Nature cardiovascular research
Bernier-Latmani, J;Cisarovsky, C;Mahfoud, S;Ragusa, S;Dupanloup, I;Barras, D;Renevey, F;Nassiri, S;Anderle, P;Squadrito, ML;Siegert, S;Davanture, S;González-Loyola, A;Fournier, N;Luther, SA;Benedito, R;Valet, P;Zhou, B;De Palma, M;Delorenzi, M;Sempoux, C;Petrova, TV;
PMID: 35602406 | DOI: 10.1038/s44161-022-00061-5
Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.
Parker, C;Chambers, AC;Flanagan, DJ;Ho, JWY;Collard, TJ;Ngo, G;Baird, DM;Timms, P;Morgan, RG;Sansom, OJ;Williams, AC;
PMID: 35468497 | DOI: 10.1016/j.dnarep.2022.103331
The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.
Klingler, S;Hsu, KS;Hua, G;Martin, ML;Adileh, M;Baslan, T;Zhang, Z;Paty, PB;Fuks, Z;Brown, AM;Kolesnick, R;
PMID: 35260534 | DOI: 10.1172/jci.insight.153793
Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5-positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane-dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury-induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.
Fatehullah, A;Terakado, Y;Sagiraju, S;Tan, TL;Sheng, T;Tan, SH;Murakami, K;Swathi, Y;Ang, N;Rajarethinam, R;Ming, T;Tan, P;Lee, B;Barker, N;
PMID: 34857912 | DOI: 10.1038/s41556-021-00793-9
Gastric cancer is among the most prevalent and deadliest of cancers globally. To derive mechanistic insight into the pathways governing this disease, we generated a Claudin18-IRES-CreERT2 allele to selectively drive conditional dysregulation of the Wnt, Receptor Tyrosine Kinase and Trp53 pathways within the gastric epithelium. This resulted in highly reproducible metastatic, chromosomal-instable-type gastric cancer. In parallel, we developed orthotopic cancer organoid transplantation models to evaluate tumour-resident Lgr5+ populations as functional cancer stem cells via in vivo ablation. We show that Cldn18 tumours accurately recapitulate advanced human gastric cancer in terms of disease morphology, aberrant gene expression, molecular markers and sites of distant metastases. Importantly, we establish that tumour-resident Lgr5+ stem-like cells are critical to the initiation and maintenance of tumour burden and are obligatory for the establishment of metastases. These models will be invaluable for deriving clinically relevant mechanistic insights into cancer progression and as preclinical models for evaluating therapeutic targets.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Vaughan-Shaw, PG;Blackmur, JP;Grimes, G;Ooi, LY;Ochocka-Fox, AM;Dunbar, K;von Kriegsheim, A;Rajasekaran, V;Timofeeva, M;Walker, M;Svinti, V;Din, FVN;Farrington, SM;Dunlop, MG;
PMID: 34918389 | DOI: 10.1096/fj.202101430RR
Vitamin D deficiency is associated with risk of several common cancers, including colorectal cancer (CRC). Here we have utilized patient derived epithelial organoids (ex vivo) and CRC cell lines (in vitro) to show that calcitriol (1,25OHD) increased the expression of the CRC tumor suppressor gene, CDH1, at both the transcript and protein level. Whole genome expression analysis demonstrated significant differential expression of a further six genes after 1,25OHD treatment, including genes with established links to carcinogenesis GADD45, EFTUD1 and KIAA1199. Furthermore, gene ontologies relevant to carcinogenesis were enriched by 1,25OHD treatment (e.g., 'regulation of Wnt signaling pathway', 'regulation of cell death'), with common enriched processes across in vitro and ex vivo cultures including 'negative regulation of cell proliferation', 'regulation of cell migration' and 'regulation of cell differentiation'. Our results identify genes and pathways that are modifiable by calcitriol that have links to CRC tumorigenesis. Hence the findings provide potential mechanism to the epidemiological and clinical trial data indicating a causal association between vitamin D and CRC. We suggest there is strong rationale for further well-designed trials of vitamin D supplementation as a novel CRC chemopreventive and chemotherapeutic agent.
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.
Leibowitz BJ, Yang L, Wei L, Buchanan ME, Rachid M, Parise RA, Beumer JH, Eiseman JL, Schoen RE, Zhang L, Yu J.
PMID: 29437148 | DOI: 10.1126/scitranslmed.aam7610
The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type-specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy-induced loss of LGR5+ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.
