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Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626–633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.

Background Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor–stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. Methods MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. Results Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated ‘omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. Conclusions This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide and is heterogeneous both morphologically and molecularly. In an era of personalized medicine, the greatest challenge is to predict individual response to therapy and distinguish patients likely to be cured with surgical resection of tumors and systemic therapy from those resistant or non-responsive to treatment. Patients would avoid futile treatments, including clinical trial regimes and ultimately this would prevent under- and over-treatment and reduce unnecessary adverse side effects. In this review, the potential of specific biomarkers will be explored to address two key questions—1) Can the prognosis of patients that will fare well or poorly be determined beyond currently recognized prognostic indicators? and 2) Can an individual patient’s response to therapy be predicted and those who will most likely benefit from treatment/s be identified? Identifying and validating key prognostic and predictive biomarkers and an understanding of the underlying mechanisms of drug resistance and toxicity in CRC are important steps in order to personalize treatment. This review addresses recent data on biological prognostic and predictive biomarkers in CRC. In addition, patient cohorts most likely to benefit from currently available systemic treatments and/or targeted therapies are discussed in this review.

Intestinal stem cells are responsible for the extremely rapid renewal of the entire epithelial lining. The maintenance of the critical absorptive function of the gut relies on the appropriate control of the stem cell pool by the local micro-environment or niche cells. Aging causes dramatic changes in the local regulation of stem cells and alters their function, resulting in absorptive defects and development of diseases. Mechanistically, aging induces both profound cellular and molecular intrinsic alterations of the stem cell pool and mediates aberrant behavior of their associated niche cells. Importantly, some of these identified changes can be therapeutically targeted, which offers the possibility to restore gut function in aging.

Colorectal cancer stem cells have been proposed to drive disease progression, tumour recurrence and chemoresistance. However, studies ablating leucine rich repeat containing G protein-coupled receptor 5 (LGR5)-positive stem cells have shown that they are rapidly replenished in primary tumours. Following injury in normal tissue, LGR5+ stem cells are replaced by a newly defined, transient population of revival stem cells. We investigated whether markers of the revival stem cell population are present in colorectal tumours and how this signature relates to chemoresistance. We examined the expression of different stem cell markers in a cohort of patient-derived colorectal cancer organoids and correlated expression with sensitivity to 5-fluorouracil (5-FU) treatment. Our findings revealed that there was inter-tumour variability in the expression of stem cell markers. Clusterin (CLU), a marker of the revival stem cell population, was significantly enriched following 5-FU treatment and expression correlated with the level of drug resistance. Patient outcome data revealed that CLU expression is associated with both lower patient survival and an increase in disease recurrence. This suggests that CLU is a marker of drug resistance and may identify cells that drive colorectal cancer progression.

Almost a half of microRNAs (miRNAs) in mammalian cells are generated from polycistronic primary transcripts encoding more than one miRNA. Mature miRNAs from polycistronic clusters frequently regulate complementary sets of target mRNAs. How the processing of individual miRNAs within the clusters is controlled to give rise to distinct miRNA levels in vivo is not fully understood. Our investigation of SRSF3 (Serine-Arginine Rich Splicing Factor3) regulated noncoding RNAs in pluripotent cells identified miR-17-92 cluster as a key SRSF3 target, SRSF3 binding to the CNNC motif 17-18nt downstream of the miRNA stem loop. Here we show that SRSF3 binding site context, not merely the distance from the stem loop, within primary transcript is a critical determinant of the processing efficiency of distinct miRNAs derived from the miR-17-92 cluster. SRSF3 specifically enhanced the processing of two paralog miRNAs, miR-17 and miR-20a, targeting overlapping mRNAs including the cell cycle inhibitor CDKN1A/p21. Functional analysis demonstrated that SRSF3 inhibits CDKN1A expression and promotes cell cycle and self-renewal through the miRNA processing pathway both in normal pluripotent stem cells and cancer cells. Strikingly, analysis of colorectal cancer tumour-normal pairs demonstrated that the SRSF3-regulated miRNA processing pathway is present in a large proportion of colorectal cancer patients and distinguishes poorly differentiated high-grade tumours. Our research uncovers a critical role of SRSF3 in selective processing of miR-17-92 miRNAs, which mechanistically and functionally links SRSF3 to hallmark features of cancer.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types.

