Publications

<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>

Abstract Deletion 3p syndrome is associated with characteristic facial features, growth failure, and mental retardation. Typically, individuals with deletion 3p syndrome have terminal deletions that result in loss of material from 3p25 to 3pter. We present a child with a clinical phenotype consistent with deletion 3p syndrome (ptosis, microcephaly, growth retardation, and developmental delay) and a subtle interstitial deletion in the distal portion of the short arm of chromosome 3, del(3)(p25.3p26.2). Fluorescence in situ hybridization (FISH) studies using 3p subtelomeric probes confirmed the terminal region of chromosome 3 was present. Sequence tagged sites (STS)‐linked BAC clones mapping to chromosomal region 3p25‐p26 were used to characterize the interstitial deletion by FISH. The results indicate the deletion is within a region of approximately 4.5 Mb between STS markers D3S3630 and D3S1304. This interstitial deletion lies within all previously reported terminal deletions in deletion 3p syndrome individuals, and represents the smallest reported deletion associated with deletion 3p syndrome. Characterization of the deletion may help identify genes important to growth and development that contribute to the deletion 3p syndrome phenotype when present in a hemizygous state. © 2002 Wiley‐Liss, Inc.

PURPOSE: Variable uptake of the glucose analog 18 fluorodeoxyglucose (FDG) has been noticed in positron emission tomography (PET) studies of breast cancer patients, with low uptake occurring especially in lobular cancer. At present, no satisfactory biologic explanation exists for this phenomenon. This study compared 18 FDG uptake in vivo with biomarkers expected to be involved in the underlying biologic mechanisms. PATIENTS AND METHODS: Preoperative 18 FDG-PET scans were performed in 55 patients. 18 FDG activity was assessed visually by three observers using a four-point score. Tumor sections were stained by immunohistochemistry for glucose transporter-1 (Glut-1); Hexokinase (HK) I, II, and III; macrophages; hypoxia-inducible factor-1-alfa (HIF-1α); vascular endothelial growth factor (VEGF 165 ); and microvessels. Mitotic activity index (MAI), amount of necrosis, number of lymphocytes, and tumor cells/volume were assessed. RESULTS: There were positive correlations between 18 FDG uptake and Glut-1 expression (P < .001), MAI (P = .001), amount of necrosis (P = .010), number of tumor cells/volume (P = .009), expression of HK I (P = .019), number of lymphocytes (P = .032), and microvessel density (r = .373; P = .005). HIF-1α, VEGF 165 , HK II, HK III, and macrophages showed no univariate correlation with 18 FDG. In logistic regression, however, HIF-1α and HK II added value to MAI and Glut-1. CONCLUSION: 18 FDG uptake in breast cancer is a function of microvasculature for delivering nutrients, Glut-1 for transportation of 18 FDG into the cell, HK for entering 18 FDG into glycolysis, number of tumor cells/volume, proliferation rate (also reflected in necrosis), number of lymphocytes (not macrophages), and HIF-1α for upregulating Glut-1. Together, these features explain why breast cancers vary in 18 FDG uptake and elucidate the low uptake in lobular breast cancer.

Hypoxia-inducible factors (HIFs) control the transcription of genes that are crucial for the pathogenesis of cancer and other human diseases. The transcriptional activity of HIFs is rapidly increased upon exposure to hypoxia, but expression of some HIF target genes decreases during prolonged hypoxia. However, the underlying mechanism for feedback inhibition is not completely understood. Here, we report that peroxiredoxin 2 (PRDX2) and PRDX4 interact with HIF-1α and HIF-2α in vitro and in hypoxic HeLa cells. Prolonged hypoxia increases the nuclear translocation of PRDX2 and PRDX4. As a result, PRDX2 and PRDX4 impair HIF-1 and HIF-2 binding to the hypoxia response elements of a subset of HIF target genes, thereby inhibiting gene transcription in cells exposed to prolonged hypoxia. PRDX2 and PRDX4 have no effect on the recruitment of p300 and RNA polymerase II to HIF target genes and the enzymatic activity of PRDX2 and PRDX4 is not required for inhibition of HIF-1 and HIF-2. We also demonstrate that PRDX2 is a direct HIF target gene and that PRDX2 expression is induced by prolonged hypoxia. These findings uncover a novel feedback mechanism for inhibition of HIF transcriptional activity under conditions of prolonged hypoxia.

<p>Supplementary Figure S1. Cell proliferation of U87-MG subclones in vitro. 5,000 cells were seeded and cultured under 20% or 1% O2. Viable cell number was counted by trypan blue staining on days 2, 3, and 4, and normalized to the starting cell number (mean + SEM, n = 3). Supplementary Figure S2. Analysis of orthotopic tumors after injection of 1 x 104 cells into the caudate-putamen. (A) Tumor volume was determined by histological analysis 5 weeks after stereotactic injection of U87-MG parental or subclone cells into the brains of SCID mice (bar = mean, n = 7); *p < 0.05 vs shNT. (B) Depth of invasion was determined by histological analysis (mean + SEM, n = 5-7). (C) Anti-CD105 immunohistochemistry was performed and blood vessel density in tumor sections was determined (mean + SEM, n = 10-14). (D) Anti-Ki-67 immunohistochemistry was performed and stained cells were counted (mean + SEM, n = 5-7). (E) Immunohistochemistry was performed to detect active caspase-3 and stained cells were counted (mean + SEM, n = 5-7). Supplementary Figure S3. HIF-2α mRNA levels were determined by RT-qPCR (mean + SEM, n = 3) in parental, shScr, shCBS#07, and shCBS#61 subclones cultured at 20% O2.</p>

Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1alpha and HIF-1beta subunits. O2-dependent degradation of the HIF-1alpha subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1alpha is regulated by the competition of RACK1 and HSP90 for binding to HIF-1alpha. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1alpha. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1alpha. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1alpha. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1alpha degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1alpha each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1alpha. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1alpha degradation. These results indicate that intracellular calcium levels can regulate HIF-1alpha expression by modulating calcineurin activity and RACK1 dimerization.

<p>HIF-1 activates transcription of the DUSP9, REST and RCOR2 genes.</p>

<p>DUSP9 and DUSP16 reciprocally regulate the breast cancer stem cell phenotype.</p>

Significance Remote ischemic preconditioning (IPC) is a phenomenon in which brief cycles of limb ischemia and reperfusion, which can be induced in mice by clamping and unclamping the femoral artery and in human subjects by inflating and deflating a blood pressure cuff, result in protection of the heart against a prolonged episode of ischemia and reperfusion. The underlying mechanism may involve signals transmitted by the nervous system and by secreted factors, including interleukin-10. In this study, we demonstrate that the transcriptional activator hypoxia-inducible factor 1 (HIF-1) is necessary and sufficient for remote IPC in mice and for induction of Il10 gene expression in cultured mouse myocytes. Strategies designed to induce HIF-1 activity may afford cardioprotection in patients undergoing cardiac bypass surgery.

<p>HIF-1 activates transcription of the DUSP9, REST and RCOR2 genes.</p>

Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2⁺ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2⁺ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection.