Interplay between metabolic identities in the intestinal crypt supports stem cell function.

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Fibroblast activation protein identifies Consensus Molecular Subtype 4 in colorectal cancer and allows its detection by 68Ga-FAPI-PET imaging.

BACKGROUND: In colorectal cancer (CRC), the consensus molecular subtype 4 (CMS4) is associated with therapy resistance and poor prognosis. Clinical diagnosis of CMS4 is hampered by locoregional and temporal variables influencing CMS classification. Diagnostic tools that comprehensively detect CMS4 are therefore urgently needed. METHODS: To identify targets for molecular CMS4 imaging, RNA sequencing data of 3232 primary CRC patients were explored. Heterogeneity of marker expression in relation to CMS4 status was assessed by analysing 3-5 tumour regions and 91.103 single-tumour cells (7 and 29 tumours, respectively). Candidate marker expression was validated in CMS4 peritoneal metastases (PM; n = 59). Molecular imaging was performed using the 68Ga-DOTA-FAPI-46 PET tracer. RESULTS: Fibroblast activation protein (FAP) mRNA identified CMS4 with very high sensitivity and specificity (AUROC > 0.91), and was associated with significantly shorter relapse-free survival (P = 0.0038). Heterogeneous expression of FAP among and within tumour lesions correlated with CMS4 heterogeneity (AUROC = 1.00). FAP expression was homogeneously high in PM, a near-homogeneous CMS4 entity. FAPI-PET identified focal and diffuse PM that were missed using conventional imaging. Extra-peritoneal metastases displayed extensive heterogeneity of tracer uptake. CONCLUSION: FAP expression identifies CMS4 CRC. FAPI-PET may have value in the comprehensive detection of CMS4 tumours in CRC. This is especially relevant in patients with PM, for whom effective imaging tools are currently lacking.

Cancer-associated rs6983267 SNP and its accompanying long noncoding RNA CCAT2 induce myeloid malignancies via unique SNP-specific RNA mutations.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Wnt signaling regulates the lineage differentiation potential of mouse embryonic stem cells through Tcf3 down-regulation.

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.

Cancer stemness in Wnt-driven mammary tumorigenesis.

Wnt signaling plays a central role in mammary stem cell (MaSC) homeostasis and in breast cancer. In particular, epigenetic alterations at different members of the Wnt pathway have been identified among triple-negative, basal-like breast cancers. Previously, we developed a mouse model for metaplastic breast adenocarcinoma, a subtype of triple-negative breast cancer, by targeting a hypomorphic mutations in the endogenous Apc gene (Apc (1572T/+)). Here, by employing the CD24 and CD29 cell surface antigens, we have identified a subpopulation of mammary cancer stem cells (MaCSCs) from Apc (1572T/+) capable of self-renewal and differentiation both in vivo and in vitro. Moreover, immunohistochemical analysis of micro- and macrolung metastases and preliminary intravenous transplantation assays suggest that the MaCSCs underlie metastasis at distant organ sites. Expression profiling of the normal and tumor cell subpopulations encompassing MaSCs and CSCs revealed that the normal stem cell compartment is more similar to tumor cells than to their own differentiated progenies. Accordingly, Wnt signaling appears to be active in both the normal and cancer stem cell compartments, although at different levels. By comparing normal with cancer mouse mammary compartments, we identified a MaCSC gene signature able to predict outcome in breast cancer in man. Overall, our data indicate that constitutive Wnt signaling activation affects self-renewal and differentiation of MaSCs leading to metaplasia and basal-like adenocarcinomas.

Wnt/beta-catenin signaling in cancer stemness and malignant behavior.

Stem cells are defined by their intrinsic capacity to self-renew and differentiate. Cancer stem cells retain both these features but have lost homeostatic mechanisms which maintain normal cell numbers. The canonical Wnt/beta-catenin signaling pathway plays a central role in modulating the delicate balance between stemness and differentiation in several adult stem cell niches such as the hair follicles in the skin, the mammary gland, and the intestinal crypt. Accordingly, constitutive Wnt signaling activation, resulting from mutations in genes encoding its downstream components, underlies tumorigenesis in these tissues. In the majority of sporadic colorectal cancer cases, the rate-limiting event is either loss of APC function or oncogenic beta-catenin mutations. However, although the presence of these initiating mutations would predict nuclear beta-catenin accumulation throughout the tumor mass, heterogeneous intracellular distributions of this key Wnt signaling molecule are observed within primary tumors and their metastases. In particular, tumor cells located at the invasive front and those migrating into the adjacent stromal tissues show nuclear beta-catenin staining. Hence, different levels of Wnt signaling activity reflect tumor heterogeneity and are likely to account for distinct cellular activities such as proliferation and epithelial-mesenchymal transitions, which prompt tumor growth and malignant behavior, respectively. Several intrinsic (cell-autonomous and/or autocrine) and extrinsic (paracrine, derived from the tumor microenvironment) factors may explain this heterogeneity of Wnt/beta-catenin signaling activity within the tumor mass.

