ENPP2 protects cardiomyocytes from erastin-induced ferroptosis
Yu-Ting Bai a, c, Rong Chang c, Hua Wang b, Feng-Jun Xiao b, Ri-Li Ge a, d, *, Li-Sheng Wang b, **
a Research Center for High Altitude Medicine, Qinghai University, Xining, 810001, PR China
b Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, 100850, PR China
c Qinghai Provincial People’s Hospital, Xining, PR China
d Key Laboratory for Application of High Altitude Medicine in Qinghai Province, Qinghai, Xining, PR China
A R T I C L E I N F O
Received 11 March 2018
Accepted 14 March 2018 Available online xxx
Cardiomyocytes Ferroptosis Erastin
A B S T R A C T
Ferroptosis is an iron- and oxidative-dependent form of regulated cell death and may play important roles in maintaining myocardium homeostasis and pathology of cardiovascular diseases. Currently, the regulatory roles of lipid signals in regulating cardiomyocytes ferroptosis has not been explored. In this study, we show that ENPP2, as a lipid kinase involved in lipid metabolism, protects against erastin- induced ferroptosis in cardiomyocytes. The classical ferroptosis inducer erastin remarkably inhibits the growth which could be rescued by the small molecule Fer-1 in H9c2 cells. Adenovirus mediated ENPP2 overexpression modestly promotes migration and proliferation and signiﬁcantly inhibits erastin-induced ferroptosis of H9c2 cells. ENPP2 overexpression leads to increase the LPA level in supernatant of H9c2 cells. H9c2 cells express the LPAR1, LPAR3, LPAR4 and LPAR5 receptors. The supernatant of ENPP2 transduced cardiomyocytes could protects the cells from erastin-induced ferroptosis of H9c2 cells. Furthermore, we observed that ENPP2 overexpression regulates ferroptosis-associated gene GPX4, ACSL4 and NRF2 expression and modulates MAPK and AKT signal in H9c2 cells. Collectively, these ﬁndings demonstrated that ENPP2/LPA protects cardiomyocytes from erastin-induced ferroptosis through modulating GPX4, ACSL4 and NRF2 expression and enhancing AKT survival signal.
Various types of cell death such as apoptosis, ferroptosis and autophagic cell death play an important role in myocardial ho- meostasis and pathology [1,2]. Ferroptosis is a recently identiﬁed type of regulated cell death characterized by the iron-dependent accumulation of lipid ROS . It is regulated by multiple signal pathways such as a6b4 integrin and NRF2-Keap1 axis [4,5]. Many regulators such as glutathione peroxidase 4 (GPX4), NRF2, p53, heme oxygenase-1 and acyl-CoA synthetase long-chain family member 4 (ACSL4) have been identiﬁed to be involved in regulation of cell ferroptosis [6e8]. Ferroptosis has been implicated in the pathology of a variety of diseases such as tumors, tissue or organ * Corresponding author. Research Center for High Altitude Medicine, Qinghai University, Xining, 810001, PR China.
** Corresponding author. Department of Experimental Hematology, Beijing Insti- tute of Radiation Medicine, 27 Taiping Road, Beijing, 100850, PR China. injury, stroke, ischemia-reperfusion injury and kidney degenera- tion [9,10]. However, its biological roles and regulation pathways in heart diseases remain poorly understood. New insight into obser- vation of cardiomyocytes ferroptosis may provide new diagnostic and therapeutic approaches for cardiovascular diseases.
Bioactive lipids such as lysophosphatidic acid (LPA) and sphingosine-phosphate (S1P) play important roles in both physio- logical and pathophysiological processes including angiogenesis, inﬂammation, ﬁbrosis and carcinogenesis [11e13]. LPA, a bioactive phospholipids with diverse functions, acts as an autocrine/para- crine messenger by activation of its G protein-coupled receptors . Autotaxin (ATX), also termed ENPP2, is a secreted enzyme important for generating LPA . Disturbances in normal ATX-LPA signaling is associated to a range of diseases including cardiovas- cular disease. Cardiomyocytes express LPA receptor subtypes including LPA1-LPA5. These receptors mediate LPA-induced hy- pertrophy of neonatal cardiac myocytes via activation of Gi and Rho . LPA also mediated augmentation of cardiomyocytes lipopro- tein lipase and involved in both acute and chronic ischemic cardiac damage [17,18]. However, the regulatory roles of ATX-LPA signaling in regulating of cardiomyocytes characteristics have less been explored. In this study, we investigated the regulatory roles of ATX- LPA signaling in erastin-induced ferroptosis of cardiomyocytes and elucidate its mechanisms.
