Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

Our plain-English summary of Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia, by Z. Li, L. Jiang, S.H. Chew, et al (Redox Biology, 2019).

The study

• Authors: Z. Li, L. Jiang, S.H. Chew, T. Hirayama, Y. Sekido, S. Toyokuni
• Journal: Redox Biology
• Published: 2019;26:101297
• Cited by: 150 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO2, bicarbonate and H+. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O2) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O2- and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells.

Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

In plain English

This was a laboratory study of cells, not of people. The authors first re-analyzed their earlier gene data from 24 asbestos-induced rat mesotheliomas. Higher levels of the gene Ca9 (carbonic anhydrase 9) were linked with shorter survival (r = −0.52, P = 0.0095). They then grew three human malignant mesothelioma cell lines and one non-cancerous mesothelial line, MeT-5A, under low oxygen (1% O2). Under low oxygen, CA9 protein rose in all three mesothelioma lines, exceeding levels in MeT-5A. In ACC-Meso-1 cells, reactive iron, called catalytic Fe(II), also increased. Two CA9-blocking drugs, S4 and U104, reduced the number of living ACC-Meso-1 cells and slowed their movement. The drugs also slowed MeT-5A growth, but less. Blocking CA9 raised reactive iron, oxidative stress and signs of mitochondrial damage. Drugs blocking two kinds of cell death, apoptosis and ferroptosis, reduced the resulting cell death; an iron-binding drug reduced death caused by S4. The authors concluded that CA9 helps mesothelioma cells manage iron under low oxygen and is an attractive drug target. They noted the drugs are not completely specific for CA9.

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-culture study of human cell lines, plus re-analysis of the authors' previously generated gene-expression data from asbestos-induced rat mesotheliomas
• Who was studied: Three human malignant mesothelioma cell lines (epithelioid ACC-Meso-1 and NCI–H2052; sarcomatoid NCI–H2373) and one immortalized human mesothelial cell line (MeT-5A). The rat re-analysis covered asbestos-induced rat mesotheliomas: 13 sarcomatoid, 9 epithelioid and 2 biphasic, total N = 24.
• Exposure: The study did not test asbestos exposure. Cells were grown under low oxygen (1% O2) and treated with the CA9 inhibitors S4 and U104 (for example 40 to 75 μM), with siRNA to silence CA9, and with the ferroptosis inducer erastin. Cell-death blockers were also tested: deferoxamine (an iron binder), ferrostatin-1 and Z-VAD-FMK.
• Main results:
  • In the re-analyzed asbestos-induced rat mesotheliomas, higher Ca9 expression was inversely correlated with survival (Pearson's r = −0.52, P = 0.0095).
  • CA9 protein increased in all three human mesothelioma cell lines under low oxygen, and all of them had higher CA9 protein than MeT-5A cells.
  • Under low oxygen, catalytic Fe(II) signals in ACC-Meso-1 cells were significantly elevated (P < 0.01). This was measured in ACC-Meso-1 cells only.
  • S4 and U104 each significantly decreased the viability of ACC-Meso-1 cells at 48 and 72 hours under low oxygen (P < 0.001).
  • S4 and U104 also had antiproliferative effects on MeT-5A cells, but these were milder than in ACC-Meso-1 cells. A migration comparison for MeT-5A was not reported.
  • S4 and U104 reduced migration of ACC-Meso-1 cells at 24 hours under low oxygen, in a dose-dependent way (40 and 75 μM).
  • After CA9 inhibition in ACC-Meso-1 cells, the iron-transport proteins TFRC and FPN-1 increased and the iron-storage proteins FTL and FTH decreased. Catalytic Fe(II) rose significantly, with more of it located in lysosomes and mitochondria (P < 0.001; N = 20 cells).
  • Cell death caused by S4 (50 μM) or U104 (60 μM) at 72 hours was significantly reversed by the apoptosis blocker Z-VAD (P < 0.01 and P < 0.001, respectively). The ferroptosis blocker Fer-1 (3 μM) was also significantly effective (P < 0.05 and P < 0.01). Low-dose deferoxamine (0.5 μM) significantly reduced the toxic effect of S4 (P < 0.01).
  • Erastin, a known inducer of ferroptosis, produced a similar pattern of iron-related proteins in ACC-Meso-1 cells and lowered CA9 protein.
• The authors' conclusion: The authors concluded that CA9 plays a pivotal role in the metabolism of mesothelioma cells under low oxygen. In their view it supports cell growth and migration and keeps iron in balance so that the cells avoid ferroptosis and apoptosis. They considered CA9 an attractive molecular target for chemotherapy of malignant mesothelioma.
• Limitations the authors state:
  • The CA9 inhibitors are not completely specific, so the authors also used knockdown (siRNA) experiments to confirm the results.
  • Mesothelioma cells most probably developed in an iron-rich environment, so studies of other cancers are needed to confirm whether the results apply to cancer generally.

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: Apoptosis, Malignant Mesothelioma, Hypoxia, Prognosis, Carcinogenesis, Mesothelial Cells.

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