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Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells.

ABSTRACT: Glioblastoma (GBM) is the most malignant brain tumor with very limited therapeutic options. Standard multimodal treatments, including surgical resection and combined radio-chemotherapy do not target the most aggressive subtype of glioma cells, brain tumor stem cells (BTSCs). BTSCs are thought to be responsible for tumor initiation, progression, and relapse. Furthermore, they have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT) thereby inducing tumor dissemination and chemo resistance. Using high resolution proton nuclear magnetic resonance spectroscopy (1H NMR) on GBM cell cultures we provide evidence that the expression of well-known EMT activators of the ZEB, TWIST and SNAI families and EMT target genes N-cadherin and VIMENTIN is associated with aberrant choline metabolism. The cholinic phenotype is characterized by high intracellular levels of phosphocholine and total choline derivatives and was associated with malignancy in various cancers. Both genetic and pharmacological inhibition of the cardinal choline metabolism regulator choline kinase alpha (CHK?) significantly reduces the cell viability, invasiveness, clonogenicity, and expression of EMT associated genes in GBM cells. Moreover, in some cell lines synergetic cytotoxic effects were observed when combining the standard of care chemotherapeutic temozolomide with the CHK? inhibitor V-11-0711. Taken together, specific inhibition of the enzymatic activity of CHK? is a powerful strategy to suppress EMT which opens the possibility to target chemo-resistant BTSCs through impairing their mesenchymal transdifferentiation. Moreover, the newly identified EMT-oncometabolic network may be helpful to monitor the invasive properties of glioblastomas and the success of anti-EMT therapy.

SUBMITTER: Koch K

PROVIDER: S-EPMC5341988 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells.

Koch Katharina K Hartmann Rudolf R Schröter Friederike F Suwala Abigail Kora AK Maciaczyk Donata D Krüger Andrea Caroline AC Willbold Dieter D Kahlert Ulf Dietrich UD Maciaczyk Jaroslaw J

Oncotarget 20161101 45

Glioblastoma (GBM) is the most malignant brain tumor with very limited therapeutic options. Standard multimodal treatments, including surgical resection and combined radio-chemotherapy do not target the most aggressive subtype of glioma cells, brain tumor stem cells (BTSCs). BTSCs are thought to be responsible for tumor initiation, progression, and relapse. Furthermore, they have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT) th ...[more]

PMID: 27705917

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Project description:BackgroundEribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) in human breast cancer cells were investigated.MethodsTriple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER-)/progesterone receptor (PR-)/human epithelial growth receptor 2 (HER2-) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-?/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting.ResultsTreatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-? induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model.ConclusionsEribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.

Project description:BACKGROUND:Several members of the tripartite motif-containing (TRIM) protein family have been reported to serve as vital regulators of tumorigenesis. Recent studies have demonstrated an oncogenic role of TRIM 14 in multiple human cancers; however, the importance of this protein in glioblastoma remains to be elucidated. METHODS:The expression levels of TRIM14 were analyzed in a series of database and were examined in a variety of glioblastoma cell lines. Two independent TRIM14 shRNA were transfected into LN229 and U251 cells, and the effect of TRIM14 depletion was confirmed. Transwell assay and wound healing assay assay were carried out to assess the effect of TRIM14 depletion on glioblastoma cell invasion and migration. Western blotting was performed to screen the downstream gene of TRIM14. The stability analysis and Ubiquitylation assays and Orthotopic xenograft studies were also performed to investigate the role of TRIM14 and the relationship with downstream gene. Human glioblastoma tissues were obtained and immunohistochemical staining were carried out to confirm the clinical significance of TRIM14. RESULTS:In this study, we showed that TRIM14 was upregulated in human glioblastoma specimens and cell lines, and correlated with glioblastoma progression and shorter patient survival times. Functional experiments showed that decreased TRIM14 expression reduced glioblastoma cell invasion and migration. Furthermore, we identified that zinc finger E-box binding homeobox 2 (ZEB2), a transcription factor involved in epithelial-mesenchymal transition, is a downstream target of TRIM14. Further investigation revealed that TRIM14 inactivation significantly facilitated ZEB2 ubiquitination and proteasomal degradation, which led to aggressive invasion and migration. Our findings provide insight into the specific biological role of TRIM14 in tumor invasion. CONCLUSIONS:Our findings provide insight into the specific biological role of TRIM14 in tumor invasion, and suggest that targeting the TRIM14/ZEB2 axis might be a novel therapeutic approach for blocking glioblastoma.

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Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells.

Publications

Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells.

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