Search

Elevated CUB-domain containing protein 1 (CDCP1) is predictive of colorectal cancer (CRC) recurrence and poor patient survival. While CDCP1 expression identifies stem cell populations that mediate lung metastasis, mechanisms underlying the role of this cell surface receptor in CRC have not been defined. We sought to identify CDCP1 regulated processes in CRC using stem cell populations, enriched from primary cells and cell lines, in extensive in vitro and in vivo assays. These experiments, demonstrating that CDCP1 is functionally important in CRC tumor initiation, growth and metastasis, identified CDCP1 as a positive regulator of Wnt signaling. Detailed cell fractionation, immunoprecipitation, microscopy, and immunohistochemical analyses demonstrated that CDCP1 promotes translocation of the key regulators of Wnt signaling, β-catenin, and E-cadherin, to the nucleus. Of functional importance, disruption of CDCP1 reduces nuclear localized, chromatin-associated β-catenin and nuclear localized E-cadherin, increases sequestration of these proteins in cell membranes, disrupts regulation of CRC promoting genes, and reduces CRC tumor burden. Thus, disruption of CDCP1 perturbs pro-cancerous Wnt signaling including nuclear localization of β-catenin and E-cadherin.

Recent molecularly targeted approach gains advance in breast cancer treatment. However, the estimated 5-year survival rate has not met the desired expectation for improvement, especially for patients with triple-negative breast cancer (TNBC). Here we report that the lncRNA PVT1 promotes KLF5/beta-catenin signaling to drive TNBC tumorigenesis. PVT1 is upregulated in clinical TNBC tumors. Using genetic approaches targeting PVT1 in TNBC cells, we found that PVT1 depletion inhibited cell proliferation, colony formation, and orthotopic xenograft tumor growth. Mechanistically, PVT1 binds with KLF5 and increases its stability via BAP1, which upregulates beta-catenin signaling, resulting in enhanced TNBC tumorigenesis. PVT1, KLF5, and beta-catenin were also revealed to be co-expressed in clinical TNBC samples. Our findings uncover a new singaling pathway to mediate TNBC, and provide PVT1 as a new target for improving treatment of TNBC.

Cellular heterogeneity is a common feature in breast cancer, yet an understanding of the coexistence and regulation of various tumor cell subpopulations remains a significant challenge in cancer biology. In the current study, we approached tumor cell heterogeneity from the perspective of Wnt pathway biology to address how different modes of Wnt signaling shape the behaviors of diverse cell populations within a heterogeneous tumor landscape. Using a syngeneic TP53-null mouse model of breast cancer, we identified distinctions in the topology of canonical Wnt β-catenin-dependent signaling activity and non-canonical β-catenin-independent Ror2-mediated Wnt signaling across subtypes and within tumor cell subpopulations in vivo. We further discovered an antagonistic role for Ror2 in regulating canonical Wnt/β-catenin activity in vivo, where lentiviral shRNA depletion of Ror2 expression augmented canonical Wnt/β-catenin signaling activity across multiple basal-like models. Depletion of Ror2 expression yielded distinct phenotypic outcomes and divergent alterations in gene expression programs among different tumors, despite all sharing basal-like features. Notably, we uncovered cell state plasticity and adhesion dynamics regulated by Ror2, which influenced Ras Homology Family Member A (RhoA) and Rho-Associated Coiled-Coil Kinase 1 (ROCK1) activity downstream of Dishevelled-2 (Dvl2). Collectively, these studies illustrate the integration and collaboration of Wnt pathways in basal-like breast cancer, where Ror2 provides a spatiotemporal function to regulate the balance of Wnt signaling and cellular heterogeneity during tumor progression.

Tumor suppressor genes and the immune system are critical players in inhibiting cancer initiation and/or progression. However, little is known about whether a tumor suppressor gene can function through both immune-dependent and -independent mechanisms. Retinoic acid receptor responder 2 (RARRES2) is transcriptionally downregulated in multiple cancer types. Previous studies suggested that it can serve as an immune-dependent tumor suppressor by acting as a chemoattractant to recruit anticancer immune cells expressing its receptor, the chemerin chemokine receptor 1 (CMKLR1), to sites of tumor. In this study, we investigated the role of RARRES2 in adrenocortical carcinoma (ACC), a rare lethal malignancy in which aberrant Wnt/β-catenin signaling is frequently detected. We show that RARRES2 expression is downregulated in ACC as compared with normal and benign adrenocortical tissues, which is a result of CpG hypermethylation. Despite minimal CMKLR1 expression and lack of phenotypic tumor-suppressive effect with exogenous RARRES2 treatment, RARRES2 overexpression in ACC cell lines not only reduced cell proliferation, cell invasion and tumorigenicity in vitro, but also inhibited tumor growth in vivo in two immunodeficient mouse xenograft models. Mechanistically, RARRES2 overexpression in ACC cells inhibited Wnt/β-catenin pathway activity by promoting β-catenin phosphorylation and degradation, it also inhibited the phosphorylation of p38 mitogen-activated protein kinase. Thus our study identifies RARRES2 as a novel tumor suppressor for ACC, which can function through an immune-independent mechanism.

