Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma

Our plain-English summary of Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma, by S.H. Chew, Y. Okazaki, S. Akatsuka, et al (Free Radical Biology & Medicine, 2017).

The study

• Authors: S.H. Chew, Y. Okazaki, S. Akatsuka, S. Wang, L. Jiang, Y. Ohara, F. Ito, H. Saya, Y. Sekido, S. Toyokuni
• Journal: Free Radical Biology & Medicine
• Published: 2017;106:91-99
• Cited by: 21 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

CD44 exists as a standard (CD44s) isoform and different variant isoforms (CD44v) due to alternative splicing. While the complex nature of these different isoforms has not been fully elucidated, CD44v expression has been shown to exert oncogenic effects by promoting tumor progression, metastasis and resistance of tumor cells to chemotherapy. One of the CD44v isoforms, CD44v8-10, was recently shown to protect cancer cells from oxidative stress by increasing the synthesis of glutathione (GSH). However, data regarding CD44 isoform expression in malignant mesothelioma (MM) are still lacking. Here, we show that most of the MM cell lines express both the CD44s and CD44v isoforms, in contrast to non-tumorigenic mesothelial cells, which express only CD44s. Moreover, we show here that these MM cell lines are positive for CD44 variable exon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable exon 9 was found to be statistically associated with NF2 inactivation, a common occurrence in MM. Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. Our results thus suggest that the response to CD44 depletion is cell type-dependent and, in cases such as MM cells, compensatory pathway(s) might be activated rheostatically to account for the loss of CD44 and counteract enhanced oxidative stress.

Copyright © 2017 Elsevier Inc. All rights reserved.

In plain English

This was a laboratory study of cells grown in dishes. It did not study people or asbestos exposure. The authors examined forms of a cell-surface molecule, CD44, in 14 human malignant mesothelioma cell lines and in MeT-5A, a non-malignant human mesothelial cell line. All the mesothelioma lines had more CD44v8-10 messenger RNA than MeT-5A. Most also made variant CD44 protein (CD44v) besides the standard form (CD44s), but four showed none on western blot. MeT-5A cells expressed only the standard form. Using NF2 gene status from earlier publications, the authors reported a statistically significant association (p less than 0.05) between NF2 inactivation and CD44v protein expression. When CD44 was reduced in the ACC-MESO-1 cell line, the cells grew less, had less of the transporter protein xCT, and had more reactive oxygen species. Their glutathione level nonetheless rose, and they were more resistant to the drug cisplatin. The authors linked this to increased GCLC and to the Nrf2 and AP-1 factors. The authors concluded that the response to losing CD44 might be cell-type specific. They stated that only two CD44-depleted cell lines were used.

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 cell lines, including gene knockdown experiments
• Who was studied: 14 human malignant mesothelioma (MM) cell lines and the immortalized non-malignant human mesothelial cell line MeT-5A. Eight of the MM lines were Japanese lines established at Aichi Cancer Center Research Institute, Nagoya; the others came from ATCC or were gifts. CD44 knockdown was done in ACC-MESO-1 cells, and CD44 variable exon 9 siRNA was used in ACC-MESO-1 and Y-MESO-12 cells.
• Exposure: No asbestos or fiber exposure was studied. The experimental manipulation was reduction (knockdown) of CD44 using shRNA targeting all CD44 isoforms, or siRNA targeting CD44 variable exon 9. Cells were then treated with cisplatin (CDDP), with or without the glutathione synthesis inhibitor BSO.
• Main results:
  • By semiquantitative RT-PCR (messenger RNA), MM cells expressed variant CD44 (CD44v) in addition to standard CD44 (CD44s), while non-malignant MeT-5A cells expressed only CD44s.
  • All MM cell lines had higher CD44v8-10 mRNA levels than MeT-5A cells.
  • At the protein level, CD44v was found together with CD44s in the majority of MM cell lines, not all of them. On western blot, four lines showed no CD44v protein: NCI-H290, and 3 of the 4 lines with wild-type NF2 (NCI-H28, MSTO-211H and ACC-MESO-4).
  • The splicing regulator ESRP1 was generally downregulated in MM cell lines, except Y-MESO-12 and Y-MESO-14.
  • Of the 14 MM cell lines, 4 had wild-type NF2 (status taken from a literature review). CD44v protein was detected in all NF2-inactivated lines except NCI-H290. NF2 status and CD44v protein expression were statistically associated (p less than 0.05, Fisher's exact test).
  • In ACC-MESO-1 cells, CD44 knockdown reduced cell proliferation, reduced xCT protein, and increased reactive oxygen species (ROS).
  • CD44-knockdown cells had increased glutathione (GSH) and enhanced resistance to cisplatin. Pre-treatment with BSO, an inhibitor of GSH synthesis, reversed the cisplatin resistance.
  • CD44-knockdown cells showed increased GCLC expression and increased nuclear Nrf2. c-Jun was increased in both the nucleus and the cytoplasm. There was no difference in nuclear phosphorylated NF-κB.
• The authors' conclusion: The authors concluded that the response to CD44 ablation might be cell-type specific, because MM cells responded differently from other cancer cells such as gastric adenocarcinoma. They suggested that compensatory pathways might be activated to counteract increased oxidative stress.
• Limitations the authors state:
  • Only two CD44-depleted MM cell lines were used. Knockdown failed in other cell lines because of cell death after transfection or poor knockdown efficiency.
  • The authors did not characterize all of the CD44 isoforms expressed in the MM cells.

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: Oxidative Stress, Malignant Mesothelioma, Metastasis, Chemotherapy, Glutathione (GSH), Mesothelial Cells.

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