Prodigiosin, a natural red pigment produced by numerous bacterial species, has exhibited promising anticancer activity; however, the molecular mechanisms of action of prodigiosin on malignant cells remain unclear. Aberrant activation of the Wnt/β-catenin signaling cascade is associated with numerous human cancers. In this study, we identified prodigiosin as a potent inhibitor of the Wnt/ β-catenin pathway. Prodigiosin blocked Wnt/β-catenin signaling by targeting multiple sites of this pathway, including the lowdensity lipoprotein-receptor-related protein (LRP) 6, Dishevelled (DVL), and glycogen synthase kinase-3β (GSK3β). In breast cancer MDA-MB-231 and MDA-MB-468 cells, nanomolar concentrations of prodigiosin decreased phosphorylation of LRP6, DVL2, and GSK3β and suppressed β-catenin-stimulated Wnt target gene expression, including expression of cyclin D1. In MDA-MB-231 breast cancer xenografts and MMTV-Wnt1 transgenic mice, administration of prodigiosin slowed tumor progression and reduced the expression of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, active β-catenin, and cyclin D1. Through its ability to inhibit Wnt/β-catenin signaling and reduce cyclin D1 levels, prodigiosin could have therapeutic activity in advanced breast cancers.prodigiosin | Wnt/beta-catenin signaling | breast cancer | LRP6 | Dishevelled (DVL)

BackgroundGigantol is a bibenzyl compound derived from several medicinal orchids. This biologically active compound has been shown to have promising therapeutic potential against cancer cells, but its mechanism of action remains unclear.MethodsThe inhibitory effect of gigantol on Wnt/β-catenin signaling was evaluated with the SuperTOPFlash reporter system. The levels of phosphorylated low-density lipoprotein receptor related protein 6 (LRP6), total LRP6 and cytosolic β-catenin were determined by Western blot analysis. The expression of Wnt target genes was analyzed using real-time PCR. Cell viability was measured with a MTT assay. The effect of gigantol on cell migration was examined using scratch wound-healing and transwell migration assays.ResultsGigantol decreased the level of phosphorylated LRP6 and cytosolic β-catenin in HEK293 cells. In breast cancer MDA-MB-231 and MDA-MB-468 cells, treatment with gigantol reduced the level of phosphorylated LRP6, total LRP6 and cytosolic β-catenin in a dose-dependent manner, resulting in a decrease in the expression of Wnt target genes Axin2 and Survivin. We further demonstrated that gigantol suppressed the viability and migratory capacity of breast cancer cells.ConclusionGigantol is a novel inhibitor of the Wnt/β-catenin pathway. It inhibits Wnt/β-catenin signaling through downregulation of phosphorylated LRP6 and cytosolic β-catenin in breast cancer cells.

The incidence of thyroid cancer has increased significantly in the last decade, and the most frequent type of this cancer is papillary thyroid carcinoma. MicroRNAs have been demonstrated to be abnormally expressed in tumors and associated with the development of the tumors. Our aim was to analyze the role and molecular mechanisms of tumor suppressor let-7b in the papillary thyroid carcinoma. Expression of let-7b and high-mobility group A2 in papillary thyroid carcinoma tissues and cell lines was assessed using quantitative reverse transcription polymerase chain reaction and western blot analysis. To explore the role of let-7b or high-mobility group A2 in the BCPAP and TPC-1 cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell methods were used. Let-7b expression was significantly downregulated while expression of high-mobility group A2 was upregulated dramatically in papillary thyroid carcinoma tissues and cells compared with that in normal thyroid tissues and cells. In addition, overexpression of let-7b or knockdown of high-mobility group A2 inhibited cell migration and invasion compared with that of control. Besides, high-mobility group A2 was negatively regulated by let-7b in BCPAP cells. Moreover, high-mobility group A2 reintroduction reversed the anti-proliferation, anti-migration, and anti-invasion roles of let-7b. Let-7b might function as a tumor suppressor in papillary thyroid carcinoma by suppressing the expression of high-mobility group A2, and therefore might provide a promising therapeutic target for patients with papillary thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) has a poor prognosis due to its resistance to all conventional treatments. The long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) serves a critical role in cancer chemoresistance; however, whether NEAT1 is associated with chemoresistance of ATC remains unclear. In the present study, reverse transcription-quantitative PCR assays were performed to detect the expression levels of NEAT1, microRNA (miR)-9-5p and sperm-associated antigen 9 (SPAG9). Western blot analysis was conducted to assess the protein expression levels of p62, microtubule-associated proteins 1A/1B light chain 3B and SPAG9. Cell proliferation was detected using the Cell Counting kit-8 assay, and cell apoptosis was determined by flow cytometry. Dual-luciferase reporter and RNA immunoprecipitation assays were performed to verify the interaction between NEAT1 and miR-9-5p, or miR-9-5p and SPAG9. Furthermore, an animal model was used to investigate the regulatory effects of NEAT1 on cisplatin (DDP)-resistance in tumors in vivo. The present results demonstrated that NEAT1 was upregulated in ATC tissues and cell lines, and NEAT1 silencing resulted in decreased DDP-resistance of ATC cells. In addition, NEAT1 suppressed miR-9-5p expression by binding to miR-9-5p and SPAG9 was a direct target of miR-9-5p. miR-9-5p overexpression sensitized ATC cells to DDP. Notably, NEAT1 silencing exerted its inhibitory effect on DDP-resistance of ATC via the miR-9-5p/SPAG9 axis in vitro and in vivo. In conclusion, the present study demonstrated that NEAT1 silencing ameliorated DDP-resistance of ATC, at least in part by reducing miR-9-5p sponging and regulating SPAG5 expression; therefore, NEAT1 may be considered a potential therapeutic target of ATC.