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

Studies have reported that interactions between keratins (KRTs) and other proteins initiate signaling cascades that regulate cell migration, invasion, and metastasis. In the current study, we found that expression of KRT19 was specifically high in breast cancers and significantly correlated with their invasiveness. Moreover, knockdown of KRT19 led to increased proliferation, migration, invasion, drug resistance, and sphere formation in breast cancer cells via an upregulated NOTCH signaling pathway. This was owing to reduced expression of NUMB, an inhibitory protein of the NOTCH signaling pathway. In addition, we found that KRT19 interacts with β-catenin/RAC1 complex and enhances the nuclear translocation of β-catenin. Concordantly, knockdown of KRT19 suppressed the nuclear translocation of β-catenin as well as β-catenin-mediated NUMB expression. Furthermore, modulation of KRT19-mediated regulation of NUMB and NOTCH1 expression led to the repression of the cancer stem cell properties of breast cancer patient-derived CD133high/CXCR4high/ALDH1high cancer stem-like cells (CSLCs), which showed very low KRT19 and high NOTCH1 expression. Taken together, our study suggests a novel function for KRT19 in the regulation of nuclear import of the β-catenin/RAC1 complex, thus modulating the NUMB-dependent NOTCH signaling pathway in breast cancers and CSLCs, which might bear potential clinical implications for cancer or CSLC treatment.

Colon carcinogenesis is a multiple-step process involving the accumulation of a series of genetic and epigenetic alterations. The most commonly initiating event of intestinal carcinogenesis is mutation of the adenomatous polyposis coli (APC) gene, which leads to activation of the Wnt/β-catenin pathway. Olfactomedin 4 (OLFM4) has emerged as an intestinal stem-cell marker, but its biological function in the intestine remains to be determined. Here we show that Olfm4 deletion induced colon adenocarcinoma in the distal colon of ApcMin/+ mice. Mechanistically, we found that OLFM4 is a target gene of the Wnt/β-catenin pathway and can downregulate β-catenin signaling by competing with Wnt ligands for binding to Frizzled receptors, as well as by inhibition of the Akt-GSK-3β (Akt-glycogen synthase kinase-3β) pathway. We have shown that both Wnt and nuclear factor-κB (NF-κB) signaling were boosted in tumor tissues of Apc Olfm4 double-mutant mice. These data establish OLFM4 as a critical negative regulator of the Wnt/β-catenin and NF-κB pathways that inhibits colon-cancer development initiated by APC mutation. In addition, Olfm4 deletion significantly enhanced intestinal-crypt proliferation and inflammation induced by azoxymethane/dextran sodium sulfate. Thus, OLFM4 has an important role in the regulation of intestinal inflammation and tumorigenesis, and could be a potential therapeutic target for intestinal malignant tumors. Unlike the human colonic epithelium, the mouse colonic epithelium does not express OLFM4, but nevertheless, systemic OLFM4 deletion promotes colon tumorigenesis and that loss from mucosal neutrophils may have a role to play.

Metastasis begins with a subset of local tumor cells acquiring the potential to invade into surrounding tissues, and remains to be a major obstacle for cancer treatments. More than 90% of cancer patients died from tumor metastasis, instead of primary tumor growth. The canonical Wnt/β-catenin pathway plays essential roles in promoting tumor formation, yet its function in regulating tumor metastasis and the underlying mechanisms remain controversial. Here we employed well-established Drosophila tumor models to investigate the regulating mechanism of Wingless (Wg) pathway in tumor invasion. Our results showed that Wg signaling is necessary and sufficient for cell polarity disruption-induced cell migration and molecular changes reminiscent of epithelial-mesenchymal transition (EMT). Moreover, reducing Wg signaling suppressed lgl-/-/RasV12-induced tumor invasion, and cooperation between Arm and RasV12 is sufficient to induce tumor invasion. Mechanistically, we found that cell polarity disruption activates JNK signaling, which in turn upregulate wg expression through transcription factor activator protein-1 (AP-1). We identified a consensus AP-1 binding site located in the 2nd intron of wg, and confirmed that it is essential for AP-1 induced wg transcription both in vitro and in vivo. Lastly, we confirmed that the transcriptional activation of WNT by AP-1 is conserved in human cancer cells. These evidences reveal a positive role of Wnt/β-catenin pathway in tumor invasion, and provide a conserved mechanism that connects JNK and Wnt signaling in regulating tumor progression.

Cadherin subtype switching from E-cadherin to N-cadherin is associated with the epithelial-to-mesenchymal transition (EMT), a process required for invasion and dissemination of carcinoma cells. We found that N-cadherin is expressed in human and mouse pancreatic intraepithelial neoplasia (PanIN), suggesting that N-cadherin may also have a role in early-stage pancreatic cancer. To investigate the role of N-cadherin in mouse PanIN (mPanIN), we simultaneously activated oncogenic K-ras(G12D) and deleted the N-cadherin (Cdh2) gene in the murine pancreas. Genetic ablation of N-cadherin (N-cad KO) caused hyperproliferation, accelerated mPanIN progression, and early tumor development in K-ras(G12D) mice. Decreased E-cadherin and redistribution of β-catenin accompanied the loss of N-cadherin in pancreatic ductal epithelial cells (PDEC). Nuclear accumulation of β-catenin and its transcription co-activator Tcf4 led to activation of Wnt/β-catenin target genes. Unexpectedly, loss of N-cadherin in the K-ras(G12D) model resulted in increased mPanIN progression and tumor incidence. These in vivo results demonstrate for the first time that N-cadherin functions as a growth suppressor in the context of oncogenic K-ras.

