Ulf Brockmeier

New Insight into the SDF-1/CXCR4 Axis in a Breast Carcinoma Model: Hypoxia-Induced Endothelial SDF-1 and Tumor Cell CXCR4 Are Required for Tumor Cell Intravasation

The SDF-1/CXCR4 axis has been implicated in breast cancer metastasis. In contrast to its well-established role in organ-specific homing and colonization of tumor cells, the involvement in intravasation, especially in a hypoxic environment, is still poorly understood. Initially, we detected both, the chemokine SDF-1 and its receptor CXCR4 in microvessels in invasive ductal cancer samples. To elucidate the role of the SDF-1/CXCR4 axis in vascular endothelium for tumor intravasation, we evaluated the effects of CXCR4 activation in human umbilical vein and dermal microvascular endothelial cells (HUVEC and HDMEC) and in cultured mammary carcinoma cells (MDA MB231, and MCF7). We observed an upregulation of SDF-1 and CXCR4 in HUVECs in hypoxia, which led to proliferation, migration, and tube formation. Hypoxia induced adhesion of tumor cells to endothelial cells and stimulated transendothelial migration. The effects of hypoxia were dependent on the activity of the transcription factor hypoxia-inducible factor. Adhesion to and migration through a HUVEC monolayer were significantly reduced by lentiviral inhibition of CXCR4 in breast carcinoma cells or treatment of endothelial cells with an anti-SDF-1 neutralizing antibody. These data show that the interaction of SDF-1 secreted by ECs with tumor cell CXCR4 is sufficient to stimulate transendothelial migration of the tumor cells. Our results suggest that the SDF-1/CXCR4 axis is important in angiogenesis and tumor cell intravasation. Because both proteins were readily identifiable in a significant fraction of human breast cancer samples by immunohistochemistry, CXCR4 may constitute a molecular target for therapy when both, SDF-1, and CXCR4 are expressed. Mol Cancer Res; 10(8); 1021–31. ©2012 AACR.

Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2).

Summary The asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. In addition to HIF-&agr;, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, to date, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD-containing substrate. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity through interaction with partitioning defective 3 homolog (Par-3). Using mass spectrometry we identified ASPP2 as a new substrate of FIH-1 but inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of FIH-1. However, FIH-1 depletion did lead to impaired binding of Par-3 to ASPP2 while the interaction between ASPP2 and p53, apoptosis and proliferation of the cancer cells were not affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in relocation of ASPP2 from cell–cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD-containing substrates can be modified by FIH-1-dependent hydroxylation. The large cellular pool of ARD-containing proteins suggests that FIH-1 can affect a broad range of cellular functions and signalling pathways under certain conditions, for example, in response to severe hypoxia.

SDF-1 restores angiogenesis synergistically with VEGF upon LDL exposure despite CXCR4 internalization and degradation

AIMS Angiogenesis is compromised under conditions of hypercholesterolaemia. Since disturbed angiogenesis predisposes to ischaemic injuries, efforts have been made to promote angiogenesis by delivery of growth factors. How stromal cell-derived growth factor (SDF)-1 influences angiogenesis under conditions reflecting hypercholesterolaemia was unknown. METHODS AND RESULTS We investigated the effects of SDF-1, administered alone or in combination with vascular endothelial growth factor (VEGF), on angiogenesis using proliferation, transwell migration, and Matrigel-based tube formation assays with human umbilical vein endothelial cells that were exposed to low-density lipoprotein (LDL). We observed that SDF-1 dose-dependently enhanced angiogenesis, but only partly reversed the LDL-mediated suppression of angiogenesis. Reduced abundance of SDF-1s receptor, CXCR4, was noted on the surface of LDL-exposed endothelial cells. In subcellular localization studies combined with pharmacological experiments, we showed that the loss of CXCR4 resulted from its internalization and degradation. SDF-1 synergistically increased angiogenesis when combined with VEGF. As a consequence, angiogenesis was fully restored. SDF-1 reduced oxidized LDL formation and increased the anti-oxidant capacity of endothelial cells, most strongly when administered together with VEGF. CONCLUSION Combination therapies of growth factors, specifically SDF-1 and VEGF, might enhance angiogenesis more successfully than monotherapies under conditions of hypercholesterolaemia.

LDL attenuates VEGF-induced angiogenesis via mechanisms involving VEGFR2 internalization and degradation following endosome-trans-Golgi network trafficking

Considerable efforts have been made to amplify angiogenesis under conditions of hypoxia and ischemia by vascular endothelial growth factor (VEGF) delivery, so far with limited success. Ischemic vascular diseases are often associated with hypercholesterolemia. To elucidate whether the exposure to blood lipids influences VEGF responses of microvessels, we characterized effects of low density lipoprotein (LDL) exposure on the proliferation, migration and tube formation of human umbilical vein endothelial cells. By examining the expression, phosphorylation and downstream signals of VEGF’s receptor VEGFR2, we characterized mechanisms controlling angiogenic responses following LDL exposure. LDL attenuated endothelial proliferation, migration and tube formation in a dose-dependent way. Reduced abundance of VEGFR2 and VEGFR1 were noticed in LDL-exposed endothelial cells. In subcellular localization studies that we combined with pharmacological experiments, we showed that the loss of VEGFR2 resulted from its internalization and degradation, the latter of which required syntaxin-16-dependent endosome-trans-Golgi network trafficking. As a consequence, VEGFR2 phosphorylation and downstream signals -specifically Akt and ERK1/2 phosphorylation- were attenuated in response to VEGF treatment. VEGF only partly reversed the effects of LDL on angiogenesis under conditions of normoxia and hypoxia. Our results suggest that angiogenic responses to VEGF are compromised in hypercholesterolemia as a consequence of endosomal VEGFR2 degradation.

Synergistic Antitumor Effects of Endostar in Combination with Oxaliplatin via Inhibition of HIF and CXCR4 in the Colorectal Cell Line SW1116

Combination treatment with endostar, a novel modified endostatin, and cytotoxic chemotherapies showed a survival benefit in Chinese clinical trials. However, the exact mechanism for this synergism remains unclear. In this study, we report for the first time that the chemokine receptor CXCR4 and the hypoxia-inducible transcription factors (HIF)-1α and HIF-2α are involved in these synergistic antitumor effects in human colorectal cancer SW1116 cells in vitro when endostar treatment is combined with the cytotoxic drug oxaliplatin. Under normoxia, we demonstrate that endostar and oxaliplatin treatments synergize to inhibit SW1116 cell proliferation, Matrigel adhesion and invasion by reduction of CXCR4 expression. Consistently, these antitumor abilities of endostar and oxaliplatin were markedly reduced by silencing of CXCR4 in SW1116 cells. Under low oxygen conditions (hypoxia, 1% oxygen), enhanced proliferation of SW1116 cells exposed to oxaliplatin was observed due to the emergence of drug resistance. Strikingly, endostar overcame oxaliplatin-resistance, most likely as a consequence of reduced HIF-2α and CXCR4 levels. CXCR4, is only dependent on HIF-2α, which promotes more aggressive phenotype and more significant for oxaliplatin resistance in SW1116 cells. Our data not only provide clues to aid understanding of the mechanism of the synergism of endostar and chemotherapy under either normoxia or hypoxia, but also suggests a new strategy of combination endostar and chemotherapy treatments which might potentiate therapeutic efficacies and/or counteract chemotherapy resistance.

Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

Stabilisation and knockdown of HIF--two distinct ways comparably important in radiotherapy.

Background: Radiotherapy is one of the most widely used treatments for cancer. The benefit of radiation is known to be negatively affected by tumor hypoxia and the expression of hypoxia-inducible factors (HIF), respectively. HIF-1α/ β and HIF-2α/ β are transcriptional activators of oxygen-regulated genes. The aim of the study was to examine cell type-specific effects of HIF-1α and -2α knockdown or oxygen-independent HIF-stabilisation on radiosensitivity. Methods: Herein, we treated four different wildtype and HIF-1α- or HIF-2α-deficient human cancer cell lines, cultured under normoxic or hypoxic conditions, with ionising radiation in doses from 2 to 6 Gy and examined clonogenic survival. Furthermore, the cells were partly preincubated with a HIF-stabiliser (di-tert-butyroyl-oxymethyl-2,4-pyridine-dicarboxylate, tBu-2,4-PDC). Results: The results show that both hypoxia exposure and treatment withtBu-2,4-PDC increased the radioresistance of human cancer cells. The HIF-mediated decrease of radioresponsiveness induced by the chemical stabiliser emerged to be as strong as the one caused by hypoxia. Clonogenic survival assays furthermore revealed that HIF-1 expression enhanced resistance to radiation, whereas knocking-down HIF-1 increased the sensitivity to radiation under normoxic as well as under hypoxic conditions. Conclusion: These data extend previous observations of HIF-1α and broaden the view by showing HIF-2α inverse correlation between HIF expression and prognosis for the outcome of radiotherapy.

The Function of Hypoxia-Inducible Factor (HIF) Is Independent of the Endoplasmic Reticulum Protein OS-9

The protein “amplified in osteosarcoma-9” (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.

Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET-based study

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.

Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics

Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation.