Our lab is interested in the molecular mechanisms underlying breast and colon cancer development and progression. Our focus is on the role of two highly conserved transcription factors Notch and Hypoxia Inducible factor (HIF) that are both frequently implicated in many cancers. Our aim is by deciphering the molecular mechanisms controlling these pathways we can translate basic research biology into clinical application for diagnosis or treatment in patients.
I. NOTCH SIGNALING IN SELF-RENEWAL AND CANCER
NOTCH signaling is a highly conserved short-range signaling pathway involved in cell fate specification during development and homeostatic self-renewal in adult tissues. Germ line mutations in the NOTCH pathway cause several hereditary diseases, and somatic mutations are found in cancer. Notch receptors are membrane bound transcription factors that are activated through ligand binding on adjacent cells. NOTCH pathway activity is governed by ligand induced proteolytic cleavage in the extracellular domain executed by a metalloprotease (MP). This cleavage is required and rate-limiting for the consecutive transmembrane cleavage by the γ-secretase complex leading to release of the intracellular domain and target gene transcription.
A. Canonical Notch Pathway is governed by proteolysis. Full-length (FL) receptors are cleaved at S1 in the secretory pathway. Upon ligand binding S2 cleavage removes the Notch extracellular domain to produce NEXT, which is a substrate for S3 cleavage to produce NICD B) Schematic representation of Notch proteolytic products. Shown are
extracellular EGF repeats (ligand binding), LNG repeat (repression), RAM (CSL binding)
and nls (nuclear localization), ANKyrin repeats (CSL binding) and the OPA/PEST (transcriptional activation and protein degradation) domains. (see Mumm et al., Mol Cell 2000) The precise mechanism that confers ligand regulated NOTCH proteolysis and which proteases are involved is unresolved. Human cancer prone NOTCH1 receptors are characterized by increased MP-dependent cleavage and activity. Our lab is focussed on understanding how such mutant Notch proteins contribute to cancer by generating mouse models and using these mouse models to identify novel approaches to inhibit aberrant Notch signalling in cancer.
We recently established that Notch signalling plays a critical role in gut homeostasis by regulating secretory cell differentiation in the small intestine. Using lineage tracing with NIP1-Cre mice, Notch1 marks intestinal (cancer) stem cells. Using NIP1-Cre mice we are establishing whether Notch1 is also active in other stem cell like compartments. We are investigated whether Notch signalling alters the renewal capacity of different stem cell systems in vitro and in vivo.
Notch and stem cell renewal: Staining and lineage tracing for activated
Notch1 protein in crypt epithelium of the small intestine
(Radtke and Clevers 2005, Vooijs 2007)
PROJECTS
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NOTCH1 gain of function alleles (Geert van Tetering, PhD Student)
Cancer prone Notch mutants are introduced into embryonic stem cells via homologous recombination in a manner permitting tissue-specific and inducible expression in vivo. These mutant Notch proteins are ligand independent but require MP cleavage. Notch proteins are tagged with YFP fluorescent reporter, which will enable by FACS the sorting and ex-vivo analysis of subpopulations of cells expressing YFP. Using different Cre-transgenic lines we will activate Notch in the adult gut and breast epithelium. Using tetracycline we will be able to turn the Notch expression ON and OFF in mice. These mice will be crossed with tumor prone mice to investigate the role of Notch1 in the initiation or maintenance of tumor phenotypes. Finally, Notch-YFP mice will be used to monitor -by non-invasive imaging (Bioluminescence)- the efficacy of inhibitors directed against Notch proteolysis.
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Targeting Notch proteases. (Renee de Vries, PhD Student)
We are developing semi-high throughput cell based assays amendable to RNA silencing (RNAi) and small molecule screening to inhibit and isolate the cellular activities responsible for activating Notch. Using activity based protease profiling we will evaluate the importance of candidate protease activity in mouse and human tissue samples to establish a correlation between activity and clinical outcome. We have identified a common cleavage site in Notch receptors and have generated a unique tool that permits monitoring of endogenous Notch receptor cleavage in cells. Approaches are underway to further develop similar antibodies into inhibitory antibodies that prevent Notch processing.
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NOTCH in stem cell renewal and breast cancer (Cigdem Ercan, PhD Student)
We will use our mouse models to study the requirement of Notch signaling and consequence of mutant Notch protein expression by generating loss of function and gain of function alleles in vivo in adult breast tissue. Similar approaches will be pursued as described above using existing mouse models for breast cancer (E-cad/p53 or PymTag model). We are investigating whether manipulation of Notch signaling in mammary epithelial stem cells (MESC) derived from ES cells or from virgin mammary glands alters the capacity to differentiate or self renew in vitro and in vivo. In addition pharmacological approaches will be used to block Notch signaling in vitro/vivo and to study the effect on repopulation in vivo.
