Angiogenesis and Hypoxia
General information
In order for tumors to grow above a few millimetres in size they must establish a (neo-) vasculature. One of the major stimuli for the development of new blood vessels is tissue hypoxia. The aim of this research line is to study the functional genomics of hypoxia in order to optimise radiotherapy and chemotherapy, to look for new targets in angiogenic blood vessels by using genetic profiling of tumour endothelium and to develop novel therapeutics for the treatment of patients based upon laboratory models of angiogenesis.
Angiogenesis
The mission of the Angiogenesis Laboratory is two-fold. Firstly, it is aimed to unravel fundamental processes and mechanisms of angiogenesis. Secondly, it is aimed to apply research and new technology for development of novel (cancer) treatment modalities in the clinic. The mission is elaborated in several interconnective and collaborative research projects i.e.:
Genetic profiling of tumor endothelium. This research project focuses on the identification of specific targets on tumor endothelium. Several molecular biological techniques have been applied to identify a series of 17 genes that are specifically overexpressed on tumor endothelium.
Identification and development of new angiostatic agents. The angiostatic designer peptide anginex and its mimetic KM0118 have been developed in our laboratory. The current research focuses on the identification of the cellular receptors for these molecules. These compounds are planned for clinical testing in the near future.
Study of the cross-talk between the vasculature and the immune system. The major achievement of this research is the recognition that anti-angiogenesis can improve anti-tumor immunity, which implies that it can improve immunotherapy as well.
Vasculogenic mimicry. Plasticity of tumor cells can de-dedifferentiate them into endothelial-like structures. We have demonstrated that this process is highly associated to aggressiveness in Ewing's sarcoma. In addition, we propose an important role of hypoxia in this process.
Tumor Hypoxia
Poor oxygenation (hypoxia) is a common feature of solid human tumors and is associated with increased malignancy, resistance to therapy and poor prognosis. Tumor cells actively sense and respond to hypoxia by initiating changes in gene expression that affect their phenotype. In order to understand the various biological responses to hypoxia it is necessary to both understand how hypoxia effects gene expression and to identify which genes are differentially expressed. Furthermore, tumor hypoxia can be exploited with bacteria based therapy and for tumor imaging purposes. We are focusing on:
The regulation of mRNA translation during hypoxia, and have shown that overall translation is rapidly and severely inhibited during hypoxic conditions. In addition to the effects that hypoxia exhibit upon overall mRNA translation, we are particularly interested in the consequences of regulating translation for differential gene expression, since different mRNA species are affected to a different extent. We are identifying mRNAs that remain efficiently translated during hypoxia, in spite of the overall repression of translation, and study the molecular mechanisms responsible for ensuring the efficient translation of these mRNA species during hypoxia. Finally, we are identifying novel hypoxia-induced proteins through a proteomic approach. In particular we are attempting to define molecular determinants of hypoxia tolerance and tumor growth to pursue novel molecular markers and targets for clinical use.
Imaging aspects. The ability for non-invasive imaging of hypoxia or hypoxia associated molecular markers is examined. This will ultimately allow earlier detection and phenotyping of tumors and provide an opportunity to tailor therapies to individual patients. We will focus on two approaches. The first is based upon optical imaging in small animals, taking advantage of genetically modified tumors and bacteria used for gene therapy purposes. In a second approach we will take advantage of the first CPT-PET stimulator of the world available in Maastricht to extend the basic research into more clinically relevant questions concerning quantification of hypoxia treatment.
Bacteria based gene therapy. We are investigating alternative gene delivery systems based on the use of non-pathogenic bacteria. The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localize within and cause cell lysis of experimental animal tumors. One of the most important strains in that context is Clostridium. Other bacteria have also been implicated in experimental anti-cancer settings. Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising.
topAngiogenesis
The mission of the Angiogenesis Laboratory, which is part of the Department of Pathology, is two-fold. Firstly, it is aimed to unravel fundamental processes and mechanisms of angiogenesis. Secondly, it is aimed to apply research and new technology for development of novel (cancer) treatment modalities in the clinic. Four different interconnective research lines are currently ongoing.
I. Genetic profiling of tumor endothelium.
This research line focuses on the identification of specific targets on tumor endothelium. We compared the transcriptional profile, by suppression subtractive hybridization analysis, of tumor endothelial cells with that of normal resting endothelial cells, normal but angiogenically activated placenta endothelial cells, and tumor-conditioned cultured endothelial cells. Though the majority of transcripts were classified as general angiogenesis markers, we identified 17 genes that show specific overexpression in tumor endothelium. We recently showed that targeting of four cell-surface expressed or secreted products with antibodies inhibited angiogenesis in vitro and in vivo. In collaboration with Dr. Van Engeland we also identified a series of genes that are silenced in tumor endothelium. The role of these genes in angiogenesis is currently being investigated.
