Wnt Signaling in 3D: Recent Advances in the Applications of Intestinal Organoids

Intestinal organoids grown from adult stem cells have emerged as prototype 3D organotypic models for studying tissue renewal and homeostasis. Owing to their strict dependence on Wnt signaling, intestinal organoids offer an unprecedented opportunity to examine Wnt pathway regulation in normal physiology and cancer. We review how alterations in growth factor dependency and organoid morphology can be exploited to identify Wnt signaling mechanisms, characterize mutated pathway components, and predict responses of patient-derived tumors to targeted therapy. We discuss current deficits in the understanding of genotype–phenotype relationships that are to be considered when interpreting mutation-induced changes in organoid morphology

RNF43 mutations facilitate colorectal cancer metastasis via formation of a tumour-intrinsic niche

In colorectal cancer (CRC), RNF43 mutations are linked to BRAF V600E-initiated serrated adenomas that advance into mucinous adenocarcinomas with poor prognosis upon metastasis. How RNF43 mutations facilitate a metastasis-prone growth state remains unknown. Here, we addressed this issue by repairing mutant RNF43 in patient-derived BRAF-mutant CRC organoids using gene editing. Upon RNF43 correction, CRC organoids exhibit strongly decreased mucus production and, moreover, display loss of niche factor independence and metastatic capacity upon orthotopic transplantation in mice. Mechanistically, we show that mutant RNF43 promotes cancer cell lineage specification towards a non-dividing niche population that secretes essential growth factors, providing a state of self-sufficiency to the cancer epithelium. We show that phenotypic diversification into tumour-intrinsic niche cells (TINCs) and proliferative cancer stem cells depends on tuneable WNT levels enabled by mutations in RNF43, but not APC. In patient samples, enhanced TINC profiles correlate with RNF43-mutant CRC, mucinous histology and metastatic disease, thus representing a general cellular mechanism by which tumours acquire a self-sufficient, pro-metastatic growth state

Understanding and targeting Wnt receptor complex regulation in cancer

The adult human body is built of around 3.72 × 1013 cells. These cells display a great variation in shape, size and molecular composition, which reflects the morphology and functions of distinct tissues and organs. Adult human cells can be divided roughly in two categories: differentiated cells and stem cells. Differentiated cells are non-dividing, specialized cells that perform specific tasks within a given organ, for example enterocytes and goblet cells that mediate nutrient absorption and mucus production in the intestinal epithelial lining. The main activity of stem cells, on the other hand, is to divide and replenish tissues with new cells. By tight regulation of stem cell activities, adult tissues balance self-renewal and differentiation during tissue homeostasis and repair. Tissue homeostasis is stringently dependent on communication between stem cells and surrounding differentiated cells (stem cell niche cells) via several tightly regulated signaling pathways. A major driving force for tissue renewal is mediated by Wnt-induced signaling. The ability of this pathway to drive stemness and proliferative properties is often misused by cancer cells that frequently acquire mutations in Wnt pathway components that activate signaling and bypass the need for cell-to-cell communication. The overall aim of this thesis is to unearth novel mechanisms of Wnt signaling in tissue homeostasis and cancer development. By uncovering the novel roles of RNF43 and TRIP13 in Wnt signaling pathway, we gained a new prospective on how Wnt pathway perturbations affect adult stem cells fate. Finally, we apply our knowledge to rationally develop reagents capable of targeting Wnt hypersensitive tumors

Publisher Correction: Mutations and mechanisms of WNT pathway tumour suppressors in cancer

Nature Reviews Cancer (2020) https://doi.org/10.1038/s41568-020-00307-z Published online 23 October 2020 Figure 3 of the article as originally published contained a graphic editing error, whereby the publisher’s redrawn figure wrongly indicated the length of the MCR in the APC protein structure. This has been corrected in the HTML and PDF versions of the manuscript

Mutations and mechanisms of WNT pathway tumour suppressors in cancer

Mutation-induced activation of WNT-β-catenin signalling is a frequent driver event in human cancer. Sustained WNT-β-catenin pathway activation endows cancer cells with sustained self-renewing growth properties and is associated with therapy resistance. In healthy adult stem cells, WNT pathway activity is carefully controlled by core pathway tumour suppressors as well as negative feedback regulators. Gene inactivation experiments in mouse models unequivocally demonstrated the relevance of WNT tumour suppressor loss-of-function mutations for cancer growth. However, in human cancer, a far more complex picture has emerged in which missense or truncating mutations mediate stable expression of mutant proteins, with distinct functional and phenotypic ramifications. Herein, we review recent advances and challenges in our understanding of how different mutational subsets of WNT tumour suppressor genes link to distinct cancer types, clinical outcomes and treatment strategies

Three-dimensional analysis of single molecule FISH in human colon organoids

The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids.We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinctmRNAtranscripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids

RNF43 truncations trapCK1 to drive niche-independent self-renewal in cancer

Contains fulltext : 226136.pdf (publisher's version ) (Open Access

LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation

Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA)

Anti-LRP5/6 VHHs promote differentiation of Wnt-hypersensitive intestinal stem cells

Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors

Anti-LRP5/6 VHHs promote differentiation of Wnt-hypersensitive intestinal stem cells

Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors