Publications

Abstract Metastasis is responsible for the majority of breast cancer–related deaths, however, the mechanisms underlying metastasis in this disease remain largely elusive. Here we report that under hypoxic conditions, alternative splicing of MBD2 is suppressed, favoring the production of MBD2a, which facilitates breast cancer metastasis. Specifically, MBD2a promoted, whereas its lesser known short form MBD2c suppressed metastasis. Activation of HIF1 under hypoxia facilitated MBD2a production via repression of SRSF2-mediated alternative splicing. As a result, elevated MBD2a outcompeted MBD2c for binding to promoter CpG islands to activate expression of FZD1, thereby promoting epithelial-to-mesenchymal transition and metastasis. Strikingly, clinical data reveal significantly correlated expression of MBD2a and MBD2c with the invasiveness of malignancy, indicating opposing roles for MBD2 splicing variants in regulating human breast cancer metastasis. Collectively, our findings establish a novel link between MBD2 switching and tumor metastasis and provide a promising therapeutic strategy and predictive biomarkers for hypoxia-driven breast cancer metastasis. Significance: This study defines the opposing roles and clinical relevance of MBD2a and MBD2c, two MBD2 alternative splicing products, in hypoxia-driven breast cancer metastasis.

Key points Rats exposed to chronic intermittent hypoxia (CIH) exhibited imbalanced expression of hypoxia‐inducible factor (HIF)‐α isoforms and oxidative stress in brainstem regions associated with the carotid body (CB) chemoreflex, and in the adrenal medulla, an end organ of the sympathetic nervous system. Selective ablation of the CB abolished the effects of CIH on HIF‐α isoform expression and oxidative stress. In the adrenal medulla, chemoreflex‐mediated sympathetic activation regulates HIF‐α isoform expression via muscarinic acetylcholine receptor‐mediated Ca 2+ influx and the resultant activation of the mammalian target of rapamycin pathway and calpain proteases. Thus, CB neural activity regulates HIF‐α isoform expressions and redox state in the central and peripheral nervous system associated with the chemoreflex pathway under the setting of CIH. Abstract Previous studies reported that chronic intermittent hypoxia (CIH) results in an imbalanced expression of hypoxia‐inducible factor‐α (HIF‐α) isoforms and oxidative stress in rodents, which may be due either to the direct effect of CIH or indirectly via hitherto uncharacterized mechanism(s). As neural activity is a potent regulator of gene transcription, we hypothesized that carotid body (CB) neural activity contributes to CIH‐induced HIF‐α isoform expression and oxidative stress in the chemoreflex pathway. Experiments were performed on adult rats exposed to CIH for 10 days. Rats exposed to CIH exhibited: increased HIF‐1α and decreased HIF‐2α expression; increased NADPH oxidase 2 and decreased superoxide dismutase 2 expression; and oxidative stress in the nucleus tractus solitarius and rostral ventrolateral medulla as well as in the adrenal medulla (AM), a major end organ of the sympathetic nervous system. Selective ablation of the CB abolished these effects. In the AM, sympathetic activation by the CB chemoreflex mediates CIH‐induced HIF‐α isoform imbalance via muscarinic acetylcholine receptor‐mediated Ca 2+ influx, and the resultant activation of mammalian target of rapamycin pathway and calpain proteases. Rats exposed to CIH presented with hypertension, elevated sympathetic activity and increased circulating catecholamines. Selective ablation of either the CB (afferent pathway) or sympathetic innervation to the AM (efferent pathway) abolished these effects. These observations uncover CB neural activity‐dependent regulation of HIF‐α isoforms and the redox state by CIH in the central and peripheral nervous systems associated with the chemoreflex.

<p>Integrin expression in breast tumors and breast cancer cell lines.</p>

The editors of JCI and JCI Insight are revisiting our editorial processes in light of the strain that the COVID-19 pandemic places on the worldwide scientific community. Here, we discuss adjustments to our decision framework in light of restrictions placed on laboratory working conditions for many of our authors.

Digoxin and other cardiac glycosides in‐hibit hypoxia‐inducible factor‐1 (HIF‐1) transcriptional activity in cultured cells and suppress tumor xenograft growth. We tested the hypothesis that digoxin reduces HIF‐1 levels in ischemic tissue in vivo and suppresses neovascularization. Well‐established murine models of ocular neovascularization were used to test our hypothesis. In mice with ischemic retinopathy, intraocular or intraperitoneal injection of digoxin markedly reduced retinal levels of HIF‐1α protein and mRNAs encoding multiple hypoxia‐regulated proangiogenic proteins and their receptors. Daily intraperitoneal injection of 2 mg/kg starting at postnatal day (P) 12 or a single intravitreous injection of 100 ng of digoxin at P12 reduced retinal neovascularization by >70% at P17. Digoxin also reduced the number of CXCR4 + cells and F4/80 + macrophages in ischemic retina and significantly reduced choroidal neovascularization at Bruch's membrane rupture sites. Digoxin suppresses retinal and choroidal neovascularization by reducing HIF‐1α levels, which blocks several proangiogenic pathways. Since digoxin suppresses multiple pathways in addition to VEGF signaling, it may provide advantages over specific VEGF antagonists for treatment of patients with retinal and choroidal diseases complicated by neovascularization and/or excessive vascular permeability. It may also be useful for treatment of neovascular diseases in other tissues.—Yoshida, T., Zhang, H., Iwase, T., Shen, J., Semenza, G. L., Campochiaro, P. A. Digoxin inhibits retinal ischemia‐induced HIF‐1α expression and ocular neovascularization. FASEB J. 24, 1759–1767 (2010). www.fasebj.org