This research is supported by GROW and by substantial support from industry.
II. Identification and development of new angiostatic agents.
The angiostatic designer peptide anginex and its mimetic KM0118 have been developed in the Angiogenesis Laboratory in collaboration with the University of Minnesota (Prof. K.H. Mayo). The research to identify the mechanisms of action has revealed galectin-1 as the receptor that transduces the signaling by anginex. It was found that this molecule is of extreme importance in development of vasculature as well. This was demonstrated by morpholino knock-down experiments in zebrafish.
The potential application of angiostatic agents in therapy is studied by gene expression profiling. This includes known molecules involved in angiogenesis as well as newly discovered ones. The prognostic value of such angiogenesis parameters is studied in lung cancer and in pediatric tumors.
Research line II is supported by KWF, NWO/STW and NIH.
III. Study of the cross-talk between the vasculature and the immune system. Current research has revealed that angiogenic potential, as measured by proliferating endothelial cells, in colorectal cancer is a prognostic factor (see figure). Similarly, leukocyte infiltration is of prognostic value. This has therapeutic implications and is in support to future combination therapies of anti-angiogenesis and immunotherapy. The novel angiogenesis inhibitors as identified in research line II were found to improve vascular adhesion molecule expression and leukocyte infiltration into the tumor. Immunotherapy protocols will be tested in mice to develop these compounds for clinical testing.
This research line is supported by KWF/NKB and by the Coenegracht Stichting.
IV. Tumor cell plasticity and vasculogenic mimicry.
Recent work has indicated that blood lakes in Ewing sarcoma contribute to circulation and can be regarded as a sign of vasculogenic mimicry. We demonstrated that hypoxia is a driving force behind this phenomenon.
This work is financially supported by GROW.
Selected publications
Thijssen, V.L., Postel, R., Brandwijk, R.J., Dings, R.P., Nesmelova, I., Satijn, S.A., Verhofstad, N., Nakabeppu, Y., Baum, L. G., Bakkers, J., Mayo, K.H., Poirier, F., and Griffioen, A.W.
Galectin-1 is essential in angiogenesis and is a target for anti-angiogenesis therapy. Proc.Natl.Acad.Sci.USA, 103:15975-15980, 2006
Derksen, P.W., Liu, X., Saradin, F., Evers, B., Van Beijnum, J.R., Griffioen, A.W., Van der Gulden, H., Zevenhoven, J., Peterse, H., Cardiff, R., Vink, J., Krimpenfort, P., Berns, A., and Jonkers, J.
Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis.
Cancer Cell, 10:437-449, 2006
Dings, R.P., Chen, X., Nesmelova, I., Haseman, J., Hellebrekers, D.M.E.I., Maxwell, J., Van Eijk, L.I., Hoye, T.R., Griffioen, A.W., Mayo, K.H.,
Design of non-peptidic helix/sheet topomimetics: applications to inhibition of angiogenesis and tumor growth in mice.
J Natl Cancer Inst, 98:932-936, 2006.
Van Beijnum, J.R., Dings, R.P.M., Zwaans, B., Van der Linden, E., Ramaekers, F.C.S., Mayo, K.H. and Griffioen, A. W.
Gene expression of tumor angiogenesis dissected; specific targeting of colon cancer angiogenic vasculature.
Blood, 108:2339-2348, 2006.
Mulder, W.J.M., Koole, R., Brandwijk, R.J., Storm, G., Chin, P., Strijkers, G.J., Celso de Mello Donega, Nicolay, K., Griffioen, A.W.
Paramagnetic quantum dots as a bimodal molecular imaging probe for angiogenesis.
Nano Letters. 6:1-6, 2006.
Figure: Loss of galectin-1 L2 and L3 results in hemorrhaging and defective vessel formation in the zebrafish brain.
(A-D) Staining for blood (arrows). (A) wild type control (B) morpholino knock- down lgals1 L2 AT-MO, (C) lgals-1 L3 ATG-MO, (D) both lgals-1 L2 and L3 ATG- MOs. Co-injection of L2 and L3 ATG-MO results in severe hemorrhaging in the brain region (arrowheads). (E) Schematic drawing of blood vessels in the dorsal brain at 2.5 day of development. Confocal microscopy from Tg(fli1:egfp)y1 transgenic embryos at the level of the dorsal brain vessels. (F) wild type control embryo.
