First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.

Role of TCF/LEF Transcription Factors in Bone Development and Osteogenesis.

Bone formation occurs by two distinct mechanisms, namely, periosteal ossification and endochondral ossification. In both mechanisms, osteoblasts play an important role in the secretion and mineralization of bone-specific extracellular matrix. Differentiation and maturation of osteoblasts is a prerequisite to bone formation and is regulated by many factors. Recent experiments have shown that transcription factors play an important role in regulating osteoblast differentiation, proliferation, and function. Osteogenesis related transcription factors are the central targets and key mediators of the function of growth factors, such as cytokines. Transcription factors play a key role in the transformation of mesenchymal progenitor cells into functional osteoblasts. These transcription factors are closely linked with each other and in conjunction with bone-related signaling pathways form a complex network that regulates osteoblast differentiation and bone formation. In this paper, we discuss the structure of T-cell factor/lymphoid enhancer factor (TCF/LEF) and its role in embryonic skeletal development and the crosstalk with related signaling pathways and factors.

The planarian TCF/LEF factor Smed-tcf1 is required for the regeneration of dorsal-lateral neuronal subtypes.

The adult brain of the planarian Schmidtea mediterranea (a freshwater flatworm) is a dynamic structure with constant cell turnover as well as the ability to completely regenerate de novo. Despite this, function and pattern is achieved in a reproducible manner from individual to individual in terms of the correct spatial and temporal production of specific neuronal subtypes. Although several signaling molecules have been found to be key to scaling and cell turnover, the mechanisms by which specific neural subtypes are specified remain largely unknown. Here we performed a 6 day RNAseq time course on planarians that were regenerating either 0, 1, or 2 heads in order to identify novel regulators of brain regeneration. Focusing on transcription factors, we identified a TCF/LEF factor, Smed-tcf1, which was required to correctly pattern the dorsal-lateral cell types of the regenerating brain. The most severely affected neurons in Smed-tcf1(RNAi) animals were the dorsal GABAergic neurons, which failed to regenerate, leading to an inability of the animals to phototaxis away from light. Together, Smed-tcf1 is a critical regulator, required to pattern the dorsal-lateral region of the regenerating planarian brain.

Variations in T cell transcription factor gene structure and expression associated with the two disease forms of sheep paratuberculosis.

Two different forms of clinical paratuberculosis in sheep are recognised, related to the level of bacterial colonization. Paucibacillary lesions are largely composed of lymphocytes with few bacteria, and multibacillary pathology is characterized by heavily-infected macrophages. Analysis of cytokine transcripts has shown that inflammatory Th1/Th17 T cells are associated with development of paucibacillary pathology and Th2 cytokines are correlated with multibacillary disease. The master regulator T cell transcription factors TBX21, GATA3, RORC2 and RORA are critical for the development of these T cell subsets. Sequence variations of the transcription factors have also been implicated in the distinct disease forms of human mycobacterial and gastrointestinal inflammatory diseases. Relative RT-qPCR was used to compare expression levels of each transcript variant of the master regulators in the ileo-caecal lymph nodes of uninfected controls and sheep with defined paucibacillary and multibacillary pathology. Low levels of GATA3 in multibacillary sheep failed to confirm that multibacillary paratuberculosis is caused simply by a Th2 immune response. However, high levels of TBX21, RORC2 and RORC2v1 highlights the role of Th1 and Th17 activation in paucibacillary disease. Increased RORAv1 levels in paucibacillary tissue suggests a role for RORα in Th17 development in sheep; while elevated levels of RORAv4 hints that this variant might inhibit RORα function and depress Th17 development in multibacillary sheep.

BMP10 inhibited the growth and migration of gastric cancer cells.

Bone morphogenetic protein 10 (BMP10), a novel member of BMP family, has been identified as an important regulator for angiogenesis. Dysregulation of BMP has been observed in several cancer types. However, its roles in gastric cancer (GC) remain unknown. In this study, the expression of BMP10 was found to be down-regulated in GC samples. Forced expression of BMP10 in GC cells inhibited its growth and migration, while knocking down the expression of BMP10 in GC cells promoted cell growth, migration, and metastasis. BMP10 was shown to negatively regulated beta-catenin/TCF signaling by up-regulating Axin protein level. Taken together, the present study revealed the suppressive function of BMP10 in gastric cancer.

KLF2 is downregulated in pancreatic ductal adenocarcinoma and inhibits the growth and migration of cancer cells.