Whole-genome and transcriptome sequencing were used to discover novel gene fusions in a case of colon cancer. A tumor-specific LACTB2-NCOA2 fusion originating from intra-chromosomal rearrangement of chromosome 8 was identified at both DNA and RNA levels. Unlike conventional oncogenic chimeric proteins, the fusion product lacks functional domain from respective genes, indicative of an amorphic rearrangement. This chimeric LACTB2-NCOA2 transcript was detected in 6 out of 99 (6.1%) colorectal cancer (CRC) cases, where NCOA2 was significantly downregulated. Enforced expression of wild-type NCOA2 but not the LACTB2-NCOA2 fusion protein impaired the pro-tumorigenic phenotypes of CRC cells, whereas knockdown of endogenous NCOA2 in normal colonocytes had opposite effects. Mechanistically, NCOA2 inhibited Wnt/β-catenin signaling through simultaneously upregulating inhibitors and downregulating stimulators of Wnt/β-catenin pathway. Collectively, our data supports that NCOA2 is a novel negative growth regulatory gene repressing the Wnt/β-catenin pathway in CRC, where recurrent fusion with LACTB2 contributes to its disruption.

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularlyin vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice underde novoconditions and in both the strains display E-cadherin-to-N-cadherin switch, reduced expression of cellular senescence markers and reduced expression of senescence-associated secretory phenotype, indicating a genetic predisposition of these mice to increased carcinogen-induced lung tumorigenesis. Interestingly, Wnt7a induced an alternate senescence pathway, which was independent of β-catenin, and distinct from that of classical oncogene-induced senescence mediated by the well-known p16INK4aand p19ARFpathways. Mechanistically, Wnt7a induced cellular senescence via inactivation of S-phase kinase-associated protein 2, an important alternate regulator of cellular senescence. Additionally, we identified Iloprost, a prostacyclin analog, which initiates downstream signaling cascades similar to that of Wnt7a, as a novel inducer of cellular senescence, presenting potential future clinical translational strategies. Thus pro-senescence therapies using either Wnt7a or its mimic, Iloprost, might represent a new class of therapeutic treatments for lung cancer.

Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in β-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc, Axin2 and Lef1. Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (ΔNTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced β-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting β-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/β-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/β-catenin signalings during oncogenesis.

Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of Wnt signaling pathway. We aim to clarify its epigenetic inactivation, biological function and clinical implication in colon cancer. DACT2 was silenced in five out of eight colon cancer cell lines, but robustly expressed in normal colon tissues. The loss of DACT2 expression was regulated by promoter hypermethylation. Restoring DACT2 expression in colon cancer cell lines suppressed tumor cell growth by inducing cell apoptosis and inhibiting cell proliferation both in vitro and in vivo. Moreover, DACT2 overexpression effectively reduced lung metastasis of colon cancer cells in nude mice. These effects by DACT2 were attributed to inhibition of Wnt/β-catenin signaling. Reexpression of DACT2 significantly suppressed the transcriptional activity of both wild-type β-catenin and degradation-resistant form mutant β-catenin (S33Y). DACT2 could actively shuttle into and out of nuclei, with its predominant steady-state localization in the cytoplasm dependent on its nuclear export signal. Co-immunoprecipitation results indicated that DACT2 strongly associated β-catenin as well as lymphoid enhancer-binding factor 1 (LEF1) and directly disrupted the formation of the β-catenin–LEF1 complex in the nucleus. Whereas in the cytoplasm, DACT2 restored junctional localization of E-cadherin–β-catenin complexes and prevented β-catenin nuclear translocation through direct interaction with β-catenin. DACT2 methylation was detected in 43.3% (29/67) of colon cancer tissues, but none in normal controls. Multivariate analysis revealed that patients with DACT2 methylation had a significant decrease in overall survival (P=0.006). Kaplan–Meier survival curves showed that DACT2 methylation was significantly associated with shortened survival in stage I–III colon cancer patients. In conclusion, DACT2 acts as a functional tumor suppressor in colon cancer through inhibiting Wnt/β-catenin signaling. Its methylation at early stages of colon carcinogenesis is an independent prognostic factor.