Emetine, an amoebicidal drug, exerts potent anticancer activity against various solid tumors, however, the underlying molecular mechanism remains unclear. In the present study, the effects of emetine were investigated on various proteins involved in the Wnt/β-catenin signaling pathway, which has been linked to various human cancers. It was revealed that emetine blocked Wnt/β-catenin signaling by targeting components of this pathway, including the low-density lipoprotein-receptor-related protein 6 (LRP6) and disheveled (DVL). Moreover, nanomolar concentrations of emetine decreased phosphorylation of these proteins and suppressed the expression of Wnt target genes, including fibronectin, frizzled-7 (Fzd7), c-Myc, Nanog and CD133 in MDA-MB-231 and MDA-MB-468 breast cancer cells. Additionally, emetine treatment induced apoptosis and suppressed the viability, migration, invasion, and sphere formation of breast cancer cells. Collectively the present results indicated that emetine antagonizes Wnt/β-catenin signaling, providing insight into the molecular mechanism underlying the anticancer activity of emetine.

SignificanceThe phorbol ester 12-O-tetra-decanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in two-stage mouse skin carcinogenesis, but the exact mechanism by which TPA promotes tumorigenesis remains elusive. This study discovered that TPA could stabilize CK1ε, enhance its kinase activity, and induce phosphorylation of LRP6, resulting in the formation of CK1ε–LRP6–axin1 complex, which may bypass the requirement of Wnt–Fzd–Dvl complex. TPA also increased the interaction between β-catenin and TCF4E in a CK1ε/δ-dependent way, and finally led to activation of the Wnt/β-catenin pathway. Our findings reveal a pathway by which TPA activates the Wnt/β-catenin signaling cascade. This pathway may represent a common mechanism for the tumor-promoting activity of some carcinogenic agents.

Background and Purpose Salinomycin is a well‐known inhibitor of human cancer stem cells ( CSCs ). However, the molecular mechanism(s) by which salinomycin targets colorectal CSCs is poorly understood. Here, we have investigated underlying antitumour mechanisms of salinomycin in colorectal cancer cells and three tumour models. Experimental Approach The inhibitory effect of salinomycin on the Wnt/β‐catenin pathway was analysed with the SuperTopFlash reporter system. The mRNA expression of Wnt target genes was evaluated with real‐time PCR. Effects of salinomycin on β‐catenin/TCF4E interaction were examined using co‐immunoprecipitation and an in vitro GST pull‐down assay. Cell proliferation was determined by BrdU incorporation and soft agar colony formation assay. The stemness of the cells was assessed by sphere formation assay. Antitumour effects of salinomycin on colorectal cancers was evaluated with colorectal CSC xenografts, APC min/+ transgenic mice, and patient‐derived colorectal tumour xenografts. Key Results Salinomycin blocked β‐catenin/TCF4E complex formation in colorectal cancer cells and in an in vitro GST pull‐down assay, thus decreasing expression of Wnt target genes. Salinomycin also suppressed the transcriptional activity mediated by β‐catenin/LEF1 or β‐catenin/TCF4E complex and exhibited an inhibitory effect on the sphere formation, proliferation, and anchorage‐independent growth of colorectal cancer cells. In colorectal tumour xenografts and APC min/+ transgenic mice, administration of salinomycin significantly reduced tumour growth and the expression of CSC‐related Wnt target genes including LGR5. Conclusions and Implications Our study suggested that salinomycin could suppress the growth of colorectal cancer by disrupting the β‐catenin/TCF complex and thus may be a promising agent for colorectal cancer treatment.

Background: Amino-terminal enhancer of split (AES) has been identified as a tumor and metastasis suppressor in some cancers including colorectal cancer (CRC), but very little is known about the regulation of AES expression. Methods: Bioinformatics analysis was used to investigate the expression patterns of AES, CK1δ and CK1ε. The co-immunoprecipitation, GST pull-down, Western Blot, real-time PCR and immunohistochemistry were performed to study the mechanism underlying the regulation of AES expression by CK1δ/ε. The biological function was assessed by in vitro colony formation, transwell, sphere formation, tumor organoids, in vivo tumor metastasis model and patient-derived colorectal tumor xenografts (PDTX) model. Results: A strong inverse relationship was observed between the expression of AES and the expression of CK1δ/ε. Mechanically, AES could interact with CK1δ/ε and SKP2 using its Q domain. SKP2 mediated the ubiquitination and degradation of AES in a CK1δ/ε-dependent manner. CK1δ/ε phosphorylated AES at Ser121 and accelerated the SKP2-mediated ubiquitination and degradation of AES. In colon cancer cells, CK1δ/ε antagonized the effect of wild-type AES but not that of its mutant (S121A) on Wnt and Notch signaling, leading to an increase in the expression of Wnt target genes and Notch target genes. By downregulating the expression of AES, CK1δ/ε enhanced anchorage-independent growth, migration, invasion and sphere formation in colon cancer cells. CK1δ/ε also promoted the growth of APC min/+ colorectal tumor organoids and liver metastasis in colon cancer mouse models through the regulation of AES degradation. Furthermore, CK1 inhibitor SR3029 treatment suppressed tumor growth via stabilizing AES in APC min/+ colorectal tumor organoids and patient-derived colorectal tumor xenografts (PDTX). Conclusions: Our results revealed that the CK1δ/ε-AES axis is important for CRC tumorigenesis and metastasis, and targeted inhibition of this axis may be a potential therapeutic strategy for CRC.