(G) Embryos co-injected with lgals-1 L2 and -L3 ATG-MO display aberrant sprouting and misguidance of the middle cerebral vein (MCeV) into the dorsal longitudinal vein (DLV) (arrowheads). Defective angiogenic sprouting is also observed in the mesencephalic vein (arrow). (H) Co-injection of the lgals-1 L2 and L3 splice-MO shows similar defects in angiogenic sprouting of the brain vessels. DLV, dorsal longitudinal vein; MCeV, l vein; MsV mesencephalic vein; MtA, metencephalic artery; PCeV, posterior cerebral vein.
Research group
Dr. Arjan W. Griffioen, cell biologist, immunologist, project leader
Dr. Judy R. van Beijnum, molecular biologist
Dr. Freek Bot, MD, pathologist
Dr. Anne-Marie Dingemans, MD, pulmonologist
Dr. Ruud P.M. Dings, cell biologist
Dr. Bernd Granzen, MD, pediatrician
Dr. Daisy W.J. van der Schaft, cell biologist
Dr. Sebastien Tabruyn, molecular biologist.
Dr. Victor Thijssen, molecular biologist
PhD Students
Bisan Ahmed
Marcella Baldewijns
Vivian van den Boogaart
Ricardo Brandwijk
Karolien Castermans
Debby Hellebrekers
Femke Hillen
Veerle Melotte
Willem Mulder
Technicians
Loes van Eijk
Petra Hautvast
Sarah Hulsmans
Edith van der Linden
Sietske Satijn
Students
Rene Marx
Hypoxia and Gene Therapy
Hypoxia
Hypoxia (low oxygenation) is common in human tumors and results in more aggressive disease and poor patient prognosis. We have published three review articles which describe the molecular mechanisms that control gene expression during hypoxia, with focus on mRNA translation. Their role in determining the hypoxic malignant phenotype and the implications for therapeutic interventions are discussed. (Magagnin et al., Drug Resist Updates 9, 2006; Koumenis & Wouters, Mol Cancer Res 4(7) 2006; van den Beucken et al., Cancer Biol Ther 5(7) 2006).
This year, we demonstrated that mRNA translation initiation is severely inhibited during hypoxia and using genetically matched cell models showed that the molecular mechanism responsible for this inhibition switched from eIF2? at early timepoints to eIF4F during chronic hypoxia. Interestingly, individual mRNA species have different dependencies on eIF2? and eIF4F (Figure 1). Therefore, the switch of molecular control mechanism gives rise to differential gene expression during acute and chronic hypoxia (Koritzinsky et al., EMBO J 25(5) 2006). Our results indicate that activation of the molecular pathways including eIF2? and eIF4F are important for surviving the hypoxic stress that tumor cells are frequently subjected to. We are therefore evaluating the potential of proteins in these pathways as molecular targets in cancer therapy. Isogenic cell models have been created in which relevant proteins can be turned "off" or "on" as to mimic targeting in existing tumors, and these are currently being evaluated.
In an in vivo study, we evaluated the therapeutic benefit of adding an inhibitor of mRNA translation, rapamycin, during fractionated radiotherapy. Rapamycin inhibits mTOR which is an important regulator of mRNA translation that can stimulate proliferation and is regulated by hypoxia. We found that rapamycin did not significantly improve outcome of radiotherapy, possibly due to induction of thrombosis which resulted in radioresistant hypoxic cells (Weppler et al., Radiother Oncol 82(1), 96-104 2007).
As mentioned above, we have discovered that regulation of mRNA translation influences gene expression significantly during hypoxia. Therefore we have conducted large microarray and proteomic studies in which we have addressed the global impact of hypoxia on transcription and translation. Using isogenic models, we have also addressed the impact of the cells' ability to regulate eIF2a and eIF4F to these global changes in gene expression. The microarray studies have enabled us to generate "signatures" which describe the gene expression of hypoxic tumor cells. Using gene expression data sets obtained from large clinical trials, we have established that these signatures can be used to predict for poor patient prognosis. Data from these studies will be submitted for publication shortly.
Selected publication
Koritzinsky M, Magagnin MG, van den Beucken T, Savelkouls K, Koumenis C, Dostie J, Pyronnet S, Kaufman RJ, Weppler SA, Voncken JW, Lambin P, Sonenberg N, Wouters BG, Gene expression during acute and chronic hypoxia is mediated by distinct modes of translational control.
EMBO Journal 2006, Mar 8; 25(5):1114-25.