Mutations of Wnt/β-catenin signaling pathway has essential roles in development and cancer. Although β-catenin and adenomatous polyposis coli (APC) gene mutations are well established and are known to drive tumorigenesis, discoveries of mutations in other components of the pathway lagged, which hinders the understanding of cancer mechanisms. Here we report that δ-catenin (gene designation: CTNND2), a primarily neural member of the β-catenin superfamily that promotes canonical Wnt/β-catenin/LEF-1-mediated transcription, displays exonic mutations in human prostate cancer and promotes cancer cell survival adaptation and metabolic reprogramming. When overexpressed in cells derived from prostate tumor xenografts, δ-catenin gene invariably gives rise to mutations, leading to sequence disruptions predicting functional alterations. Ectopic δ-catenin gene integrating into host chromosomes is locus nonselective. δ-Catenin mutations promote tumor development in mouse prostate with probasin promoter (ARR2PB)-driven, prostate-specific expression of Myc oncogene, whereas mutant cells empower survival advantage upon overgrowth and glucose deprivation. Reprogramming energy utilization accompanies the downregulation of glucose transporter-1 and poly (ADP-ribose) polymerase cleavage while preserving tumor type 2 pyruvate kinase expression. δ-Catenin mutations increase β-catenin translocation to the nucleus and hypoxia-inducible factor 1α (HIF-1α) expression. Therefore, introducing δ-catenin mutations is an important milestone in prostate cancer metabolic adaptation by modulating β-catenin and HIF-1α signaling under glucose shortage to amplify its tumor-promoting potential.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Apc-driven tumor formation in patients and Apc-mutant mouse models is generally attributed to increased levels of beta-catenin signaling. We and others have proposed that a specific level of b-catenin signaling is required to successfully initiate tumor formation, and that each tissue prefers different dosages of signaling. This is illustrated by APC genotype - tumor phenotype correlations in cancer patients, and by the different tumor phenotypes displayed by different Apc-mutant mouse models. Apc1638N mice, associated with intermediate b-catenin signaling, characteristically develop intestinal tumors (< 10) and extra-intestinal tumors, including cysts and desmoids. Apc1572T mice associated with lower levels of b-catenin signaling are free of intestinal tumors, but instead develop mammary tumors. Although the concept of b-catenin signaling dosage and its impact on tumor growth among tissues is gaining acceptance, it has not been formally proven. Additionally, alternative explanations for Apc-driven tumor formation have been proposed. To obtain direct evidence for the dominant role of b-catenin dosage in tumor formation and tissue-specific tumor predisposition, we crossed Apc1638N mice with heterozygous b-catenin knockout mice, thereby reducing b-catenin levels. Whereas all the Apc1638N; Ctnnb1(+/+) mice developed gastrointestinal tumors, none were present in the Apc1638N; Ctnnb1(-/-) mice. Incidence of other Apc1638N-associated lesions, including desmoids and cysts, was strongly reduced as well. Interestingly, Apc1638N; Ctnnb1(-/-) females showed an increased incidence of mammary tumors, which are normally rarely observed in Apc1638N mice, and the histological composition of the tumors resembled that of Apc1572T-related tumors. Hereby, we provide in vivo genetic evidence confirming the dominant role of b-catenin dosage in tumor formation and in dictating tumor predisposition among tissues in Apc-driven cancer.

Wnt signaling is one of the key cascades regulating development and stemness, and has also been tightly associated with cancer. The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer, but aberrant Wnt signaling is observed in many more cancer entities. Here, we review current insights into novel components of Wnt pathways and describe their impact on cancer development. Furthermore, we highlight expanding functions of Wnt signaling for both solid and liquid tumors. We also describe current findings how Wnt signaling affects maintenance of cancer stem cells, metastasis and immune control. Finally, we provide an overview of current strategies to antagonize Wnt signaling in cancer and challenges that are associated with such approaches.

The Wnt/β-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated β-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/β-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of β-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that β-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that β-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that β-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of β-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

The canonical Wnt signalling pathway induces the β-catenin/lymphoid enhancer factor transcription factors. It is activated in various cancers, most characteristically carcinomas, in which it promotes metastatic spread by increasing migration and/or invasion. The Wnt/β-catenin signalling pathway is frequently activated in melanoma, but the presence of β-catenin in the nucleus does not seem to be a sign of aggressiveness in these tumours. We found that, unlike its positive role in stimulating migration and invasion of carcinoma cells, β-catenin signalling decreased the migration of melanocytes and melanoma cell lines. In vivo, β-catenin signalling in melanoblasts reduced the migration of these cells, causing a white belly-spot phenotype. The inhibition by β-catenin of migration was dependent on MITF-M, a key transcription factor of the melanocyte lineage, and CSK, an Src-inhibitor. Despite reducing migration, β-catenin signalling promoted lung metastasis in the NRAS-driven melanoma murine model. Thus, β-catenin may have conflicting roles in the metastatic spread of melanoma, repressing migration while promoting metastasis. These results highlight that metastasis formation requires a series of successful cellular processes, any one of which may not be optimally efficient.

Dysregulation of β-catenin turnover due to mutations of its regulatory proteins including adenomatous polyposis coli (APC) and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated β-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, has a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of β-catenin by inducing β-catenin degradation via proteasomal degradation pathway that is glycogen synthase kinase 3β and Siah-1 independent. Nur77 induction of β-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for β-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with β-catenin, and induced β-catenin degradation, demonstrating that Nur77-mediated β-catenin degradation was independent of its DNA binding and transactivation, and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and β-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing β-catenin degradation. Together, our results demonstrate that β-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for β-catenin degradation.

Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP–chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes. Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP-chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes.Oncogene advance online publication, 3 October 2011; doi:10.1038/onc.2011.453.

