Protocol for rapid assessment of the efficacy of novel Wnt inhibitors using zebrafish models.

Dysregulation of Wnt signaling is a hallmark of many cancers, and the development of effective, non-toxic small-molecule Wnt inhibitors is desirable. Off-target toxicities of new compounds are typically tested in mouse models, which is both costly and time consuming. Here, we present a rapid and inexpensive protocol to determine the in vivo toxicity and efficacy of novel Wnt inhibitors in zebrafish using a combination of a fluorescence reporter assay as well as eye rescue and fin regeneration assays. These experiments are completed within 1 week to rapidly narrow drug candidates before moving to more expensive pre-clinical testing. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020).

A RNA-seq approach for exploring the protective effect of ginkgolide B on glutamate-induced astrocytes injury.

ETHNOPHARMACOLOGICAL RELEVANCE: There is substantial experimental evidence to support the view that Ginkgo biloba L. (Ginkgoaceae), a traditional Chinese medicine known to treating stroke, has a protective effect on the central nervous system and significantly improves the cognitive dysfunction caused by disease, including alzheimer disease (AD), vascular dementia, and diabetic encephalopathy. Although a number of studies have reported that ginkgolide B (GB), a diterpenoid lactone compound extracted from Ginkgo biloba leaves, has neuroprotective effects, very little research has been performed to explore its potential pharmacological mechanism on astrocytes under abnormal glutamate (Glu) metabolism in the pathological environment of AD. AIM OF THE STUDY: We investigated the protective effect and mechanism of GB on Glu-induced astrocytes injury. METHODS: Astrocytes were randomly divided into the control group, Glu group, GB group, and GB + IWP-4 group.The CCK-8 assay was used to determine relative cell viability in vitro. Furthermore, RNA sequencing (RNA-seq) was performed to assess the preventive effects of GB in the Glu-induced astrocyte model and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to validate the possible molecular mechanisms. The effects of GB on the Glu transporter and Glu-induced apoptosis of astrocytes were studied by RT-qPCR and western blot. RESULTS: GB attenuated Glu-induced apoptosis in a concentration-dependent manner, while the Wnt inhibitor IWP-4 reversed the protective effect of GB on astrocytes. The RNA-seq results revealed 4,032 differential gene expression profiles; 3,491 genes were up-regulated, and 543 genes were down-regulated in the GB group compared with the Glu group. Differentially expressed genes involved in a variety of signaling pathways, including the Hippo and Wnt pathways have been associated with the development and progression of AD. RT-qPCR was used to validate 14 key genes, and the results were consistent with the RNA-seq data. IWP-4 inhibited the regulation of GB, disturbed the apoptosis protective effect on astrocytes, and promoted Glu transporter gene and protein expression caused by Glu. CONCLUSION: Our findings demonstrate that GB may play a protective role in Glu-induced astrocyte injury by regulating the Hippo and Wnt pathways. GB was closely associated with the Wnt pathway by promoting expression of the Glu transporter and inhibiting Glu-induced injury in astrocytes.

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells.

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-ß (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.

New insights into the core Hippo signaling and biological macromolecules interactions in the biology of solid tumors.

As an evolutionarily conserved pathway, Hippo signaling pathway impacts different pathology and physiology processes such as wound healing, tissue repair/size and regeneration. When some components of Hippo signaling dysregulated, it affects cancer cells proliferation. Moreover, the relation Hippo pathway with other signaling including Wnt, TGFß, Notch, and EGFR signaling leaves effect on the proliferation of cancer cells. Utilizing a number of therapeutic approaches, such as siRNAs and long noncoding RNA (lncRNA) to prevent cancer cells through the targeting of Hippo pathways, can provide new insights into cancer target therapy. The purpose of present review, first of all, is to demonstrate the importance of Hippo signaling and its relation with other signaling pathways in cancer. It also tries to demonstrate targeting Hippo signaling progress in cancer therapy.

High-Throughput Drug Screening Identifies a Potent Wnt Inhibitor that Promotes Airway Basal Stem Cell Homeostasis.

Mechanisms underpinning airway epithelial homeostatic maintenance and ways to prevent its dysregulation remain elusive. Herein, we identify that ß-catenin phosphorylated at Y489 (p-ß-cateninY489) emerges during human squamous lung cancer progression. This led us to develop a model of airway basal stem cell (ABSC) hyperproliferation by driving Wnt/ß-catenin signaling, resulting in a morphology that resembles premalignant lesions and loss of ciliated cell differentiation. To identify small molecules that could reverse this process, we performed a high-throughput drug screen for inhibitors of Wnt/ß-catenin signaling. Our studies unveil Wnt inhibitor compound 1 (WIC1), which suppresses T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activity, reduces ABSC proliferation, induces ciliated cell differentiation, and decreases nuclear p-ß-cateninY489. Collectively, our work elucidates a dysregulated Wnt/p-ß-cateninY489 axis in lung premalignancy that can be modeled in vitro and identifies a Wnt/ß-catenin inhibitor that promotes airway homeostasis. WIC1 may therefore serve as a tool compound in regenerative medicine studies with implications for restoring normal airway homeostasis after injury.

Wnt Signaling and Drug Resistance in Cancer.

Wnts are secreted proteins that bind to cell surface receptors to activate downstream signaling cascades. Normal Wnt signaling plays key roles in embryonic development and adult tissue homeostasis. The secretion of Wnt ligands, the turnover of Wnt receptors, and the signaling transduction are tightly regulated and fine-tuned to keep the signaling output "just right." Hyperactivated Wnt signaling due to recurrent genetic alterations drives several human cancers. Elevated Wnt signaling also confers resistance to multiple conventional and targeted cancer therapies through diverse mechanisms including maintaining the cancer stem cell population, enhancing DNA damage repair, facilitating transcriptional plasticity, and promoting immune evasion. Different classes of Wnt signaling inhibitors targeting key nodes of the pathway have been developed and show efficacy in treating Wnt-driven cancers and subverting Wnt-mediated therapy resistance in preclinical studies. Several of these inhibitors have advanced to clinical trials, both singly and in combination with other existing US Food and Drug Administration-approved anti-cancer modalities. In the near future, pharmacological inhibition of Wnt signaling may be a real choice for patients with cancer. SIGNIFICANCE STATEMENT: The latest insights in Wnt signaling, ranging from basic biology to therapeutic implications in cancer, are reviewed. Recent studies extend understanding of this ancient signaling pathway and describe the development and improvement of anti-Wnt therapeutic modalities for cancer.

Blocking Wnt as a therapeutic target in mice model of skin cancer.

Wnt pathway plays an important role in controlling metabolism in cancer cells. It acts as positive modulator for both cell inflammation, through activation of NFκB, and fibrosis, through activation of TGF-ß. Therefore, the aim of this study is to investigate the therapeutic effects of blocking Wnt pathway by IWP12 on skin cancer by studying its effects on skin cancer-induced inflammation and fibrosis in a mice model of skin cancer. Skin cancer was induced by application of 7,12-dimethylbenz[a]anthracene (DMBA) and croton oil on the dorsal skin of mice. Dorsal skin was removed for estimation of gene and protein expression of Wnt, ß-catenin, SMAD, TGF-ß, NFκB, TNF-α, IL-4 and IL-10. Part of the skin is stained with hematoxylin/eosin for assessment of cell structure. Treatment of mice with IWP12 completely blocked Wnt in skin cancer mice without affecting the control mice. Skin of tumorigenic mice showed marked skin hyperkeratosis, parakeratosis, acanthosis and dysplasia. Treatment with IWP12 markedly attenuated epidermal atypia and hyperplasia. In addition, IWP12 reduced expression of ß-catenin, SMAD, TGF-ß, NFκB and TNF-α associated with increase in the expression of IL-4 and IL-10. In conclusion, blocking Wnt production ameliorated skin cancer via blocking pro-inflammatory cytokines and enhancing the anti-inflammatory cytokines. Moreover, blocking Wnt attenuated skin cancer-induced activation of fibrosis pathway.

