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Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells

Tasmia Tahsin, Darius K. McPhail Orcid Logo, Jesse D. Champion, Mohammad A. M. Alzahrani, Madeleine L. Hilditch, Alexandre Faris-Orr, Brian L. Calver, James Cronin Orcid Logo, Juan C. Mareque-Rivas, Darren W. Sexton Orcid Logo, Stephen Fôn Hughes Orcid Logo, Steve Conlan Orcid Logo, David Mark Davies, Andrew R. Tee Orcid Logo

Cancers, Volume: 17, Issue: 16, Start page: 2714

Swansea University Authors: James Cronin Orcid Logo, Steve Conlan Orcid Logo

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DOI (Published version): 10.3390/cancers17162714

Abstract

Background/Objectives: Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and holds promise as a therapeutic strategy against cancers with elevated iron metabolism. However, many tumors evade ferroptosis through the upregulation of specialized antioxidant defe...

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Published in: Cancers
ISSN: 2072-6694
Published: MDPI AG 2025
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URI: https://cronfa.swan.ac.uk/Record/cronfa70650
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However, many tumors evade ferroptosis through the upregulation of specialized antioxidant defense mechanisms. Here, we investigated ferroptosis susceptibility and resistance mechanisms in TSC models and in ovarian and breast cancer cell lines, aiming to identify potential therapeutic targets. Methods: Ferroptosis sensitivity was assessed using RSL3 and erastin. We explored the contribution of ferroptosis defense pathways using inhibitors of NRF2 (ML385) and FSP1 (iFSP1). RNA sequencing was performed to evaluate the expression of ferroptosis resistance genes and to explore NRF2-regulated transcriptional programs. Results: TSC2-deficient cells were resistant to RSL3- and erastin-induced ferroptosis. This resistance correlated with upregulation of ferroptosis defense genes, including NRF2 and its downstream targets. Pharmacological inhibition of NRF2 resensitized TSC2-deficient cells to ferroptosis, confirming a protective role for NRF2. However, FSP1 inhibition did not restore ferroptosis sensitivity in TSC2-deficient angiomyolipoma cells. In contrast, FSP1 knockdown significantly enhanced ferroptosis sensitivity in ovarian (PEO1, PEO4, OVCAR3) and breast (MDA-MB-436) cancer cells. Notably, in MDA-MB-436 cells, FSP1 knockdown was more effective than NRF2 inhibition to enhance ferroptosis sensitivity. FSP1 expression was not regulated by NRF2, suggesting that NRF2-targeted therapies alone may be insufficient to overcome ferroptosis resistance in certain cancer contexts. Conclusions: TSC2-deficient cells resist ferroptosis via an adaptive antioxidant response that protects against elevated iron-mediated lipid peroxidation. Our findings identify NRF2 and FSP1 as key, but mechanistically distinct, regulators of ferroptosis resistance. The differential efficacy of targeting these pathways across cancer types highlights the potential need for patient stratification. Dual targeting of NRF2 and FSP1 may offer an effective therapeutic strategy for iron-dependent, ferroptosis-resistant cancers.</abstract><type>Journal Article</type><journal>Cancers</journal><volume>17</volume><journalNumber>16</journalNumber><paginationStart>2714</paginationStart><paginationEnd/><publisher>MDPI AG</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint/><issnElectronic>2072-6694</issnElectronic><keywords>ferroptosis; tuberous sclerosis complex; mTOR; cancer resistance; NRF2; FSP1; RSL3</keywords><publishedDay>21</publishedDay><publishedMonth>8</publishedMonth><publishedYear>2025</publishedYear><publishedDate>2025-08-21</publishedDate><doi>10.3390/cancers17162714</doi><url/><notes/><college>COLLEGE NANME</college><department>Medical School</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>MEDS</DepartmentCode><institution>Swansea University</institution><apcterm>Another institution paid the OA fee</apcterm><funders>This research was funded by Welsh Government&#x2019;s European Social Fund (ESF) convergence programme for West Wales and the Valleys KESS II and BCUHB to T.T., S.F.H., R.S.C., D.M.D. and A.R.T.; Cancer Research Wales PhD studentship, grant number 2504 to D.K.M., D.M.D. and A.R.T.; Cancer Research Wales Fellowship, grant number 2527 to D.K.M., J.G.C., J.C.M., D.M.D. and A.R.T.; Tuberous Sclerosis Association, grant number 2018-S04 to J.D.C., D.M.D. and A.R.T.; King Fahd Security College/Ministry of Interior/Saudi Arabia, grant number 1050795978 to M.A.M.A. and A.R.T.; and Health and Care Research Wales (Wales Gene Park), grant number R21CA263133 to A.R.T.</funders><projectreference/><lastEdited>2025-12-12T11:46:13.7387384</lastEdited><Created>2025-10-13T17:12:38.0168453</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Biomedical Science</level></path><authors><author><firstname>Tasmia</firstname><surname>Tahsin</surname><order>1</order></author><author><firstname>Darius K.