Figure: Regulation of gene-specific mRNA translation is dependent on eIF2a phosphorylation.
Acute hypoxia causes inhibition of CA9 mRNA translation (left panel) and stimulation of GADD34 mRNA translation (right panel) in wild-type mouse embryo fibroblasts (MEFs). MEFs which carry a mutation at the eIF2a phosphorylation site (S51A) are not able to regulate mRNA translation in response to hypoxia.
Koritzinsky et al. EMBO J, 2006.
Research group
Prof.dr. Bradly G. Wouters, project leader
Dr. Marianne Koritzinsky, project leader
Prof.dr. Philippe Lambin
Post-doctoral fellows
Dr. Kasper Rouschop
Dr. Renaud Seigneuric
PhD students
Twan van den Beucken
Michael G. Magagnin
Maud Starmans
Sherry A. Weppler
Technicians
Mieke Duysinx
Kim Savelkouls
Gene Therapy
Prokaryotic-based tumor-targeted therapy is an area of growing interest for the treatment of solid tumors. Upon systemic administration, non-pathogenic obligate anaerobes and facultative anaerobes (with Clostridium and modified Salmonella strains as prototypical agents) have been shown to infiltrate and selectively replicate within the regions of hypoxia and necrosis in solid tumors. These vectors can be safely administered and we and others have proven their potential to deliver therapeutic proteins specifically to tumors in vivo.
Clostridium sporogenes
We have developed a reliable Clostridium transformation method and used it to manipulate a superior tumor colonizing strain, C. sporogenes, to express a novel nitroreductase enzyme with improved kinetic properties. We demonstrated that multiple treatment cycles using recombinant bacteria combined with prodrug administration, resulted in sustained growth delay. In addition, use of non-invasive imaging techniques allowed us to evaluate prodrug conversion in real time. In collaboration with Nigel Minton (University Nottingham, UK), important developments with regards to integration of the recombinant gene into the clostridial chromosome have taken place. In combination with newly synthesised vectors and therapeutic proteins with optimised codon usage, this is an essential step in order to move forward to clinical applications.
Salmonella VNP20009
Although studies with VNP20009 have established the feasibility to deliver therapeutic proteins to tumors, they also demonstrated that normal tissues could be colonized. Since this can cause undesirable side-effects, we developed a vector in which gene expression is controlled by a hypoxia-responsive HIP-1 promoter. Use of HIP-1 resulted in induction levels up to 200-fold. Using non-invasive imaging, we showed that gene expression can be significantly induced relative to a constitutive promoter in vivo when tumors are made hypoxic. We also successfully used another imaging technique, 19F MRS, to predict the response of tumors following CDase recombinant Salmonella/5-FC treatment (see fig.). Using the data originating from an extensive series of Salmonella micro-array experiments following hypoxia and ionizing radiation, we are currently creating a novel and powerful inducible promoter system, that will allow us to express context-dependent therapeutic proteins. Importantly, analogous to the situation with Clostridium, we are developing the necessary integration technology for cloning the gene of interest into the chromosome.
Selected publication 2006
J. Theys, O. Pennington, L. Dubois, W. Landuyt, J. Anné, P. Burke, G. Anlezark, P. D?rre, B.G. Wouters, N.P. Minton and P. Lambin.
Repeated systemic treatment cycles of Clostridium-directed enzyme prodrug therapy results in sustained anti-tumour effects in vivo.
Brit J Cancer (2006), 95, 1212-1219.
Figure: 19F-MRS can predict tumor response following CDase recombinant Salmonella/5-FC treatment.
(A) Growth delay for ‘responder' (9/12; ?) and ‘non-responder' (3/12; ?) animals. Sham saline-treated animals are indicated with ?. Animals in which a 5-FU signal could be detected using in vivo 19F-MRS during the follow-up period were marked as ‘responders', the others as ‘non-responders'. The 100% value represents the tumour volume at day 0 (i.e. volume at the start of 5-FC administration). Statistical significance at p < 0.001 using linear regression analysis is indicated (*). Data are shown as mean ± SEM.
(B) Representative spectra (13 min/spectrum; TR = 0.75s; NA = 1024; LB = 6 Hz; zerofilling to 4096 points) of a ‘non-responder' (top) and a ‘responder' (bottom) animal.
Research group
Dr. Jan Theys , project leader
Prof.dr. Philippe Lambin
Prof.dr. Bradly G. Wouters
Post-doctoral fellow
Willy Landuyt
PhD students
Asferd Mengesha
Ludwig Dubois
Technicians
Kim Paesmans
Students
Felix Kolk
Tom Keulers