!!Hans Clevers - Interests and research

!Research summary

__Lgr5 stem cells, Wnt signaling & cancer__
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''Tcf as Wnt effector''\\
In 1991, we reported the cloning of a T cell specific transcription factor that we termed TCF1 (1). Related
genes exist in genomes throughout the animal kingdom.We have shown in frogs (4), flies (7) and worms (11) that the TCF proteins constitute the effectors of the canonical Wnt pathway. Upon Wnt signaling, ßcatenin
binds and activates nuclear TCFs by providing a trans-activation domain. For these studies, we
designed the widely used pTOPFLASH Wnt reporters. In the absence of Wnt signaling, we found that Tcf
factors associate with proteins of the Groucho family of transcriptional repressors to repress target gene
transcription (9).
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''Wnt signaling in cancer''
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The tumor suppressor protein APC forms the core of a cytoplasmic complex which binds ß-catenin and
targets it for degradation in the proteasome. In APC-deficient colon carcinoma cells, we demonstrated
that ß-catenin accumulates and is constitutively complexed with the TCF family member TCF4, providing
a molecular explanation for the initiation of colon cancer (5).
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''Wnt signaling in adult stem cells''
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In mammals, physiological Wnt signaling is intimately involved with the biology of adult stem cells and
self-renewing tissues (18,19). We were the first to link Wnt signaling with adult stem cell biology, when we
showed that TCF4 gene disruption leads to the abolition of crypts of the small intestine (8), and that TCF1
gene knockout severely disables the stem cell compartment of the thymus (2). The Tcf4-driven target
gene program in colorectal cancer cells is the malignant counterpart of a physiological gene program in
selfrenewing crypts (13, 14, 21).
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''Lgr5 as adult stem cell marker''
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Amongst the Wnt target genes, we found the Lgr5 gene to be unique in that it marks small cycling cells at
crypt bottoms. These cells represent the epithelial stem cells of the small intestine and colon (23), the hair
follicle (24), the stomach (28) and -probably- all other epithelial stem cell types of the mammalian body.
They also represent the cells-of-origin of adenomas in the gut (25) and within adenomas Lgr5 stem cells
act as adenoma stem cells (36). The related Lgr6 marks multipotent skin stem cells (29).
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''Lgr5 stem cell biology''
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Lgr5 crypt stem cells behave in unanticipated ways: Against common belief, they divide constantly. Stem
cells numbers remain fixed because stem cells compete 'neutrally' for niche space. Thus, they do not
divide asymmetrically (31), a phenomenon that was confirmed by in vivo imaging (43). Daughters of the
small intestinal stem cells, the Paneth cells, serve as crypt niche cells by providing Wnt, Notch and EGF
signals (30).
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The Wnt target gene encoding the transcription factor Achaete scute-like 2 controls the fate of the
intestinal stem cell (26).
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''Lgr5 is the R-spondin receptor''
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Lgr5 resides in Wnt receptor complexes and mediates signaling of the R-spondin Wnt agonists (32),
explaining the unique dependence of Lgr5 stem cells of various epithelia on R-spondins in vivo and in
vitro. Two other Wnt target genes, RNF43 and ZNRF3, encode stem cell-specific E3 ligases that
downregulate Wnt receptors. They serve in a negative feedback loop to control the size of the stem cell
zone (34). Independent work by the Feng Cong lab has first shown that R-spondin, when bound to Lgr5,
captures and inactivates RNF43/ZNRF3.
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''Long-term clonal culturing of organoids from Lgr5 stem cells''
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Wnt signaling intimately interacts with the BMP and Notch cascades to drive proliferation and inhibit
differentiation in intestinal crypts and adenomas (17, 20). Based on these combined insights, we have
established Lgr5/R-spondin-based culture systems that allow the outgrowth of single mouse or human
Lgr5 stem cells into ever-expanding mini-guts (27), mini-stomachs (28), liver organoids (38, 45), prostate
organoids (44), breast cancer organoids (52) and organoids representing multiple other adult tissues.
These epithelial organoid cultures are genetically and phenotypically extremely stable, allowing
transplantation of the cultured offspring of a single stem cell, as well as disease modeling by growing
organoids directly from diseased patient tissues (45). The direct cloning of multiple individual cells from
primary tumors allows molecular and functional analysis of tumor heterogeneity with an unprecedented
resolution (53). Human organoids are readily amenable to CRISP-mediated genome modification to
model for instance malignant transformation (47) and mutagenesis upon faulty DNA repair (51).
As proof-of-concept, the CFTR locus was repaired in single gut stem cells from two Cystic Fibrosis
patients, using CRISPR/Cas9 technology in conjunction with homologous recombination. Repaired stem
cells were clonally expanded into mini-guts and shown -in a swelling assay- to contain a functional CFTR
channel (42). The organoid-based swelling assay has meanwhile become clinical practice in the
Netherlands to identify patient with rare mutations that respond to the Vertex drugs. To this end, we
founded the non-for-profit HUB foundation which currently builds a biobank of all 1500 Dutch CF patients
funded by our national insurance companies. The HUB also maintains large biobanks of colon-, breast-,
lung- and pancreas cancer organoids, accessible by academia and industry.
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