic deletion of Rbpj, in hematopoietic stem cells (HSCs). Our data identified that loss of canonical notch results in normal adult HSC pool, at steady state conditions. However, HSC maintenance and functions in response to radiation-, chemotherapy-, and cytokine- induced stress were compromised in the absence of canonical notch. Rbpj deficient HSCs exhibit decreased proliferation rates and elevated expression of p57Kip2. Surprisingly, loss of Rbpj resulted in upregulation of key notch target genes and augmented binding of Hes1 to p57 and Gata2 promoters. Further molecular analyses identified an increase in notch activity, elevated expression and nuclear translocation of Hif proteins, and augmented binding of Hif1α to Hes1 promoter in the absence of Rbpj. These studies, for the first time, identify a previously unknown role for non-canonical notch signaling and establish a functional link between Hif and Notch pathways in hematopoiesis.

Hypoxia-Induced LXRα Contributes to the Migration and Invasion of Gastric Cancer Cells

Gastric cancer is characterized by the presence of high invasion ability, hypoxia and chemoresistance. Previous studies reported that liver X receptor α (LXRα) was involved in epithelial-mesenchymal transition (EMT) of gastric cancer cells. However, hypoxia-mediated EMT and the role of LXRα in gastric cancer remained elusive. In this study, we demonstrated that LXRa mRNA and protein levels were up-regulated by hypoxia treatment and LXRα played an important role in HIF-1 dimer induced-EMT. The putative HIF-1α binding site was identified in the LXRa promoter. Expression of LXRα and HIF-1α was significantly up-regulated in gastric cancer tissues compared to that in normal tissues. More importantly, we noticed that the expression of LXRα and HIF-1α was significantly correlated. Taken together, these data suggested that LXRα is regulated by the activity and accumulation of HIF-1α and contributes to EMT of gastric cancer cells. This suggests that targeting LXRα might be a potential approach for improving survival of gastric cancer patients.

Cardiac-specific overexpression of HIF-1α during acute myocardial infarction ameliorates cardiomyocyte apoptosis via differential regulation of hypoxia-inducible pro-apoptotic and anti-oxidative genes

HIF-1α acts as the cellular rheostat for oxygen sensing in cardiomyocytes. Overexpression of HIF-1α in the heart during acute myocardial infarction (MI) is known to attenuate cardiac dysfunction by upregulating pro-angiogenic HIF-1α target genes. However, the effect of HIF-1α overexpression on hypoxic cardiomyocyte apoptosis still remains obscure. In this study, we report for the first time that myocardium-targeted nanotized HIF-1α overexpression during MI downregulates cardiomyocyte apoptosis. HIF-1α overexpression attenuates bnip3-mediated apoptosis indirectly by promoting HO-1-induced anti-oxidant response. Chromatin immunoprecipitation experiment revealed that HIF-1α overexpression in hypoxic cardiomyocytes increases binding of HIF-1α to the hypoxia-responsive element in the promoter of its target anti-oxidant gene ho-1 which is known to attenuate ROS accumulation. ROS accumulation in hypoxic cardiomyocytes causes cysteine oxidation of the DNA-binding p50 subunit of NFκB, which hampers NFκB binding to κB-responsive genes like bnip3. Downregulated oxidative stress due to HIF-1α overexpression leads to decline in cysteine oxidation of NFκBp50, causing NFκB to bind to the promoter of bnip3 as a transcriptional repressor. Therefore HIF-1α overexpression-mediated attenuation of cardiomyocyte apoptosis occurs by transcriptional repression of bnip3 by NFκB. Our study thus reveals that downregulation of bnip3-mediated cardiomyocyte apoptosis occurs via ho-1 upregulation upon HIF-1α overexpression during MI, despite both being HIF-1α target genes. The cross-regulation of HIF-1α and NFκB-mediated pathways effectively downregulates apoptosis due to HIF-1α overexpression during MI, which can be exploited for possible therapeutic intervention.

HIF-1α Directly Controls WNT7A Expression During Myogenesis.

