RSPO3 expands intestinal stem cell and niche compartments and drives tumorigenesis

Objective The gross majority of colorectal cancer cases results from aberrant Wnt/ß-catenin signalling through adenomatous polyposis coli (APC) or CTNNB1 mutations. However, a subset of human colon tumours harbour, mutually exclusive with APC and CTNNB1 mutations, gene fusions in RSPO2 or RSPO3, leading to enhanced expression of these R-spondin genes. This suggested that RSPO activation can substitute for the most common mutations as an alternative driver for intestinal cancer. Involvement of RSPO3 in tumour growth was recently shown in RSPO3-fusion-positive xenograft models. The current study determines the extent into which solely a gain in RSPO3 actually functions as a driver of intestinal cancer in a direct, causal fashion, and addresses the in vivo activities of RSPO3 in parallel. Design We generated a conditional Rspo3 transgenic mouse model in which the Rspo3 transgene is expressed upon Cre activity. Cre is provided by cross-breeding with Lgr5-GFP-CreERT2 mice. Results Upon in vivo Rspo3 expression, mice rapidly developed extensive hyperplastic, adenomatous and adenocarcinomatous lesions throughout the intestine. RSPO3 induced the expansion of Lgr5+ stem cells, Paneth cells, non-Paneth cell label-retaining cells and Lgr4+ cells, thus promoting both intestinal stem cell and niche compartments. Wnt/ß-catenin signalling was modestly increased upon Rspo3 expression and mutant Kras synergised with Rspo3 in hyperplastic growth. Conclusions We provide in vivo evidence that RSPO3 stimulates the crypt stem cell and niche compartments and drives rapid intestinal tumorigenesis. This establishes RSPO3 as a potent driver of intestinal cancer and proposes RSPO3 as a candidate target for therapy in patients with colorectal cancer harbouring RSPO3 fusions

Bone Morphogenetic Protein Pathway Antagonism by Grem1 Regulates Epithelial Cell Fate in Intestinal Regeneration

BACKGROUND & AIMS In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. METHODS A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease