YB-1 Knockdown Inhibits the Proliferation of Mesothelioma Cells through Multiple Mechanisms

Our plain-English summary of YB-1 Knockdown Inhibits the Proliferation of Mesothelioma Cells through Multiple Mechanisms, by T.G. Johnson, K. Schelch, K. Lai, et al (Cancers, 2020).

The study

• Authors: T.G. Johnson, K. Schelch, K. Lai, K.A. Marzec, M. Kennerson, M. Grusch, G. Reid, A. Burgess
• Journal: Cancers
• Published: 2020;12(8)
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Y-box binding protein-1 (YB-1) is a multifunctional oncoprotein that has been shown to regulate proliferation, invasion and metastasis in a variety of cancer types. We previously demonstrated that YB-1 is overexpressed in mesothelioma cells and its knockdown significantly reduces tumour cell proliferation, migration, and invasion. However, the mechanisms driving these effects are unclear. Here, we utilised an unbiased RNA-seq approach to characterise the changes to gene expression caused by loss of YB-1 knockdown in three mesothelioma cell lines (MSTO-211H, VMC23 and REN cells). Bioinformatic analysis showed that YB-1 knockdown regulated 150 common genes that were enriched for regulators of mitosis, integrins and extracellular matrix organisation. However, each cell line also displayed unique gene expression signatures, that were differentially enriched for cell death or cell cycle control. Interestingly, deregulation of STAT3 and p53-pathways were a key differential between each cell line. Using flow cytometry, apoptosis assays and single-cell time-lapse imaging, we confirmed that MSTO-211H, VMC23 and REN cells underwent either increased cell death, G1 arrest or aberrant mitotic division, respectively. In conclusion, this data indicates that YB-1 knockdown affects a core set of genes in mesothelioma cells. Loss of YB-1 causes a cascade of events that leads to reduced mesothelioma proliferation, dependent on the underlying functionality of the STAT3/p53-pathways and the genetic landscape of the cell.

In plain English

This was a laboratory study of malignant pleural mesothelioma (MPM) cancer cell lines, not of patients. The authors used small RNA molecules (siRNA) to lower levels of a protein called YB-1. Five of six MPM cell lines grew significantly less. In three lines studied further (MSTO-211H, VMC23 and REN), growth fell by 40–60% with the main siRNA. Gene-activity measurements (RNA-seq) found 150 genes lowered in all three lines, many linked to cell division, but the lines differed, especially in the p53 and STAT3 pathways. Cell death increased in MSTO-211H and REN cells but not significantly in VMC23 cells. When single cells were filmed over 72 hours, average divisions fell from 2 to 1 in MSTO-211H and VMC23 and from 3 to 1 in REN. REN cells took on average 127.95 minutes longer to divide. The authors concluded that YB-1 loss lowers a core set of cell cycle and migration genes, and that each line's STAT3 and p53 pathway function shaped whether cells stopped dividing, died or divided abnormally. They noted their flow cytometry could not clearly confirm the predicted cell cycle arrests.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of human mesothelioma cell lines using gene knockdown, RNA sequencing, apoptosis assays, flow cytometry and live-cell time-lapse imaging
• Who was studied: Malignant pleural mesothelioma cell lines grown in the laboratory. Six lines (MSTO-211H, H226, VMC20, VMC23, MMO5, REN) were screened, and three (MSTO-211H, VMC23 and REN) were selected for detailed analysis. Lines came from the ATCC, the Medical University of Vienna, The Prince Charles Hospital (Brisbane) and the University of Pennsylvania.
• Exposure: Knockdown of YB-1 using 5 nM of YB-1-specific siRNA (siYB-1 #1, and siYB-1 #2 for validation), compared with control siRNA, mostly assessed 96 hours after transfection. No asbestos exposure was studied.
• Main results:
  • In the screen, five of six MPM cell lines showed significantly reduced proliferation after YB-1 knockdown, three of them below 60% of control.
  • In MSTO-211H, VMC23 and REN cells, proliferation was significantly reduced by 40–60% with siYB-1 #1.
  • More genes were significantly downregulated than upregulated: MSTO-211H 955 down vs 528 up; VMC23 586 down vs 395 up; REN 903 down vs 772 up.
  • 150 genes were downregulated in all three cell lines (8.3% of the total), enriched for mitosis and cell cycle pathways.
  • p53-dependent pathways differed by cell line: significant activation in MSTO-211H, weak activation in VMC23, and robust downregulation in REN.
  • In apoptosis assays, MSTO-211H cells showed a significant increase in late apoptotic cells after YB-1 knockdown.
  • In apoptosis assays, VMC23 cells showed no significant increase in apoptotic or necrotic cells after YB-1 knockdown.
  • In apoptosis assays, REN cells showed a significant increase in necrotic cells after YB-1 knockdown.
  • In live-cell imaging, average divisions fell significantly from 2 to 1 in MSTO-211H and VMC23 cells, and from 3 to 1 in REN cells.
  • In REN cells, mitosis took on average 127.95 minutes longer than in controls.
  • Cells undergoing a normal cell cycle were significantly reduced by 38% (MSTO-211H), 42% (VMC23) and 56% (REN).
  • Sequence analysis suggested all three lines likely express wild-type p53, with MSTO-211H and VMC23 carrying the P72R polymorphism.
• The authors' conclusion: The authors concluded that YB-1 loss drove downregulation of a core set of cell cycle and migration genes. Combined with the functionality of each cell's STAT3 and p53 pathways, this determined whether cells arrested, underwent apoptosis, or progressed through an aberrant mitosis.
• Limitations the authors state:
  • The single end-point flow cytometry data could not clearly validate the cell cycle arrests predicted by RNA-seq.
  • The flow cytometry results did not explain the G2/M arrest predicted for REN cells.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Metastasis, Apoptosis.

Related studies

• PIM1 knockdown inhibits cell proliferation and invasion of mesothelioma cells — A.S. Mawas, V.J. Amatya, et al. (2017)
• Turning back the Wheel: Inducing Mesenchymal to Epithelial Transition via Wilms Tumor 1 Knockdown in Human Mesothelioma Cell Lines to Influence Proliferation, Invasiveness, and Chemotaxis — T. Plönes, M. Fischer, et al. (2017)
• Cilengitide inhibits attachment and invasion of malignant pleural mesothelioma cells through antagonism of integrins αvβ3 and αvβ5 — N.C. Cheng, N. van Zandwijk, et al. (2014)
• Paclitaxel-releasing mesenchymal stromal cells inhibit in vitro proliferation of human mesothelioma cells — F. Petrella, V. Coccè, et al. (2017)
• Raltitrexed Inhibits HepG2 Cell Proliferation via G0/G1 Cell Cycle Arrest — H. Zhao, Y. Zhang, et al. (2016)
• Gefitinib targets EGFR dimerization and ERK1/2 phosphorylation to inhibit pleural mesothelioma cell proliferation — R.E. Favoni, A. Pattarozzi, et al. (2010)

Browse more research in Cancers, or return to medical research.