Herein we unveil that Hypoxia-inducible factor-1α (HIF-1α) directly regulates WNT7A expression during myogenesis. In fact, chromatin immunoprecipitation (ChiP) and site-directed mutagenesis experiments revealed two distinct hypoxia response elements (HREs) that are specific HIF-1α binding sites on the WNT7A promoter. Remarkably, a pharmacological activation of HIF-1α induced WNT7A expression and enhanced muscle differentiation. On the other hand, silencing of WNT7A using CRISPR/Cas9 genome editing blocked the effects of HIF-1α activation on myogenesis. Finally, treatment with prolyl hydroxylases (PHDs) inhibitors improved muscle regeneration in vitro and in vivo in a cardiotoxin (CTX)-induced muscle injury mouse model, paving the way for further studies to test its efficacy on acute and chronic muscular pathologies.

Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity

Innate lymphoid cells (ILCs) and CD4+ T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4+ T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4+ T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4+ T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4+ T cells and ILCs to maintain intestinal homeostasis.

Reactivation of Epstein-Barr Virus by HIF-1α Requires p53.

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

Abstract 1482: PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α

Abstract An essential process for the growth and dissemination of solid tumors is angiogenesis, or the formation of new blood vessels. Overexpression of Proviral Integration site for Moloney murine leukemia virus (PIM)-1, a serine-threonine kinase, has been implicated as a driver of aggressive prostate and colon cancer. PIM1 is known to promote tumor growth and survival, and we recently uncovered a role for PIM1 in promoting resistance to anti-angiogenic agents. Here, we identify a novel signaling pathway that directly links PIM to tumor angiogenesis. Immunohistochemical staining of prostate cancer tissue revealed a significant positive correlation between PIM1 and CD31 expression, a marker of endothelial cells. Using prostate cancer as a model, in vitro models of endothelial tube formation and DCE-MRI confirmed that PIM1 overexpression increases tumor angiogenesis. Gene expression analysis of PIM1 overexpressing cells showed that PIM kinases regulates the expression levels of many hypoxia inducible factor-1α (HIF-1α) target genes in normoxic conditions. In vitro, in vivo and kinase assays discovered a novel phosphorylation site within the oxygen-dependent degradation domain (ODDD) of HIF-1α that is a direct target of PIM1. Biochemical analysis demonstrates that phosphorylation of this site increases HIF-1α stability by decreasing PHD binding, hydroxylation, and subsequent proteasomal degradation of HIF-1α. Two CRISPR-generated cell lines with point mutations that mimic phosphorylation of this site were used in xenograft models of colon cancer to confirm the significance of this site to the identified PIM-HIF signaling and tumor angiogenesis. Importantly, we show that the anti-angiogenic and cytotoxic effects of PIM inhibitors are largely dependent on their ability to downregulate HIF-1α signaling. In summary, we uncovered a novel role for PIM1 in promoting tumor angiogenesis and identified a novel phosphorylation site that controls the stability of HIF-1α in normoxic and hypoxic conditions.This research yields important insight into the role of PIM signaling in solid tumors and provides preclinical evidence that to improve the translation of PIM kinase inhibitors in solid tumors. Citation Format: Andrea L. Casillas, Shailender S. Chauhan, Rachel K. Toth, Corbin C. Jensen, Noel A. Warfel. PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1482.

Carbonic anhydrase III is a new target of HIF1α in prostate cancer model.

Carbonic Anhydrase III (CAIII) belongs to a member of the alpha Carbonic Anhydrase (CA) family. Although some CA members are strongly up-regulated by HIF1-α, it is not known about the transcriptional regulation of CAIII in prostate cancer cells, PCa. Therefore, we aimed to identify regulatory regions important for the regulation of CAIII gene under hypoxic conditions in human prostate cancer cells (PC3). The present study, for the first time, demonstrated that the chemically mimicked hypoxic condition led to the induced CAIII mRNA and protein expression in prostate cancer cells. Transcriptional regulation of CAIII was investigated by transient transfection assay that indicates that the most active promoter activity was in the region of P2 -699/+86. Hypoxic condition also upregulates the basal activity of for P1;-941/+86 and P2;-699/+86 constructs containing putative Hypoxia Response Element (HRE) region located in -268/-252. EMSA analysis of HRE located in -268/-252 bases, showed one DNA-protein binding complexes. Competition assays indicated this complex is resulted from HIF1α interactions. In addition, site-directed mutagenesis of potential HIF1α binding sites diminished a DNA-protein complex. These findings suggest that CAIII is a hypoxia-regulated gene and valuable for targeting of prostate cancer tumors in hypoxic condition.

Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.

BRG1 Stimulates Endothelial Derived Alarmin MRP8 to Promote Macrophage Infiltration in an Animal Model of Cardiac Hypertrophy.

Endothelial cell derived angiocrine factors contribute to the disruption of homeostasis and the pathogenesis of cardiovascular diseases in response to stress stimuli. In the present study we investigated the role of BRG1, a key component of the chromatin remodeling complex, in the regulation of angiocrine signaling. We report that angiotensin II (Ang II) induced pathological cardiac hypertrophy was attenuated in mice with endothelial-specific ablation of BRG1 (ecKO) compared to the control mice (WT). Mitigation of cardiac hypertrophy as a result of BRG1 deficiency was accompanied by decreased macrophage homing to the hearts. This could be explained by the observation that the ecKO mice exhibited down-regulation of myeloid-related protein 8 (MRP8), a well-established chemokine for macrophages, in vascular endothelial cells compared to the WT mice. Further analysis revealed that BRG1 mediated the activation of MRP8 expression by Ang II treatment in endothelial cells to promote macrophage migration. BRG1 was recruited to the MRP8 promoter by interacting with hypoxia-inducible factor 1 (HIF-1α). Reciprocally, BRG1 facilitated the binding of HIF-1α to the MRP8 promoter by sequentially recruiting histone acetyltransferase p300 and histone demethylase KDM3A. Depletion of either p300 or KDM3A repressed the induction of MRP8 expression by Ang II and ameliorated macrophage migration. In conclusion, our data delineate a novel epigenetic pathway whereby Ang II stimulates MRP8 production and macrophage homing to promote cardiac hypertrophy.

RETRACTED ARTICLE: LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis

BackgroundLong noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are aberrantly expressed in various cancers. However, the functional roles of lncRNAs in breast cancer remain largely unknown.MethodsBased on public databases and integrating bioinformatics analyses, the overexpression of lncRNA BCRT1 in breast cancer tissues was detected and further validated in a cohort of breast cancer tissues. The effects of lncRNA BCRT1 on proliferation, migration, invasion and macrophage polarization were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA BCRT1, miR-1303, and PTBP3. Chromatin immunoprecipitation (ChIP) and RT-PCR were used to evaluate the regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on lncRNA BCRT1.ResultsLncRNA BCRT1 was significantly upregulated in breast cancer tissues, which was correlated with poor prognosis in breast cancer patients. LncRNA BCRT1 knockdown remarkably suppressed tumor growth and metastasis in vitro and in vivo. Mechanistically, lncRNA BCRT1 could competitively bind with miR-1303 to prevent the degradation of its target gene PTBP3, which acts as a tumor-promoter in breast cancer. LncRNA BCRT1 overexpression could promote M2 polarization of macrophages, mediated by exosomes, which further accelerated breast cancer progression. Furthermore, lncRNA BCRT1 was upregulated in response to hypoxia, which was attributed to the binding of HIF-1α to HREs in the lncRNA BCRT1 promoter.ConclusionsCollectively, these results reveal a novel HIF-1α/lncRNA BCRT1/miR-1303/PTBP3 pathway for breast cancer progression and suggest that lncRNA BCRT1 might be a potential biomarker and therapeutic target for breast cancer.

