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.
topHypoxia 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