2. Materials and methods
2.1. Cell culture
The rat embryonic cardiomyoblast H9c2 cells were purchased from the ATCC. The H9c2 cells were maintained in DMEM medium (Gibico, Carlsbad, CA, USA) supplemented with 10% FBS (Hyclone, Logan, UT) and propagated at 37 ◦C under 5% CO2.
2.2. Cell viability assays
H9c2 cells were cultured in 96-well plates at 5000 cells/well and treated with erastin (Tocris, Minneapolis, MN, USA) or Ferrostatin-1 (Fer-1) (Sigma-Aldrich, St Louis, MO, USA) at different concentra- tions for 24 h. Then CCK-8 (Dojindo Molecular Technologies, Kumamoto, Japan) were added and re-incubated at 37 ◦C under 5% CO2 for 3 h. The absorbance at a wavelength of 450 nm was measured by a microplate reader. The percentage of cell viability was calculated according to the following formula: Percent of cell viability ¼ OD treatment group/OD control group × 100%.
2.3. Adenovirus production and transduction of H9c2 cells
Adenovirus vector carrying the ENPP2 gene (Ad.ENPP2) was purchased from ViGene Biosciences(Shandong, China). The H9c2 cells were infected with Ad.ENPP2 and control vector (Ad.Null) at 20 multiplicity of infection (MOI) for 48 h, then these cells were applied for further assays including proliferation, migration and ferroptosis.
2.4. RNA extraction and real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA was extracted from H9c2 cells by using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and cDNA was synthesized using RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientiﬁc, Wilmington, DE) according to the manufac- turer’s instructions. The LPAR1-5 primers were designed and syn- thesized in Tsingke (Beijing, China) and the primers were as follows: LPAR1, sense 50-tcttctgggccattttcaa-30 and antisense 50- gccgttggggttctcgtt-30; LPAR2, sense 50-caatctgccgcttgactgga-30 and antisense 50-taaccagcaggttggtcagcaata-30; LPAR3, sense 50-tcttag- gagccttcgtggtgt-30 and antisense 50-gctgatgctgtcctccaggta-30; LPAR4, sense 50-agtgcgagttgcccgtttac-30 and antisense 50-ttagtt- caaaccaaactctgacacc-30; LPAR5, sense 50-tgccaattcttcagccaaca-30 and antisense 50-ggaagacccagagagccaga-30; b-actin, sense 50-gga- gattactgccctggctccta-30 and antisense 50-gactcatcgtactcctgcttgctg. qPCR was performed for amplifying target genes by using the SYBR® Premix Ex Taq kit (TaKaRa, Dalian, China).
The primers of ENPP2 and GAPDH were purchased from Applied Biosystems (ENPP2 ID: Hs00905125_m1; GAPDH ID:
Hs02758991_g1) and were carried out according to protocol of the TaqMan® Gene Expression Assays (Applied Biosystems, FosterCity, CA, USA).
2.5. Western blotting
Total proteins were extracted from H9c2 cells using RIPA buffer containing 1% Phenylmethane sulfonylﬂuoride (PMSF). The con- centrations of protein were determined by a BCA Protein Assay Kit (Thermo Fisher Scientiﬁc, Rockford, IL, USA). Equivalent amounts of proteins were loaded on 10% SDS polyacrylamide gels for electro- phoresis and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline for 2 h and then labeled with primary antibodies (anti-GAPDH [Cell Signaling], anti-ENPP2 [abcam], anti-GPX4 [abcam], anti-ACSL4 [abcam], anti- NRF2 [abcam], anti-AKT [Cell Signaling], anti-P-AKT [C31E5E, Cell Signaling], anti-p44/42 MAPK [Erk1/2, Cell Signaling], anti-P-p44/ 42 MAPK [T202/Y204, Cell Signaling]). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibodies (ZhongShan Golden Bridge Biotechnology, Beijing, China) and visualized using enhanced chemiluminescence (Thermo Fisher Scientiﬁc, Rockford, USA).