Epidermal growth factor (EGF) maintains intestinal stem cell (ISC) proliferation and is a key component of organoid growth media yet is dispensable for intestinal homeostasis, suggesting roles for multiple EGF family ligands in ISC function. Here, we identified neuregulin 1 (NRG1) as a key EGF family ligand that drives tissue repair following injury. NRG1, but not EGF, is upregulated upon damage and is expressed in mesenchymal stromal cells, macrophages, and Paneth cells. NRG1 deletion reduces proliferation in intestinal crypts and compromises regeneration capacity. NRG1 robustly stimulates proliferation in crypts and induces budding in organoids, in part through elevated and sustained activation of mitogen-activated protein kinase (MAPK) and AKT. Consistently, NRG1 treatment induces a proliferative gene signature and promotes organoid formation from progenitor cells and enhances regeneration following injury. These data suggest mesenchymal-derived NRG1 is a potent mediator of tissue regeneration and may inform the development of therapies for enhancing intestinal repair after injury.

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient‐derived) Kras G12D ‐driven LAC models, the specific blockade of ADAM17, including with a non‐toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro‐tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL‐6R, to release soluble IL‐6R that drives IL‐6 trans‐signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in Kras G12D ‐driven LAC was independent of bone marrow‐derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho‐ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS ‐driven LAC and provide the rationale to employ ADAM17‐based therapeutic strategies for targeting KRAS mutant cancers.

Interleukin (IL)-22 activates STAT (signal transducer and activator of transcription) 3 and antiapoptotic and proproliferative pathways; but beyond this, the molecular mechanisms by which IL-22 promotes carcinogenesis are poorly understood. Characterizing the molecular signature of IL-22 in human DLD-1 colon carcinoma cells, we observed increased expression of 26 genes, including NNMT (nicotinamide N-methyltransferase, ≤10-fold) and CEA (carcinoembryonic antigen, ≤7-fold), both known to promote intestinal carcinogenesis. ERP27 (endoplasmic reticulum protein-27, function unknown, ≤5-fold) and the proinflammatory ICAM1 (intercellular adhesion molecule-1, ≤4-fold) were also increased. The effect on CEA was partly STAT3-mediated, as STAT3-silencing reduced IL-22-induced CEA by ≤56%. Silencing of CEA or NNMT inhibited IL-22-induced proliferation/migration of DLD-1, Caco-2, and SW480 colon carcinoma cells. To validate these results in primary tissues, we assessed IL-22-induced gene expression in organoids from human healthy colon and colon cancer patients, and from normal mouse small intestine and colon. Gene regulation by IL-22 was similar in DLD-1 cells and human and mouse healthy organoids. CEA was an exception with no induction by IL-22 in organoids, indicating the 3-dimensional organization of the tissue may produce signals absent in 2D cell culture. Importantly, augmentation of NNMT was 5-14-fold greater in human cancerous compared to normal organoids, supporting a role for NNMT in IL-22-mediated colon carcinogenesis. Thus, NNMT and CEA emerge as mediators of the tumor-promoting effects of IL-22 in the intestine. These data advance our understanding of the multifaceted role of IL-22 in the gut and suggest the IL-22 pathway may represent a therapeutic target in colon cancer.

Mouse models of intestinal carcinogenesis are very powerful for studying the impact of specific mutations on tumour initiation and progression. Mutations can be studied both singularly and in combination using conditional alleles that can be induced in a temporal manner. The steps in intestinal carcinogenesis are complex and can be challenging to image in live animals at a cellular level. The ability to culture intestinal epithelial tissue in three-dimensional organoids in vitro provides an accessible system that can be genetically manipulated and easily visualised to assess specific biological impacts in living tissue. Here, we describe methodology for conditional mutation of genes in organoids from genetically modified mice via induction of Cre recombinase induced by tamoxifen or by transient exposure to TAT-Cre protein. This methodology provides a rapid platform for assessing the cellular changes induced by specific mutations in intestinal tissue.