Members of the Kruppel-like factor (KLF) family have been considered as the tumor suppressors for their inhibitory effects on cell proliferation. Dysregulation of KLF2, a member of KLF family, has been observed in various cancer types. However, its expression pattern and functions in the pancreatic ductal adenocarcinoma (PDAC) are unknown. In this study, we examined the expression of KLF2 in PDAC clinical samples and evaluated the functions of KLF2 in the progression of PDAC. KLF2 is shown to be downregulated in PDAC clinical samples and overexpression of KLF2 inhibits the growth, migration, and metastasis of PDAC cancer cells. KLF2 interacts with beta-catenin and negatively regulates the beta-catenin/TCF signaling. Taken together, this study suggests the suppressive functions of KLF2 in PDAC.

Up-regulation of SRPK1 in non-small cell lung cancer promotes the growth and migration of cancer cells.

Dys-regulation of serine-arginine protein kinase 1 (SRPK1) has been reported in non-small cell lung cancer (NSCLC). However, its functions in the progression of NSCLC remain poorly understood. In this study, the expression of SRPK1 in NSCLC tissues was determined using real-time PCR, and the roles of SRPK1 in the progression of NSCLC were investigated. It was found that both the mRNA level and the protein level of SRPK1 were up-regulated in NSCLC tissues. Forced expression of SRPK1 promoted the growth and migration of NSCLC cells, while knocking down the expression of SRPK1 inhibited the growth, migration, and tumorigenicity of NSCLC cells. Mechanism studies showed that SRPK1 activated the transcriptional activity of beta-catenin/T-cell factor (TCF) complex, and knocking down the expression of SRPK1 attenuated the expression of target genes of beta-catenin/T-cell factor (TCF) complex. In addition, silencing the expression of SRPK1 down-regulated the phosphorylation of GSK3beta. Taken together, SRPK1 might play an oncogenic role in NSCLC, and SRPK1 might be a therapeutic target for NSCLC.

A functional connectome: regulation of Wnt/TCF-dependent transcription by pairs of pathway activators.

BACKGROUND: Wnt/ß-catenin signaling is often portrayed as a simple pathway that is initiated by Wnt ligand at the cell surface leading, via linear series of interactions between 'core pathway' members, to the induction of nuclear transcription from genes flanked by ß-catenin/TCF transcription factor binding sites. Wnt/ß-catenin signaling is also regulated by a much larger set of 'non-core regulators'. However the relationship between 'non-core regulators' is currently not well understood. Aberrant activation of the pathway has been shown to drive tumorgenesis in a number of different tissues. METHODS: Mammalian cells engineered to have a partially-active level of Wnt/ß-catenin signaling were screened by transfection for proteins that up or down-regulated a mid-level of TCF-dependent transcription induced by transient expression of an activated LRP6 Wnt co-receptor (∆NLRP). RESULTS: 141 novel regulators of TCF-dependent transcription were identified. Surprisingly, when tested without ∆NLRP activation, most up-regulators failed to alter TCF-dependent transcription. However, when expressed in pairs, 27 % (466/1170) functionally interacted to alter levels of TCF-dependent transcription. When proteins were displayed as nodes connected by their ability to co-operate in the regulation of TCF-dependent transcription, a network of functional interactions was revealed. In this network, 'core pathway' components (Eg. ß-catenin, GSK-3, Dsh) were found to be the most highly connected nodes. Activation of different nodes in this network impacted on the sensitivity to Wnt pathway small molecule antagonists. CONCLUSIONS: The 'functional connectome' identified here strongly supports an alternative model of the Wnt pathway as a complex context-dependent network. The network further suggests that mutational activation of highly connected Wnt signaling nodes predisposed cells to further context-dependent alterations in levels of TCF-dependent transcription that may be important during tumor progression and treatment.

Wnt5a-induced Wnt1-inducible secreted protein-1 suppresses vascular smooth muscle cell apoptosis induced by oxidative stress.

OBJECTIVE: Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the atherosclerotic plaque fibrous cap and is thereby associated with myocardial infarction. Wnt protein activation of ß-catenin regulates numerous genes that are associated with cell survival. We therefore investigated Wnt/ß-catenin survival signaling in VSMCs and assessed the presence of this pathway in human atherosclerotic plaques at various stages of the disease process. APPROACH AND RESULTS: Wnt5a induced ß-catenin/T-cell factor signaling and retarded oxidative stress (H2O2)-induced apoptosis in mouse aortic VSMCs. Quantification of mRNA levels revealed a >4-fold (P<0.05; n=9) increase in the expression of the Wnt/ß-catenin responsive gene, Wnt1-inducible secreted protein-1 (WISP-1), which was dependent on cAMP response element-binding protein and sustained in the presence of H2O2. Exogenous WISP-1 significantly reduced H2O2-induced apoptosis by 43% (P<0.05; n=3) and was shown using silencing small interfering RNA, to be important for Wnt5a-dependent survival responses to H2O2 (P<0.05; n=3). WISP-1 protein levels were significantly lower (≈50%) in unstable atherosclerosis compared with stable plaques (n=11 and n=14). CONCLUSIONS: These results indicate for the first time that Wnt5a induces ß-catenin survival signaling in VSMCs via WISP-1. The deficiency of the novel survival factor, WISP-1 in intimal VSMCs of unstable coronary plaques, suggests that there is altered Wnt/ß-catenin/ T-cell factor signaling with progressive atherosclerosis, and restoration of WISP-1 protein might be an effective stabilization factor for vulnerable atherosclerotic plaques.

