Inhibition of the PI3K-AKT-mTOR pathway suppresses the adipocyte-mediated proliferation and migration of breast cancer cells.

Objective: Although it is well known that adipocyte significantly affects breast cancer progression, its mechanism has not been fully understood. Here, we analyzed the effect of adipocytes on breast cancer progression including cell proliferation and migration. Materials and Methods: We treated the conditioned media obtained from mouse 3T3-L1-derived or human adipose tissue-derived mesenchymal stem cells (hAMSC)-derived adipocytes to breast cancer cells, MCF-7 and MDA-MB-231. And then, cells viability and proliferation were analyzed using MTT assays and colony forming assays, respectively. Also mRNA expression of inflammatory cytokines and proteins expression in main signal pathway were analyzed by RT-qPCR and immunoblotting, respectively. Results: Adipocyte-derived conditioned media increased the proliferation and migration of MCF-7 and MDA-MB-231 cells while little effects in a human normal immortalized mammary epithelial cell line MCF10A. In addition, adipocyte-derived conditioned media induced phosphorylation of AKT and mTOR and upregulated the expression of target genes of the PI3K-AKT-mTOR pathway including IL6, IL1ß, IL1α and TNFα in breast cancer cells. Furthermore, BEZ235 a dual inhibitor of PI3K and mTOR significantly decreased the adipocyte-mediated the proliferation and migration of breast cancer cells. Conclusion: Adipocyte-derived conditioned media enhance the proliferation and migration of breast cancer cells through the PI3K-AKT-mTOR pathway, supporting the importance of heterotypic interactions between breast cancer cells and adipocytes in the tumor microenvironment.

CCR4­NOT transcription complex subunit 2 regulates TRAIL sensitivity in non­small­cell lung cancer cells via the STAT3 pathway.

TRAIL is an attractive candidate for anticancer therapy in a variety of tumors since it targets only tumors and not normal tissue. However, a remaining major hurdle is that the majority of tumors exhibit a resistance mechanism against the effects of TRAIL via the induction of anti­apoptotic signaling pathways. In this study, we aimed to evaluate whether the modulation of CCR4­NOT transcription complex subunit 2 (CNOT2) function can promote TRAIL sensitivity in non­small­cell lung cancer (NSCLC) cells. CNOT2 depletion partially decreased colony numbers and the proliferation of NSCLC cells. When combined with TRAIL, the suppression of CNOT2 expression markedly decreased the survival rate and increased apoptosis, as compared with TRAIL treatment alone in TRAIL­resistant NSCLC cells. Of note, CNOT2 overexpression in TRAIL­sensitive H460 cells enhanced the survival rate and decreased apoptosis when compared with TRAIL treatment alone. Gene expression analysis indicated that genes involved in the signal transducer and activator of transcription 3 (STAT3) signaling pathway were dominantly altered in the CNOT2­depleted A549 cells. Under this condition, Src homology region 2 domain containing phosphatase­1 (SHP1) was significantly upregulated and subsequently increased apoptosis. On the whole, the findings of this study demonstrate that CNOT2 participates in TRAIL sensitivity in NSCLC cells via the regulation of the STAT3 signaling pathway, and suggest that combination therapy with CNOT2 depletion and TRAIL treatment may prove to be a useful strategy for overcoming TRAIL resistance in NSCLC.

