GAS5 long non-coding RNA in malignant pleural mesothelioma

Our plain-English summary of GAS5 long non-coding RNA in malignant pleural mesothelioma, by A. Renganathan, J. Kresoja-Rakic, N. Echeverry, et al (Molecular Cancer, 2014).

The study

• Authors: A. Renganathan, J. Kresoja-Rakic, N. Echeverry, G. Ziltener, B. Vrugt, I. Opitz, R.A. Stahel, E. Felley-Bosco
• Journal: Molecular Cancer
• Published: 2014;13:119
• Cited by: 81 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA "sponge". Our aim was to investigate the possible role of the GAS5 in the growth of MPM.

Methods: Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry.

Results: GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression.

Conclusions: The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.

In plain English

This laboratory study examined GAS5, a long non-coding RNA (an RNA that does not make a protein), in malignant pleural mesothelioma. It did not study asbestos exposure. The authors cited earlier mouse research by others in which a deleted region that includes this gene was found in asbestos-induced mesothelioma. GAS5 levels were lower in 22 mesothelioma cell lines than in 7 normal mesothelial cell lines. Drugs that halt cell growth (HhAntag and NVP-BEZ235) raised GAS5 levels and GAS5 promoter activity. Partly silencing GAS5 raised two glucocorticoid-responsive genes and shortened the cell cycle from 55–77 hours to 40 to 47 hours. GAS5 was about twice as abundant in the nucleus as in the cytosol. Unexpectedly, tumor tissue (116 samples) had six-fold higher GAS5 than non-tumoral tissue (10 samples). In tumors, GAS5-expressing tumor cells were Ki-67 negative (not dividing). GAS5 correlated positively with podoplanin but not with mesothelin or calretinin. The authors concluded that GAS5 plays a role in mesothelioma biology.

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 study of mesothelioma cells grown in culture and of mesothelioma tumor tissue. It is not a study of people's asbestos exposure.
• Who was studied: 22 malignant pleural mesothelioma cell lines and 7 normal mesothelial cell lines, including primary cultures established from surgical specimens. Also tumor tissue (116 samples) compared with non-tumoral pleural tissue (10 samples, from patients having thoracic surgery unrelated to mesothelioma) at Zurich University Hospital. Dates not given.
• Exposure: No asbestos exposure was studied. Cells were treated with growth-arrest-inducing drugs: the Hedgehog signaling inhibitor HhAntag and the dual PI3K/mTOR inhibitor NVP-BEZ235. GAS5 was also silenced with doxycycline-inducible shRNA and with siRNA. Asbestos appears only in the background, which cites an earlier study by others that found a deleted region including the gas5 locus in asbestos-induced murine mesothelioma.
• Main results:
  • GAS5 expression was lower in the 22 mesothelioma cell lines than in the 7 normal mesothelial cell lines, and it increased when growth arrest was induced by drugs.
  • Drug-induced growth arrest increased GAS5 expression in primary mesothelioma cells (ZL55SPT and SDM103T2) and in cell lines (ZL55 and ACC-Meso4).
  • GAS5 promoter activity rose significantly in ZL55SPT and ZL55 cells after treatment with HhAntag and NVP-BEZ235.
  • In ZL55SPT clones, silencing GAS5 (a 35% and 25% decrease in the two clones) increased the glucocorticoid-responsive genes GILZ (in shGAS5-2) and SGK1 (in shGAS5-1).
  • Cell cycle length was 55–77 hours in uninduced shGAS5 clones and 40 to 47 hours in GAS5-silenced cells.
  • In ZL55SPT cells, GAS5 was twice as abundant in the nucleus as in the cytosol without HhAntag. It increased with HhAntag dose and remained predominantly nuclear.
  • Tumor tissue (n = 116) had a significant six-fold higher GAS5 level than non-tumoral samples (n = 10), p less than 0.0001. The authors described this as unexpected.
  • In situ hybridization in a subset of tumor samples showed that GAS5-expressing tumor cells were Ki-67 negative, meaning quiescent (not dividing).
  • In tumor tissue, GAS5 levels correlated positively with podoplanin (p less than 0.05). No correlation was seen with mesothelin or calretinin.
• The authors' conclusion: The authors concluded that their experiments show GAS5 expression plays a role in malignant pleural mesothelioma biology. The abstract words this as findings that 'support a role'.
• Limitations the authors state:
  • The authors could not test in cultured cells whether cells with high nuclear GAS5 are quiescent, because nuclear RNA in situ hybridization is not compatible with Ki-67 staining in the same specimen.
  • The primers used detected only 20 of the 29 GAS5 splice variants.
  • The idea that GAS5 acts as a miRNA sponge protecting podoplanin was not tested. The authors state further work is needed.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Overall Survival, Mesothelioma, Podoplanin (D2-40), Ki-67, Immunohistochemistry, Mesothelial Cells.

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