ß-Catenin-regulated myeloid cell adhesion and migration determine wound healing.

A ß-catenin/T cell factor-dependent transcriptional program is critical during cutaneous wound repair for the regulation of scar size; however, the relative contribution of ß-catenin activity and function in specific cell types in the granulation tissue during the healing process is unknown. Here, cell lineage tracing revealed that cells in which ß-catenin is transcriptionally active express a gene profile that is characteristic of the myeloid lineage. Mice harboring a macrophage-specific deletion of the gene encoding ß-catenin exhibited insufficient skin wound healing due to macrophage-specific defects in migration, adhesion to fibroblasts, and ability to produce TGF-ß1. In irradiated mice, only macrophages expressing ß-catenin were able to rescue wound-healing deficiency. Evaluation of scar tissue collected from patients with hypertrophic and normal scars revealed a correlation between the number of macrophages within the wound, ß-catenin levels, and cellularity. Our data indicate that ß-catenin regulates myeloid cell motility and adhesion and that ß-catenin-mediated macrophage motility contributes to the number of mesenchymal cells and ultimate scar size following cutaneous injury.

Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF.

Active canonical Wnt signaling results in recruitment of ß-catenin to DNA by TCF/LEF family members, leading to transcriptional activation of TCF target genes. However, additional transcription factors have been suggested to recruit ß-catenin and tether it to DNA. Here, we describe the genome-wide pattern of ß-catenin DNA binding in murine intestinal epithelium, Wnt-responsive colorectal cancer (CRC) cells and HEK293 embryonic kidney cells. We identify two classes of ß-catenin binding sites. The first class represents the majority of the DNA-bound ß-catenin and co-localizes with TCF4, the prominent TCF/LEF family member in these cells. The second class consists of ß-catenin binding sites that co-localize with a minimal amount of TCF4. The latter consists of lower affinity ß-catenin binding events, does not drive transcription and often does not contain a consensus TCF binding motif. Surprisingly, a dominant-negative form of TCF4 abrogates the ß-catenin/DNA interaction of both classes of binding sites, implying that the second class comprises low affinity TCF-DNA complexes. Our results indicate that ß-catenin is tethered to chromatin overwhelmingly through the TCF/LEF transcription factors in these three systems.

Physiological role of ß-catenin/TCF signaling in neurons of the adult brain.

Wnt/ß-catenin pathway, the effectors of which are transcription factors of the LEF1/TCF family, is primarily associated with development. Strikingly, however, some of the genes of the pathway are schizophrenia susceptibility genes, and the proteins that are often mutated in neurodegenerative diseases have the ability to regulate ß-catenin levels. If impairment of this pathway indeed leads to these pathologies, then it likely plays a physiological role in the adult brain. This review provides an overview of the current knowledge on this subject. The involvement of ß-catenin and LEF1/TCF factors in adult neurogenesis, synaptic plasticity, and the function of thalamic neurons are discussed. The data are still very preliminary and often based on circumstantial or indirect evidence. Further research might help to understand the etiology of the aforementioned pathologies.

Visualization and exploration of Tcf/Lef function using a highly responsive Wnt/ß-catenin signaling-reporter transgenic zebrafish.

Evolutionarily conserved Tcf/Lef transcription factors (Lef1, Tcf7, Tcf7l1, and Tcf7l2) mediate gene expression regulated by Wnt/ß-catenin signaling, which has multiple roles in early embryogenesis, organogenesis, adult tissue homeostasis, and tissue regeneration. However, the spatiotemporal dynamics of Tcf/Lef activity during these events remain poorly understood. We generated stable transgenic zebrafish lines carrying a new Wnt/ß-catenin signaling reporter, Tcf/Lef-miniP:dGFP. The reporter revealed the transcriptional activities of four Tcf/Lef members controlled by Wnt/ß-catenin signaling, which were expressed in known Wnt/ß-catenin signaling-active sites during embryogenesis, organ development and growth, and tissue regeneration. We used the transgenic lines to demonstrate the contribution of Tcf/Lef-mediated Wnt/ß-catenin signaling to the development of the anterior lateral line, dorsal and secondary posterior lateral lines, and gill filaments. Thus, these reporter lines are highly useful tools for studying Tcf/Lef-mediated Wnt/ß-catenin signaling-dependent processes.

