Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors.

Previously, we found that Wnt and Notch signaling govern stem cells of clear cell kidney cancer (ccRCC) in patients. To mimic stem cell responses in the normal kidney in vitro in a marker-unbiased fashion, we have established tubular organoids (tubuloids) from total single adult mouse kidney epithelial cells in Matrigel and serum-free conditions. Deep proteomic and phosphoproteomic analyses revealed that tubuloids resembled renewal of adult kidney tubular epithelia, since tubuloid cells displayed activity of Wnt and Notch signaling, long-term proliferation and expression of markers of proximal and distal nephron lineages. In our wish to model stem cell-derived human ccRCC, we have generated two types of genetic double kidney mutants in mice: Wnt-ß-catenin-GOF together with Notch-GOF and Wnt-ß-catenin-GOF together with a most common alteration in ccRCC, Vhl-LOF. An inducible Pax8-rtTA-LC1-Cre was used to drive recombination specifically in adult kidney epithelial cells. We confirmed mutagenesis of ß-catenin, Notch and Vhl alleles on DNA, protein and mRNA target gene levels. Surprisingly, we observed symptoms of chronic kidney disease (CKD) in mutant mice, but no increased proliferation and tumorigenesis. Thus, the responses of kidney stem cells in the tubuloid and genetic systems produced different phenotypes, i.e. enhanced renewal versus CKD.

Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses.

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8+ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.

Targeting Wnt/ß-Catenin Signaling by TET1/FOXO4 Inhibits Metastatic Spreading and Self-Renewal of Cancer Stem Cells in Gastric Cancer.

Metastasis is the main cause of death for patients suffering gastric cancer. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critical attributes of metastasis, both of which are regulated tightly by DNA methylation and Wnt/ß-catenin signaling. Here, we studied the functions of DNA dioxygenase TET1 in regulating Wnt signaling and in gastric cancer metastasis. Knocking-down and overexpressing TET1 in gastric cancer cells promoted and inhibited metastatic spreading to the liver in immune-deficient mice, respectively. TET1 showed inhibitory effects on metastasis-related features -EMT and CSC, which were reversed by interfering with Wnt/ß-catenin signaling. RNA-sequencing identified FOXO4 as a direct transactivating target of TET1. FOXO4 directly interacted with ß-catenin and recruited it in the cytoplasm, so as to inhibit ß-catenin-mediated transcription of Wnt target genes, including CSC marker EpCAM. Moreover, modulation of FOXO4 could reverse the effects of TET1 manipulation on EMT and self-renewal of CSCs. The analysis with clinical samples confirmed the value of FOXO4 as an independent prognostic predictor of patients' overall survival. Taken together, regulation of Wnt signaling by TET1/FOXO4 is essential for metastasis-associated cellular properties, and targeting TET1/FOXO4/ß-catenin pathway may serve as promising therapeutics in the prevention and treatment of gastric cancer metastasis.

Targeting SHP2 phosphatase in breast cancer overcomes RTK-mediated resistance to PI3K inhibitors.

BACKGROUND: PI3K signaling is frequently activated in breast cancer and is targeted by PI3K inhibitors. However, resistance of tumor cells to PI3K inhibition, often mediated by activated receptor tyrosine kinases, is commonly observed and reduces the potency of PI3K inhibitors. Therefore, new treatment strategies to overcome resistance to PI3K inhibitors are urgently needed to boost their efficacy. The phosphatase SHP2, which plays a crucial role in mediating signal transduction between receptor tyrosine kinases and both the PI3K and MAPK pathways, is a potential target for combination treatment. METHODS: We tested combinations of PI3K and SHP2 inhibitors in several experimental breast cancer models that are resistant to PI3K inhibition. Using cell culturing, biochemical and genetic approaches, we evaluated tumor cell proliferation and signaling output in cells treated with PI3K and SHP2 inhibitors. RESULTS: Combination treatment with PI3K and SHP2 inhibitors counteracted both acquired and intrinsic breast cancer cell resistance to PI3K inhibition that is mediated by activated receptor tyrosine kinases. Dual PI3K and SHP2 inhibition blocked proliferation and led to sustained inactivation of PI3K and MAPK signaling, where resistant cells rapidly re-activated these pathways upon PI3K inhibitor monotreatment. In addition, we demonstrate that overexpression of SHP2 induced resistance to PI3K inhibition, and that SHP2 was frequently activated during the development of PI3K inhibitor resistance after prolonged treatment of sensitive cells. CONCLUSIONS: Our results highlight the importance of SHP2 as a player in resistance to PI3K inhibitors. Combination treatment with PI3K and SHP2 inhibitors could pave the way for significant improvements in therapies for breast cancer.

