Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model

Our plain-English summary of Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model, by H. Sudo, A.B. Tsuji, A. Sugyo, et al (Genomics, 2010).

The study

• Authors: H. Sudo, A.B. Tsuji, A. Sugyo, M. Kohda, C. Sogawa, C. Yoshida, Y.N. Harada, O. Hino, T. Saga
• Journal: Genomics
• Published: 2010;95(4):210-6
• Cited by: 59 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes-COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A-would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.

Copyright 2010 Elsevier Inc. All rights reserved.

In plain English

This was a laboratory study using cells and mice. It did not study people or asbestos exposure. The authors used a library of 8,589 small interfering RNAs (siRNAs), which switch off individual genes, to find genes a human mesothelioma cell line called 211H needs to keep growing. Switching off 383 genes left fewer than 50% of cells alive compared with mock-transfected cells, and switching off 78 of these left fewer than 20%. On retesting, 39 genes reduced survival with both of two different siRNAs. For seven of these genes, including COPA, switching the gene off significantly increased programmed cell death (apoptosis) by 48 hours. COPA protein was highly expressed in three of four mesothelioma cell lines but not in a pleural mesothelial cell line. Switching off COPA left 5.4% and 4.6% of 211H cells alive compared with control, against 67.1% and 61.2% of the pleural mesothelial cells. In nude mice with 211H tumors under the skin, COPA siRNAs significantly slowed tumor growth and increased dying tumor cells. The authors concluded that COPA may have potential as a therapeutic target and a new diagnostic marker of 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 study: genome-wide siRNA (gene knockdown) screen in a human mesothelioma cell line, follow-up experiments in cell lines, and an animal experiment in mice carrying human mesothelioma tumors
• Who was studied: Human malignant mesothelioma cell lines 211H, H226, H2052 and H2452, and the human pleural mesothelial cell line MeT-5A, all from the American Type Culture Collection. Female nude mice aged 5 to 6 weeks from CLEA Japan carried subcutaneous 211H tumors: n=5 in the pretreatment experiment and n=4 tumors in the local injection experiment.
• Exposure: No asbestos exposure was studied. The experimental treatment was siRNA knockdown of genes: 8,589 siRNAs in the screen, and COPA-specific siRNAs (si1 and si2) in cells and mice, compared with a negative control siRNA.
• Main results:
  • In the primary screen in 211H cells, siRNAs against 383 genes left fewer than 50% viable cells compared with mock-transfected cells. For 78 of these genes, fewer than 20% of cells remained viable.
  • In the secondary screen, which used two distinct siRNAs per gene for the 78 genes, both siRNAs reduced 211H cell viability for 39 genes.
  • For seven selected genes (COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, RPL18A), the share of apoptotic 211H cells was significantly higher than with the negative control at 48 hours after transfection (P less than 0.01). At 24 hours this was true for five genes but not for COPA or COPB2.
  • COPA protein was highly expressed in mesothelioma cell lines 211H, H2052 and H2452. It was not highly expressed in mesothelioma line H226 or in the pleural mesothelial line MeT-5A.
  • After COPA knockdown with the two siRNAs, viable cells compared with the negative control were 5.4% and 4.6% in 211H cells, and 19.2% and 31.7% in H2052 cells.
  • In the pleural mesothelial cell line MeT-5A, 67.1% and 61.2% of cells survived COPA knockdown compared with control.
  • In nude mice, 211H cells treated with COPA siRNAs before injection grew tumors significantly more slowly than control (P less than 0.01). COPA siRNAs injected around established 211H tumors also significantly inhibited tumor growth compared with control siRNA (P less than 0.05).
  • In 211H mouse tumors, the two COPA siRNAs produced 12-fold and 10-fold increases in TUNEL-positive (apoptotic) cells compared with the negative control siRNA (P less than 0.01).
• The authors' conclusion: The authors concluded that knockdown of 39 genes suppressed mesothelioma cell proliferation and that at least seven are associated with anti-apoptotic function. They concluded that COPA may hold promise for new therapy for mesotheliomas that highly express COPA, and may have potential as a therapeutic target and a new diagnostic marker of mesothelioma.
• Limitations the authors state:
  • For genes where only one of the two siRNAs reduced viability, the authors could not tell whether this reflected insufficient knockdown or an off-target effect. They said further work with additional siRNAs is needed.
  • The pretreatment protocol worked better than local injection in mice. The authors attributed this probably to lower transfection efficiency of siRNAs in mice than in dishes.
  • COPA siRNAs did not reduce COPA mRNA enough in the H226 and H2452 mesothelioma cell lines, so those lines were not used in the proliferation assay.

Topics

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

Medical terms in this study, explained in our Lexicon: Apoptosis, Mesothelioma, Malignant Mesothelioma, Serosal Surface, Pleura, Mesothelial Cells.

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