Axis formation in hydra.

A hydra has a simple structure consisting of a head, body column, and foot along a single axis called the oral-aboral axis. The tissue dynamics of a hydra consist of a steady state of production and loss of tissue involving the entire animal. Axis formation and its maintenance is controlled by the head organizer, which is located at the apex of the animal. The head organizer produces two signals, the head activator and head inhibitor, which are transmitted to, and are distributed in, descending gradients among the epithelial cells along the body column. The two gradients control axial patterning along the oral-aboral axis. In the context of the tissue dynamics of the adult hydra, these three elements controlling axis formation and axial patterning are in a steady state of production and loss. The canonical Wnt pathway plays a major role in setting up and maintaining the head organizer.

Inhibition of glycogen synthase kinase-3 increases the cytotoxicity of enzastaurin.

Cutaneous T-cell lymphomas (CTCL) represent a spectrum of several distinct non-Hodgkin's lymphomas that are characterized by an invasion of the skin by malignant, clonal lymphocytes. Our laboratory has previously demonstrated that the protein kinase C (PKC) ß inhibitor Enzastaurin increases apoptosis in malignant lymphocytes of CTCL. These results directly led to a clinical trial for Enzastaurin in CTCL in which it was well tolerated and showed modest activity. To ascertain a means of improving the efficacy of Enzastaurin, we investigated complementary signaling pathways and identified glycogen synthase kinase-3 (GSK3) as important in survival signaling in CTCL. Enzastaurin combined with GSK3 inhibitors demonstrated an enhancement of cytotoxicity. Treatment with a combination of Enzastaurin and the GSK3 inhibitor AR-A014418 resulted in upregulation of ß-catenin total protein and ß-catenin-mediated transcription. Inhibition of ß-catenin-mediated transcription or small hairpin RNA (shRNA) knockdown of ß-catenin decreased the cytotoxic effects of Enzastaurin plus AR-A014418. In addition, treatment with Enzastaurin and AR-A014418 decreased the mRNA levels and surface expression of CD44. shRNA knockdown of ß-catenin also restored CD44 surface expression. Our observations provide a rationale for the combined targeting of PKC and GSK3 signaling pathways in CTCL to enhance the therapeutic outcome.

[Cytokines in bone diseases. Wnt signaling and osteoporosis-pseudoglioma syndrome].

Wnt signaling system plays essential roles in development, cancer and bone metabolism. Canonical wnt signaling, which involves wnt ligands, receptor named frizzled and co-receptors LRP5/6, beta-catenin and transcription factors named LEF/TCF is well characterized and its defect causes bone abnormalities. The loss-of-function type of the LRP5 gene mutation is responsible for osteoporosis-pseudoglioma syndrome. In addition, the LRP6 gene mutation leads to osteoporosis and metabolic syndrome. Thus, wnt signaling system is one of determinant factors for bone mineral density.

Resveratrol modulates angiogenesis through the GSK3ß/ß-catenin/TCF-dependent pathway in human endothelial cells.

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis due to its potent and specific ability to promote the proliferation and migration of endothelial cells. Resveratrol has been shown to have many health-benefiting effects, including the protection of cardiovascular system. In this study we examined the effect of resveratrol on angiogenesis in human umbilical vein endothelial cells (HUVECs). We observed that resveratrol was able to modulate the expression of VEGF and the formation of vascular network in a biphasic pattern. While resveratrol at low concentrations, from 1 to 10µM, up-regulated the expression of VEGF and promoted angiogenesis, it had opposite effect at high concentrations (20µM and higher). The biphasic effect of resveratrol on angiogenesis was confirmed by chick chorioallantoic membrane assay. Up-regulation of VEGF expression depended on the nuclear accumulation and transcriptional activity of ß-catenin. Correspondingly, GSK3ß, a negative regulator of ß-catenin, turned into a less active state (phosphorylated at Ser9) in cells exposed to 5µM of resveratrol, but became more active at 20µM. We demonstrated that both Akt and ERK signaling pathways, which are known to be critical for angiogenesis, became activated in response to 5µM of resveratrol and functioned to inactivate GSK3ß. Our findings may have implications in the management of cardiovascular diseases and other conditions such as cancer by the use of resveratrol.