OR31-04 Androgen Signaling Regulates Female Fertility Through Modulation of H3K27me3 in Granulosa Cells

Androgens play a major role in female fertility and in women’s health, in general. However, the underlying mechanism and downstream physiological targets of androgen are not fully understood. Androgen actions are mediated by “nuclear” transcriptional signals or “extra-nuclear” kinase actions. Primary androgen receptor (AR) target genes are those at which AR occupies a genomic androgen response element (ARE) and regulates gene transcription. Subsequently, androgens also regulate other genes in an AR-ARE independent fashion, involving membrane-initiated androgen signaling. In the last couple of years, we have reported that androgens may also influence gene expression through histone modifications. We have found that H3K27me3 (tri-methyl lysine 27 histone3) is a downstream target of androgen actions. H3K27me3 is a gene silencing mark, regulated by Enhancer of Zeste Homologue 2 (Ezh2), a histone methyltransferase that promotes tri-methylation of K27 and androgens inhibit the expression and activity of Ezh2. In this study, we report that androgens also regulate the expression of a histone demethylase called Jumonji domain containing protein 3 (JMJD3/KDM6B), that is responsible of removing the H3K27me3 mark. We find that in granulosa cells (GCs), androgen through the PI3K/Akt pathway, in a transcription-independent fashion, increases hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. ChIP studies reveal increased binding of HIF1α on the JMJD3 promoter region with DHT treatment. To understand the global impact of androgens on ovarian gene expression and the contribution of androgen-induced decrease of H3K27me3, we have performed RNA-seq and ChIP-seq analysis with H3K27me3 antibody in primary mouse GCs treated with DHT or vehicle. Results show 190 significantly differentially expressed genes in DHT treated sample vs vehicle, out of which 129 and 61 genes were significantly up- and downregulated, respectively. Moreover, comparison of the RNA-seq and ChIP-seq data reveals that a number of upregulated genes have significantly lower H3K27me3 enrichment in the enhancer and/or promoter region in the DHT treated samples vs vehicle. This establishes that in the ovary, androgen-induced modulation of H3K27me3 mark through regulation Ezh2 and JMJD3 expression/activity is an important regulatory mechanism for ovarian gene expression. To delineate the physiological importance of JMJD3 in normal ovarian function and female fertility, we have also developed a GC-specific JMJD3 knockout mice. We find that GC-specific JMJD3KO mice are sub-fertile with longer estrous cycles and dysregulated follicular development. This phenotype is very similar to GC-specific ARKO mice. Thus, we propose that one of the critical actions of androgens in regulating follicular function and female fertility is through the regulation of JMJD3 expression.

Short chain fatty acids regulate T cell metabolism to promote IL-22 production and gut immunity

Abstract Interleukin-22 (IL-22) is critical in gut immunity. Both innate lymphocyte cells (ILCs) and CD4+ T cells produce IL-22. ILCs provide a rapid source of IL-22 that is essential for early protection of epithelial barrier function upon enteric infection and inflammatory insult, while T cells become the dominant source of IL-22 in the intestinal lamina propria during chronic intestinal inflammation. Microbiota is central in IL-22 production in the intestines, however, the factors that regulate IL-22 production in T cells and ILCs in the intestines, and the mechanisms involved remain unclear. We found that microbiota-derived short-chain fatty acids (SCFAs) promoted IL-22 production in T cells and ILCs through their inhibition of HDAC activity but not GPR43. Additionally, SCFAs upregulated IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor (HIF)1α expression, which were differentially regulated by mTOR and Stat3 pathways. Furthermore, HIF1α directly bind to the il22 promoter, and SCFAs increased HIF1α binding to the il22 promoter through histone modification. SCFAs enhanced oxygen consumption and glycolysis, which mediated SCFA-induced IL-22 production. Hypixic condition enhanced the SCFAs induction of IL-22, and glucose facilitated SCFAs induction of IL-22 production in a dose-dependent manner. SCFA supplementation protected intestines from Citrobacter Rodentium infection and dextran sodium sulfate (DSS) insult, which was mediated by enhanced IL-22. SCFAs promoted T cell IL-22 production from patients with Crohn’s disease through inducing AHR and HIF1α. These findings establish the roles of SCFAs in inducing IL-22 production in T cells and ILCs to maintain the intestinal homeostasis.

