scholarly journals Assessing methods to quantitatively validate TGFβ-dependent autophagy

Biology Open ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. bio055103
Author(s):  
Charles B. Trelford ◽  
Gianni M. Di Guglielmo
Keyword(s):  
Cell Lines ◽  
Transforming Growth Factor Beta ◽  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
Nsclc Cell ◽  
Serum Starvation ◽  
Autophagic Flux ◽  
Mesenchymal Transition ◽  
Protein Levels ◽  
Lc3 Puncta

ABSTRACTTransforming growth factor beta (TGFβ) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGFβ may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGFβ1 modulates autophagy related (ATG) gene expression and ATG protein levels. We also assessed microtubule-associated protein light chain 3 (LC3) lipidation, LC3 puncta formation and autophagosome-lysosome co-localization in non-small cell lung cancer (NSCLC) cell lines. These experimental approaches were validated using pharmacological autophagy inhibitors (chloroquine and spautin-1) and an autophagy activator (MG132). We found that TGFβ1, chloroquine and MG132 had little effect on ATG protein levels but increased LC3 lipidation, LC3 puncta formation and autophagosome-lysosome co-localization. Since similar outcomes were observed using chloroquine and MG132, we concluded that several techniques employed to assess TGFβ-dependent autophagy may not differentiate between the activation of autophagy versus lysosomal inhibition. Thus, NSCLC cell lines stably expressing a GFP-LC3-RFP-LC3ΔG autophagic flux probe were used to assess TGFβ-mediated autophagy. Using this approach, we observed that TGFβ, MG132 and serum starvation increased autophagic flux, whereas chloroquine and spautin-1 decreased autophagic flux. Finally, we demonstrated that ATG5 and ATG7 are critical for TGFβ-dependent autophagy in NSCLC cells. The application of this model will fuel future experiments to characterize TGFβ-dependent autophagy, which is necessary to understand the molecular processes that link, TGFβ, autophagy and tumorigenesis.

scholarly journals Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Yngvild Bjørlykke ◽  
Anne M. Søviknes ◽  
Laurence Hoareau ◽  
Heidrun Vethe ◽  
Andreas F. Mathisen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transforming Growth Factor Beta ◽  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
Colony Morphology ◽  
Unstable State ◽  
Protein Markers ◽  
Mesenchymal Transition ◽  
Differentiation Capacity

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity.

scholarly journals The TGFβ-induced phosphorylation and activation of p38 mitogen-activated protein kinase is mediated by MAP3K4 and MAP3K10 but not TAK1

Open Biology ◽  
2013 ◽  
Vol 3 (6) ◽  
pp. 130067 ◽  
Author(s):  
Gopal P. Sapkota
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Transforming Growth Factor Beta ◽  
Molecular Mechanisms ◽  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Phosphorylation ◽  
Mesenchymal Transition ◽  
Mitogen Activated Protein

The signalling pathways downstream of the transforming growth factor beta (TGFβ) family of cytokines play critical roles in all aspects of cellular homeostasis. The phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in TGFβ-induced epithelial-to-mesenchymal transition and apoptosis. The precise molecular mechanisms by which TGFβ cytokines induce the phosphorylation and activation of p38 MAPK are unclear. In this study, I demonstrate that TGFβ-activated kinase 1 (TAK1/MAP3K7) does not play a role in the TGFβ-induced phosphorylation and activation of p38 MAPK in MEFs and HaCaT keratinocytes. Instead, RNAi -mediated depletion of MAP3K4 and MAP3K10 results in the inhibition of the TGFβ-induced p38 MAPK phosphorylation. Furthermore, the depletion of MAP3K10 from cells homozygously knocked-in with a catalytically inactive mutant of MAP3K4 completely abolishes the TGFβ-induced phosphorylation of p38 MAPK, implying that among MAP3Ks, MAP3K4 and MAP3K10 are sufficient for mediating the TGFβ-induced activation of p38 MAPK.

scholarly journals Loss of BRMS1 Promotes a Mesenchymal Phenotype through NF-κB-Dependent Regulation ofTwist1