Epithelial stem cells from a variety of tissues have been shown to express genes linked to mesenchymal cell states. The Snail family of transcriptional factors has long been regarded as a marker of mesenchymal cells, however recent studies have indicated an involvement in regulation of epithelial stem cell populations. Snai1 is expressed in the stem cell population found at the base of the mouse small intestinal crypt that is responsible for generating all differentiated cell types of the intestinal epithelium. We utilized an inducible Cre recombinase approach in the intestinal epithelium combined with a conditional floxed Snai1 allele to induce knockout of gene function in the stem cell population. Loss of Snai1 resulted in loss of crypt base columnar cells and a failure to induce a proliferative response following radiation damage. We induced Snai1 loss in cultured organoids that had been derived from epithelial cells and compared gene expression to organoids with functional Snai1. Here we describe in detail the methods for generation of knockout organoids and analysis of microarray data that has been deposited in Gene Expression Omnibus (GEO):GSE65005.

Brg1 is a chromatin remodeling factor involved in mediation of a plethora of signaling pathways leading to its participation in various physiological processes both during development and in adult tissues. Among other signaling pathways, the Wnt pathway has been proposed to require Brg1 for transactivation of its target genes. Given the pivotal role of the Wnt pathway in the maintenance of normal intestinal homeostasis, we aimed to investigate the effects of Brg1 loss on the intestinal physiology. To this end, we deleted Brg1 in the murine small and large intestinal epithelia using a range of transgenic approaches. Pan-epithelial loss of Brg1 in the small intestine resulted in crypt ablation, while partial Brg1 deficiency led to gradual repopulation of the intestinal mucosa with wild-type cells. In contrast, Brg1 loss in the large intestinal epithelium was compensated by upregulation of Brm. We propose that while Brg1 is dispensable for the survival and function of the progenitor and differentiated cells in the murine intestinal epithelium, it is essential for the maintenance of the stem cell population in a tissue-specific manner.

Background & Aims: Mutations in components of the Wnt signaling pathway, including β-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. Methods: Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of β-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. Results: Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G2/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of β-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of β-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. Conclusions: Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of β-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC.

Adiponectin is an adipose tissue-derived hormone, expressed almost exclusively in adipose tissue, with significant antidiabetic, anti-atherosclerotic, anti-inflammatory and anti-proliferative properties. The anti-carcinogenic effects of adiponectin result from two main mechanisms: a modulation in the signaling pathways involved in proliferation process and a subtle regulation of the apoptotic response. In this review, we present recent findings on the association of adiponectin with the risk of several malignancies (breast, colorectal, liver and prostate cancers), as well as data on underlying molecular mechanisms by which adiponectin plays a substantial role in cancer pathogenesis.

Background and aims: LKB1 is a serine-threonine kinase, mutation of which can lead to the development of multiple benign intestinal hamartomas (Peutz–Jeghers syndrome). In this study, the authors investigate the mechanisms underlying this phenotype by exploring the transcriptional changes associated with Lkb1 deletion in intestinal epithelium. Methods: The authors used mice with Lkb1 deleted in the intestinal epithelium using a Cyp1a1-specific inducible Cre recombinase and used Affymetrix (Santa Clara, California, USA) microarray analysis to examine the transcriptional changes occurring immediately after Lkb1 loss. The authors also generated crypt–villus organoid culture to analyse Lkb1 role in intestinal responses to exogenous stimuli. Results: Affymetrix analysis identified the most significant change to be in Ren1 expression, a gene encoding a protease involved in angiotensinogen processing. Lkb1 deletion also enhanced ACE expression and subsequently angiotensin II (AngII) production in the mouse intestine. Intestinal apoptosis induced by Lkb1 deficiency was suppressed by ACE inhibitor captopril. Lkb1-deficient intestinal epithelium showed dynamic changes in AngII receptor type 1, suggesting a possible compensatory response to elevated AngII levels. A similar reduction in epithelial AngII receptor type 1 was also observed in human Peutz–Jeghers syndrome tumours contrasting with high expression of the receptor in the tumour stroma. Mechanistically, the authors showed two pieces of data that position Lkb1 in renin expression regulation, and they implied the importance of Lkb1 in linking cell responses with nutrient levels. First, the authors showed that Lkb1 deletion in isolated epithelial organoid culture resulted in renin upregulation only when the organoids were challenged with external cues such as AngII; second, that renin upregulation was dependent upon the MEK/ERK pathway in a circadian fashion and corresponded to active feeding time when nutrient levels were high. Conclusions: Taken together, these data reveal a novel role for Lkb1 in regulation of the gastrointestinal renin–angiotensin system.

