Hans Clevers - Interests and research#

Research summary#

Lgr5 stem cells, Wnt signaling & cancer

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).

Wnt signaling in cancer

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).

Wnt signaling in adult stem cells

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).

Lgr5 as adult stem cell marker

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).

Lgr5 stem cell biology

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).

The Wnt target gene encoding the transcription factor Achaete scute-like 2 controls the fate of the intestinal stem cell (26).

Lgr5 is the R-spondin receptor

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.

Long-term clonal culturing of organoids from Lgr5 stem cells

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.

References

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