Establishment and characterization of CRISPR/Cas9-mediated NF2-/- human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma

Our plain-English summary of Establishment and characterization of CRISPR/Cas9-mediated NF2-/- human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma, by M. Wahiduzzaman, S. Karnan, A. Ota, et al (Cancer Science, 2019).

The study

• Authors: M. Wahiduzzaman, S. Karnan, A. Ota, I. Hanamura, H. Murakami, A. Inoko, M.L. Rahman, T. Hyodo, H. Konishi, S. Tsuzuki, Y. Hosokawa
• Journal: Cancer Science
• Published: 2019;110(1):180-193
• Cited by: 19 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

In plain English

This was a laboratory study that did not examine asbestos exposure. Researchers in Japan used the CRISPR/Cas9 gene-editing tool to disable the NF2 gene in MeT-5A, an immortalized normal human mesothelial cell line. NF2 is a tumor suppressor gene that is often altered in mesothelioma. The NF2-knockout cells grew faster, formed more colonies and migrated more than unedited cells. Putting NF2 back significantly reduced these changes. The knockout cells also made more FGFR2, a growth-factor receptor, and disabling FGFR2 as well slowed their growth. The team stained a purchased set of 23 malignant mesothelioma and 10 normal mesothelium tissue samples. The tumors came from the pleura, abdominal cavity, pericardium and other sites. FGFR2 was detected in 11 of 12 NF2-negative tumors (91.7%), in 2 of 11 NF2-positive tumors (18.2%), and in 1 of 10 normal samples. The authors concluded that FGFR2 signaling might play a pivotal role in the growth of malignant pleural mesothelioma cells. They proposed FGFR2 as a candidate for developing treatment and diagnostic strategies for malignant pleural mesothelioma with NF2 loss. They said it remains unclear how NF2 loss raises FGFR2.

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) cell-line experiments using gene knockout, plus immunohistochemical staining of a purchased mesothelioma tissue array
• Who was studied: Human cell lines: the immortalized normal pleural mesothelial line MeT-5A (main model), the immortalized normal omental mesothelial lines HOMC-A4 and HOMC-D4, and the mesothelioma line NCI-H2052. Also a commercial tissue array (US Biomax) of 23 malignant mesothelioma and 10 normal mesothelium samples. The article calls the tumors MPM tissues, but its Table 1 lists sites including the abdominal cavity, retroperitoneum, heart, lung, mediastinum, pleura and cardiac pericardium.
• Exposure: No asbestos or fiber exposure was studied. The experimental 'exposure' was genetic: NF2 (and in some experiments FGFR2) was disabled with CRISPR/Cas9, and NF2 was restored by adding it back.
• Main results:
  • In MeT-5A cells, the NF2-knockout clones grew significantly faster than the parental cells and the unedited (NF2-WT) clones. They also formed more soft-agar colonies and showed higher migration and wound-healing activity. Restoring NF2 significantly suppressed these changes.
  • Microarray gene-expression testing of the NF2-knockout clones found 17 genes upregulated more than 5-fold and 77 genes downregulated (fold change below 0.2), compared with the NF2-WT and parental cells.
  • FGFR2 protein increased in the NF2-knockout MeT-5A clones, and restoring NF2 reduced it. Knocking out NF2 in HOMC-A4 and HOMC-D4 cells also increased FGFR2. Adding NF2 to the NCI-H2052 mesothelioma line decreased FGFR2.
  • Disabling FGFR2 in NF2-knockout cells significantly decreased their growth. Disabling FGFR2 alone in otherwise normal MeT-5A cells did not significantly change growth compared with parental cells.
  • In the tissue array, FGFR2 staining was positive in 13 of 23 malignant mesothelioma samples (3 strong, 8 moderate, 2 weak). Only 1 of 10 normal mesothelium samples was positive, and its staining was weak.
  • Among the malignant mesothelioma samples, FGFR2 positivity was 11 of 12 (91.7%) in NF2-negative tissues versus 2 of 11 (18.2%) in NF2-positive tissues. The authors reported this difference as significant.
  • The authors analyzed two public gene-expression datasets (GSE2549 and GSE29354). The results are shown only in a supplementary figure, and no numbers are given in the text. In both datasets, MPM patients with high FGFR2 expression had shorter overall survival than those with low FGFR2 expression.
• The authors' conclusion: The authors concluded that FGFR2 signaling might play a pivotal role in the proliferation of MPM cells. They proposed FGFR2 as a candidate molecule for developing therapeutic and diagnostic strategies for MPM with NF2 loss.
• Limitations the authors state:
  • The authors state that the molecular mechanism by which NF2 loss raises FGFR2 remains unclear.
  • The YAP experiments gave mixed results: one YAP knockdown vector lowered FGFR2 and the other did not. The authors say their data could not exclude a role for YAP.

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: Mesothelial Cells, Fibroblast, Pleural Mesothelioma, Quality of Life, Tumor Suppressor Gene, Mesothelioma, Tumorigenesis, Mesothelium.

Related studies

• Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers — J.C. Pache, Y.M. Janssen, et al. (1998)
• Expression of the Wilms' tumor gene WT1 in human malignant mesothelioma cell lines and relationship to platelet-derived growth factor A and insulin-like growth factor 2 expression — A.W. Langerak, K.A. Williamson, et al. (1995)
• Loss of NF2 Induces TGFβ Receptor 1-mediated Noncanonical and Oncogenic TGFβ Signaling: Implication of the Therapeutic Effect of TGFβ Receptor 1 Inhibitor on NF2 Syndrome — J.H. Cho, A.Y. Oh, et al. (2018)
• Establishment and characterization of five human malignant mesothelioma cell lines derived from pleural effusions — L.S. Manning, D. Whitaker, et al. (1991)
• Establishment and characterization of 20 human non-small cell lung cancer cell lines in a serum-free defined medium (ACL-4) — N. Masuda, M. Fukuoka, et al. (1991)
• Characterization of preneoplastic and neoplastic rat mesothelial cell lines: the involvement of TETs, DNMTs, and 5-hydroxymethylcytosine — D. Roulois, S. Deshayes, et al. (2016)

Browse more research in Cancer Science, or return to medical research.