Targeting Cancer Stem Cells for Chemoprevention of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is one of the deadliest cancers worldwide and the fourth leading cause of cancer-related deaths in United States. Regardless of the advances in molecular pathogenesis and consequential efforts to suppress the disease, this cancer remains a major health problem in United States. By 2030, the projection is that pancreatic cancer will be climb up to be the second leading cause of cancer-related deaths in the United States. Pancreatic cancer is a rapidly invasive and highly metastatic cancer, and does not respond to standard therapies. Emerging evidence supports that the presence of a unique population of cells called cancer stem cells (CSCs) as potential cancer inducing cells and efforts are underway to develop therapeutic strategies targeting these cells. CSCs are rare quiescent cells, and with the capacity to self-renew through asymmetric/symmetric cell division, as well as differentiate into various lineages of cells in the cancer. Studies have been shown that CSCs are highly resistant to standard therapy and also responsible for drug resistance, cancer recurrence and metastasis. To overcome this problem, we need novel preventive agents that target these CSCs. Natural compounds or phytochemicals have ability to target these CSCs and their signaling pathways. Therefore, in the present review article, we summarize our current understanding of pancreatic CSCs and their signaling pathways, and the phytochemicals that target these cells including curcumin, resveratrol, tea polyphenol EGCG (epigallocatechin- 3-gallate), crocetinic acid, sulforaphane, genistein, indole-3-carbinol, vitamin E δ- tocotrienol, Plumbagin, quercetin, triptolide, Licofelene and Quinomycin. These natural compounds or phytochemicals, which inhibit cancer stem cells may prove to be promising agents for the prevention and treatment of pancreatic cancers.

High-throughput targeted screening in triple-negative breast cancer cells identifies Wnt-inhibiting activities in Pacific brittle stars.

Pro-proliferative oncogenic signaling is one of the hallmarks of cancer. Specific targeting of such signaling pathways is one of the main approaches to modern anti-cancer drug discovery, as opposed to more traditional search for general cytotoxic agents. Natural products, especially from marine sources, represent a largely untapped source of chemical diversity, which so far have mostly been screened for cytotoxicity. Here we present a pioneering pipeline of high-throughput screening of marine-based activities targeted against the Wnt signaling pathway, which is one of the key factors in oncogenic transformation, growth and metastasis in different cancers, including the devastating triple-negative breast cancer (TNBC) currently lacking any targeted therapies. This pipeline consisted of collection and characterization of numerous invertebrates during the SokhoBio expedition to the Kuril Basin in North Pacific, preparation of extracts from these specimen, and their screening in dedicated assays monitoring Wnt signaling in TNBC cells. This approach yielded a number of promising hits, including highly specific anti-Wnt activities targeting multiple levels within the Wnt pathway from Ophiura irrorata and other Pacific brittle stars.

Polyphyllin I suppresses human osteosarcoma growth by inactivation of Wnt/ß-catenin pathway in vitro and in vivo.

Osteosarcoma is the most common primary bone cancer in children and adolescents. In spite of aggressive treatment, osteosarcoma has a high mortality rate with minimal improvements in survival over past few decades. Polyphyllin I (PPI), a component in the traditional Chinese medicinal herb Paris polyphylla Smith, has been shown to have anti-tumor properties. However, its mechanism as an anti-osteosarcoma agent has not been well elucidated. In this study, we found that PPI suppressed osteosarcoma cell viability, arrested cell cycle in G2/M phase, induced apoptosis and inhibited invasion and migration of osteosarcoma cells. Moreover, PPI significantly suppressed intratibial primary tumor growth in xenograft orthotopic mouse model without any obvious side effects. These therapeutic efficacies were associated with inactivation of Wnt/ß-catenin pathway, as PPI treatment decreased the amount of p-GSK-3ß, leading to down-regulated levels of active ß-catenin. PPI induced inhibition of osteosarcoma cell viability was abolished upon addition of GSK-3ß specific inhibitor, CHIR99021, while PPI induced inhibition of osteosarcoma cell viability and migration were potentiated by ß-catenin silencing. These findings suggested that, in vitro and in vivo, PPI treatment inhibited osteosarcoma, at least in part, via the inactivation of Wnt/ß-catenin pathway. Thus, PPI could serve a novel therapeutic option for osteosarcoma patients.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of ß-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by ß-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when ß-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/ß-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/ß-catenin signaling pathway.