The mammary gland undergoes numerous developmental processes postnatally, from the elongation of the ductal tree-like structure to pregnancy-induced lobulo-alveolar development. Mammary epithelial stem cells have been suggested to be central to the control of enormous tissue expansion and remodelling during phases of mammary development. The Wnt signalling pathway plays a critical role in these biological steps and is suggested to be involved in the maintenance of the stem cell population. This review provides insight into recent findings on the activity of Wnt signalling during ductal and lobular mammary development and discusses the potential interplay between Wnt signals and mammary stem cells in mice.

Summary The isolation of pure populations of mouse intestinal stem cells (ISCs) is essential to facilitate functional studies of tissue homeostasis, tissue regeneration, and intestinal diseases. However, the purification of ISCs has relied predominantly on the use of transgenic reporter alleles in mice. Here, we introduce a combinational cell surface marker-mediated strategy that allows the isolation of an ISC population transcriptionally and functionally equivalent to the gold standard Lgr5-GFP ISCs. Used on reporter-free mice, this strategy allows the isolation of functional, transcriptionally distinct ISCs uncompromised by Lgr5 haploinsufficiency., Graphical Abstract, Highlights • Reporter-free method to purify intestinal stem cells (ISCs) • Cells share molecular signature with gold standard Lgr5-GFP high cells • Multidimensional FACS data analysis reveals structure of intestinal crypt • Lgr5 haploinsufficiency with functional consequences in ISCs from reporter mice, Polo, Abud, and colleagues introduce a cell surface marker-mediated strategy that allows isolation of an intestinal stem cell (ISCs) population transcriptionally and functionally equivalent to the gold standard Lgr5-GFP ISCs. When used on reporter-free mice, this strategy allows the isolation of functional, transcriptionally distinct ISCs uncompromised by Lgr5 haploinsufficiency.

The fungicidal and bactericidal actions of the essential oil (EO) of Melaleuca alternifolia seem well established, but their anti-inflammatory and antioxidative effects remain unclear. This study investigated in vitro the possible role of whole Melaleuca alternifolia EO as a modulator of the inflammatory/non-specific immune response by exploring the chemotaxis and kinetic radical oxygen species (ROS) production of leukocytes and cytokine secretion in peripheral blood mononuclear cells (PBMCs) in humans. The influence of Melaleuca alternifolia EO on the chemotaxis under agarose of isolated neutrophils (PMNs) was evaluated. The kinetics of ROS production by stimulated total circulating leukocytes was followed over 2 h by recording the fluorescence intensity of oxidized dihydrorhodamine 123. The effects of this EO on pro-(interleukin IL-2) and anti-(IL-4 and IL10) inflammatory cytokine secretions were determined by ELISA following incubation of PBMCs with the EO for 24 h. Melaleuca alternifolia EO was inefficient on the chemotaxis of PMNs. It exerted an antioxidant effect, reducing ROS production throughout the kinetic study. Melaleuca alternifolia EO inhibited PBMC proliferation, as revealed by a reduction in IL-2 secretion by stimulated lymphocytes. This EO at 0.1% directly increased the secretion of the anti-inflammatory cytokine IL-4 compared with IL-4 secretion without EO (18.5 +/- 10.0 vs 3.3 +/- 1, p < 0.05), and also increased IL-10 secretion at 0.01% (94.9 +/- 38.7 vs 44.1 +/- 18, ns). Melaleuca alternifolia EO may not only act as an anti-inflammatory mediator through its antioxidant activity but may also efficiently protect the organism by reducing the proliferation of inflammatory cells without affecting their capacity to secrete anti-inflammatory cytokines.

Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.

Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.

International audience; Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, a hormone excessively produced during obesity, is suggested to be involved in breast cancer. The aim of the study was to investigate procarcinogenic potential of leptin by evaluating influence of leptin on cell proliferation, cell cycle, apoptosis, and signaling on numerous breast cells lines, including 184B5 normal cells, MCF10A fibrocystic cells and MCF-7, MDA-MB-231, and T47D cancer cells. Expressions of leptin and Ob-R were analyzed using qRT-PCR and immunohistochemistry, proliferation using fluorimetric resazurin reduction test and xCELLigence system, apoptosis and cell cycle by flow cytometry, and effect of leptin on different signalling pathways using qRT-PCR and Western blot. Cells were exposed to increasing concentrations of leptin. All cell lines expressed mRNA and protein of leptin and Ob-R. Leptin stimulated proliferation of all cell lines except for 184B5 and MDA-MB-231 cells. Leptin inhibited apoptosis but didn't alter proportion of cells within cell cycle in MCF7 cells. Leptin induced overexpression of leptin, Ob-R, estrogen receptor, and aromatase mRNA in MCF-7 and T47D cells. Autoregulation induced by leptin, relationship with estrogen pathway, and proliferative and antiapoptic activity in breast cancer cells may explain that obesity-associated hyperleptinemia may be a breast cancer risk factor.

Although significant progress has been made in understanding the importance of Wnt signaling in the initiation of colorectal cancer, less is known about responses that accompany the reversal of oncogenic Wnt signaling. The aim of this study was to analyze in vivo and in vitro responses to an ‘ideal’ Wnt pathway inhibitor as a model for the therapeutic targeting of the pathway. A tetracycline-inducible transgenic mouse model expressing truncated β-catenin (ΔN89β-catenin) that exhibited a strong intestinal hyperplasia was analysed during the removal of oncogenic β-catenin expression both in 3D ‘crypt culture’ and in vivo. Oncogenic Wnt signaling was rapidly and completely reversed. The strongest inhibition of Wnt target gene expression occurred within 24 hours of doxycycline removal at which time the target genes Ascl2, Axin2 and C-myc were down-regulated to levels below that in the control intestine. In vitro, the small molecule Wnt inhibitor CCT036477 induced a response within 4 hours of treatment. By 7 days following doxycycline withdrawal, gene expression, cell proliferation and tissue morphology were undistinguishable from control animals. In conclusion, these results demonstrate that the reversal of Wnt signaling by inhibitors should ideally be studied within hours of treatment. The reversible system described, involving medium throughput in vitro approaches and rapid in vivo responses, should allow the rapid advance of early stage compounds into efficacy models that are more usually considered later in the drug discovery pipeline.

Obesity is a recognized breast cancer risk factor in postmenopausal women. A recent hypothesis suggests a major role for adipose tissue in carcinogenesis. During many years, the adipose tissue was only considered as a fat storage of energy. This tissue is now described as an endocrine organ secreting a large range of molecules called adipokines. Among these adipokines, adiponectin may play a major role in breast cancer. Plasma adiponectin levels were found to be decreased in cases of breast cancer and in obese patients. Adiponectin may act directly on breast cancer cells by inhibiting proliferation and angiogenesis or by stimulating apoptosis. Increasing adiponectin levels may be of major importance in the prevention and/or the treatment of breast cancer. This therapeutic approach may be of particular significance for obese patients. The beneficial effects of adiponectin and its possible therapeutic applications will be discussed in this review.