Berberine Protects Against Simulated Ischemia/Reperfusion Injury-Induced H9C2 Cardiomyocytes Apoptosis In Vitro and Myocardial Ischemia/Reperfusion-Induced Apoptosis In Vivo by Regulating the Mitophagy-Mediated HIF-1α/BNIP3 Pathway.

Berberine (BBR) has a variety of pharmacological activities and is widely used in Asian countries. However, the clinical application of BBR still lacks scientific basis, what protective mechanism of BBR against myocardial ischemia-reperfusion injury (MIRI). In vitro experiments, BBR pretreatment regulated autophagy-related protein expression, induced cell proliferation and autophagosome formation, and reduced the mitochondrial membrane potential (ΔΨm) increase in H9C2 cells. In vivo experiments, BBR reduced the myocardial infarct size, decreased cardiomyocyte apoptosis, and markedly decreased myocardial enzyme (CK-MB, LDH, and AST) activity-induced I/R. In addition, upon BNIP3 knockdown, the regulatory effects of BBR on the above indicators were weakened both in H9C2 cells and in vivo. Luciferase reporter and ChIP assays indicated that BBR mediated BNIP3 expression by enhancing the binding of HIF-1α to the BNIP3 promoter. BBR protects against myocardial I/R injury by inducing cardiomyocytes proliferation, inhibiting cardiomyocytes apoptosis, and inducing the mitophagy-mediated HIF-1α/BNIP3 pathway. Thus, BBR may serve as a novel therapeutic drug for myocardial I/R injury.

Neem Leaf Glycoprotein Restrains VEGF Production by Direct Modulation of HIF1α-Linked Upstream and Downstream Cascades.

Neem Leaf Glycoprotein (NLGP) is a natural immunomodulator, have shown sustained tumor growth restriction as well as angiogenic normalization chiefly by activating CD8+ T cells. Here, we have investigated the direct role of NLGP as a regulator of tumor microenvironmental hypoxia and associated vascular endothelial growth factor (VEGF) production. We observed a significant reduction in VEGF level in both in vivo murine tumor and in vitro cancer cells (B16Mel, LLC) and macrophages after NLGP treatment. Interestingly, NLGP mediated VEGF downregulation in tumor cells or macrophages within hypoxic chamber was found at an early 4 h and again at late 24 h in mRNA level. Our data suggested that NLGP prevented hypoxia-induced strong binding of HIF1α with its co-factors, CBP/p300 and Sp3, but not with Sp1, which eventually limit the binding of HIF1α-transcriptional complex to hypoxia responsive element of VEGF promoter and results in restricted early VEGF transcription. On the otherhand, suppressed phosphorylation of Stat3 by NLGP results reduction of HIF1α at 24 h of hypoxia that further support sustained VEGF down-regulation. However, NLGP fails to regulate VHL activity as observed by both in vivo and in vitro studies. Therefore, this study for the first time reveals a mechanistic insight of NLGP mediated inhibition of angiogenesis by suppressing VEGF, which might help in vascular normalization to influence better drug delivery.

HIF-1α and HDAC1 Mediated Down-Regulation of FAM99A Contributes to Hypoxia Induced HCC Metastasis