Therapeutic Potential of Targeting Wnt/ß-Catenin Pathway in Treatment of Colorectal Cancer: Rational and Progress.

Wnt/ß-catenin pathway is one of the main/frequent dysregulated pathways in several tumor types, including colon cancer. Aberrant activation of this pathway is associated with cell proliferation, invasive behaviors, and cell resistance, suggesting its potential value as a therapeutic target in treatment of CRC. Several agents have been developed for targeting of this pathway (e.g, natural agents: curcumin, 3,3-diindolylmethane, phytoestrogen; Synthetic/small Wnt inhibitors: Rofecoxib; PRI-724, CWP232291; and monoclonal antibody against frizzled receptors, Vanituctumab). This review summarizes the current knowledge about the therapeutic potential of targeting Wnt pathway with particular emphasis on preclinical/clinical studies in treatment of colorectal cancer. J. Cell. Biochem. 118: 1979-1983, 2017. © 2017 Wiley Periodicals, Inc.

Direct inhibition of osteoblastic Wnt pathway by fibroblast growth factor 23 contributes to bone loss in chronic kidney disease.

Bone loss and increased fractures are common complications in chronic kidney disease. Because Wnt pathway activation is essential for normal bone mineralization, we assessed whether Wnt inhibition contributes to high-phosphorus-induced mineralization defects in uremic rats. By week 20 after 7/8 nephrectomy, rats fed a high-phosphorus diet had the expected high serum creatinine, phosphorus, parathyroid hormone, and fibroblast growth factor 23 (FGF23) levels and low serum calcium. There was a 15% reduction in tibial mineral density and a doubling of bone cortical porosity compared to uremic rats fed a normal-phosphorus diet. The decreases in tibial mineral density were preceded by time-dependent increments in gene expression of bone formation (Osteocalcin and Runx2) and resorption (Cathepsin K) markers, which paralleled elevations in gene expression of the Wnt inhibitors Sfrp1 and Dkk1 in bone. Similar elevations of Wnt inhibitors plus an increased phospho-ß-catenin/ß-catenin ratio occurred upon exposure of the osteoblast cell line UMR106-01 either to uremic serum or to the combination of parathyroid hormone, FGF23, and soluble Klotho, at levels present in uremic serum. Strikingly, while osteoblast exposure to parathyroid hormone suppressed the expression of Wnt inhibitors, FGF23 directly inhibited the osteoblastic Wnt pathway through a soluble Klotho/MAPK-mediated process that required Dkk1 induction. Thus, the induction of Dkk1 by FGF23/soluble Klotho in osteoblasts inactivates Wnt/ß-catenin signaling. This provides a novel autocrine/paracrine mechanism for the adverse impact of high FGF23 levels on bone in chronic kidney disease.

Huaier aqueous extract inhibits stem-like characteristics of MCF7 breast cancer cells via inactivation of hedgehog pathway.

The theory of targeting cancer stem-like cells (CSCs) provides novel strategy for cancer treatment. In the present study, we examined the inhibitory effect of Huaier aqueous extract on eradicating breast cancer stem cells and explored the underlying mechanisms. Our data demonstrated that various concentrations of Huaier extract significantly decreased the viabilities, numbers, and sizes of mammospheres. After incubation with Huaier extract for 24 h, the clonogenicity of MCF7 cell line was obviously impaired, along with less holoclones. In addition, Huaier extract reduced the number of cells expressing CD44+/CD24- and decreased the level of stem cell markers (OCT-4, NESTIN, and NANOG). The hedgehog (Hh), notch, and Wnt/ß-catenin pathways were essential stem cell signaling pathways involved in regulating CSC renewal and maintenance. We reported that the inhibitory effect of Huaier extract was partly depended on the inactivation of Hh pathway. These findings provided experimental evidence that Huaier extract was a promising therapeutic drug for eliminating the breast cancer stem cells.