</firstname><surname>McPhail</surname><orcid>0009-0006-4490-2972</orcid><order>2</order></author><author><firstname>Jesse D.</firstname><surname>Champion</surname><order>3</order></author><author><firstname>Mohammad A. M.</firstname><surname>Alzahrani</surname><order>4</order></author><author><firstname>Madeleine L.</firstname><surname>Hilditch</surname><order>5</order></author><author><firstname>Alexandre</firstname><surname>Faris-Orr</surname><order>6</order></author><author><firstname>Brian L.</firstname><surname>Calver</surname><order>7</order></author><author><firstname>James</firstname><surname>Cronin</surname><orcid>0000-0002-0590-9462</orcid><order>8</order></author><author><firstname>Juan C.</firstname><surname>Mareque-Rivas</surname><order>9</order></author><author><firstname>Darren W.</firstname><surname>Sexton</surname><orcid>0000-0003-3344-3150</orcid><order>10</order></author><author><firstname>Stephen F&#xF4;n</firstname><surname>Hughes</surname><orcid>0000-0001-6558-9037</orcid><order>11</order></author><author><firstname>Steve</firstname><surname>Conlan</surname><orcid>0000-0002-2562-3461</orcid><order>12</order></author><author><firstname>David Mark</firstname><surname>Davies</surname><order>13</order></author><author><firstname>Andrew R.</firstname><surname>Tee</surname><orcid>0000-0002-5577-4631</orcid><order>14</order></author></authors><documents><document><filename>70650__35806__f552311686034e1c9ce157e18776e051.pdf</filename><originalFilename>70650.VoR.pdf</originalFilename><uploaded>2025-12-12T11:44:00.1286096</uploaded><type>Output</type><contentLength>4650576</contentLength><contentType>application/pdf</contentType><version>Version of Record</version><cronfaStatus>true</cronfaStatus><documentNotes>&#xA9; 2025 by the authors. 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spelling 2025-12-12T11:46:13.7387384 v2 70650 2025-10-13 Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells 9cfd17551c0d1f7438895121e4fbb6e8 0000-0002-0590-9462 James Cronin James Cronin true false 0bb6bd247e32fb4249de62c0013b51cb 0000-0002-2562-3461 Steve Conlan Steve Conlan true false 2025-10-13 MEDS Background/Objectives: Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and holds promise as a therapeutic strategy against cancers with elevated iron metabolism. However, many tumors evade ferroptosis through the upregulation of specialized antioxidant defense mechanisms. Here, we investigated ferroptosis susceptibility and resistance mechanisms in TSC models and in ovarian and breast cancer cell lines, aiming to identify potential therapeutic targets. Methods: Ferroptosis sensitivity was assessed using RSL3 and erastin. We explored the contribution of ferroptosis defense pathways using inhibitors of NRF2 (ML385) and FSP1 (iFSP1). RNA sequencing was performed to evaluate the expression of ferroptosis resistance genes and to explore NRF2-regulated transcriptional programs. Results: TSC2-deficient cells were resistant to RSL3- and erastin-induced ferroptosis. This resistance correlated with upregulation of ferroptosis defense genes, including NRF2 and its downstream targets. Pharmacological inhibition of NRF2 resensitized TSC2-deficient cells to ferroptosis, confirming a protective role for NRF2. However, FSP1 inhibition did not restore ferroptosis sensitivity in TSC2-deficient angiomyolipoma cells. In contrast, FSP1 knockdown significantly enhanced ferroptosis sensitivity in ovarian (PEO1, PEO4, OVCAR3) and breast (MDA-MB-436) cancer cells. Notably, in MDA-MB-436 cells, FSP1 knockdown was more effective than NRF2 inhibition to enhance ferroptosis sensitivity. FSP1 expression was not regulated by NRF2, suggesting that NRF2-targeted therapies alone may be insufficient to overcome ferroptosis resistance in certain cancer contexts. Conclusions: TSC2-deficient cells resist ferroptosis via an adaptive antioxidant response that protects against elevated iron-mediated lipid peroxidation. Our findings identify NRF2 and FSP1 as key, but mechanistically distinct, regulators of ferroptosis resistance. The differential efficacy of targeting these pathways across cancer types highlights the potential need for patient stratification. Dual targeting of NRF2 and FSP1 may offer an effective therapeutic strategy for iron-dependent, ferroptosis-resistant cancers. Journal Article Cancers 17 16 2714 MDPI AG 2072-6694 ferroptosis; tuberous sclerosis complex; mTOR; cancer resistance; NRF2; FSP1; RSL3 21 8 2025 2025-08-21 10.3390/cancers17162714 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University Another institution paid the OA fee This research was funded by Welsh Government’s European Social Fund (ESF) convergence programme for West Wales and