Hypoxia-inducible factor 1 recruits FACT and RNF20/40 to mediate histone ubiquitination and transcriptional activation of target genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.

Targeting intratumoral hypoxia to enhance anti-tumor immunity.

Cancers express a large battery of genes by which they establish an immunosuppressive tumor microenvironment. Many of these genes are induced by intratumoral hypoxia through transcriptional activation mediated by hypoxia-inducible factors HIF-1 and HIF-2. This review summarizes several recent reports describing hypoxia-induced mechanisms of immune evasion in sarcoma and breast, colorectal, hepatocellular, prostate and uterine cancer. These studies point to several novel therapeutic approaches to improve anti-tumor immunity and increase responses to immunotherapy.

Mechanisms of Breast Cancer Stem Cell Specification and Self-Renewal Mediated by Hypoxia-Inducible Factor 1.

Many advanced human cancers contain regions of intratumoral hypoxia, with O2 gradients extending to anoxia. Hypoxia-inducible factors (HIFs) are activated in hypoxic cancer cells and drive metabolic reprogramming, vascularization, invasion, and metastasis. Hypoxia induces breast cancer stem cell (BCSC) specification by inducing the expression and/or activity of the pluripotency factors KLF4, NANOG, OCT4, and SOX2. Recent studies have identified HIF-1-dependent expression of PLXNB3, NARF, and TERT in hypoxic breast cancer cells. PLXNB3 binds to and activates the MET receptor tyrosine kinase, leading to activation of the SRC non-receptor tyrosine kinase and subsequently focal adhesion kinase, which promotes cancer cell migration and invasion. PLXNB3-MET-SRC signaling also activates STAT3, a transcription factor that mediates increased NANOG gene expression. Hypoxia-induced NARF binds to OCT4 and serves as a coactivator by stabilizing OCT4 binding to the KLF4, NANOG, and SOX2 genes and by stabilizing the interaction of OCT4 with KDM6A, a histone demethylase that erases repressive trimethylation of histone H3 at lysine 27, thereby increasing KLF4, NANOG, and SOX2 gene expression. In addition to increasing pluripotency factor expression by these mechanisms, HIF-1 directly activates expression of the TERT gene encoding telomerase, the enzyme required for maintenance of telomeres, which is required for the unlimited self-renewal of BCSCs. HIF-1 binds to the TERT gene and recruits NANOG, which serves as a coactivator by promoting the subsequent recruitment of USP9X, a deubiquitinase that inhibits HIF-1α degradation, and p300, a histone acetyltransferase that mediates acetylation of H3K27, which is required for transcriptional activation.

Plexin-B3 expression stimulates MET signaling, breast cancer stem cell specification, and lung metastasis.

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Disruption of the HIF-1 pathway in individuals with Ollier disease and Maffucci syndrome.

Ollier disease (OD) and Maffucci Syndrome (MS) are rare disorders characterized by multiple enchondromas, commonly causing bone deformities, limb length discrepancies, and pathological fractures. MS is distinguished from OD by the development of vascular anomalies. Both disorders are cancer predisposition syndromes with malignancies developing in ~50% of the individuals with OD or MS. Somatic gain-of-function variants in IDH1 and IDH2 have been described in the enchondromas, vascular anomalies and chondrosarcomas of approximately 80% of the individuals with OD and MS. To date, however, no investigation of germline causative variants for these diseases has been comprehensively performed. To search for germline causative variants, we performed whole exome sequencing or whole genome sequencing of blood or saliva DNA in 94 unrelated probands (68 trios). We found that 7 had rare germline missense variants in HIF1A, 6 had rare germline missense variants in VHL, and 3 had IDH1 variants including 2 with mosaic IDH1-p.Arg132His variant. A burden analysis using 94 probands assigned as cases and 2,054 unrelated individuals presenting no OD- or MS-related features as controls, found that variants in HIF1A, VHL, and IDH1 were all significantly enriched in cases compared to controls. To further investigate the role of HIF-1 pathway in the pathogenesis of OD and MS, we performed RNA sequencing of fibroblasts from 4 probands with OD or MS at normoxia and at hypoxia. When cultured in hypoxic conditions, both proband and control cells showed altered expression of a subset of HIF-1 regulated genes. However, the set of differentially expressed genes in proband fibroblasts included a significantly reduced number of HIF-1 regulated genes compared to controls. Our findings suggest that germline or early post-zygotic variants identified in HIF1A, VHL, and IDH1 in probands with OD and MS underlie the development of the phenotypic abnormalities in a subset of individuals with OD and MS, but extensive functional studies are needed to further confirm it.

NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification.

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.

Hypoxia-inducible factors: cancer progression and clinical translation.

Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis that match O2 supply and demand for each of the 50 trillion cells in the adult human body. Cancer cells co-opt this homeostatic system to drive cancer progression. HIFs activate the transcription of thousands of genes that mediate angiogenesis, cancer stem cell specification, cell motility, epithelial-mesenchymal transition, extracellular matrix remodeling, glucose and lipid metabolism, immune evasion, invasion, and metastasis. In this Review, the mechanisms and consequences of HIF activation in cancer cells are presented. The current status and future prospects of small-molecule HIF inhibitors for use as cancer therapeutics are discussed.

ANGPTL4 influences the therapeutic response of patients with neovascular age-related macular degeneration by promoting choroidal neovascularization.

Most patients with neovascular age-related macular degeneration (nvAMD), the leading cause of severe vision loss in elderly US citizens, respond inadequately to current therapies targeting a single angiogenic mediator, vascular endothelial growth factor (VEGF). Here, we report that aqueous fluid levels of a second vasoactive mediator, angiopoietin-like 4 (ANGPTL4), can help predict the response of patients with nvAMD to anti-VEGF therapies. ANGPTL4 expression was higher in patients who required monthly treatment with anti-VEGF therapies compared with patients who could be effectively treated with less-frequent injections. We further demonstrate that ANGPTL4 acts synergistically with VEGF to promote the growth and leakage of choroidal neovascular (CNV) lesions in mice. Targeting ANGPTL4 expression was as effective as targeting VEGF expression for treating CNV in mice, while simultaneously targeting both was more effective than targeting either factor alone. To help translate these findings to patients, we used a soluble receptor that binds to both VEGF and ANGPTL4 and effectively inhibited the development of CNV lesions in mice. Our findings provide an assay that can help predict the response of patients with nvAMD to anti-VEGF monotherapy and suggest that therapies targeting both ANGPTL4 and VEGF will be a more effective approach for the treatment of this blinding disease.

HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy.

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide and available therapies, including immunotherapies, are ineffective for many patients. HCC is characterized by intratumoral hypoxia, and increased expression of hypoxia-inducible factor 1α (HIF-1α) in diagnostic biopsies is associated with patient mortality. Here we report the development of 32-134D, a low-molecular-weight compound that effectively inhibits gene expression mediated by HIF-1 and HIF-2 in HCC cells, and blocks human and mouse HCC tumor growth. In immunocompetent mice bearing Hepa1-6 HCC tumors, addition of 32-134D to anti-PD1 therapy increased the rate of tumor eradication from 25% to 67%. Treated mice showed no changes in appearance, behavior, body weight, hemoglobin, or hematocrit. Compound 32-134D altered the expression of a large battery of genes encoding proteins that mediate angiogenesis, glycolytic metabolism, and responses to innate and adaptive immunity. This altered gene expression led to significant changes in the tumor immune microenvironment, including a decreased percentage of tumor-associated macrophages and myeloid-derived suppressor cells, which mediate immune evasion, and an increased percentage of CD8+ T cells and natural killer cells, which mediate antitumor immunity. Taken together, these preclinical findings suggest that combining 32-134D with immune checkpoint blockade may represent a breakthrough therapy for HCC.

HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30-60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

Breakthrough science: hypoxia-inducible factors, oxygen sensing, and disorders of hematopoiesis.

Hypoxia-inducible factors (HIFs) were discovered as activators of erythropoietin gene transcription in response to reduced oxygen (O2) availability. O2-dependent hydroxylation of HIFs on proline and asparagine residues regulates protein stability and transcriptional activity, respectively. Mutations in genes encoding components of the O2-sensing pathway cause familial erythrocytosis. Several small-molecule inhibitors of HIF prolyl hydroxylases are currently in clinical trials as erythropoiesis-stimulating agents. HIFs are overexpressed in bone marrow neoplasms, and the development of HIF inhibitors may improve outcomes in these disorders.

HIF-1-regulated expression of calreticulin promotes breast tumorigenesis and progression through Wnt/ß-catenin pathway activation.

Calreticulin (CALR) is a multifunctional protein that participates in various cellular processes, which include calcium homeostasis, cell adhesion, protein folding, and cancer progression. However, the role of CALR in breast cancer (BC) is unclear. Here, we report that CALR is overexpressed in BC compared with normal tissue, and its expression is correlated with patient mortality and stemness indices. CALR expression was increased in mammosphere cultures, CD24-CD44+ cells, and aldehyde dehydrogenase-expressing cells, which are enriched for breast cancer stem cells (BCSCs). Additionally, CALR knockdown led to BCSC depletion, which impaired tumor initiation and metastasis and enhanced chemosensitivity in vivo. Chromatin immunoprecipitation and reporter assays revealed that hypoxia-inducible factor 1 (HIF-1) directly activated CALR transcription in hypoxic BC cells. CALR expression was correlated with Wnt/ß-catenin pathway activation, and an activator of Wnt/ß-catenin signaling abrogated the inhibitory effect of CALR knockdown on mammosphere formation. Taken together, our results demonstrate that CALR facilitates BC progression by promoting the BCSC phenotype through Wnt/ß-catenin signaling in an HIF-1-dependent manner and suggest that CALR may represent a target for BC therapy.

Heritable disorders of oxygen sensing.

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis.

HIF-1 recruits NANOG as a coactivator for TERT gene transcription in hypoxic breast cancer stem cells.

Breast cancer stem cells (BCSCs) play essential roles in tumor formation, drug resistance, relapse, and metastasis. NANOG is a protein required for stem cell self-renewal, but the mechanisms by which it performs this function are poorly understood. Here, we show that hypoxia-inducible factor 1α (HIF-1α) is required for NANOG-mediated BCSC enrichment. Mechanistically, NANOG is recruited by HIF-1 to cooperatively activate transcription of the TERT gene encoding the telomerase reverse transcriptase that maintains telomere length, which is required for stem cell self-renewal. NANOG stimulates HIF-1 transcriptional activity by recruitment of the deubiquitinase USP9X, which inhibits HIF-1α protein degradation, and by stabilizing HIF-1α interaction with the coactivator p300, which mediates histone acetylation. Our results delineate a cooperative transcriptional mechanism by which HIF-1 and NANOG mediate BCSC self-renewal.

Histone citrullination by PADI4 is required for HIF-dependent transcriptional responses to hypoxia and tumor vascularization.

Hypoxia-inducible factors (HIFs) activate transcription of target genes by recruiting coactivators and chromatin-modifying enzymes. Peptidylarginine deiminase 4 (PADI4) catalyzes the deimination of histone arginine residues to citrulline. Here, we demonstrate that PADI4 expression is induced by hypoxia in a HIF-dependent manner in breast cancer and hepatocellular carcinoma cells. PADI4, in turn, is recruited by HIFs to hypoxia response elements (HREs) and is required for HIF target gene transcription. Hypoxia induces histone citrullination at HREs that is PADI4 and HIF dependent. RNA sequencing revealed that almost all HIF target genes in breast cancer cells are PADI4 dependent. PADI4 is required for breast and liver tumor growth and angiogenesis in mice. PADI4 expression is correlated with HIF-1α expression and vascularization in human breast cancer biopsies. Thus, HIF-dependent recruitment of PADI4 to target genes and local histone citrullination are required for transcriptional responses to hypoxia.

HIF-1α and HIF-2α redundantly promote retinal neovascularization in patients with ischemic retinal disease.

Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2-specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.