Osteocyte- and late Osteoblast-derived NOTUM Reduces Cortical Bone Mass in Mice
American journal of physiology. Endocrinology and metabolism
Nilsson, KH;Henning, P;El Shahawy, M;Wu, J;Koskela, A;Tuukkanen, J;Perret, C;Lerner, UH;Ohlsson, C;Movérare-Skrtic, S;
PMID: 33749332 | DOI: 10.1152/ajpendo.00565.2020
Osteoporosis is a common skeletal disease, with increased risk of fractures. Currently available osteoporosis treatments reduce the risk of vertebral fractures, mainly dependent on trabecular bone, whereas the effect on non-vertebral fractures, mainly dependent on cortical bone, is less pronounced. WNT signaling is a crucial regulator of bone homeostasis, and the activity of WNTs is inhibited by NOTUM, a secreted WNT lipase. We previously demonstrated that conditional inactivation of NOTUM in all osteoblast lineage cells increases the cortical but not the trabecular bone mass. The aim of the present study was to determine if NOTUM increasing cortical bone is derived from osteoblast precursors/early osteoblasts or from osteocytes/late osteoblasts. First, we demonstrated Notum mRNA expression in Dmp1-expressing osteocytes and late osteoblasts in cortical bone using in situ hybridization. We then developed a mouse model with inactivation of NOTUM in Dmp1 expressing osteocytes and late osteoblasts (Dmp1-creNotumflox/flox mice). We observed that the Dmp1-creNotumflox/flox mice displayed a substantial reduction of Notum mRNA in cortical bone, resulting in increased cortical bone mass and decreased cortical porosity in femur, but no change in trabecular bone volume fraction (BV/TV) in femur or in the lumbar vertebrae L5 in Dmp1-creNotumflox/flox mice as compared to control mice. In conclusion, osteocytes and late osteoblasts are the principal source of NOTUM in cortical bone, and NOTUM derived from osteocytes/late osteoblasts reduces cortical bone mass. These findings demonstrate that inhibition of osteocyte/late osteoblast-derived NOTUM might be an interesting pharmacological target to increase cortical bone mass and reduce non-vertebral fracture risk.
van Neerven, SM;Smit, WL;van Driel, MS;Kakkar, V;de Groot, NE;Nijman, LE;Elbers, CC;Léveillé, N;Heijmans, J;Vermeulen, L;
PMID: 36321561 | DOI: 10.15252/emmm.202216194
The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.
Liu, Y;Guerrero-Juarez, C;Xiao, F;Shettigar, N;Ramos, R;Kuan, C;Lin, Y;de Jesus Martinez Lomeli, L;Park, J;Oh, J;Liu, R;Lin, S;Tartaglia, M;Yang, R;Yu, Z;Nie, Q;Li, J;Plikus, M;
| DOI: 10.1016/j.devcel.2022.06.005
Hair follicle stem cells are regulated by dermal papilla fibroblasts, their principal signaling niche. Overactivation of Hedgehog signaling in the niche dramatically accelerates hair growth and induces follicle multiplication in mice. On single-cell RNA sequencing, dermal papilla fibroblasts increase heterogeneity to include new Wnt5ahigh states. Transcriptionally, mutant fibroblasts activate regulatory networks for Gli1, Alx3, Ebf1, Hoxc8, Sox18, and Zfp239. These networks jointly upregulate secreted factors for multiple hair morphogenesis and hair-growth-related pathways. Among these is non-conventional TGF-β ligand Scube3. We show that in normal mouse skin, Scube3 is expressed only in dermal papillae of growing, but not in resting follicles. SCUBE3 protein microinjection is sufficient to induce new hair growth, and pharmacological TGF-β inhibition rescues mutant hair hyper-activation phenotype. Moreover, dermal-papilla-enriched expression of SCUBE3 and its growth-activating effect are partially conserved in human scalp hair follicles. Thus, Hedgehog regulates mesenchymal niche function in the hair follicle via SCUBE3/TGF-β mechanism.
Linssen, JDG;van Neerven, SM;Aelvoet, AS;Elbers, CC;Vermeulen, L;Dekker, E;
PMID: 35962368 | DOI: 10.1186/s12876-022-02442-3
Familial adenomatous polyposis (FAP) is a rare autosomal dominant disease characterized by germline mutations in the Adenomatous Polyposis Coli (APC) gene, resulting in the development of numerous colorectal adenomas. As these patients have a high risk of developing colorectal cancer (CRC), guidelines suggest prophylactic colectomy during early adulthood, however, adenoma development is still observed in the remaining intestinal tract. Therefore, FAP patients would benefit from chemoprevention strategies reducing the development of adenomas. Recent work in mice reveals a chemopreventive effect of lithium on the development of adenomas by inhibiting the expansion of Apc mutated intestinal stem cells (ISCs) within the crypts of normal intestinal mucosa. Here, we aim to investigate the effect of lithium on the spread of APC mutant cells within the human intestinal epithelium.This prospective phase II single arm trial has a duration of 18 months. FAP patients (18-35 years) with a genetically confirmed APC mutation who did not undergo colectomy will be treated with lithium carbonate orally achieving a serum level of 0.2-0.4 mmol/l between month 6 and 12. Colonoscopy with biopsies of normal intestinal mucosa will be performed at baseline and every six months. The primary endpoint is the effect of lithium on the spread of APC mutant cells within intestinal crypts over time by using APC specific marker NOTUM in situ hybridization. Secondary endpoints include change in adenoma burden, patient reported side effects and safety-outcomes. Total sample size is 12 patients and recruitment will take place in the Amsterdam UMC, location AMC in the Netherlands.The outcome of this study will function as a proof-of-concept for the development of novel chemoprevention approaches that interfere with the competition between normal and mutant ISCs.ClinicalTrials.gov ( https://clinicaltrials.gov/ ): NCT05402891 (June 1, 2022) and the EU Clinical Trials Register: EuraCT 2022-000240-30 (January 1, 2022).