Periplocin from Cortex periplocae inhibits cell growth and down-regulates survivin and c-myc expression in colon cancer in vitro and in vivo via beta-catenin/TCF signaling.

Cancer of the colon and rectum is the third most commonly diagnosed cancer and accounts for approximately 10% of all cancer-related deaths. Although surgical resection or radiotherapy are potentially curative for localized disease, advanced colon cancer is currently associated with poor prognosis. Therefore, the development of a new and effective chemotherapeutic agent is required to target critical pathways to induce responsiveness of colon cancer cells to death signals. Dysregulation of the beta-catenin/TCF pathway plays a central role in early activities of colorectal carcinogenesis. In this study, human colon cancer SW480 cells were used to investigate the effect of CPP (periplocin from Cortex periplocae) on the modulation of the beta-catenin/TCF signaling pathway. Our research results showed that CPP caused a dose- and time-dependent inhibition of cell growth as assessed by MTT assay and an induction in apoptosis as measured by flow cytometry and transmission electron microscopy. Furthermore, the CPP- treated cells were characterized by a decreased expression of beta-catenin protein in the total cell lysates and cytosolic and nuclear extracts. This expression alleviates the binding activity of T-cell factor (Tcf) complexes to its specific DNA-binding sites. Thus, the protein expression of the downstream elements survivin and c-myc was down-regulated. To determine the precise inhibitory mechanisms involved, further in-depth in vivo studies of CPP are warranted. In conclusion, our data suggest that CPP wields a multi-prong strategy to target the beta-catenin/Tcf signaling pathway, leading to the induction of apoptosis and inhibition of growth of colon cancer cells in vitro and in vivo. Therefore, CPP may become a potential agent against colon cancer.

The role of Wnt/beta-catenin signaling in renal carcinogenesis: lessons from cadmium toxicity studies.

Wnt/beta-catenin signaling plays a crucial role during embryogenesis. However, this signaling pathway also plays a role in normal adult tissues and in carcinogenesis, including cadmium (Cd2+) induced nephrocarcinogenesis, which is the topic of this review. Wnt/beta-catenin signaling is tightly regulated in mature epithelia to balance cell proliferation, differentiation and death. This is accomplished by modulating phosphorylation of the multifunctional protein beta-catenin which in turn determines its preference for a particular fate, i.e. cell-cell adhesion by binding to E-cadherin, proteasomal degradation, or co-activation of the transcription factor Tcf/Lef. The pivotal role of beta-catenin is not limited to Wnt signaling, but can be challenged by other transcription factors under stress conditions (e.g. FOXO, HIF-1alpha, NF-kappaB, c-jun), where beta-catenin acts as a molecular switch in response to the cellular redox status. Aberrant Wnt/beta-catenin signaling can contribute to carcinogenesis of intestinal, lung or kidney epithelia, either by mutations of its signaling components and/or disruption of linked signaling networks. The nephrotoxic metal Cd2+ causes renal cancer in humans. Because it is not genotoxic Cd2+ is thought to induce mutations and carcinomas indirectly: Possible mechanisms include oxidative stress, inhibition of DNA repair, aberrant gene expression, deregulation of cell proliferation, resistance to apoptosis, and/or disruption of cell adhesion. Wnt signaling may contribute to Cd2+ carcinogenesis because Cd2+ disrupts the junctional E-cadherin/beta-catenin complex, resulting in excessive nuclear translocation of beta-catenin and activation of Tcf4. Up-regulation of target genes of the beta-catenin/Tcf4 complex, such as c-myc, cyclin D1 and the multidrug transporter P-glycoprotein (MDR1/ABCB1), leads to increased proliferation, evasion of apoptosis, adaptation to Cd2+ toxicity and thereby promotes the selection of mutated and pre-neoplastic cells.

Cutting edge: granulocyte-macrophage colony-stimulating factor is the major CD8+ T cell-derived licensing factor for dendritic cell activation.

During priming, CD8(+) T lymphocytes can induce robust maturation of dendritic cells (DCs) in a CD40-independent manner by secreting licensing factor(s). In this study, we isolate this so-far elusive licensing factor and identify it, surprisingly, as GM-CSF. This provides a new face for an old factor with a well-known supporting role in DC development and recruitment. Signaling through the GM-CSFR in ex vivo-purified DCs upregulated the expression of costimulatory molecules more efficiently than did any tested TLR agonist and provided a positive feedback loop in the stimulation of CD8(+) T cell proliferation. Combined with a variety of microbial stimuli, GM-CSF supports the formation of potent "effector" DCs capable of secreting a variety of proinflammatory cytokines that guide the differentiation of T cells during the immune response.