Chronic hypoxia (CH) causes pulmonary vasoconstriction because of increased pulmonary arterial smooth muscle cell (PASMC) contraction and proliferation. We previously demonstrated that intracellular Ca 2+ concentration ([Ca 2+ ] i ) was elevated in PASMCs from chronically hypoxic rats because of Ca 2+ influx through pathways other than L-type Ca 2+ channels and that development of hypoxic pulmonary hypertension required full expression of the transcription factor hypoxia inducible factor 1 (HIF-1). In this study, we examined the effect of CH on the activity and expression of store-operated Ca 2+ channels (SOCCs) and the regulation of these channels by HIF-1. Capacitative Ca 2+ entry (CCE) was enhanced in PASMCs from intrapulmonary arteries of rats exposed to CH (10% O 2 ; 21 days), and exposure to Ca 2+ -free extracellular solution or SOCC antagonists (SKF96365 or NiCl 2 ) decreased resting [Ca 2+ ] i in these cells. Expression of TRPC1 and TRPC6, but not TRPC4, mRNA and protein was increased in PASMCs from rats and wild-type mice exposed to CH, in PASMCs from normoxic animals cultured under hypoxic conditions (4% O 2 ; 60 hours), and in PASMCs in which HIF-1 was overexpressed under nonhypoxic conditions. Hypoxia-induced increases in basal [Ca 2+ ] i and TRPC expression were absent in mice partially deficient for HIF-1. These results suggest that increased TRPC expression, leading to enhanced CCE through SOCCs, may contribute to hypoxic pulmonary hypertension by facilitating Ca 2+ influx and increasing basal [Ca 2+ ] i in PASMCs and that this response is mediated by HIF-1.

<p>Supplementary Figure S1. Analysis of G6PD mRNA expression. Supplementary Figure S2. Analysis of MCF-7 Subclones. Supplementary Figure S3. Effect of PHGDH knockdown on cell growth. Supplementary Figure S4. Effect of carboplatin on cell viability. Supplementary Figure S5. Effect of doxorubicin treatment. Supplementary Figure S6. Clinical correlates.</p>

Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress. Iron chelators are well established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton chemistry and the formation of highly reactive hydroxyl radicals. Here, we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1alpha and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1alpha and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2alpha levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1alpha and CREB; and that HIF-2alpha, not HIF-1alpha, regulates susceptibility to normoxic oxidative neuronal death.

<p>PDF file - 98K, Figure S1. HIF-1α is required for PLOD induction under hypoxia in mouse embryo fibroblasts.</p>

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.

Many lung diseases induce HPH, leading to increased mortality. HPH, caused by vascular contraction and remodeling, is associated with increased intracellular pH (pH i ) and calcium concentration ([Ca 2+ ] i ) in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia‐inducible factor 1 (HIF‐1), a transcription factor, plays a critical role in the development of HPH. Recently, digoxin (Dig) was found to inhibit HIF‐1. We hypothesized that Dig could attenuate the development of HPH. Mice were weighed and injected daily with saline or Dig (low = 0.2 mg/kg or high = 1.0 mg/kg; i.p.) and kept in hypoxia (10% O 2 ) or room air. After 21 days, right ventricular (RV) pressures were measured via closed‐chest puncture. Hypoxia increased RV pressures in mice receiving saline. Low Dig reduced, and high Dig normalized, RV pressure in hypoxic mice. RV hypertrophy (RVH) increased in hypoxic mice treated with saline or low Dig, but was reduced in hypoxic mice receiving high Dig. Hypoxia increased hematocrit in mice treated with saline or low Dig, whereas high Dig reduced the hypoxia‐induced increase in hematocrit. Both pH i and [Ca 2+ ] i increased in PASMCs from saline‐treated hypoxic mice, and treatment with low or high Dig prevented these changes in ion homeostasis. From these results, we conclude that digoxin attenuates pulmonary vascular responses to chronic hypoxia and may be a possible candidate for treatment of HPH. HL67191, GM008074

The level of HIF-1 alpha increases as the pathologic stage increases and is higher in poorly differentiated lesions than in the corresponding type of well-differentiated lesions. Increased levels of HIF-1 alpha are associated with increased proliferation and increased expression of ER and VEGF. Thus, increased levels of HIF-1 alpha are potentially associated with more aggressive tumors.