Publications

<div>Abstract<p>Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor–positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers. <i>Cancer Res; 76(15); 4430–42. ©2016 AACR</i>.</p></div>

Significance Cancer cells release from their cell surface membrane-lined microvesicles (MVs), which contain proteins, mRNAs, and microRNAs that can be taken up by other cells. We report that breast cancer cells exposed to decreased oxygen availability (hypoxia) increase their production of MVs, which stimulate invasion and metastasis by recipient breast cancer cells. Increased MV shedding by hypoxic cells requires expression of hypoxia-inducible factors (HIFs), which activate transcription of the RAB22A gene, and expression of the small GTPase RAB22A, which is a protein that localizes to budding MVs. Our results delineate a molecular mechanism by which hypoxia increases invasion and metastasis by stimulating MV shedding and provide further evidence that addition of HIF inhibitors to current treatment regimens may improve clinical outcome.

Abstract Sleep‐disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro‐oxidant enzymes, especially NADPH oxidase‐2 (Nox2). Previous studies showed that (i) IH activates hypoxia‐inducible factor 1 (HIF‐1) in a ROS‐dependent manner and (ii) HIF‐1 is required for IH‐induced ROS generation, indicating the existence of a feed‐forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH‐induced expression of Nox2 is mediated by HIF‐1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild‐type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF‐1 activity through RNA interference or pharmacologic inhibition (digoxin or YC‐1) or by genetic knockout of HIF‐1α in MEFs. Increasing HIF‐1α expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF‐1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild‐type mice to IH (8 h/day for 10 days) up‐regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a +/− mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF‐1, ROS generation, and cardiovascular pathology in response to IH. J. Cell. Physiol. 226: 2925–2933, 2011. © 2011 Wiley‐Liss, Inc.

<p>Epistasis analysis of the signaling pathway induced by chemotherapy in TNBC cells.</p>

<p>More detailed description of the abbreviated Materials and Methods that appears in the main text.</p>

Abstract Sleep‐disordered breathing with recurrent apnea (periodic cessation of breathing) results in chronic intermittent hypoxia (IH), which leads to cardiovascular and respiratory pathology. Molecular mechanisms underlying IH‐evoked cardio‐respiratory co‐morbidities have not been delineated. Mice with heterozygous deficiency of hypoxia‐inducible factor 1α (HIF‐1α) do not develop cardio‐respiratory responses to chronic IH. HIF‐1α protein expression and HIF‐1 transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH‐evoked HIF‐1α accumulation. PC12 cells were exposed to aerobic conditions (20% O 2 ) or 60 cycles of IH (30 sec at 1.5% O 2 followed by 5 min at 20% O 2 ). Our results show that IH‐induced HIF‐1α accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS‐dependent Ca 2+ signaling pathways involving phospholipase Cγ (PLCγ) and protein kinase C activation are required for IH‐evoked HIF‐1α accumulation. IH leads to activation of mTOR and S6 kinase (S6K) and rapamycin partially inhibited IH‐induced HIF‐1α accumulation. IH also decreased hydroxylation of HIF‐1α protein and anti‐oxidants as well as inhibitors of Ca +2 signaling prevented this response. Thus, both increased mTOR‐dependent HIF‐1α synthesis and decreased hydroxylase‐dependent HIF‐1α degradation contribute to IH‐evoked HIF‐1α accumulation. Following IH, HIF‐1α, and phosphorylated mTOR levels remained elevated during 90 min of re‐oxygenation despite re‐activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF‐1α levels during re‐oxygenation indicating that mTOR‐dependent protein synthesis is required for the persistent elevation of HIF‐1α levels during re‐oxygenation. J. Cell. Physiol. 217: 674–685, 2008. © 2008 Wiley‐Liss, Inc.

Human ether‐a‐go‐go‐related (hERG) K + channel regulates cell excitability in a variety of cells. hERG protein is synthesized in the endoplasmic reticulum (ER) as an immature core glycosylated protein (cg) which is exported to the Golgi apparatus for glycosylation and eventually transported to the plasma membrane as fully glycosylated mature protein (fg). Sustained hypoxia (SH) down regulates the fg form with accumulation of the cg form, resulting in defective trafficking. Systemic and cellular responses to SH require activation of hypoxia‐inducible factors 1 and 2 (HIF‐1 and HIF‐2), which are composed of HIF‐1α or HIF‐2α heterodimerized with HIF‐1β. In the present study, we examined the role of HIFs in SH‐induced hERG trafficking defect. Experiments were performed on SH‐SY5Y neuroblastoma cells exposed to either 4 days of SH (1.5% O 2 ) or normoxia (20% O 2 ). Silencing HIF‐2α but not HIF‐1α by short‐hairpin RNA (shRNA) blocked ER accumulation. Co‐immunoprecipitation assay revealed physical interaction of HIF‐2α with hERG cg form. Silencing HIF‐1β, which is required for HIF‐2 transcriptional activity, did not rescue the SH‐induced hERG trafficking defect, suggesting that HIF‐2α has a non‐transcriptional role in regulating hERG trafficking during hypoxia. Supported by NIH‐HL‐090554