Alterations in Wnt-ß-catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression.

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-ß-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear ß-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-ß-catenin and Pten signalling pathways in endometrial cancer onset and progression.

Cancer stem cells and metastasis.

Cancer stem cells (CSCs) represent a subpopulation of tumour cells endowed with self-renewal and multi-lineage differentiation capacity but also with an innate resistance to cytotoxic agents, a feature likely to pose major clinical challenges towards the complete eradication of minimal residual disease in cancer patients. Operationally, CSCs are defined by their tumour-propagating ability when serially transplanted into immune-compromised mice and by their capacity to fully recapitulate the original heterogeneity of cell types observed in the primary lesions they are derived from. CSCs were first identified in haematopoietic malignancies and later in a broad spectrum of solid tumours including those of the breast, colon and brain. Notably, several CSC characteristics are relevant to metastasis, such as motility, invasiveness and, as mentioned above, resistance to DNA damage-induced apoptosis. Here, we have reviewed the current literature on the relation between CSCs and metastasis formation. Preliminary studies on cancer cell lines and patient-derived material suggest a rate-limiting role for stem-like cells in the processes of tumour cell dissemination and metastasis formation. However, additional studies are needed to deliver formal proof of their identity as the cell of origin of recurrences at distant organ sites. Nevertheless, several studies have already provided pre-clinical evidence of the efficacy of novel therapies directed against disseminated CSCs.

Concepts of metastasis in flux: the stromal progression model.

The ability of tumor cells to leave a primary tumor, to disseminate through the body, and to ultimately seed new secondary tumors is universally agreed to be the basis for metastasis formation. An accurate description of the cellular and molecular mechanisms that underlie this multistep process would greatly facilitate the rational development of therapies that effectively allow metastatic disease to be controlled and treated. A number of disparate and sometimes conflicting hypotheses and models have been suggested to explain various aspects of the process, and no single concept explains the mechanism of metastasis in its entirety or encompasses all observations and experimental findings. The exciting progress made in metastasis research in recent years has refined existing ideas, as well as giving rise to new ones. In this review we survey some of the main theories that currently exist in the field, and show that significant convergence is emerging, allowing a synthesis of several models to give a more comprehensive overview of the process of metastasis. As a result we postulate a stromal progression model of metastasis. In this model, progressive modification of the tumor microenvironment is equally as important as genetic and epigenetic changes in tumor cells during primary tumor progression. Mutual regulatory interactions between stroma and tumor cells modify the stemness of the cells that drive tumor growth, in a manner that involves epithelial-mesenchymal and mesenchymal-epithelial-like transitions. Similar interactions need to be recapitulated at secondary sites for metastases to grow. Early disseminating tumor cells can progress at the secondary site in parallel to the primary tumor, both in terms of genetic changes, as well as progressive development of a metastatic stroma. Although this model brings together many ideas in the field, there remain nevertheless a number of major open questions, underscoring the need for further research to fully understand metastasis, and thereby identify new and effective ways of treating metastatic disease.

Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.

Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer.

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion.

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling.

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.

Long-term effect of resistant starch on cancer risk in carriers of hereditary colorectal cancer: an analysis from the CAPP2 randomised controlled trial.

BACKGROUND: Observational studies report that higher intake of dietary fibre (a heterogeneous mix including non-starch polysaccharides and resistant starches) is associated with reduced risk of colorectal cancer, but no randomised trials with prevention of colorectal cancer as a primary endpoint have been done. We assessed the effect of resistant starch on the incidence of colorectal cancer. METHODS: In the CAPP2 study, individuals with Lynch syndrome were randomly assigned in a two-by-two factorial design to receive 600 mg aspirin or aspirin placebo or 30 g resistant starch or starch placebo, for up to 4 years. Randomisation was done with a block size of 16. Post-intervention, patients entered into double-blind follow-up; participants and investigators were masked to treatment allocation. The primary endpoint for this analysis was development of colorectal cancer in participants randomly assigned to resistant starch or resistant-starch placebo with both intention-to-treat and per-protocol analyses. This study is registered, ISRCTN 59521990. FINDINGS: 463 patients were randomly assigned to receive resistant starch and 455 to receive resistant-starch placebo. At a median follow-up 52·7 months (IQR 28·9-78·4), 53 participants developed 61 primary colorectal cancers (27 of 463 participants randomly assigned to resistant starch, 26 of 455 participants assigned to resistant-starch placebo). Intention-to-treat analysis of time to first colorectal cancer showed a hazard ratio (HR) of 1·40 (95% CI 0·78-2·56; p=0·26) and Poisson regression accounting for multiple primary events gave an incidence rate ratio (IRR) of 1·15 (95% CI 0·66-2·00; p=0·61). For those completing 2 years of intervention, per-protocol analysis yielded a HR of 1·09 (0·55-2·19, p=0·80) and an IRR of 0·98 (0·51-1·88, p=0·95). No information on adverse events was gathered during post-intervention follow-up. INTERPRETATION: Resistant starch had no detectable effect on cancer development in carriers of hereditary colorectal cancer. Dietary supplementation with resistant starch does not emulate the apparently protective effect of diets rich in dietary fibre against colorectal cancer. FUNDING: European Union, Cancer Research UK, Bayer Corporation, National Starch and Chemical Co, UK Medical Research Council, Newcastle Hospitals Trustees, Cancer Council of Victoria Australia, THRIPP South Africa, The Finnish Cancer Foundation, SIAK Switzerland, and Bayer Pharma.

