Research interest
We are using the zebrafish as an experimental model system to elucidate the developmental roles of tumor suppressors and oncogenes and to dissect molecular mechanisms that when subverted contribute to cancer.
We are following three main lines of research:
Analysis of energy metabolism and hypoxia during normal development and cancer
We have generated two mutant zebrafish lines in the serine-threonine kinase LKB1. LKB1 is a tumor suppressor in humans but the precise biochemical pathways affected via loss of LKB1 leading to cancer are not well understood. LKB1 is regulating AMP-activated protein kinase (AMPK), which is the major "energy sensor" of the cell. AMPK activation leads to inhibition of mTOR signaling and “shutting-down” of transcription and other energy-consuming processes in order to preserve energy. Activation of mTOR leads to proliferation and this pathway is often aberrantly activated in cancer. LKB1 is also involved in establishing epithelial polarity. Lkb1 mutant zebrafish exhibit hallmarks of premature starvation and die at day 7 post-fertilization presumably due to organ failure. AMPK is not phosphorylated, indicating that lkb1 mutants are incapable of sensing energetic stress. However, zTOR pathway activity is not affected by loss of LKB1 and treatment with the TOR-inhibitor rapamycin does not rescue the phenotype. Preliminary experiments show that genes involved in lipid and glucose metabolism are differentially expressed in lkb1 mutants compared to wild-type siblings and that some hypoxia-associated genes are overexpressed in lkb1 mutants. We are currently exploring further the connection between LKB1, energy metabolism and hypoxia. This investigation will lead to a better understanding of the adaptation of metabolic processes in order to meet energy demand that tumor cells employ in order to cope with decreasing nutrient supply and reduced oxygen supply within the tumor. We aim to gain insight into these processes at the organim level during normal development and during cancer formation.
Analysis of the developmental roles of the Polycomb group proteins (Collaboration with Maarten van Lohuizen).
Polycomb group (PcG) protein complexes, which function in the epigenetic regulation of gene expression, control numerous developmental processes. Epigenetic silencing mediated by PcG is implicated in stem-cell fate maintenance and cancer. The PcG member Ring1b is an ubiquitin ligase that ubiquitinates histone H2A. This mark correlates with the repression of gene expression of PcG target genes.
Disruption of Ring1b in mice leads to very early lethality. Fortuitously, zebrafish embryos in which Ring1b function has been reduced survive gastrulation. This gives us the opportunity to study the role of Ring1b during vertebrate development. Morpholino-mediated knock-down of Ring1b function leads to heart laterality defects and blood circulation abnormalities. We have recently succeeded in generating a stable mutant in Ring1b by using Zinc Finger Endonuclease technology and are currently characterizing the phenotype in detail.
Study of deubiquitination enzymes acting on the Wnt signaling pathway in zebrafish (Collaboration with Huib Ovaa)
More recently, I have initiated a collaborative project within the NKI with the aim to decipher the developmental roles of deubiquitinating enzymes (DUBS) in zebrafish. Deregulation of ubiquitin-conjugation and deconjugation of proteins leads to disease, including cancer. Indeed, several of the DUBs have been identified as oncogenes or tumor suppressors. Despite the importance of DUBs, little is known regarding their functions in vivo.
Emphasis will be given to the identification and functional characterization of DUBs acting on the Wnt signaling pathway, employing our apc mutant fish.
We employ HA-tagged Ub-derived active site directed probes to isolate and identify differentially active DUBs in apc embryos. We have initiated these studies and have confirmed that the HA-tagged active Ub probes recognize specific DUBS in zebrafish protein lysates. We next would like to compare this activity profile to the one from apc embryos. Once we identify the specific DUBS, we will down-regulate their function by morpholinos in wt and apc embryos. The embryonic heart phenotype will serve as a robust readout of the effects of the DUB knockdown in apc embryos.
We recently have tested in zebrafish a set of chemical inhibitors against USP7, the DUB that deubiquitinates p53. We have shown that the USP7-inhibitors are not toxic to zebrafish embryos and a couple of them cause a specific developmental defect. We are pursuing further these studies.
Key publications
Muncan V, Faro A, Haramis APG, Hurlstone AF, Wienholds E, van Es J, Korving J, Begthel H, Zivkovic D, Clevers H (2007). T-cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine. EMBO Rep. 8(10), 966-973.
Haramis APG, Perrakis A (2006). Selectivity and promiscuity in Eph receptors. Structure. 2006 Feb;14(2):169-71.
Haramis APG*, Hurlstone A, van der Velden Y, Begthel H, van den Born M, Offerhaus GJ, Clevers HC (2006). Adenomatous polyposis coli-deficient zebrafish are susceptible to digestive tract neoplasia. EMBO Rep. 7(4), 444-449.
Haramis APG, Begthel H, van de Born M, van Es J, Jonkers S, Offerhaus GJA, and Clevers H (2004). De novo crypt formation and Juvenile Polyposis on BMP inhibition in mouse intestine. Science. 303, 1684-1686.
Zuniga A, Michos O, Spitz F, Haramis APG, Panman L, Galli A, Vintersten K, Klasen C, Mansfield W, Kuc S, Duboule D, Dono R, Zeller R (2004). Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression. Genes Dev. 18(13), 1553-1564
Haramis APG*, Hurlstone AF*, Wienholds E, Begthel H, Korving J, van Eeden F, Cuppen E, Zivkovic D, Plasterk RHA and Clevers H (2003). The Wnt/beta-catenin pathway regulates cardiac valve formation. Nature. 425, 633-637.
van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis APG, Tjon-Pon-Fong M, Moerer P, van den Born M, Soete G, Pals S, Eilers M, Medema R, and Hans Clevers (2002). The b-Catenin/TCF-4 Complex Imposes a Crypt Progenitor Phenotype on Colorectal Cancer Cells. Cell. 111, 241-250.
Haramis APG*, Zuniga A*, McMahon AP, and Zeller R (1999). Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature. 401, 598-602.
More publications by Anna Haramis on PubMed
Biographic sketch
Anna-Pavlina Haramis did her doctoral work at European Molecular Biology Laboratory (EMBL) in Heidelberg in the lab of Rolf Zeller studying limb pattern formation in mice. She received her PhD from the University of Heidelberg in 1998 (magna cum laude). For her postdoctoral studies, she joined the lab of Nigel Holder and Steve Wilson at UCL in London as an EEC Marie-Curie fellow. There, she participated in forward genetic screens aimed at identifying mutants in developmental processes in zebrafish. She then worked as a postdoc in the lab of Hans Clevers in the Hubrecht Institute in Utrecht. She identified and characterized the zebrafish apc mutant, and unravelled a role for Wnt/beta-catenin signaling in regulating cardiac valve formation. She also validated the apc/+ zebrafish as a model to study cancer by demonstrating their high susceptibility to digestive tract neoplasia. In parallel, she analyzed the role of BMP4 signaling in murine intestinal development. She showed that inhibition of BMP signaling in the intestine resulted in a cancer predisposition syndrome that phenocopies Juvenile Polyposis (JP) in humans . In 2005, she obtained a Vidi grant from NWO and joined the NKI as research associate. Since January 2008 she is junior group leader analyzing the developmental roles of tumor suppressor and oncogenes in zebrafish.
Co-workers
- Yme van der Velden, Msc graduate student
- Liqin Wang, Technican
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