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt-signaling pathway by targeting the proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription cofactor for genes required for cell proliferation such as cyclin-D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and the cytoplasm, with nuclear APC implicated in the inhibition of Wnt target gene expression. Adopting a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals (NLSs) of Apc (Apc(mNLS)). Apc(mNLS/mNLS) mice are viable and fractionation of mouse embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc as compared with Apc(+/+) MEFs. The levels of Apc and β-catenin protein were not significantly altered in small intestinal epithelia from Apc(mNLS/mNLS) mice. Compared with Apc(+/+) mice, Apc(mNLS/mNLS) mice showed increased proliferation in epithelial cells from the jejunum, ileum and colon. These same tissues from Apc(mNLS/mNLS) mice showed more mRNA from three genes upregulated in response to canonical Wnt signal, c-Myc, axin-2 and cyclin-D1, and less mRNA from Hath-1, which is downregulated in response to Wnt. These observations suggest a role for nuclear Apc in the inhibition of canonical Wnt signaling and the control of epithelial proliferation in intestinal tissue. Furthermore, we found Apc(Min/+) mice, which harbor a mutation that truncates Apc, to have an increased polyp size and multiplicity if they also carry the Apc(mNLS) allele. Taken together, this analysis of the novel Apc(mNLS) mouse model supports a role for nuclear Apc in the control of Wnt target genes, intestinal epithelial cell proliferation and polyp formation.

WNT, PI3K or RAS signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector beta-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRASG12D dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRASG12D in granulosa cells, we sought to determine if KRASG12D would block precancerous lesion and tumor formation in follicles of the CTNNB1 mutant mice. Unexpectedly, transgenic Ctnnb1;Kras mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten mutant mice. Microarray and RT-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, 1) other components of these pathways may be altered or mutated, 2) these mutations may relate to juvenile GCTs or 3) they may occur in tumors of other tissues where CTNNB1 is mutated. Importantly, our results provide strong evidence that CTNNB1 is the driver in these contexts and that KRASG12D and Pten loss promote the program set in motion by the CTNNB1.

Using a screen for Wnt/β-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe(2+) with a dissociation constant of ∼10(-19) M, with much weaker binding to Fe(3+) and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific β-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on β-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse β-catenin-independent cell lines. HQBA is a promising specific intracellular Fe(2+) chelator with activity against spontaneous mouse mammary cancers.

Cell-type-specific signalling determines cell fate under physiological conditions, but it is increasingly apparent that also in cancer development the impact of any given oncogenic pathway on the individual cancer pathology is dependent on cell-lineage-specific molecular traits. For instance in colon and liver cancer canonical Wnt signalling produces increased cytoplasmic and nuclear localised beta-catenin, which correlates with invasion and poor prognosis. In contrast, in melanoma increased cytoplasmic and nuclear beta-catenin is currently emerging as a marker for good prognosis, and thus seems to have a different function compared with other cancer types; however, this function is unknown. We discovered that in contrast to its function in other cancers, in melanoma, beta-catenin blocks invasion. We demonstrate that this opposing role of nuclear beta-catenin in melanoma is mediated through MITF, a melanoma-specific protein that defines the lineage background of this cancer type. Downstream of beta-catenin MITF not only suppresses the Rho-GTPase-regulated cell morphology of invading melanoma cells, but also interferes with beta-catenin-induced expression of the essential collagenase MT1-MMP, thus affecting all aspects of an invasive phenotype. Importantly, overexpression of MITF in invasive colon cancer cells modifies beta-catenin-directed signalling and induces a 'melanoma phenotype'. In summary, the cell-type-specific presence of MITF in melanoma affects beta-catenin's pro-invasive properties otherwise active in colon or liver cancer. Thus our study reveals the general importance of considering cell-type-specific signalling for the accurate interpretation of tumour markers and ultimately for the design of rational therapies.

Deregulation of the pRb/E2F or Wnt/β-catenin pathway occurs frequently in human cancers, which is often associated with inappropriate cell proliferation. Although the oncogenic roles of pRb/E2F1 and Wnt/β-catenin pathways have been well studied, the functional interaction between the two pathways has only recently been characterized. In particular, E2F1 has been recently reported to negatively regulate Wnt/β-catenin activity in human colorectal cancers, though the mechanism underlying this regulation is not fully understood. Here we provide evidence that β-catenin interacting protein 1 (CTNNBIP1), also known as ICAT (inhibitor of β-catenin and TCF4), functions as a crucial node to mediate the cross talk between E2F1 and β-catenin signaling. We show that ICAT is a direct transcriptional target of E2F1, and that activation of ICAT by E2F1 is required for E2F1 to inhibit β-catenin activity. This study provides a mechanistic insight into the antagonistic interaction between E2F1 and β-catenin signaling.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling, and activation of β-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosing and treating metastasis at early stages.

β-Catenin is constantly degraded through the ubiquitin-proteasomal pathway. In this study, we report that a different type of β-catenin degradation is causally involved in epidermal cell death. We observed that reactive oxygen species (ROS) caused β-catenin degradation in the epidermal cells through a caspase-dependent mechanism, which results in disruption of cell adhesion. Disruption of cell adhesion increased ROS and activated caspases. Upregulation of the intact β-catenin blocked ROS accumulation and caspase activation. These results indicate that a feed-forward loop consisting of ROS, caspases activation and β-catenin degradation induces epidermal cell death.

Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/β-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/β-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/β-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.

