A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Meyer-Schaller, Nathalie

doi:10.18632/oncotarget.8418

Cited by 20 publications

(19 citation statements)

References 65 publications

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“…EMT is mediated by the binding of various stimuli, such as TGFβ, to specific receptors, which initiates intracellular signaling that regulates the transcription of genes involved in cell-cell adhesion, reorganization of the cytoskeleton, and survival mechanisms to support metastasis (Heerboth et al, 2015;Lotz-Jenne et al, 2016). However, our results suggest that distinct signaling pathways are used for EMT in CAMSAP3-deficient and TGF-β-treated cells.…”

Section: Camsap2 Expression Level Did Not Change In Correlation Withmentioning

confidence: 67%

Loss of CAMSAP3 promotes EMT via the modification of microtubule–Akt machinery

Pongrakhananon

Wattanathamsan

Takeichi

et al. 2018

Journal of Cell Science

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Epithelial-to-mesenchymal transition (EMT) plays pivotal roles in a variety of biological processes, including cancer invasion. Although EMT involves alterations of cytoskeletal proteins such as microtubules, the role of microtubules in EMT is not fully understood. Microtubule dynamics are regulated by microtubule-binding proteins, and one such protein is CAMSAP3, which binds the minus-end of microtubules. Here, we show that CAMSAP3 is important to preserve the epithelial phenotypes in lung carcinoma cells. Deletion of CAMSAP3 in human lung carcinoma-derived cell lines showed that CAMSAP3-deficient cells acquired increased mesenchymal features, mostly at the transcriptional level. Analysis of the mechanisms underlying these changes demonstrated that tubulin acetylation was dramatically increased following CAMSAP3 removal, leading to the upregulation of Akt proteins (also known as protein kinase B proteins, hereafter Akt) activity, which is known to promote EMT. These findings suggest that CAMSAP3 functions to protect lung carcinoma cells against EMT by suppressing Akt activity via microtubule regulation and that CAMSAP3 loss promotes EMT in these cells. This article has an associated First Person interview with the first author of the paper.

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Section: Camsap2 Expression Level Did Not Change In Correlation Withmentioning

confidence: 67%

Loss of CAMSAP3 promotes EMT via the modification of microtubule–Akt machinery

Pongrakhananon

Wattanathamsan

Takeichi

et al. 2018

Journal of Cell Science

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“…1b ). Subsequently, mesenchymal cell characteristics were monitored by high-content fluorescence microscopy analysis and quantified, including the deposition of the extracellular matrix protein fibronectin and the formation of focal adhesions and of actin stress fibres 23 . Nine out of 32 differentially expressed miRNAs were able to block or at least delay the EMT process: miR-125b-5p, miR-181b-2-3p, miR-1247-3p, miR-200a-3p, miR-200b-3p, miR-429-3p, miR-1199-5p, miR-145a-3p and miR-504-5p.…”

Section: Resultsmentioning

confidence: 99%

“…The general setup of the screen has been described recently 23 . In brief, NMuMG/E9 cells were plated as duplicates in a 96-well plate and reverse transfected with 20 nM pre-miR miRNA precursors (Ambion; Supplementary Table 4 ) using Lipofectamine RNAiMax (Invitrogen) 2 days prior to the addition of TGFβ for 4 days.…”

Section: Methodsmentioning

confidence: 99%

“…For immunofluorescence analysis, cells were processed as described above and mesenchymal characteristics, such as formation of focal adhesions (paxillin), actin stress fibres (phalloidin) and fibronectin deposition, were stained as described in ref. 23 . Images of cells in a 96-well plate were taken with an Operetta HCS microscope (Perkin Elmers).…”

Section: Methodsmentioning

confidence: 99%

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miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis

et al. 2017

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Epithelial tumour cells can gain invasive and metastatic capabilities by undergoing an epithelial–mesenchymal transition. Transcriptional regulators and post-transcriptional effectors like microRNAs orchestrate this process of high cellular plasticity and its malignant consequences. Here, using microRNA sequencing in a time-resolved manner and functional validation, we have identified microRNAs that are critical for the regulation of an epithelial–mesenchymal transition and of mesenchymal tumour cell migration. We report that miR-1199-5p is downregulated in its expression during an epithelial–mesenchymal transition, while its forced expression prevents an epithelial–mesenchymal transition, tumour cell migration and invasion in vitro, and lung metastasis in vivo. Mechanistically, miR-1199-5p acts in a reciprocal double-negative feedback loop with the epithelial–mesenchymal transition transcription factor Zeb1. This function resembles the activities of miR-200 family members, guardians of an epithelial cell phenotype. However, miR-1199-5p and miR-200 family members share only six target genes, indicating that, besides regulating Zeb1 expression, they exert distinct functions during an epithelial–mesenchymal transition.

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“…RhoA, an important member of the Rho family, regulates numerous downstream genes, alters actin structure and morphology and regulates cell proliferation, apoptosis, metastasis and other biological processes (38). A recent study demonstrated that ROCK, an important RhoA regulator, is associated with cell apoptosis, EMT, migration and metastasis (39). In the present study, it was revealed that knockdown of URG11 suppresses TGF-β-induced RhoA and ROCK1 expression levels, indicating that URG11 regulates BPH progression via the RhoA/ROCK1 signaling pathway.…”

Section: Discussionmentioning

confidence: 99%

Silencing of URG11 expression inhibits the proliferation and epithelial‑mesenchymal transition in benign prostatic hyperplasia cells via the RhoA/ROCK1 pathway

et al. 2018

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Upregulated gene 11 (URG11) represents a gene upregulated by hepatitis B virus X protein and is involved in the biological processes of multifarious tumors. The present study aimed to investigate the protective effects and regulatory mechanisms of URG11 in benign prostatic hyperplasia (BPH). URG11, Ras homolog family member A (RhoA) and Rho‑associated protein kinase 1 (ROCK1) expression was detected in patients with BPH using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Furthermore, URG11 expression was silenced using URG11‑targeting small interfering RNAs. In addition, cell viability was determined by performing a Cell Counting Kit‑8 assay, and the effect of URG11 on the cell cycle was investigated by flow cytometry. Expression levels of cyclin D1, p27, E‑cadherin, N‑cadherin, vimentin, RhoA and ROCK1 were investigated by RT‑qPCR and western blotting. The results revealed that the expression levels of URG11, RhoA and ROCK1 were enhanced in patients with BPH‑1 cells compared with matched healthy controls. Furthermore, it was demonstrated that transforming growth factor‑β (TGF‑β) induced the proliferation of BPH‑1 cells in vitro, and silencing of URG11 inhibited the effects of TGF‑β on BPH‑1 cell proliferation and the cell cycle. In addition, silencing of URG11 altered the expression levels of cell cycle‑associated genes, epithelial‑mesenchymal transition‑associated genes, and RhoA and ROCK1 protein levels. Thus, the results of the present study suggest that URG11 may be a potential therapeutic target, which may be important to inhibit the development and progression of prostatic hyperplasia.

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A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

Cited by 20 publications

References 65 publications

Loss of CAMSAP3 promotes EMT via the modification of microtubule–Akt machinery

Loss of CAMSAP3 promotes EMT via the modification of microtubule–Akt machinery

miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis

Silencing of URG11 expression inhibits the proliferation and epithelial‑mesenchymal transition in benign prostatic hyperplasia cells via the RhoA/ROCK1 pathway

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