Publications

<p>Legends for Supplementary Figures S1-S6</p>

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists of two subunits, HIF-1alpha, which is degraded during normoxia, and HIF-1beta, which is constitutively expressed. Activated HIF-1 induces the expression of genes involved in angiogenesis, erythropoiesis, and glucose metabolism. We have previously reported that insulin stimulates vascular endothelial growth factor (VEGF) expression (). In this study, we show that insulin activates HIF-1, leading to VEGF expression in retinal epithelial cells. Insulin activates HIF-1alpha protein expression in a dose-dependent manner with a maximum reached within 6 h. The expression of HIF-1alpha is correlated with the activation of HIF-1 DNA binding activity and the transactivation of a HIF-1-dependent reporter gene. Insulin does not appear to affect HIF-1alpha mRNA transcription but regulates HIF-1alpha protein expression through a translation-dependent pathway. The expression of an active form of protein kinase B and treatment of cells with specific inhibitors of phosphatidylinositol 3-kinase (PI3K), MAPK, and target of rapamycin (TOR) show that mainly PI3K and to a lesser extent TOR are required for insulin-induced HIF-1alpha expression. HIF-1 activity and VEGF expression are also dependent on PI3K- and TOR-dependent signaling. In conclusion, we show here that insulin regulates HIF-1 action through a PI3K/TOR-dependent pathway, resulting in increased VEGF expression.

<p>Expression of DUSP9 and DUSP16 mRNA is correlated with HIF target gene expression.</p>

Vascular remodeling resulting from altered pulmonary arterial smooth muscle cell (PASMC) growth is a contributing factor to the pathogenesis of hypoxic pulmonary hypertension. PASMC growth requires an alkaline shift in intracellular pH (pH i ) and we previously showed that PASMCs isolated from mice exposed to chronic hypoxia exhibited increased Na + /H + exchanger (NHE) expression and activity, which resulted in increased pH i . However, the mechanism by which hypoxia caused these changes was unknown. In this study we tested the hypothesis that hypoxia-induced changes in PASMC pH homeostasis are mediated by the transcriptional regulator hypoxia-inducible factor 1 (HIF-1). Consistent with previous results, increased NHE isoform 1 (NHE1) mRNA and protein, enhanced NHE activity, and an alkaline shift in pH i were observed in PASMCs isolated from wild-type mice exposed to chronic hypoxia (3 wk at 10% O 2 ). In contrast, these changes were absent in PASMCs isolated from chronically hypoxic mice with partial deficiency for HIF-1. Exposure of PASMCs to hypoxia ex vivo (48 h at 4% O 2 ) or overexpression of HIF-1 in the absence of hypoxia also increased NHE1 mRNA and protein expression. Our results indicate that full expression of HIF-1 is essential for hypoxic induction of NHE1 expression and changes in PASMC pH homeostasis and suggest a novel mechanism by which HIF-1 mediates pulmonary vascular remodeling during the pathogenesis of hypoxic pulmonary hypertension.

<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>

Breast cancer stem cells (BCSCs), which are characterized by a capacity for unlimited self-renewal and for generation of the bulk cancer cell population, play a critical role in cancer relapse and metastasis. Hypoxia is a common feature of the cancer microenvironment that stimulates the specification and maintenance of BCSCs. In this study, we found that hypoxia increased expression of adenosine receptor 2B (A2BR) in human breast cancer cells through the transcriptional activity of hypoxia-inducible factor 1. The binding of adenosine to A2BR promoted BCSC enrichment by activating protein kinase C-δ, which phosphorylated and activated the transcription factor STAT3, leading to increased expression of interleukin 6 and NANOG, two key mediators of the BCSC phenotype. Genetic or pharmacological inhibition of A2BR expression or activity decreased hypoxia- or adenosine-induced BCSC enrichment in vitro, and dramatically impaired tumor initiation and lung metastasis after implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice. These data provide evidence that targeting A2BR might be an effective strategy to eradicate BCSCs.

Signaling through glutamate receptors has been reported in human cancers, but the molecular mechanisms are not fully delineated. We report that in hepatocellular carcinoma and clear cell renal carcinoma cells, increased activity of hypoxia-inducible factors (HIFs) due to hypoxia or VHL loss-of-function, respectively, augmented release of glutamate, which was mediated by HIF-dependent expression of the SLC1A1 and SLC1A3 genes encoding glutamate transporters. In addition, HIFs coordinately regulated expression of the GRIA2 and GRIA3 genes, which encode glutamate receptors. Binding of glutamate to its receptors activated SRC family kinases and downstream pathways, which stimulated cancer cell proliferation, apoptosis resistance, migration and invasion in different cancer cell lines. Thus, coordinate regulation of glutamate transporters and receptors by HIFs was sufficient to activate key signal transduction pathways that promote cancer progression.

Significance The recruitment of host stromal cells, such as macrophages and mesenchymal stem cells (MSCs), to the primary tumor is a critical step toward cancer malignancy. We have identified signals that are exchanged between breast cancer cells (BCCs) and MSCs. This signaling increases the recruitment of both MSCs and macrophages to primary tumors and increases metastasis of BCCs to lymph nodes and lungs. Reduced oxygen levels (hypoxia) in breast cancers are associated with increased risk of metastasis and decreased patient survival. We show that hypoxia stimulates signaling between BCCs and MSCs due to the activity of hypoxia-inducible factors (HIFs). Drugs that block HIF activity prevent signaling and macrophage recruitment, which suggests that they may be useful additions to breast cancer therapy.