Herbal Medicines Showing Synergistic Effects with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) against A549 TRAIL-resistant Lung Cancer Cells: A Screening Study.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that activates apoptosis through death receptors on the cell surface and is regarded as a potential anticancer agent. However, many cancer cells are resistant to TRAIL-induced apoptosis. OBJECTIVE: The aim is to identify the herbal medicines that could help overcome resistance in TRAIL-resistant lung cancer cells. MATERIALS AND METHODS: TRAIL-resistant A549 cells and 13 herbal medicines with known apoptosis-related anticancer effects were used in this study: Clematidis Radix, Corydalis Tuber Rhizoma, Paeoniae Radix Rubra, Corni Fructus, Curcumae longae Rhizoma (CLR), Moutan Cortex, Salviae miltiorrhizae Radix, Phellodendri Cortex, Farfarae Flos, Paeoniae Radix Alba, Angelicae gigantis Radix, Coptidis Rhizoma (CR), and Taraxaci Herba. Cytotoxic effects were investigated after a 48-h incubation, using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, to identify the herbal medicines with the most potent synergistic effects with TRAIL. RESULTS: The majority of the 13 medicines exhibited concentration-dependent cytotoxicity against A549 cells. Among them, CR and CLR showed the most potent cytotoxic effects, based on the IC50. We then investigated the use of these two medicines in combination with TRAIL and identified synergistic cytotoxic effects against TRAIL-resistant A549 cells. CONCLUSION: Synergistic cytotoxic effects of the combination of TRAIL and herbal medicines, in particular, CR and CLR, were confirmed in A549 cells. Therefore, CR and CLR showed potential to be used as candidates to overcome TRAIL resistance. Future studies to identify their underlying mechanism of action are required. SUMMARY: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive anticancer agent which can induce apoptosis in tumor cells without causing cytotoxicity to normal cellsHowever, resistance to TRAIL is often observed in some tumor cells, including nonsmall cell lung cancers, which may limit its cytotoxic efficacy in cancer treatmentThe combination treatment of TRAIL and herbal medicines, particularly Coptidis Rhizoma (CR) and Curcumae longae Rhizoma (CLR), can induce the synergistic cytotoxic effects against TRAIL-resistant A549 cells, indicating that TRAIL resistance was reduced by combination therapy. Abbreviations used: TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand; CLR: Curcumae longae Rhizoma; CR: Coptidis Rhizoma; NSCLC: non-small cell lung cancer.

Zinc finger protein 746 promotes colorectal cancer progression via c-Myc stability mediated by glycogen synthase kinase 3ß and F-box and WD repeat domain-containing 7.

To elucidate the underlying oncogenic mechanism of zinc finger protein 746 (ZNF746), current study was conducted in colorectal cancers (CRCs). Herein, ZNF746 was overexpressed in HCT116, SW620, and SW480 cells, which was supported by CRC tissue microarray and TCGA analysis. Also, DNA microarray revealed the differentially expressed gene profile particularly related to cell cycle genes and c-Myc in ZNF746 depleted HCT116 cells. Furthermore, ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization by immunoprecipitation and immunofluorescence, while ZNF746 and c-Myc exist mainly in nucleoplasm. Conversely, ZNF746 depletion attenuated phosphorylation of c-Myc (S62) and glycogen synthase kinase 3ß (GSK3ß) (S9) and also activated p-c-Myc (T58), which was reversed by GSK3 inhibitors such as SB-216763 and Enza. Also, c-Myc degradation by ZNF746 depletion was blocked by knockdown of F-box/WD repeat-containing protein 7 (FBW7) ubiquitin ligase or proteosomal inhibitor MG132. Additionally, the growth of ZNF746 depleted HCT116 cancer cells was retarded with decreased expression of ZNF746 and c-Myc. Overall, these findings suggest that ZNF746 promotes CRC progression via c-Myc stability mediated by GSK3 and FBW7.

Tanshinone IIA induces TRAIL sensitization of human lung cancer cells through selective ER stress induction.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promised anticancer medicine targeting only the tumor, most cancers show resistance to TRAIL-induced apoptosis. For this reason, new therapeutic strategies to overcome the TRAIL resistance are required for more effective tumor treatment. In the present study, potential of tanshinone IIA as a TRAIL sensitizer was evaluated in human non-small cell lung cancer (NSCLC) cells. NSCLC cells showed resistance to TRAIL-mediated cell death, but combination treatment of Tanshinone IIA and TRAIL synergistically decreased cell viability and increased apoptosis in TRAIL-resistant NSCLC cells. Tanshinone IIA greatly induced death receptor 5 (DR5), but not death receptor 4 (DR4). Furthermore, DR5 knockdown attenuated the combination treatment of tanshinone IIA with TRAIL-mediated cell death in human NSCLC cells. Tanshinone IIA also increased CHOP and activated the PERK-ATF4 pathway suggesting that tanshinone IIA increased DR5 and CHOP by activating the PERK-ATF4 pathway. Tanshinone IIA also downregulated phosphorylation of STAT3 and expression of survivin. Taken together, these results indicate that tanshinone IIA increases TRAIL-induced cell death via upregulating DR5 and downregulating survivin mediated by, respectively, selective activation of PERK/ATF4 and inhibition of STAT3, suggesting combinatorial intervention of tanshinone IIA and TRAIL as a new therapeutic strategy for human NSCLC.