Background: Hypoxic microenvironment is clinically associated with metastasis and poor prognosis of numerous cancers. Previous studies have shown that many protein-coding genes and microRNAs are regulated upon hypoxia and involved in the progression of cancer. However, the roles of lncRNAs in the hypoxia-responsive gene networks and how lncRNA-related signaling network regulates hypoxia-induced tumor metastasis is still not clear. Methods: The expression of lncRNA FAM99A was analyzed in 103 paired HCC tissues by RT-qPCR and the correlation between FAM99A expression and clinical pathology features was analyzed. The function of FAM99A on HCC cell metastasis was analyzed by transwell, wound healing assay and tail vein injection metastasis model in mice. Downstream microRNA targets of FAM99A was studied by bioinformatics analysis and luciferase reporter assays. The binding of HIF-1α, HDAC1 to the promoter region of FAM99A and the histone acetylation of FAM99A promoter were analyzed by ChIP assays and luciferase reporter assays. Findings: LncRNA FAM99A is down-regulated in HCC and correlates with poor prognosis. Moreover, FAM99A inhibits HCC metastasis by acting as a miR-92a sponge both in vitro and in vivo. More importantly, HIF-1α induces the down-regulation of FAM99A via hypoxic-environment. Mechanism wise, HIF-1α binds to the hypoxia response elements on the FAM99A promoter and recruits HDAC1 to repress FAM99A expression by reducing the histone acetylation of FAM99A promoter region. Interpretation: Hypoxic microenvironment down-regulates FAM99A expression via HIF-1α–HDAC1 mediated histone acetylation modification, resulting in the upregulation of miR-92a and downregulation of E-cadherin mediated signaling pathways to facilitate HCC metastasis. Funding: The National Natural Science Foundation of China (81672376, 81972714, 81602102), National Key R&D Program of China (2018ZX10723204), Joint Funds of Fujian Provincial Health and Education Research (2019-WJ-19), Fuzhou Health and Family Planning Science and Technology Project (2017-S-wt2), the Startup Fund for scientific research, Fujian Medical University (2018QH1201, 2018QH1199), the Scientific Foundation of Fujian Province (2019D003, 2019J01140). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: Specimen collection and usage were approved by the Institutional Review Board (IRB) of the Mengchao Hepatobiliary of Fujian Medical University.

Investigations of the underlying mechanisms of HIF-1α and CITED2 binding to TAZ1

The TAZ1 domain of CREB binding protein is crucial for transcriptional regulation and recognizes multiple targets. The interactions between TAZ1 and its specific targets are related to the cellular hypoxic negative feedback regulation. Previous experiments reported that one of the TAZ1 targets, CITED2, is an efficient competitor of another target, HIF-1α. Here, by developing the structure-based models of TAZ1 complexes, we have uncovered the underlying mechanisms of the competitions between the two intrinsic disordered proteins (IDPs) HIF-1α and CITED2 binding to TAZ1. Our results support the experimental hypothesis on the competition mechanisms and the apparent affinity. Furthermore, the simulations locate the dominant position of forming TAZ1-CITED2 complex in both thermodynamics and kinetics. For thermodynamics, TAZ1-CITED2 is the lowest basin located on the free energy surface of binding in the ternary system. For kinetics, the results suggest that CITED2 binds to TAZ1 faster than HIF-1α. In addition, the analysis of contact map and Φ values is important for guiding further experimental studies to understand the biomolecular functions of IDPs.

HIF-1α or HOTTIP/CTCF Promotes Head and Neck Squamous Cell Carcinoma Progression and Drug Resistance by Targeting HOXA9

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequently diagnosed cancer worldwide. However, the clinical outcomes remain unsatisfactory. The aim of this study is to unravel the functional role and regulatory mechanism of HOXA9 in HNSCC. A cohort of 25 HNSCC tumor tissues and normal tissue counterparts was collected. qRT-PCR and western blotting were performed to determine the levels of HOXA9 and epithelial-mesenchymal transition (EMT)-related markers. Cell Counting Kit-8 (CCK-8) and colony formation assays were conducted to monitor cell viability and cytotoxicity. Transwell and wound healing assays were used to determine cell migration and invasion. Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) staining was performed to detect cell apoptosis. Bioinformatic analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assays were performed to investigate the direct binding between HIF-1α or CCCTC binding factor (CTCF) and HOXA9. Glutathione S-transferase (GST) pull-down and RNA pull-down assays were used to validate the interaction between CTCF and HOTTIP. HOXA9 was upregulated in HNSCC tissues and cells. Knockdown of HOXA9 inhibited cell proliferation, migration, invasion, and chemoresistance but promoted apoptosis in CAL-27 and KB cells. Knockdown of HOXA9 also regulated EMT-related marker via targeting YAP1/β-catenin. Silencing of HOTTIP or CTCF exerted similar tumor-suppressive effects in HNSCC. Mechanistically, HIF-1α or CTCF transcriptionally regulated HOXA9, and HOTTIP/CTCF cooperatively regulated HOXA9 in KB cells. HIF-1α or HOTTIP/CTCF transcriptionally modulates HOXA9 expression to regulate HNSCC progression and drug resistance.