MLL1 is required for maintenance of intestinal stem cells.

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

Oxidized LDL-dependent pathway as new pathogenic trigger in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is hallmarked by ventricular fibro-adipogenic alterations, contributing to cardiac dysfunctions and arrhythmias. Although genetically determined (e.g., PKP2 mutations), ACM phenotypes are highly variable. More data on phenotype modulators, clinical prognosticators, and etiological therapies are awaited. We hypothesized that oxidized low-density lipoprotein (oxLDL)-dependent activation of PPARγ, a recognized effector of ACM adipogenesis, contributes to disease pathogenesis. ACM patients showing high plasma concentration of oxLDL display severe clinical phenotypes in terms of fat infiltration, ventricular dysfunction, and major arrhythmic event risk. In ACM patient-derived cardiac cells, we demonstrated that oxLDLs are major cofactors of adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL in a Pkp2 heterozygous knock-out mice through high-fat diet feeding, we confirmed in vivo the oxidized lipid dependency of cardiac adipogenesis and right ventricle systolic impairment, which are counteracted by atorvastatin treatment. The modulatory role of oxidized lipids on ACM adipogenesis, demonstrated at cellular, mouse, and patient levels, represents a novel risk stratification tool and a target for ACM pharmacological strategies.

Beneficial effect of voluntary physical exercise in Plakophilin2 transgenic mice.

Arrhythmogenic right ventricular cardiomyopathy is a hereditary, rare disease with an increased risk for sudden cardiac death. The disease-causing mutations are located within the desmosomal complex and the highest incidence is found in plakophilin2. However, there are other factors playing a role for the disease progression unrelated to the genotype such as inflammation or exercise. Competitive sports have been identified as risk factor, but the type and extend of physical activity as cofactor for arrhythmogenesis remains under debate. We thus studied the effect of light voluntary exercise on cardiac health in a mouse model. Mice with a heterozygous PKP2 loss-of-function mutation were given the option to exercise in a running wheel which was monitored 24 h/d. We analyzed structural and functional development in vivo by echocardiography which revealed that neither the genotype nor the exercise caused any significant structural changes. Ejection fraction and fractional shortening were not influenced by the genotype itself, but exercise did cause a drop in both parameters after 8 weeks, which returned to normal after 16 weeks of training. The electrophysiological analysis revealed that the arrhythmogenic potential was slightly higher in heterozygous animals (50% vs 18% in wt littermates) and that an additional stressor (isoprenaline) did not lead to an increase of arrhythmogenic events pre run or after 8 weeks of running but the vulnerability was increased after 16 weeks. Exercise-induced alterations in Ca handling and contractility of isolated myocytes were mostly abolished in heterozygous animals. No fibrofatty replacements or rearrangement of gap junctions could be observed. Taken together we could show that light voluntary exercise can cause a transient aggravation of the mutation-induced phenotype which is abolished after long term exercise indicating a beneficial effect of long term light exercise.

High Yap and Mll1 promote a persistent regenerative cell state induced by Notch signaling and loss of p53.

Specified intestinal epithelial cells reprogram and contribute to the regeneration and renewal of the epithelium upon injury. Mutations that deregulate such renewal processes may contribute to tumorigenesis. Using intestinal organoids, we show that concomitant activation of Notch signaling and ablation of p53 induce a highly proliferative and regenerative cell state, which is associated with increased levels of Yap and the histone methyltransferase Mll1. The induced signaling system orchestrates high proliferation, self-renewal, and niche-factor-independent growth, and elevates the trimethylation of histone 3 at lysine 4 (H3K4me3). We demonstrate that Yap and Mll1 are also elevated in patient-derived colorectal cancer (CRC) organoids and control growth and viability. Our data suggest that Notch activation and p53 ablation induce a signaling circuitry involving Yap and the epigenetic regulator Mll1, which locks cells in a proliferative and regenerative state that renders them susceptible for tumorigenesis.

YAP and ß-Catenin Cooperate to Drive Oncogenesis in Basal Breast Cancer.

Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in ß-catenin target genes, suggesting that YAP is required for nuclear ß-catenin activity. Mechanistically, nuclear YAP interacted with ß-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, ß-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/ß-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. SIGNIFICANCE: These findings show that YAP cooperates with ß-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2116/F1.large.jpg.

Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling.

Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ-Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50∼50 µM) and Western blotting of ß-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

The epigenetic regulator Mll1 is required for Wnt-driven intestinal tumorigenesis and cancer stemness.

Wnt/ß-catenin signaling is crucial for intestinal carcinogenesis and the maintenance of intestinal cancer stem cells. Here we identify the histone methyltransferase Mll1 as a regulator of Wnt-driven intestinal cancer. Mll1 is highly expressed in Lgr5+ stem cells and human colon carcinomas with increased nuclear ß-catenin. High levels of MLL1 are associated with poor survival of colon cancer patients. The genetic ablation of Mll1 in mice prevents Wnt/ß-catenin-driven adenoma formation from Lgr5+ intestinal stem cells. Ablation of Mll1 decreases the self-renewal of human colon cancer spheres and halts tumor growth of xenografts. Mll1 controls the expression of stem cell genes including the Wnt/ß-catenin target gene Lgr5. Upon the loss of Mll1, histone methylation at the stem cell promoters switches from activating H3K4 tri-methylation to repressive H3K27 tri-methylation, indicating that Mll1 sustains stem cell gene expression by antagonizing gene silencing through polycomb repressive complex 2 (PRC2)-mediated H3K27 tri-methylation. Transcriptome profiling of Wnt-mutated intestinal tumor-initiating cells reveals that Mll1 regulates Gata4/6 transcription factors, known to sustain cancer stemness and to control goblet cell differentiation. Our results demonstrate that Mll1 is an essential epigenetic regulator of Wnt/ß-catenin-induced intestinal tumorigenesis and cancer stemness.

From Pyrazolones to Azaindoles: Evolution of Active-Site SHP2 Inhibitors Based on Scaffold Hopping and Bioisosteric Replacement.

The tyrosine phosphatase SHP2 controls the activity of pivotal signaling pathways, including MAPK, JAK-STAT, and PI3K-Akt. Aberrant SHP2 activity leads to uncontrolled cell proliferation, tumorigenesis, and metastasis. SHP2 signaling was recently linked to drug resistance against cancer medications such as MEK and BRAF inhibitors. In this work, we present the development of a novel class of azaindole SHP2 inhibitors. We applied scaffold hopping and bioisosteric replacement concepts to eliminate unwanted structural motifs and to improve the inhibitor characteristics of the previously reported pyrazolone SHP2 inhibitors. The most potent azaindole 45 inhibits SHP2 with an IC50 = 0.031 µM in an enzymatic assay and with an IC50 = 2.6 µM in human pancreas cells (HPAF-II). Evaluation in a series of cellular assays for metastasis and drug resistance demonstrated efficient SHP2 blockade. Finally, 45 inhibited proliferation of two cancer cell lines that are resistant to cancer drugs and diminished ERK signaling.

Tracing tumorigenesis in a solid tumor model at single-cell resolution.

Characterizing the complex composition of solid tumors is fundamental for understanding tumor initiation, progression and metastasis. While patient-derived samples provide valuable insight, they are heterogeneous on multiple molecular levels, and often originate from advanced tumor stages. Here, we use single-cell transcriptome and epitope profiling together with pathway and lineage analyses to study tumorigenesis from a developmental perspective in a mouse model of salivary gland squamous cell carcinoma. We provide a comprehensive cell atlas and characterize tumor-specific cells. We find that these cells are connected along a reproducible developmental trajectory: initiated in basal cells exhibiting an epithelial-to-mesenchymal transition signature, tumorigenesis proceeds through Wnt-differential cancer stem cell-like subpopulations before differentiating into luminal-like cells. Our work provides unbiased insights into tumor-specific cellular identities in a whole tissue environment, and emphasizes the power of using defined genetic model systems.

Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients.

