Publications

Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.

<p>Supplementary Table S1. Oligonucleotide sequences of shRNAs targeting PHGDH. Table S2. Oligonucleotide sequence of RT-qPCR primers.</p>

Xing, Dongmei MD, PhD; Marti, Guy MD; Liu, Lixin PhD; Zhang, Xianjie MD, PhD; Reinblatt, Maura MD; Semenza, Gregg L. MD, PhD; Harmon, John W. MD, FACS Author Information

Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER + /PR + breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.

Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1alpha and constitutively expressed HIF-1beta. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based beta-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1alpha inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1alpha activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.

Chronic systemic hypertension causes cardiac pressure overload leading to increased myocardial O 2 consumption. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of O 2 homeostasis. Mouse embryos lacking expression of the O 2 -regulated HIF-1α subunit die at midgestation with severe cardiac malformations and vascular regression. Here we report that Hif1a f/f ;Tie2-Cre conditional knockout mice, which lack HIF-1α expression only in Tie2 + lineage cells, develop normally, but when subjected to pressure overload induced by transaortic constriction (TAC), they manifest rapid cardiac decompensation, which is accompanied by excess cardiac fibrosis and myocardial hypertrophy, decreased myocardial capillary density, increased myocardial hypoxia and apoptosis, and increased TGF-β signaling through both canonical and noncanonical pathways that activate SMAD2/3 and ERK1/2, respectively, within endothelial cells of cardiac blood vessels. TAC also induces dilatation of the proximal aorta through enhanced TGF-β signaling in Hif1a f/f ;Tie2-Cre mice. Inhibition of TGF-β signaling by treatment with neutralizing antibody or pharmacologic inhibition of MEK–ERK signaling prevented TAC-induced contractile dysfunction and pathological remodeling. Thus, HIF-1 plays a critical protective role in the adaptation of the heart and aorta to pressure overload by negatively regulating TGF-β signaling in endothelial cells. Treatment of wild-type mice with digoxin, which inhibits HIF-1α synthesis, resulted in rapid cardiac failure after TAC. Although digoxin has been used for decades as an inotropic agent to treat heart failure, it does not improve survival, suggesting that the countertherapeutic effects of digoxin observed in the TAC mouse model may have clinical relevance.

Abstract A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1α mutant, or a short hairpin RNA directed against HIF-1α. In human RCC biopsies, expression of E-cadherin and HIF-1α was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1–dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin. (Cancer Res 2006; 66(5): 2725-31)

Long‐term (30 days) exposure of rats to intermittent hypoxia (LT‐IH) results in persistent elevation of reactive oxygen species levels in central and peripheral nervous system ( J. Nanduri et al., J. Physiol.2016,2018 ). HIF‐ regulates pro‐oxidant enzyme (POE) genes during IH. Present study examined the effect of LT‐IH on HIF‐1α protein and NADPH oxidase (Nox) 4 mRNA abundances in the carotid body (CB) and adrenal medullae (AM) of LT‐IH treated rats. CB and AM of LT‐IH treated rats exhibited robust increase in HIF‐1α protein, Nox4 mRNA and ROS levels. Remarkably, these effects persisted after recovery in room air for 30d. The effects of LT‐IH on HIF‐1α, Nox 4 mRNA, and ROS abundance were absent in rats treated with digoxin, an inhibitor of HIF‐1α during LT‐IH as well as in mice with partial deficiency of HIF‐1a (HIF‐1α heterozygote mice). These findings suggest that HIF‐1 dependent upregulation of POE genes contributes to LT‐IH induced persistent oxidative stress in the CB and AM. Support or Funding Information Supported by NIH‐ HL‐90554. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

<div>Abstract<p>Metastasis is the leading cause of breast cancer mortality. Previous studies have implicated hypoxia-induced changes in the composition and stiffness of the extracellular matrix (ECM) in the metastatic process. Therefore, the contribution of potential ECM-binding receptors in this process was explored. Using a bioinformatics approach, the expression of all integrin receptor subunits, in two independent breast cancer patient datasets, were analyzed to determine whether integrin status correlates with a validated hypoxia-inducible gene signature. Subsequently, a large panel of breast cancer cell lines was used to validate that hypoxia induces the expression of integrins that bind to collagen (ITGA1, ITGA11, ITGB1) and fibronectin (ITGA5, ITGB1). Hypoxia-inducible factors (HIF-1 and HIF-2) are directly required for ITGA5 induction under hypoxic conditions, which leads to enhanced migration and invasion of single cells within a multicellular 3D tumor spheroid but did not affect migration in a 2D microenvironment. ITGB1 expression requires HIF-1α, but not HIF-2α, for hypoxic induction in breast cancer cells. ITGA5 (α<sub>5</sub> subunit) is required for metastasis to lymph nodes and lungs in breast cancer models, and high ITGA5 expression in clinical biopsies is associated with an increased risk of mortality.</p><p><b>Implications:</b> These results reveal that targeting ITGA5 using inhibitors that are currently under consideration in clinical trials may be beneficial for patients with hypoxic tumors. <i>Mol Cancer Res; 15(6); 723–34. ©2017 AACR</i>.</p></div>