Moreno, R;Buehler, D;Lambert, PF;
PMID: 36016373 | DOI: 10.3390/v14081751
Murine papillomavirus, MmuPV1, causes natural infections in laboratory mice that can progress to squamous cell carcinoma (SCC) making it a useful preclinical model to study the role of papillomaviruses in cancer. Papillomavirus can infect cells within hair follicles, which contain multiple epithelial progenitor cell populations, including Lgr5+ progenitors, and transgenic mice expressing human papillomavirus oncogenes develop tumors derived from Lgr5 progenitors. We therefore tested the hypothesis that Lgr5+ progenitors contribute to neoplastic lesions arising in skins infected with MmuPV1 by performing lineage tracing experiments. Ears of 6-8-week-old Lgr5-eGFP-IRES-CreERT2/Rosa26LSLtdTomato mice were treated topically with 4-OH Tamoxifen to label Lgr5+ progenitor cells and their progeny with tdTomato and, 72 h later, infected with MmuPV1. Four months post-infection, tissue at the infection site was harvested for histopathological analysis and immunofluorescence to determine the percentage of tdTomato+ cells within the epithelial lesions caused by MmuPV1. Squamous cell dysplasia showed a low percentage of tdTomato+ cells (7%), indicating that it arises primarily from non-Lgr5 progenitor cells. In contrast, cutaneous SCC (cSCC) was substantially more positive for tdTomato+ cells (42%), indicating that cSCCs preferentially arise from Lgr5+ progenitors. Biomarker analyses of dysplasia vs. cSCC revealed further differences consistent with cSCC arising from LGR5+ progenitor cells.
British journal of cancer
Takeda, T;Yokoyama, Y;Takahashi, H;Okuzaki, D;Asai, K;Itakura, H;Miyoshi, N;Kobayashi, S;Uemura, M;Fujita, T;Ueno, H;Mori, M;Doki, Y;Fujii, H;Eguchi, H;Yamamoto, H;
PMID: 34707247 | DOI: 10.1038/s41416-021-01579-4
KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC).In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter.We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples.Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.
ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation
Sun, T;Annunziato, S;Bergling, S;Sheng, C;Orsini, V;Forcella, P;Pikiolek, M;Kancherla, V;Holwerda, S;Imanci, D;Wu, F;Meylan, LC;Puehringer, LF;Waldt, A;Oertli, M;Schuierer, S;Terracciano, LM;Reinker, S;Ruffner, H;Bouwmeester, T;Sailer, AW;George, E;Roma, G;de Weck, A;Piscuoglio, S;Lohmann, F;Naumann, U;Liberali, P;Cong, F;Tchorz, JS;
PMID: 34129813 | DOI: 10.1016/j.stem.2021.05.013
AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.
Senger S, Sapone A, Fiorentino MR, Mazzarella G, Lauwers GY, Fasano A.
PMID: 26649570 | DOI: 10.1371/journal.pone.0144634
Abstract
BACKGROUND:
In celiac disease (CD), intestinal epithelium damage occurs secondary to an immune insult and is characterized by blunting of the villi and crypt hyperplasia. Similarities between Hedgehog (Hh)/BMP4 downregulation, as reported in a mouse model, and CD histopathology, suggest mechanistic involvement of Hh/BMP4/WNT pathways in proliferation and differentiation of immature epithelial cells in the context of human intestinal homeostasis and regeneration after damage. Herein we examined the nature of intestinal crypt hyperplasia and involvement of Hh/BMP4 in CD histopathology.
METHODS AND FINDINGS:
Immunohistochemistry, qPCR and in situ hybridization were used to study a cohort of 24 healthy controls (HC) and 24 patients with diagnosed acute celiac disease (A-CD) intestinal biopsies. In A-CD we observed an increase in cells positive for Leucin-rich repeat-containing G protein-coupled receptor 5 (LGR5), an epithelial stem cell specific marker and expansion of WNT responding compartment. Further, we observed alteration in number and distribution of mesenchymal cells, predicted to be part of the intestinal stem cells niche. At the molecular level we found downregulation of indian hedgehog (IHH) and other components of the Hh pathway, but we did not observe a concurrent downregulation of BMP4. However, we observed upregulation of BMPs antagonists, gremlin 1 and gremlin 2.
CONCLUSIONS:
Our data suggest that acute CD histopathology partially recapitulates the phenotype reported in Hh knockdown models. Specifically, Hh/BMP4 paradigm appears to be decoupled in CD, as the expansion of the immature cell population does not occur consequent to downregulation of BMP4. Instead, we provide evidence that upregulation of BMP antagonists play a key role in intestinal crypt hyperplasia. This study sheds light on the molecular mechanisms underlying CD histopathology and the limitations in the use of mouse models for celiac disease.
Hilkens J, Timmer NC, Boer M, Ikink GJ, Schewe M, Sacchetti A, Koppens MA, Song JY, Bakker ER.
PMID: 27511199 | DOI: 10.1136/gutjnl-2016-311606
Abstract
OBJECTIVE:
The gross majority of colorectal cancer cases results from aberrant Wnt/β-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/β-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.