Immune cells are exposed to low oxygen tensions as they develop and migrate between blood and different tissues, but the mechanisms by which lymphocytes adapt to hypoxia are poorly understood. Studies reported here of hypoxia-inducible factor 1α (HIF-1α) in lymphocyte development and functions suggest that it has a critical role in regulation of these processes. HIF-1α deficiency in Hif1α −/− → Rag2 −/− chimeric mice results in dramatic and cell lineage-specific defects, which include appearance of abnormal peritoneal B-1-like lymphocytes, with high expression of B220 (CD45) receptor-associated protein tyrosine phosphatase and autoimmunity (accumulation of anti-dsDNA antibodies and rheumatoid factor in serum, deposits of IgG and IgM in kidney and proteinuria) as well as distortions of maturation of B-2 lymphocytes in bone marrow.

<p>Epistasis analysis of the signaling pathway induced by chemotherapy in TNBC cells.</p>

<p>Supplementary Data PDF file 764K, Supplementary Figure S1, HIF-1 dependent P4HA1 and P4HA2 induction Supplementary Figure S2, P4HA1 and P4HA2 knockdown inhibits breast cancer growth and metastasis Supplementary Figure S3, Collagen hydroxylase knockdown decreases collagen deposition in vivo Supplementary Figure S4, P4HA1 and P4HA2 knockdown inhibits growth and metastasis of MDA-MB-435 cells Supplementary Figure S5, Picrosirius red staining of mouse lungs Supplementary Figure S6, P4HA re-expression in HIF-deficient cells rescues collagen deposition and metastasis Supplementary Figure S7, DHB treatment of MDA-MB-231 Cells Supplementary Table S1, Real-time qPCR primers. Supplemental Table S2, Comparison of human P4HA1 mRNA versus mouse P4HA1 mRNA expression in tumors. Supplemental Table S3, Correlation of P4HA1 and P4HA2 mRNA expression with receptor status</p>

<div>Abstract<p><b>Purpose:</b> This study characterized the therapeutic efficacy of a systemically administered formulation of 3-bromopyruvate (3-BrPA), microencapsulated in a complex with β-cyclodextrin (β-CD), using an orthotopic xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC).</p><p><b>Experimental Design:</b> The presence of the β-CD–3-BrPA complex was confirmed using nuclear magnetic resonance spectroscopy. Monolayer as well as three-dimensional organotypic cell culture was used to determine the half-maximal inhibitory concentrations (IC<sub>50</sub>) of β-CD–3-BrPA, free 3-BrPA, β-CD (control), and gemcitabine in MiaPaCa-2 and Suit-2 cell lines, both in normoxia and hypoxia. Phase-contrast microscopy, bioluminescence imaging (BLI), as well as zymography and Matrigel assays were used to characterize the effects of the drug <i>in vitro</i>. An orthotopic <i>luc</i>MiaPaCa-2 xenograft tumor model was used to investigate the <i>in vivo</i> efficacy.</p><p><b>Results:</b> β-CD–3-BrPA and free 3-BrPA demonstrated an almost identical IC<sub>50</sub> profile in both PDAC cell lines with higher sensitivity in hypoxia. Using the Matrigel invasion assay as well as zymography, 3-BrPA showed anti-invasive effects in sublethal drug concentrations. <i>In vivo</i>, animals treated with β-CD–3-BrPA demonstrated minimal or no tumor progression as evident by the BLI signal as opposed to animals treated with gemcitabine or the β-CD (60-fold and 140-fold signal increase, respectively). In contrast to animals treated with free 3-BrPA, no lethal toxicity was observed for β-CD–3-BrPA.</p><p><b>Conclusion:</b> The microencapsulation of 3-BrPA represents a promising step towards achieving the goal of systemically deliverable antiglycolytic tumor therapy. The strong anticancer effects of β-CD–3-BrPA combined with its favorable toxicity profile suggest that clinical trials, particularly in patients with PDAC, should be considered. <i>Clin Cancer Res; 20(24); 6406–17. ©2014 AACR</i>.</p></div>