International audience; We investigated the effect of relative changes in dietary nitrogen (N) and energy supply and the subsequent variations in net portal appearance (NPA) of nitrogenous and energy nutrients on the net amino acid (AA) uptake by the liver and net N supply to the peripheral tissues. Six lambs were catheterised across the splanchnic tissues and received, in a replicated Latin square, one of three dietary treatments. The diets were formulated to either match the requirements of N and energy (C), or supply only 0.8 of the N requirement (LN) or 0.8 of the energy requirement (LE). Net fluxes of AA and urea-N were measured across the portal-drained viscera, and estimation of arterial hepatic flow allowed the estimation of hepatic fluxes. Catheters were implanted into the portal and hepatic veins as well as in the abdominal aorta for the measurement of AA fluxes. Animals fed the LN diet showed more efficient N retention (0.59 of digested N) than did the C and LE diet (0.50 and 0.33, respectively; P < 0.001). The NPA of total AA-N for the LN diet was only 0.60 of the value measured for the control (C) diet (P < 0.01). Despite this, the total estimated AA-N net splanchnic fluxes were not significantly different across the three diets (3.3, 1.9 and 2.6 g total AA-N/day for C, LN and LE, respectively, P = 0.52). Thus, different metabolic regulations must have taken place across the liver between the three experimental diets. A combination of decreased net uptake of total AA-N by the liver of animals in the LN diet (0.61 of the C diet; P = 0.002) and reduced urinary urea-N production (0.52 of the C diet; P = 0.001) spared AA from catabolism in the LN diet relative to the other two diets. For the LE diet, the urinary urea-N output was 1.3 times the value of the C diet (P = 0.01). This may relate to an increased catabolism of AA by the muscle and/or, to a lesser extent, to an increased utilisation of AA for gluconeogenesis in the liver. These effects may explain the reduced whole body protein retention observed with the LE diet.

Obesity is associated with an increased risk of breast cancer in postmenopausal women. Accumulating evidence suggests that adipose tissue, which is an endocrine organ producing a large range of factors, may interfere with breast cancer development. Leptin and adiponectin are two major adipocyte-secreted hormones. The pro-carcinogenic effect of leptin and conversely, the anti-carcinogenic effect of adiponectin result from two main mechanisms: a modulation in the signalling pathways involved in proliferation process and a subtle regulation of the apoptotic response. This review provides insight into recent findings on the molecular mechanisms of leptin and adiponectin in mammary tumours, and discusses the potential interplay between these two adipokines in breast cancer.

Obesity is associated with an increased risk of breast cancer. Leptin, a hormone synthesised essentially by adipose tissue, may be involved in cancer development. We examined the expression of leptin and leptin receptor (Ob-R) in human primary breast cancer and adjacent non-cancerous tissue. We also analysed their relationships with histological variables such as the oestrogen and progesterone receptors, Ki67 proliferation factor and tumour size. The expressions of leptin and Ob-R were investigated by immunohistochemical staining in 35 primary breast cancers and 17 adjacent non-cancerous tissues. Samples and histological features were obtained from the Anti-Cancer Centre. Expressions of leptin and Ob-R were detected in, respectively, 85 and 75% of the primary breast cancer cases studied. The expression of leptin was significantly correlated with Ob-R detection (p=0.008). In addition, Ob-R expression in primary breast carcinoma was positively correlated with oestrogen receptor expression (p=0.028) and tumour size (p=0.045) but not with Ki67 or progesterone receptor expressions. However, the expression of leptin showed no statistical correlation with these variables. First, the co-expression of leptin and Ob-R in primary breast cancer shows that leptin acts on mammary tumour cells via an autocrine pathway. Second, the co-expression of Ob-R and oestrogen receptors suggests a possible interaction between leptin and oestrogen systems to promote breast carcinogenesis. Finally, the fact that Ob-R expression was positively correlated with tumour size may point to a potential role of leptin as a growth factor and of Ob-R as a new prognostic factor.