Our previous studies have found that activation of Wnt/β-Catenin signaling resulted in mouse prostatic intraepithelial neoplasia (mPIN). In the large probasin promoter directed SV40-Large T-antigen (LPB-Tag) expressing mouse prostate, mPIN forms with rare areas of adenocarcinoma. Combining expression of both Wnt-signaling and Tag expression in the mouse prostate, we have studied the role of Wnt/β-Catenin signaling in the progression from mPIN to adenocarcinoma. Our results show that the prostates of mice expressing Tag alone or nuclear β-Catenin alone developed mPIN while the activation of both Tag and the Wnt/β-Catenin pathway resulted in invasive prostate adenocarcinoma. Also, Foxa2, a forkhead transcription factor, was induced by active Wnt/β-Catenin signaling; and the expression of Foxa2 was associated with the invasive phenotype in the primary prostate cancer. In the LPB-Tag/dominant active (D.A.) β-Catenin prostates, MMP7, a Wnt/β-Catenin target gene, was up-regulated. Furthermore, we also assessed AR and AR signaling pathway in these LPB-Tag/D.A. β-Catenin mice. Although β-Catenin is a well known AR co-activator in vitro, our study provides strong in vivo evidences indicating that both AR protein and the AR pathway were down-regulated in the prostate of LPB-Tag/D.A. β-Catenin mice. Histological analysis shows that prostate sections derived from the LPB-Tag/D.A. β-Catenin mice display neuroendocrine differentiation (NED) but NE cancer does not develop. Together, our findings indicate that Wnt/β-Catenin signaling plays an important role in the progression of mPIN to prostate adenocarcinoma.

Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of β-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE(+) β-catenin(exon3/+)). Expression of this activated form of β-catenin led to the formation of localized hyperproliferative lesions by 3 months, which did not progress to malignancy. These lesions were characterized by a marked increase of the phosphatase and tensin homologue (PTEN) tumour suppressor protein. This appears to be a direct consequence of activating Wnt signalling in the bladder as conditional deletion of the adenomatous polyposis coli (Apc) gene within the adult bladder led rapidly to coincident β-catenin and PTEN expression. This PTEN expression blocked proliferation. Next, we combined PTEN deficiency with β-catenin activation and found that this caused papillary UCC. These tumours had increased pAKT signalling and were dependent on mammalian target of rapamycin (mTOR). Importantly, in human UCC, there was a significant correlation between high levels of β-catenin and pAKT (and low levels of PTEN). Taken together these data show that deregulated Wnt signalling has a critical role in promoting UCC, and suggests that human UCC that have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition.

Although Wnt-Frizzled (Fzd) signaling is critical in the pathophysiology of carcinomas, its role in human breast cancer has been difficult to establish. We show here that the adaptor protein Na(+)/H(+) exchange regulatory factor1 (NHERF1), a protein abundantly expressed in normal mammary epithelium, regulates Wnt signaling, maintaining low levels of β-catenin activation. NHERF1's effects are mediated by direct interactions between one of its PSD-95/drosophila discs large/ZO-1 (PDZ) domains and the C-terminus of a subset of Fzd receptors. Loss of NHERF1 in breast cancer cell lines enhances canonical Wnt signaling and Wnt-dependent cell proliferation. Furthermore, the mammary glands of NHERF1-knockout mice exhibit increased mammary duct density accompanied by increased proliferation and β-catenin activity. Finally, we demonstrate a negative correlation between NHERF1 expression and nuclear β-catenin in human breast carcinomas. Taken together, these results provide a novel insight into the regulation of Wnt signaling in normal and neoplastic breast tissues, and identify NHERF1 as an important regulator of the pathogenesis of breast tumors.

Wnt/beta-catenin signaling plays a pivotal role in modulating cellular proliferation, differentiation, tissue organization and embryonic development. Earlier, we found that the endocytic adaptor disabled-2 (Dab2) could attenuate Wnt/beta-catenin signaling by stabilizing Axin and preventing its translocation to the membrane. Recently, protein phosphatase 1 (PP1) has been shown to interact with, and dephosphorylate Axin, leading to its destabilization. Here, we show that Dab2 functions upstream of PP1 to block the interaction between Axin and PP1, inhibiting Axin dephosphorylation and thereby stabilizing its expression, ultimately leading to inhibition of Wnt/beta-catenin. We show that Dab2 acts as a competitive inhibitor of PP1 by binding to the same C-terminal domain of Axin. Both PP1 and Axin bind to the N-terminus of Dab2 and a Dab2 truncation mutant consisting of the N-terminal phosphotyrosine binding domain blocks PP1-Axin interactions and inhibits Wnt signaling. We confirm the inhibitory effect of Dab2 on Wnt/beta-catenin signaling in zebrafish embryos, showing that its ectopic expression phenocopies Axin overexpression resulting in altered dorsoventral patterning. We conclude that Dab2 stabilizes Axin and attenuates Wnt/beta-catenin signaling by preventing PP1 from binding Axin.