the Valleys KESS II and BCUHB to T.T., S.F.H., R.S.C., D.M.D. and A.R.T.; Cancer Research Wales PhD studentship, grant number 2504 to D.K.M., D.M.D. and A.R.T.; Cancer Research Wales Fellowship, grant number 2527 to D.K.M., J.G.C., J.C.M., D.M.D. and A.R.T.; Tuberous Sclerosis Association, grant number 2018-S04 to J.D.C., D.M.D. and A.R.T.; King Fahd Security College/Ministry of Interior/Saudi Arabia, grant number 1050795978 to M.A.M.A. and A.R.T.; and Health and Care Research Wales (Wales Gene Park), grant number R21CA263133 to A.R.T. 2025-12-12T11:46:13.7387384 2025-10-13T17:12:38.0168453 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Tasmia Tahsin 1 Darius K. McPhail 0009-0006-4490-2972 2 Jesse D. Champion 3 Mohammad A. M. Alzahrani 4 Madeleine L. Hilditch 5 Alexandre Faris-Orr 6 Brian L. Calver 7 James Cronin 0000-0002-0590-9462 8 Juan C. Mareque-Rivas 9 Darren W. Sexton 0000-0003-3344-3150 10 Stephen Fôn Hughes 0000-0001-6558-9037 11 Steve Conlan 0000-0002-2562-3461 12 David Mark Davies 13 Andrew R. Tee 0000-0002-5577-4631 14 70650__35806__f552311686034e1c9ce157e18776e051.pdf 70650.VoR.pdf 2025-12-12T11:44:00.1286096 Output 4650576 application/pdf Version of Record true © 2025 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. true eng https://creativecommons.org/ licenses/by/4.0/
title Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
spellingShingle Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
James Cronin
Steve Conlan
title_short Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
title_full Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
title_fullStr Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
title_full_unstemmed Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
title_sort Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells
author_id_str_mv 9cfd17551c0d1f7438895121e4fbb6e8
0bb6bd247e32fb4249de62c0013b51cb
author_id_fullname_str_mv 9cfd17551c0d1f7438895121e4fbb6e8_***_James Cronin
0bb6bd247e32fb4249de62c0013b51cb_***_Steve Conlan
author James Cronin
Steve Conlan
author2 Tasmia Tahsin
Darius K. McPhail
Jesse D. Champion
Mohammad A. M. Alzahrani
Madeleine L. Hilditch
Alexandre Faris-Orr
Brian L. Calver
James Cronin
Juan C. Mareque-Rivas
Darren W. Sexton
Stephen Fôn Hughes
Steve Conlan
David Mark Davies
Andrew R. Tee
format Journal article
container_title Cancers
container_volume 17
container_issue 16
container_start_page 2714
publishDate 2025
institution Swansea University
issn 2072-6694
doi_str_mv 10.3390/cancers17162714
publisher MDPI AG
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str 1
active_str 0
description Background/Objectives: Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and holds promise as a therapeutic strategy against cancers with elevated iron metabolism. However, many tumors evade ferroptosis through the upregulation of specialized antioxidant defense mechanisms. Here, we investigated ferroptosis susceptibility and resistance mechanisms in TSC models and in ovarian and breast cancer cell lines, aiming to identify potential therapeutic targets. Methods: Ferroptosis sensitivity was assessed using RSL3 and erastin. We explored the contribution of ferroptosis defense pathways using inhibitors of NRF2 (ML385) and FSP1 (iFSP1). RNA sequencing was performed to evaluate the expression of ferroptosis resistance genes and to explore NRF2-regulated transcriptional programs. Results: TSC2-deficient cells were resistant to RSL3- and erastin-induced ferroptosis. This resistance correlated with upregulation of ferroptosis defense genes, including NRF2 and its downstream targets. Pharmacological inhibition of NRF2 resensitized TSC2-deficient cells to ferroptosis, confirming a protective role for NRF2. However, FSP1 inhibition did not restore ferroptosis sensitivity in TSC2-deficient angiomyolipoma cells. In contrast, FSP1 knockdown significantly enhanced ferroptosis sensitivity in ovarian (PEO1, PEO4, OVCAR3) and breast (MDA-MB-436) cancer cells. Notably, in MDA-MB-436 cells, FSP1 knockdown was more effective than NRF2 inhibition to enhance ferroptosis sensitivity. FSP1 expression was not regulated by NRF2, suggesting that NRF2-targeted therapies alone may be insufficient to overcome ferroptosis resistance in certain cancer contexts. Conclusions: TSC2-deficient cells resist ferroptosis via an adaptive antioxidant response that protects against elevated iron-mediated lipid peroxidation. Our findings identify NRF2 and FSP1 as key, but mechanistically distinct, regulators of ferroptosis resistance. The differential efficacy of targeting these pathways across cancer types highlights the potential need for patient stratification. Dual targeting of NRF2 and FSP1 may offer an effective therapeutic strategy for iron-dependent, ferroptosis-resistant cancers.
published_date 2025-08-21T05:33:13Z
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