2014 ◽  
Vol 35 (1) ◽  
pp. 303-317 ◽  
Author(s):  
Yuan Liu ◽  
Marty W. Mayo ◽  
Aizhen Xiao ◽  
Emily H. Hall ◽  
Elianna B. Amin ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Cancer Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
Breast Cancer Metastasis ◽  
Metastasis Suppressor ◽  
Mesenchymal Phenotype ◽  
Mesenchymal Transition ◽  
Metastasis Suppressor 1 ◽  
Promoter Occupancy

Breast cancer metastasis suppressor 1 (BRMS1) is downregulated in non-small cell lung cancer (NSCLC), and its reduction correlates with disease progression. Herein, we investigate the mechanisms through which loss of theBRMS1gene contributes to epithelial-to-mesenchymal transition (EMT). Using a short hairpin RNA (shRNA) system, we show that loss of BRMS1 promotes basal and transforming growth factor beta-induced EMT in NSCLC cells. NSCLC cells expressingBRMS1shRNAs (BRMS1knockdown [BRMS1KD]) display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers. Mesenchymal phenotypes observed inBRMS1KDcells are dependent on RelA/p65, the transcriptionally active subunit of nuclear factor kappa B (NF-κB). In addition, chromatin immunoprecipitation analysis demonstrates that loss ofBRMS1increasesTwist1promoter occupancy of RelA/p65 K310—a key histone modification associated with increased transcription. Knockdown ofTwist1results in reversal ofBRMS1KD-mediated EMT phenotypic changes. Moreover, in our animal model,BRMS1KD/Twist1KDdouble knockdown cells were less efficient in establishing lung tumors thanBRMS1KDcells. Collectively, this study demonstrates that loss of BRMS1 promotes malignant phenotypes that are dependent on NF-κB-dependent regulation ofTwist1. These observations offer fresh insight into the mechanisms through which BRMS1 regulates the development of metastases in NSCLC.

scholarly journals GCC2 is a New Biomarker for Diagnosis of Early Non-Small Cell Lung Cancer and A Potential Target to Reverse Epithelial to Mesenchymal Transition

2020 ◽  
Author(s):  
Hyesun Jeong ◽  
Byeong Hyeon Choi ◽  
Jik-han Jung ◽  
Hyunku Shin ◽  
JinA Park ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Treatment ◽  
Small Cell Lung Cancer ◽  
Cell Lines ◽  
Epithelial To Mesenchymal Transition ◽  
Small Cell ◽  
Nsclc Cell ◽  
Small Cell Lung ◽  
Mesenchymal Transition ◽  
Expression Levels

Abstract Background: Nano-sized exosomes (30–150 nm) are cell membrane-encapsulated vesicles that contain nucleic acids and proteins. Specific markers detecting non-small cell lung cancer (NSCLC) cell-derived exosomes in the blood circulation remain unidentified. Here, we report a new biomarker distinguishing cancer from non-cancer exosomes that also involved in epithelial to mesenchymal transition for cancer treatment.Methods: Exosomes were isolated from plasma of patients with various pathological stages of NSCLC and NSCLC cell lines, human pulmonary alveolar epithelial cells by size exclusion chromatography and characterized by Nanoparticle Tracking Analysis and western-blotting. The exosomes were lysed and applied to proteomic analysis. The expression levels of the GCC2 proteins from NSCLC patients were analyzed by ELISA assays, and the effects by GCC2 shRNA were analyzed by real-time RT-PCR, cell migration and colony formation assays.Results: A protein GRIP and coiled-coil domain-containing 2 (GCC2), which is involved in endosome-to-Golgi transport, was identified by the proteomics analysis of exosomes isolated from NSCLC cell lines. The GCC2 protein expression levels were increased in the exosomes derived from patients with early-stage NSCLC compared with healthy controls. The receiver operating characteristic curve of exosomal GCC2 revealed 94.74% sensitivity and 75.00% specificity, and AUC of 0.875. GCC2 knockdown experiments by GCC2 shRNA showed reduced exosome secretion in cancer cell lines, which altered the molecular and cellular properties, such as the expression levels of mesenchymal-to-epithelial genes, and cellular growth and motility.Conclusion: GCC2 represents a promising biomarker for early diagnosis of NSCLC and a therapeutic target for future cancer treatment.

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