II. HYPOXIA, HIF AND BREAST CANCER METASTASIS
Metastases formation is a major factor in disease progression and accounts for the majority of cancer deaths. The molecular mechanisms controlling invasion, dissemination to blood or lymphatic systems and spread of tumor cells to distant organs are still poorly understood. Recent observations indicate that the metastatic phenotype may already be present during the angiogenic switch of tumors. Hypoxia (oxygen deficiency) in tumors is primarily a pathophysiologic consequence of the high growth rate with lagging angiogenesis and structurally and functionally abnormal blood vessels and triggers the angiogenic switch. Cancer cells can undergo genetic and adaptive changes that allow them to survive and proliferate in hypoxic environments. These survival mechanisms contribute to the malignant phenotype, result in increasingly aggressive tumors and lead to resistance to chemo and radiation therapy. Intratumoral hypoxia results in gene expression changes that either increase O2 delivery or allow metabolic adaptation to reduced O2 availability in tumor cells.
Working Hypothesis: Increasing evidence
implicates HIF function in metastatic cell
characteristics, like epithelial to mesenchymal transition,
cell detachment, invasion and tumor cell seeding.
The hypoxic gene profile in primary tumors may
therefore be a driving force in tumor progression (Gort et al., 2008). The Hypoxia Inducible transcription Factor (HIF) is a critical mediator of this response and plays a central-but currently poorly defined-role in oncogenic transformation. Mounting evidence indicates that intratumoral hypoxia and increased Hypoxia Inducible Factor (HIF) activity is commonly associated with epithelial cancer progression of the head and neck, bladder, colon, breast and lung and correlates with therapy resistance and poor clinical outcome. Therefore, tumor hypoxia is a hallmark of solid cancer development. Understanding the molecular alterations underlying breast cancer development is necessary to improve early diagnosis and effective treatment. We hypothesize that the key events underlying the response to hypoxia may provide therapeutic targets in breast cancer management.
Perinecrotic HIF-1a staining in breast Cancer
(Bos 2001, 2003 Gort 2008) HIF is a highly conserved heterodimeric bHLH transcription factor consisting of an α and a β subunit. The β subunit (ARNT) is constitutively expressed, whereas the activity of the α subunit is regulated an oxygen dependent hydroxylases. At normoxia HIF-α is hydroxylated on highly conserved Proline and Asparagine residues which enables the Von Hippel Lindau (VHL) tumor suppressor protein to bind to HIF-α. VHL binding causes ubiquitination of HIF-α resulting in degradation of HIF-1α by the proteasome. Under hypoxic conditions, the oxygen-dependent hydroxylases are inactive, and the stabilized HIF-αtranslocates to the nucleus, where it binds to HIF-1α. The HIF-1 complex binds to a specific sequence elements in the promoter of genes, the hypoxia responsive element (HRE), thereby inducing transcription. Mammalian cells have three HIF-α subunits that can form dimers with HIF-β. Little is known about their unique and overlapping functions in the hypoxic response.
We are using cell-based approaches and model organisms to identify the role of the HIF in the tumorigenic/metastatic phenotype of cancer cells. Our goal is develop new biomarkers that may be of diagnostic or prognostic significance in breast cancer management.
PROJECTS
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Invasion and metastasis. (Iordanka Ivanova postdoc)
We have identified several novel genes in the hypoxic-HIF response pathway using a genetic suppressor screen in C-elegans. Among these genes is the mesodermal fate gene TWIST1. TWIST1 is an oncogene previously implicated in breast cancer metastasis and plays an important role in epithelial to mesenchymal transition (EMT). Our working model is that through TWIST1, hypoxia/HIF induces EMT in breast cancer, which induces invasive/metastatic phenotypes. We are using cell-based assays to investigate the requirement of TWIST1 in hypoxia mediated tumor invasion/migration
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Direct HIF- targets. (Alexey Nabatov postdoc)
Using chromatin immunoprecipitation followed by DNA micro array analysis (ChiP-on-chip) we are conducting a whole genome survey of DNA sequences bound by HIF molecules under hypoxia. Preliminary results predict novel HIF1 binding sites with high confidence. We will test if these elements respond to hypoxia in transcription assays. We will conduct similar studies with the HIF2 homolog to dissect the unique and overlapping targets in the hypoxic response.
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Hypoxia regulated cell surface markers. (Jeroen Vermeulen, PhD Student)
We are using phage display to isolate novel single domain variable heavy chain antibodies (VHH) derived from camellids in screens to discriminate between hypoxic and normoxic breast epithelial cells. When successful these markers will be employed for non-invasive molecular imaging of tumor hypoxia in preclinical mouse models.
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Clostridium directed antibody therapy. (Jeroen Vermeulen, PhD Student)
We are exploring the use of obligate anaerobic bacterium Clostridium as a vehicle to deliver therapeutic antibodies to hypoxic tumors. We demonstrated that VHH can be expressed in Clostridium and are functional. New targets that arise from the above will be used to generate inhibitory VHH that will be validated for the targeted treatment of hypoxic tumors in rodents.
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Biomarkers. (Karijn Suijkerbuijk, MD,PhD Student)
We are developing methylation marks as new diagnostic tool to detect breast cancer at an early stage. These biomarkers are evaluated for their application in screening women at high risk for breast cancer using non-invasively obtained nipple fluid.