In human hepatocellular carcinoma (HCC), epithelial to mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. We employed a model of EMT based on immortalized p19(ARF) null hepatocytes (MIM), which display tumor growth upon expression of oncogenic Ras and undergo EMT through the synergism of Ras and transforming growth factor (TGF)-beta. Here, we show that the interleukin-related protein interleukin-like EMT inducer (ILEI), a novel EMT-, tumor- and metastasis-inducing protein, cooperates with oncogenic Ras to cause TGF-beta-independent EMT. Ras-transformed MIM hepatocytes overexpressing ILEI showed cytoplasmic E-cadherin, loss of ZO-1 and induction of alpha-smooth muscle actin as well as platelet-derived growth factor (PDGF)/PDGF-R isoforms. As shown by dominant-negative PDGF-R expression in these cells, ILEI-induced PDGF signaling was required for enhanced cell migration, nuclear accumulation of beta-catenin, nuclear pY-Stat3 and accelerated growth of lung metastases. In MIM hepatocytes expressing the Ras mutant V12-C40, ILEI collaborated with PI3K signaling resulting in tumor formation without EMT. Clinically, human HCC samples showed granular or cytoplasmic localization of ILEI correlating with well and poorly differentiated tumors, respectively. In conclusion, these data indicate that ILEI requires cooperation with oncogenic Ras to govern hepatocellular EMT through mechanisms involving PDGF-R/beta-catenin and PDGF-R/Stat3 signaling.

The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC- and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits beta-catenin destruction complex proteins, including APC, beta-catenin, glycogen synthase kinase-3-beta (GSK3-beta) and casein kinase-1-alpha (CK1-alpha). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for beta-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for beta-catenin degradation. An axinDeltaDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of beta-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for beta-catenin destruction.

The Wnt signaling pathway is essential for embryonic development and carcinogenesis. Upon Wnt stimulation, beta-catenin is stabilized and associates with T-cell factor or lymphoid enhancing factor, thereby activating transcription of target genes. In the absence of Wnt stimulation, the level of beta-catenin is reduced via glycogen synthase kinase (GSK)-3beta-mediated phosphorylation and subsequent proteasome-dependent degradation. Here, we report the identification of Ajuba as a negative regulator of the Wnt signaling pathway. Ajuba is a member of LIM domain-containing proteins that contribute to cell fate determination and regulate cell proliferation and differentiation. We found that enforced expression of Ajuba destabilized beta-catenin and suppressed target gene expression. Ajuba promoted GSK-3beta-mediated phosphorylation of beta-catenin by reinforcing the association between beta-catenin and GSK-3beta. Furthermore, Wnt stimulation induced both accumulation of beta-catenin and destabilization of Ajuba. Our findings suggest that Ajuba is important for regulation of the Wnt signaling pathway.

Siah-1 (seven in absentia homolog) is known to cause indirect degradation of beta-catenin through formation of a complex with Siah-interacting protein (SIP), Skp1 and Ebi. Here, we report the characterization of a novel splice variant of human Siah-1, designated Siah-1S, which is produced by an alternative splicing mechanism. The novel intron/exon junctions used to generate Siah-1S follow a non-conventional CT-AC rule. Siah-1S exhibits an even shorter half-life than Siah-1 and is able to catalyse self-ubiquitination that results in its subsequent degradation by proteasome. Siah-1S is shown to upregulate beta-catenin-dependent Tcf/Lef transcriptional activation and antagonize Siah-1's potentiation effect on the apoptosis induced by etoposide in MCF-7 cells. Additionally, Siah-1S is found to interact with Siah-1 to form heterodimer or with itself to form homodimer. Unlike homodimer Siah-1*Siah-1, neither Siah-1*Siah-1S nor Siah-1S*Siah-1S is able to bind to Siah-1-interacting protein, which may explain the underlying mechanism for Siah-1S's dominant negative effect on Siah-1. Importantly, results from in vitro soft agar assay demonstrated that Siah-1S displays a promotion effect on cells tumorigenicity.

De Clian M.-C., Pauper, E., and R. Bandiera contributed equally to this work. Notice à reprendre en Chantier Qualité avec la version finale de l’Éditeur; International audience; R-spondin1 is a secreted regulator of WNT signaling, involved in both embryonic development and homeostasis of adult organs. It can have a dual role, acting either as a mitogen or as a tumor suppressor. During ovarian development, Rspo1 is a key factor required for sex determination and differentiation of the follicular cell progenitors, but is downregulated after birth. In human, increased RSPO1 expression is associated with ovarian carcinomas, but it is not clear whether it is a cause or a consequence of the tumorigenic process. To address the role of Rspo1 expression in adult ovaries, we generated an Rspo1 gain-of-function mouse model. Females were hypofertile and exhibited various ovarian defects, ranging from cysts to ovarian tumors. Detailed phenotypical characterization showed anomalies in the ovulation process. Although follicles responded to initial follicle-stimulating hormone stimulation and developed normally until the pre-ovulatory stage, they did not progress any further. Although non-ovulated oocytes degenerated, the surrounding follicular cells did not begin atresia. RSPO1-induced expression not only promotes canonical WNT signaling but also alters granulosa cell fate decisions by maintaining epithelial-like traits in these cells. This prevents follicle cells from undergoing apoptosis, leading to the accumulation of granulosa cell tumors that reactivates the epithelial program from their progenitors. Taken together, our data demonstrate that activation of RSPO1 is sufficient in promoting ovarian tumors and thus supports a direct involvement of this gene in the commencement of ovarian cancers.