Backbone Dynamics of the TAZ1 Domain of the Creb-Binding Protein Modulate Competition Between Disordered Ligands

Intrinsically disordered proteins (IDPs) play important roles in a multitude of cellular processes; however, the mechanisms by which disordered proteins carry out their diverse roles as modulators of cellular signaling are not yet completely understood. IDPs utilize a wide range of mechanisms to interact with folded proteins within the cell, and in many cases, must compete for binding of protein interaction partners. Yet, little is known about the contributions of the folded proteins to the binding and specificity of interactions with multiple disordered ligands. Are folded protein interaction partners simply scaffolds for IDP binding or do they undergo structural and dynamic changes to accommodate different disordered ligands? Using the folded TAZ1 domain of the transcriptional coactivator CBP and the disordered proteins HIF-1α and CITED2 as an example, we demonstrate that conformational dynamics and structural changes of TAZ1 are critical for binding and exchange of different IDPs. CITED2 competes with HIF-1α for TAZ1 binding through an extremely efficient unidirectional allosteric switch that relies not only on the physicochemical properties of the disordered proteins but also on the structure and dynamics of TAZ1. To gain further mechanistic insight into this process, we used NMR relaxation methods to obtain a detailed description of the backbone dynamics that contribute to TAZ1 binding and competition. We found that TAZ1 displays altered backbone dynamics in its free and bound states, with changes in TAZ1 dynamics observed not only in the HIF-1α and CITED2 binding sites but also in regions that differ in structure when bound to HIF-1α or CITED2. These data suggest that backbone dynamics in TAZ1 play a role in modulating binding of disordered ligands and highlight the importance of characterizing both folded and disordered partners in molecular interactions.

Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells.

In this report, we demonstrated that inorganic arsenic (iAs) induces generation of the cancer stem-like cells (CSCs) through Nrf2-dependent HIF1α activation, and the subsequent metabolic reprogramming from mitochondrial oxidative phosphorylation to glycolysis in epithelial cells.Methods: Genome-wide ChIP-seq analysis was performed to investigate the global binding of Nrf2 and/or HIF1α on the genome in the cells treated with iAs. Both untargeted metabolomics and UDP-13C-glucose flux were applied to determine metabolic reprogramming in the iAs-induced CSCs. The role of Nrf2 on iAs-induced HIF1α and other stemness gene expression was validated by lentiviral transfection of Nrf2 inhibitor Keap1 and CRISPR-Cas9-mediated Nrf2 gene knockout, respectively.Results: The CSCs induced by iAs exhibit a diminished mitochondrial oxidative phosphorylation and an enhanced glycolysis that is actively shunted to the hexosamine biosynthetic pathway (HBP) and serine/glycine pathway. ChIP-seq data revealed that treatment of the cells with iAs amplified Nrf2 enrichment peaks in intergenic region, promoter and gene body. In contrast, a shift of the HIF1α peaks from distal intergenic region to gene promoter and the first exon was noted. Both Nrf2 and HIF1α are responsible for the iAs-induced expression of the glycolytic genes and the genes important for the stemness of the CSCs. Intriguingly, we also discovered a mutual transcriptional regulation between Nrf2 and HIF1α. Inhibition of Nrf2 by lentiviral infection of Keap1, or knockout of Nrf2 by CRISPR-Cas9 gene editing, not only blocked iAs-induced HIF1α activation, but reduced the expression of the key stemness genes for the formation of CSCs also.Conclusion: We demonstrated that Nrf2 activation is an initiating signal for iAs-induced HIF1α activation, and Nrf2 and HIF1α played a concerted role on inducing metabolic reprogramming and the CSCs.