Genes associated with prognosis after surgery for malignant pleural mesothelioma promote tumor cell survival in vitro

Our plain-English summary of Genes associated with prognosis after surgery for malignant pleural mesothelioma promote tumor cell survival in vitro, by G.J. Gordon, R. Bueno, D.J. Sugarbaker (BMC Cancer, 2011).

The study

• Authors: G.J. Gordon, R. Bueno, D.J. Sugarbaker
• Journal: BMC Cancer
• Published: 2011;11:169
• Cited by: 16 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype.

Methods: We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination.

Results: Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene.

Conclusions: We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.

In plain English

This laboratory study looked at four genes (ARHGDIA, COBLL1, PKM2 and TM4SF1) that the authors had earlier used in a test to predict how patients with malignant pleural mesothelioma fare after surgery. The study did not involve patients, mesothelioma cells or asbestos. The authors used small RNA molecules to reduce the activity of each gene, with three different molecules per gene. They did this in two lung-derived cell lines grown in the laboratory: normal fibroblasts (WI38) and lung tumor cells (A549). Cells were examined 72 hours later for programmed cell death (apoptosis) and for cell division. In the tumor cells, reducing ARHGDIA, COBLL1 or TM4SF1 was followed by about 2- to 4-fold more apoptosis and by fewer cells. In the normal cells, only ARHGDIA had a statistically significant effect on apoptosis. Reducing PKM2 produced no statistically significant change, and no gene significantly changed cell division. The authors concluded that three of the genes appear to help protect tumor cells from apoptosis and, with further validation, may be potential treatment targets. They noted that neither cell line came from mesothelioma.

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) gene knockdown experiment in cultured cell lines, using small inhibitory RNAs (siRNAs) and automated microscopy; each experiment run in triplicate
• Who was studied: Two commercially purchased human cell lines, not from patients and not mesothelioma-derived: WI38 normal lung-derived fibroblasts and A549 lung (alveolar) tumor cells
• Exposure: No asbestos exposure was studied. The experimental treatment was reducing (knocking down) the expression of four mesothelioma prognostic genes, ARHGDIA, COBLL1, PKM2 and TM4SF1, using three different siRNAs per gene at 30 nM. Results were compared with scrambled siRNA and mock-transfection controls, and with positive control genes KIF11 and PLK1.
• Main results:
  • In normal cells, expression of the targeted genes was reduced by about 50 to 80%. Knockdown was slightly more efficient in tumor cells.
  • In normal (WI38) cells, apoptosis was elevated about 2- to 4-fold after ARHGDIA knockdown compared with negative control (siRNA1 P = 0.00067, siRNA2 P = 0.00098, siRNA3 P = 8.7 × 10-10). Other genes did not reach statistical significance in normal cells.
  • In tumor (A549) cells, apoptosis increased 2- to 4-fold after knockdown of ARHGDIA, COBLL1 and TM4SF1, with all three siRNAs for each gene statistically significant. PKM2 knockdown did not have this effect.
  • No experimental gene knockdown produced a statistically significant change in cell division (Mitotic Index) relative to negative controls.
  • In tumor cells, condensed chromatin (an early sign of apoptosis) rose modestly after knockdown of ARHGDIA, COBLL1 and TM4SF1. The rise was statistically significant only for ARHGDIA.
  • In tumor cells, cell numbers decreased after knockdown of ARHGDIA, COBLL1 and TM4SF1. The decrease was statistically significant for all siRNAs (uncorrected P less than 0.05).
  • No gene knockdown produced a statistically significant difference in aberrantly shaped nuclei compared with negative control.
  • PKM2 knockdown was associated with a modest (about 15 to 20%) decrease in tumor cell number in two of three siRNA experiments. This decrease was not statistically significant.
• The authors' conclusion: The authors concluded that three of the four prognostic genes (ARHGDIA, COBLL1, TM4SF1) are involved in some way in protecting tumor cells from apoptosis. They concluded that, with further validation, these genes may provide potential targets for biological therapy of mesothelioma.
• Limitations the authors state:
  • Neither cell line was derived from mesothelioma.
  • No normal pleural mesothelial cell line was available as a control. WI38 was used instead, which the authors describe as not ideal.
  • Readouts were collected at a single time point only (72 hours after transfection), so early apoptotic events may have been missed.
  • The PKM2 cell-number results were not statistically significant, mainly because of excessive variability.

Topics

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

Medical terms in this study, explained in our Lexicon: Prognosis, Pleural Mesothelioma, Mesothelioma, Neoplasm, Cytoreductive Surgery, Apoptosis.

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