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecological malignancies owing to poor screening methods, non-specific symptoms and limited knowledge of the cellular targets that contribute to the disease. Cyclin G2 is an unconventional cyclin that acts to oppose cell cycle progression. Dysregulation of the cyclin G2 gene (CCNG2) in a variety of human cancers has been reported; however, the role of cyclin G2 in tumorigenesis remains unclear. In this study, we investigated the function of cyclin G2 in EOC. In vitro and in vivo studies using several EOC-derived tumor cell lines revealed that cyclin G2 inhibited cell proliferation, migration, invasion and spheroid formation, as well as tumor formation and invasion. By interrogating cDNA microarray data sets, we found that CCGN2 mRNA is reduced in several large cohorts of human ovarian carcinoma when compared with normal ovarian surface epithelium or borderline tumors of the ovary. Mechanistically, cyclin G2 was found to suppress epithelial-to-mesenchymal transition (EMT), as demonstrated by the differential regulation of various EMT genes, such as Snail, Slug, vimentin and E-cadherin. Moreover, cyclin G2 potently suppressed the Wnt/β-catenin signaling pathway by downregulating key Wnt components, namely LRP6, DVL2 and β-catenin, which could be linked to inhibition of EMT. Taken together, our novel findings demonstrate that cyclin G2 has potent tumor-suppressive effects in EOCs by inhibiting EMT through attenuating Wnt/β-catenin signaling.

Adenomatous polyposis coli (APC) mutation is the most common genetic change in sporadic colorectal cancer (CRC). Although deregulations of miRNAs have been frequently reported in this malignancy, APC-regulated miRNAs have not been extensively documented. Here, by using an APC-inducible cell line and array analysis, we identified a total of 26 deregulated miRNAs. Among them, members of miR-17-92 cluster were dramatically inhibited by APC and induced by enforced expression of β-catenin. Furthermore, we demonstrate that activated β-catenin resulted from APC loss binds to and activates the miR-17-92 promoter. Notably, enforced expression of miR-19a overrides APC tumor suppressor activity, and knockdown of miR-19a in cancer cells with compromised APC function reduced their aggressive features in vitro. Finally, we observed that expression of miR-19a significantly correlates with β-catenin levels in colorectal cancer specimens, and it is associated to the aggressive stage of tumor progression. Thus, our study reveals that miR-17-92 cluster is directly regulated by APC/β-catenin pathway and could be a potential therapeutic target in colon cancers with aberrant APC/β-catenin signaling.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.522.

The canonical Wnt pathway (TCF4/β-catenin) has important roles during normal differentiation and in disease. Some Wnt functions depend on signaling gradients requiring the pathway to be tightly regulated. A key Wnt target is the transcription factor ZEB1 whose expression by cancer cells promotes tumor invasiveness by repressing the expression of epithelial specification markers and activating mesenchymal genes, including a number of Wnt targets such as LAMC2 and uPA. The ability of ZEB1 to activate/repress its target genes depends on its recruitment of corepressors (CtBP, BRG1) or coactivators (p300) although conditions under which ZEB1 binds these cofactors are not elucidated. Here, we show that TCF4 and ZEB1 reciprocally modulate each other's transcriptional activity: ZEB1 enhances TCF4/β-catenin-mediated transcription and, in turn, Wnt signaling switches ZEB1 from a repressor into an activator. In colorectal cancer (CRC) cells with active Wnt signaling, ZEB1 enhances transcriptional activation of LAMC2 and uPA by TCF4/β-catenin. However, in CRC cells with inactive Wnt, ZEB1 represses both genes. Reciprocal modulation of ZEB1 and TCF4 activities involves their binding to DNA and mutual interaction. Wnt signaling turns ZEB1 into an activator by replacing binding of CtBP/BRG1 in favor of p300. Using a mouse model of Wnt-induced intestinal tumorigenesis, we found that downregulation of ZEB1 reduces the expression of LAMC2 in vivo. These results identify a mechanism through which Wnt and ZEB1 transcriptional activities are modulated, offering new approaches in cancer therapy.

Overexpressed or activated hepatocyte growth factor receptor, encoded by the MET proto-oncogene, was found in the majority of colorectal carcinomas (CRCs), whose stepwise progression to malignancy requires transcriptional activation of beta-catenin. We here demonstrate that a functional crosstalk between Met and beta-catenin signaling sustains and increases CRC cell invasive properties. Hepatocyte growth factor (HGF) stimulation prompts beta-catenin tyrosine phosphorylation and dissociation from Met, and upregulates beta-catenin expression via the phosphatidylinositol 3-kinase pathway in conditions that mimic those found by the invading and metastasizing cells. Additionally, a transcriptionally active form of beta-catenin, known to be oncogenic, enhances Met expression. Furthermore, HGF treatment increases the activity of the beta-catenin-regulated T-cell factor transcription factor in cells expressing the wild-type or the oncogenic beta-catenin. In the mirror experiments, either Met or beta-catenin knocking down also reduces their protein level. In biological assays, beta-catenin knocking down abrogates the HGF-induced motile phenotype, whereas active beta-catenin fosters ligand-independent cell scattering. Met and beta-catenin also cooperate in promoting entry into the cell cycle and in protecting cells from apoptosis. In conclusion, Met and beta-catenin pathways are mutually activated in CRC cells. This might generate a self-amplifying positive feedback loop resulting in the upregulation of the invasive growth properties of CRC cells.