Current treatments for clear cell renal cell cancer (ccRCC) are insufficient because two-thirds of patients with metastases progress within two years. Here we report the identification and characterization of a cancer stem cell (CSC) population in ccRCC. CSCs are quantitatively correlated with tumor aggressiveness and metastasis. Transcriptional profiling and single cell sequencing reveal that these CSCs exhibit an activation of WNT and NOTCH signaling. A significant obstacle to the development of rational treatments has been the discrepancy between model systems and the in vivo situation of patients. To address this, we use CSCs to establish non-adherent sphere cultures, 3D tumor organoids, and xenografts. Treatment with WNT and NOTCH inhibitors blocks the proliferation and self-renewal of CSCs in sphere cultures and organoids, and impairs tumor growth in patient-derived xenografts in mice. These findings suggest that our approach is a promising route towards the development of personalized treatments for individual patients.

SHP-2 in Lymphocytes' Cytokine and Inhibitory Receptor Signaling.

Somewhat counterintuitively, the tyrosine phosphatase SHP-2 (SH2 domain-containing protein tyrosine phosphatase-2) is crucial for the activation of extracellular signal-regulated kinase (ERK) downstream of various growth factor receptors, thereby exerting essential developmental functions. This phosphatase also deploys proto-oncogenic functions and specific inhibitors have recently been developed. With respect to the immune system, the role of SHP-2 in the signaling of cytokines relevant for myelopoiesis and myeloid malignancies has been intensively studied. The function of this phosphatase downstream of cytokines important for lymphocytes is less understood, though multiple lines of evidence suggest its importance. In addition, SHP-2 has been proposed to mediate the suppressive effects of inhibitory receptors (IRs) that sustain a dysfunctional state in anticancer T cells. Molecules involved in IR signaling are of potential pharmaceutical interest as blockade of these inhibitory circuits leads to remarkable clinical benefit. Here, we discuss the dichotomy in the functions ascribed to SHP-2 downstream of cytokine receptors and IRs, with a focus on T and NK lymphocytes. Further, we highlight the importance of broadening our understanding of SHP-2's relevance in lymphocytes, an essential step to inform on side effects and unanticipated benefits of its therapeutic blockade.

Decreased microglial Wnt/ß-catenin signalling drives microglial pro-inflammatory activation in the developing brain.

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/ß-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

Spatially Restricted Stromal Wnt Signaling Restrains Prostate Epithelial Progenitor Growth through Direct and Indirect Mechanisms.

Cell-autonomous Wnt signaling has well-characterized functions in controlling stem cell activity, including in the prostate. While niche cells secrete Wnt ligands, the effects of Wnt signaling in niche cells per se are less understood. Here, we show that stromal cells in the proximal prostatic duct near the urethra, a mouse prostate stem cell niche, not only produce multiple Wnt ligands but also exhibit strong Wnt/ß-catenin activity. The non-canonical Wnt ligand Wnt5a, secreted by proximal stromal cells, directly inhibits proliefration of prostate epithelial stem or progenitor cells whereas stromal cell-autonomous canonical Wnt/ß-catenin signaling indirectly suppresses prostate stem or progenitor activity via the transforming growth factor ß (TGFß) pathway. Collectively, these pathways restrain the proliferative potential of epithelial cells in the proximal prostatic ducts. Human prostate likewise exhibits spatially restricted distribution of stromal Wnt/ß-catenin activity, suggesting a conserved mechanism for tissue patterning. Thus, this study shows how distinct stromal signaling mechanisms within the prostate cooperate to regulate tissue homeostasis.

Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells.

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.

A C/EBPα-Wnt connection in gut homeostasis and carcinogenesis.

We explored the connection between C/EBPα (CCAAT/enhancer-binding protein α) and Wnt signaling in gut homeostasis and carcinogenesis. C/EBPα was expressed in human and murine intestinal epithelia in the transit-amplifying region of the crypts and was absent in intestinal stem cells and Paneth cells with activated Wnt signaling. In human colorectal cancer and murine APCMin/+ polyps, C/EBPα was absent in the nuclear ß-catenin-positive tumor cells. In chemically induced intestinal carcinogenesis, C/EBPα KO in murine gut epithelia increased tumor volume. C/EBPα deletion extended the S-phase cell zone in intestinal organoids and activated typical proliferation gene expression signatures, including that of Wnt target genes. Genetic activation of ß-catenin in organoids attenuated C/EBPα expression, and ectopic C/EBPα expression in HCT116 cells abrogated proliferation. C/EBPα expression accompanied differentiation of the colon cancer cell line Caco-2, whereas ß-catenin stabilization suppressed C/EBPα. These data suggest homeostatic and oncogenic suppressor functions of C/EBPα in the gut by restricting Wnt signaling.