CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition

Our plain-English summary of CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition, by G. Pinton, Z. Wang, C. Balzano, et al (Frontiers in Oncology, 2021).

The study

• Authors: G. Pinton, Z. Wang, C. Balzano, S. Missaglia, D. Tavian, R. Boldorini, D.A. Fennell, M. Griffin, L. Moro
• Journal: Frontiers in Oncology
• Published: 2021;11:678447
• Cited by: 15 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant pleural mesothelioma is an aggressive cancer, heterogeneous in its presentation and behaviour. Despite an increasing knowledge about molecular markers and their diagnostic and prognostic value, they are not used as much as they might be for treatment allocation. It has been recently reported that mesothelioma cells that lack BAP1 (BRCA1 Associated Protein) are sensitive to inhibition of the EZH2 (Enhancer of Zeste Homolog 2) histone methyltransferase. Since we observed strong H3K27me3 (histone H3 lysine 27 trimetylation) immunoreactivity in BAP1 wild-type mesothelioma biopsies, we decided to characterize in vitro the response/resistance of BAP1 wild-type mesothelioma cells to the EZH2 selective inhibitor, EPZ-6438. Here we demonstrate that BAP1 wild-type mesothelioma cells were rendered sensitive to EPZ-6438 upon SIRT1 (Sirtuin 1) silencing/inhibition or when cultured as multicellular spheroids, in which SIRT1 expression was lower compared to cells grown in monolayers. Notably, treatment of spheroids with EPZ-6438 abolished H3K27me3 and induced the expression of CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A), causing cell growth arrest. EPZ-6438 treatment also resulted in a rapid and sustained induction of the genes encoding HIF2α (Hypoxia Inducible Factor 2α), TG2 (Transglutaminase 2) and IL-6 (Interleukin 6). Loss of CDKN2 is a common event in mesothelioma. CDKN2A silencing in combination with EPZ-6438 treatment induced apoptotic death in mesothelioma spheroids. In a CDKN2A wild-type setting apoptosis was induced by combining EPZ-6438 with 1-155, a TG2 selective and irreversible inhibitor. In conclusion, our data suggests that the expression of CDKN2A predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis.

Copyright © 2021 Pinton, Wang, Balzano, Missaglia, Tavian, Boldorini, Fennell, Griffin and Moro.

In plain English

This was a laboratory study of malignant pleural mesothelioma (MPM) cells. It did not study asbestos exposure or treat patients. The authors stained eight biphasic MPM biopsies and saw strong BAP1 and H3K27me3 staining in most tumor cells of every sample. They then tested EPZ-6438 (tazemetostat), a drug that blocks the enzyme EZH2, on BAP1 wild-type mesothelioma cell lines, mainly MSTO-211H and also BR95. The drug did not affect the viability of MSTO-211H cells grown as flat layers. It significantly reduced the size of spheroids (three-dimensional cell clusters), which contained fewer cells. It also slowed flat-layer cells when the protein SIRT1 was blocked or silenced. In spheroids, the drug switched on the CDKN2A gene and stopped cell growth. Both returned to control levels 48 hours after the drug was removed. When CDKN2A was silenced, the drug caused cell death (apoptosis). In CDKN2A-expressing spheroids, combining it with 1-155, a TG2 blocker, also caused apoptosis. The authors concluded that CDKN2A expression predicts how these cells respond to EZH2 inhibition and could potentially help sort tumors likely to undergo apoptosis.

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, with immunohistochemistry staining of a small set of human tumor biopsies
• Who was studied: Eight biphasic malignant pleural mesothelioma biopsies (collected with approval of Ospedale Maggiore della Carità, Novara, Italy; dates not given). Also two mesothelioma cell lines: MSTO-211H (biphasic, from a patient's pleural effusion) and BR95 (epithelioid).
• Exposure: No asbestos exposure was studied. The cells were treated with the EZH2 inhibitor EPZ-6438 (tazemetostat). Some treatments combined it with the SIRT1 inhibitor EX-527, SIRT1 or CDKN2A gene silencing, or the TG2 inhibitor 1-155.
• Main results:
  • All eight biopsies showed strong BAP1 and H3K27me3 nuclear staining in the majority of tumor cells (3+ in more than 67%).
  • In MSTO-211H cells grown as flat (2D) monolayers, EPZ-6438 did not affect cell viability.
  • In MSTO-211H multicellular spheroids, EPZ-6438 significantly reduced spheroid size and changed their architecture.
  • Cell counts showed that the treated spheroids were smaller because they contained fewer cells, not because the cells were packed more tightly.
  • In MSTO-211H monolayer cells, EPZ-6438 combined with the SIRT1 inhibitor EX-527 significantly reduced growth rate. SIRT1-silenced cells showed a comparable anti-proliferative effect with G1 phase cell cycle arrest.
  • In EPZ-6438-treated MSTO-211H spheroids, CDKN2A was strongly induced within 24 h and increased gradually at 48 and 72 h. Comparable results were seen in BR95 spheroids.
  • In MSTO-211H spheroids, cell growth, architecture and CDKN2A expression returned to control levels 48 h after EPZ-6438 was removed.
  • In MSTO-211H spheroids with CDKN2A silenced, EPZ-6438 induced apoptosis. The authors note this was contrary to their hypothesis.
  • At 48 h, EPZ-6438 induced expression of EPAS1 (HIF2 alpha), TGM2 (TG2) and IL-6 in MSTO-211H and BR95 spheroids.
  • In CDKN2A-expressing MSTO-211H spheroids, EPZ-6438 combined with the TG2 inhibitor 1-155 induced apoptosis and abrogated the EPZ-6438-mediated increase in IL-6 expression.
• The authors' conclusion: The authors concluded that CDKN2A expression predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis. They suggested that tumors with homozygous CDKN2A deletion should respond with apoptosis. They suggested that CDKN2A wild-type tumors would show growth arrest, with apoptosis possibly augmented by TG2 inhibition.
• Limitations the authors state:
  • Further investigation is needed to link differences in H3K27me3 staining intensity to the epithelioid or sarcomatoid parts of the tumors.
  • The mechanism behind the different responses of CDKN2A wild-type and CDKN2A-silenced spheroids to EPZ-6438 needs further investigation.

Topics

Medical terms in this study, explained in our Lexicon: CDKN2A, Mesothelioma, Pleural Mesothelioma, BAP1, Hypoxia, Apoptosis.

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