p53 regulates nuclear GSK-3 levels through miR-34-mediated Axin2 suppression in colorectal cancer cells.

p53 is a bona fide tumor suppressor gene whose loss of function marks the most common genetic alteration in human malignancy. Although the causal link between loss of p53 function and tumorigenesis has been clearly demonstrated, the mechanistic links by which loss of p53 potentiates oncogenic signaling are not fully understood. Recent evidence indicates that the microRNA-34 (miR-34) family, a transcriptional target of the p53, directly suppresses a set of canonical Wnt genes and Snail, resulting in p53-mediated suppression of Wnt signaling and the EMT process. In this study, we report that p53 regulates GSK-3ß nuclear localization via miR-34-mediated suppression of Axin2 in colorectal cancer. Exogenous miR-34a decreases Axin2 UTR-reporter activity through multiple binding sites within the 5' and 3' UTR of Axin2. Suppression of Axin2 by p53 or miR-34 increases nuclear GSK-3ß abundance and leads to decreased Snail expression in colorectal cancer cells. Conversely, expression of the non-coding UTR of Axin2 causes depletion of endogenous miR-34 via the miR-sponge effect together with increased Axin2 function, supporting that the RNA-RNA interactions with Axin2 transcripts act as an endogenous decoy for miR-34. Further, RNA transcripts of miR-34 target were correlated with Axin2 in clinical data set of colorectal cancer patients. Although the biological relevance of nuclear GSK-3 level has not been fully studied, our results demonstrate that the tumor suppressor p53/miR-34 axis plays a role in regulating nuclear GSK-3 levels and Wnt signaling through the non-coding UTR of Axin2 in colorectal cancer.

p53 and microRNA-34 are suppressors of canonical Wnt signaling.

Although loss of p53 function and activation of canonical Wnt signaling cascades are frequently coupled in cancer, the links between these two pathways remain unclear. We report that p53 transactivated microRNA-34 (miR-34), which consequently suppressed the transcriptional activity of ß-catenin-T cell factor and lymphoid enhancer factor (TCF/LEF) complexes by targeting the untranslated regions (UTRs) of a set of conserved targets in a network of genes encoding elements of the Wnt pathway. Loss of p53 function increased canonical Wnt signaling by alleviating miR-34-specific interactions with target UTRs, and miR-34 depletion relieved p53-mediated Wnt repression. Gene expression signatures reflecting the status of ß-catenin-TCF/LEF transcriptional activity in breast cancer and pediatric neuroblastoma patients were correlated with p53 and miR-34 functional status. Loss of p53 or miR-34 contributed to neoplastic progression by triggering the Wnt-dependent, tissue-invasive activity of colorectal cancer cells. Further, during development, miR-34 interactions with the ß-catenin UTR affected Xenopus body axis polarity and the expression of Wnt-dependent patterning genes. These data provide insight into the mechanisms by which a p53-miR-34 network restrains canonical Wnt signaling cascades in developing organisms and human cancer.

A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial-mesenchymal transition.

Snail1 is a zinc finger transcriptional repressor whose pathological expression has been linked to cancer cell epithelial-mesenchymal transition (EMT) programs and the induction of tissue-invasive activity, but pro-oncogenic events capable of regulating Snail1 activity remain largely uncharacterized. Herein, we demonstrate that p53 loss-of-function or mutation promotes cancer cell EMT by de-repressing Snail1 protein expression and activity. In the absence of wild-type p53 function, Snail1-dependent EMT is activated in colon, breast, and lung carcinoma cells as a consequence of a decrease in miRNA-34 levels, which suppress Snail1 activity by binding to highly conserved 3' untranslated regions in Snail1 itself as well as those of key Snail1 regulatory molecules, including ß-catenin, LEF1, and Axin2. Although p53 activity can impact cell cycle regulation, apoptosis, and DNA repair pathways, the EMT and invasion programs initiated by p53 loss of function or mutation are completely dependent on Snail1 expression. These results identify a new link between p53, miR-34, and Snail1 in the regulation of cancer cell EMT programs.