Docosahexaenoic acid inhibited the Wnt/ß-catenin pathway and suppressed breast cancer cells in vitro and in vivo.

N-3 fatty acids (FAs) are essential FAs necessary for human health and are known to possess anticancer properties. However, the relationship between n-3 FAs and ß-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, 4T1 mouse breast cancer cells were exposed to a representative n-3 FA, docosahexaenoic acid (DHA), to investigate the relationship between n-3 FAs and the Wnt/ß-catenin signaling pathway in vivo and in vitro. In vitro studies showed that DHA strongly inhibited cell growth, and induced G1 cell cycle arrest both in 4T1 mouse breast cells and MCF-7 human breast cells. DHA reduced ß-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity in 4T1 mouse breast cells. In addition, DHA down-regulated the expression of downstream target genes such as c-myc and cyclinD1. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. We found that feeding mouse the 5% fish oil-supplemented diet for 30 days significantly reduced the growth of 4T1 mouse breast cancer in vivo through inhibition of cancer cell proliferation as well as induction of apoptosis. Feeding animals a 5% fish oil diet significantly induced down-regulation of ß-catenin in tumor tissues with a notable increase in apoptosis. In addition, fish oil-supplemented diet decreased lung metastases of breast cancer. These observations suggested that DHA exerted its anticancer activity through down-regulation of Wnt/ß-catenin signaling. Thus, our data call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer.

Alternative signaling pathways as potential therapeutic targets for overcoming EGFR and c-Met inhibitor resistance in non-small cell lung cancer.

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC50 concentrations of resistant lines exhibited a 4-5 and 11-22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2-4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.

Discovery of inhibitors of the Wnt and Hedgehog signaling pathways through the catalytic enantioselective synthesis of an iridoid-inspired compound collection.

Cousins you can count on: An iridoid-inspired compound collection was synthesized efficiently by the resolution of cyclic enones in an asymmetric cycloaddition with azomethine ylides. The collection contained novel potent inhibitors of the Wnt and Hedgehog signaling pathways.

[Current status of molecular targeted therapies in hepatocellular carcinoma].

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in Korea. Curative treatment is only possible when the disease is diagnosed at the early stage. The prognosis of patients with HCC is even dismal in advanced stages. No systemic cytotoxic chemotherapy has proven to be beneficial in overall survival. Recently, the understanding of the molecular pathogenesis led to the development of new therapies. With the evidence of dysregulation of critical genes associated with cellular proliferation, growth factor signaling, cell cycling, apoptosis, and angiogenesis in HCC, a number of molecular target agents are under clinical trials. Sorafenib is the first systemic anticancer drug which has proven to gain survival benefit in the global as well as Asia-Pacific trials. However, the survival gain is still modest, and further efforts to improve outcomes in patients with HCC are necessary by developing novel drugs or combining other forms of therapies. This article will review signaling pathways in HCC and introduce molecular target agents under investigation currently.

Canonical Wnt signaling is required for Panax notoginseng saponin-mediated attenuation of the RANKL/OPG ratio in bone marrow stromal cells during osteogenic differentiation.

Panax notoginseng saponins (PNS) are known to regulate the osteogenic differentiation of bone marrow stromal cells (BMSCs). In the present study, we investigated whether PNS could promote the osteogenic differentiation of BMSCs through modulating the Wnt/ß-catenin signaling pathways, which are implicated in BMSCs osteogenesis. We found that PNS enhanced the mRNA expression of OPG, ß-catenin, and cyclin D1 while decreased the mRNA expression of RANKL and PPARγ2. The actions of PNS on BMSCs were reversed (or partially) by DKK-1, a classical inhibitor of Wnt/ß-catenin signaling. These results suggest that PNS stimulating bone formation by promoting the proliferation and osteogenic differentiation of BMSCs, and could also protect the skeletal system by decreasing bone resorption through reduction of RANKL/OPG expression via Wnt/ß-catenin signaling pathways.