Oncogenic BRAF, unrestrained by TGFβ-receptor signalling, drives right-sided colonic tumorigenesis
Leach, JDG;Vlahov, N;Tsantoulis, P;Ridgway, RA;Flanagan, DJ;Gilroy, K;Sphyris, N;Vázquez, EG;Vincent, DF;Faller, WJ;Hodder, MC;Raven, A;Fey, S;Najumudeen, AK;Strathdee, D;Nixon, C;Hughes, M;Clark, W;Shaw, R;S:CORT consortium, ;van Hooff, SR;Huels, DJ;Medema, JP;Barry, ST;Frame, MC;Unciti-Broceta, A;Leedham, SJ;Inman, GJ;Jackstadt, R;Thompson, BJ;Campbell, AD;Tejpar, S;Sansom, OJ;
PMID: 34103493 | DOI: 10.1038/s41467-021-23717-5
Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.
Apc-mutant cells act as supercompetitors in intestinal tumour initiation
van Neerven, SM;de Groot, NE;Nijman, LE;Scicluna, BP;van Driel, MS;Lecca, MC;Warmerdam, DO;Kakkar, V;Moreno, LF;Vieira Braga, FA;Sanches, DR;Ramesh, P;Ten Hoorn, S;Aelvoet, AS;van Boxel, MF;Koens, L;Krawczyk, PM;Koster, J;Dekker, E;Medema, JP;Winton, DJ;Bijlsma, MF;Morrissey, E;Léveillé, N;Vermeulen, L;
PMID: 34079128 | DOI: 10.1038/s41586-021-03558-4
A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3β. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.
Cell Rep. 2019 Jan 8;26(2):394-406.e5.
Mizrak D, Levitin HM, Delgado AC, Crotet V, Yuan J, Chaker Z, Silva-Vargas V, Sims PA, Doetsch F.
PMID: 30625322 | DOI: 10.1016/j.celrep.2018.12.044
The ventricular-subventricular zone (V-SVZ) harbors adult neural stem cells. V-SVZ neural stem cells exhibit features of astrocytes, have a regional identity, and depending on their location in the lateral or septal wall of the lateral ventricle, generate different types of neuronal and glial progeny. We performed large-scale single-cell RNA sequencing to provide a molecular atlas of cells from the lateral and septal adult V-SVZ of male and female mice. This revealed regional and sex differences among adult V-SVZ cells. We uncovered lineage potency bias at the single-cell level among lateral and septal wall astrocytes toward neurogenesis and oligodendrogenesis, respectively. Finally, we identified transcription factor co-expression modules marking key temporal steps in neurogenic and oligodendrocyte lineage progression. Our data suggest functionally important spatial diversity in neurogenesis and oligodendrogenesis in the adult brain and reveal molecular correlates of adult NSC dormancy and lineage specialization.
NOTUM from Apc-mutant cells biases clonal competition to initiate cancer
Flanagan, DJ;Pentinmikko, N;Luopajärvi, K;Willis, NJ;Gilroy, K;Raven, AP;Mcgarry, L;Englund, JI;Webb, AT;Scharaw, S;Nasreddin, N;Hodder, MC;Ridgway, RA;Minnee, E;Sphyris, N;Gilchrist, E;Najumudeen, AK;Romagnolo, B;Perret, C;Williams, AC;Clevers, H;Nummela, P;Lähde, M;Alitalo, K;Hietakangas, V;Hedley, A;Clark, W;Nixon, C;Kirschner, K;Jones, EY;Ristimäki, A;Leedham, SJ;Fish, PV;Vincent, JP;Katajisto, P;Sansom, OJ;
PMID: 34079124 | DOI: 10.1038/s41586-021-03525-z
The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.
Proceedings of the National Academy of Sciences of the United States of America
Jean, P;Wong Jun Tai, F;Singh-Estivalet, A;Lelli, A;Scandola, C;Megharba, S;Schmutz, S;Roux, S;Mechaussier, S;Sudres, M;Mouly, E;Heritier, AV;Bonnet, C;Mallet, A;Novault, S;Libri, V;Petit, C;Michalski, N;
PMID: 37339214 | DOI: 10.1073/pnas.2221744120
Functional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type-specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments.