Paneth cells as the origin of intestinal cancer in the context of inflammation.

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response1,2. Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a "revival stem cell" (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract3. Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous dedifferentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and postmitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

The origin of intestinal cancer in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, was shown to suppress intestinal stemness. Here, we employed Paneth cells (PCs) as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation. Upon inflammation, PC-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in inflammatory bowel disease (IBD) patients but also of a larger fraction of sporadic colon cancers. The latter is likely due to the inflammatory consequences of Western-style dietary habits, the major colon cancer risk factor. Computational methods designed to predict the cell-of-origin of cancer confirmed that, in a substantial fraction of sporadic colon cancers the cells-of-origin are secretory lineages and not stem cells.

The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs.

Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1-3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity.

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer.

Background: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation. Methods: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation. Results: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFß1, VEGFA and lactate, and potently inhibited T cell proliferation. Conclusion: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.

Generation and characterization of an inducible transgenic model for studying mouse esophageal biology.

BACKGROUND: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. RESULTS: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7-10 days. CONCLUSIONS: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7-10 days.

Long-term effect of aspirin on cancer risk in carriers of hereditary colorectal cancer: an analysis from the CAPP2 randomised controlled trial.

BACKGROUND: Observational studies report reduced colorectal cancer in regular aspirin consumers. Randomised controlled trials have shown reduced risk of adenomas but none have employed prevention of colorectal cancer as a primary endpoint. The CAPP2 trial aimed to investigate the antineoplastic effects of aspirin and a resistant starch in carriers of Lynch syndrome, the major form of hereditary colorectal cancer; we now report long-term follow-up of participants randomly assigned to aspirin or placebo. METHODS: In the CAPP2 randomised trial, carriers of Lynch syndrome were randomly assigned in a two-by-two factorial design to 600 mg aspirin or aspirin placebo or 30 g resistant starch or starch placebo, for up to 4 years. Randomisation was in blocks of 16 with provision for optional single-agent randomisation and extended postintervention double-blind follow-up; participants and investigators were masked to treatment allocation. The primary endpoint was development of colorectal cancer. Analysis was by intention to treat and per protocol. This trial is registered, ISRCTN59521990. RESULTS: 861 participants were randomly assigned to aspirin or aspirin placebo. At a mean follow-up of 55·7 months, 48 participants had developed 53 primary colorectal cancers (18 of 427 randomly assigned to aspirin, 30 of 434 to aspirin placebo). Intention-to-treat analysis of time to first colorectal cancer showed a hazard ratio (HR) of 0·63 (95% CI 0·35-1·13, p=0·12). Poisson regression taking account of multiple primary events gave an incidence rate ratio (IRR) of 0·56 (95% CI 0·32-0·99, p=0·05). For participants completing 2 years of intervention (258 aspirin, 250 aspirin placebo), per-protocol analysis yielded an HR of 0·41 (0·19-0·86, p=0·02) and an IRR of 0·37 (0·18-0·78, p=0·008). No data for adverse events were available postintervention; during the intervention, adverse events did not differ between aspirin and placebo groups. INTERPRETATION: 600 mg aspirin per day for a mean of 25 months substantially reduced cancer incidence after 55·7 months in carriers of hereditary colorectal cancer. Further studies are needed to establish the optimum dose and duration of aspirin treatment. FUNDING: European Union; Cancer Research UK; Bayer Corporation; National Starch and Chemical Co; UK Medical Research Council; Newcastle Hospitals trustees; Cancer Council of Victoria Australia; THRIPP South Africa; The Finnish Cancer Foundation; SIAK Switzerland; Bayer Pharma.