2.6. Wound healing assays
Scratched a line across the monolayer with a sterile pipette tip after transfection cells were conﬂuent on 6-well plates. Cell debris were washed and obtained the images of the wound. Cells migrating into the scratched area were acquired, counted and imaged after cells were incubated for 6 h and 12 h.
2.7. Cell migration
Cell migration was also performed using transwell chambers (8 mM membrane pores; Cambridge, MA, USA). After transfection, cells were resuspended with serum-free medium at 2 104/well in the top well. DMEM with 15% FBS was added to the lower chamber. After incubation for 10 h, cells that migrated to the lower of the ﬁlter membranes were ﬁxed and stained with crystal violet. Then the number of cells was counted under microscope.
2.8. Measurement of reactive oxygen species production
1 × 10^5 cells/well were seeded in 6-well plates. Cells were treated with DMSO (control), erastin (5 mM) and Ferrostatin-1 (1 mM) for 6 h. Cells were harvested by trypsinization and collected by centrifuge, then washed with Hanks Balanced Salt Solution (HBSS, Gibico) and resuspended in HBSS containing C11- BODIPY581/591 (1 mM, ThermoFisher Scientiﬁc) incubated for 10 min at 37 ◦C. ROS were assayed using ﬂuorescence activated cell sorting (FACS Calibur) and analyzed using Cell Quest software (BD Biosciences, San Jose, CA).
2.9. ELISA analysis
Supernatants of H9c2 cells with or without ENPP2 over- expression were subjected to ELISA analysis for their content of LPA. LPA ELISA kits purchased from Guduo Biotechnology (Shanghai, China) were used according to the manufacturer’s instructions.
2.10. Statistical analyses
All experiments were repeated at least three times. The results are presented as mean ± SD. ANOVA and Student’s t-tests were performed using Graphpad Prism software (Graphpad Software, La Jolla, CA, USA). A probability level of 0.05 (P < 0.05) was considered to be statistically signiﬁcant. 3. Results 3.1. Erastin induces ferroptosis of H9c2 cardiomyocytes Erastin is a classic ferroptosis activator and triggers cell ferrop- tosis in a variety of cell types. To establish the ferroptosis model of cardiomyocytes, we treated H9c2 cells with erastin at different concentrations in the absence or presence of the small molecule Fer-1 and determined their viability by CCK8 assay. Treatment of H9c2 cells with erastin results in signiﬁcant reduced cell viability which could be rescued by Fer-1 (Fig. 1A). Whereas H2O2 induced growth inhibition could not be rescued by Fer-1 (Fig. 1B). It is demonstrated that erastin-induced growth inhibition is dependent on Fe. To further verify that erastin-induced cell growth inhibition was due to ferroptosis, we checked the morphology changes of erastin treated cells. These cells were characterized by cytological changes, including cell shrinkage and increased mitochondrial membrane densities which is different from H2O2 treated cells (Fig. 1C). Ferroptosis is a novel form of regulated cell death that is dependent on iron and reactive oxygen species (ROS). We further checked the ROS generation in erastin-treated cells. Erastin treat- ment induces a rapid generation of ROS in H9c2 cells, which could be reversed by Fer-1 (Fig. 1D). Thus, it is demonstrated that erastin induces ferroptosis of H9c2 cardiomyocytes. 3.2. Adenovirus-mediated ENPP2 overexpression modestly promotes migration and proliferation of H9c2 cells To investigate the effect of ENPP2 overexpression on car- diomyocytes ferroptosis, we transduced H9c2 cells with Ad.ENPP2 and control vector. Western blot analysis showed that both the ENPP2 mRNA and protein are increased in ENPP2 transduced cells (Fig. 2AeB). The proliferation and migration of ENPP2 transduced cells were further checked. ENPP2 overexpression increases the migration ability of H9c2 cells in both transwell and would healing assays (Fig. 2CeD). ENPP2 overexpression also modestly promotes the proliferation ability under normal culture conditions (Fig. 2E). It is indicated that ENPP2 overexpression modestly promotes migration and proliferation of H9c2 cells. 3.3. ENPP2 overexpression inhibits erastin-induced ferroptosis of H9c2 cardiomyocytes To investigate the effect of ENPP2 overexpression on car- diomyocytes ferroptosis, we further checked the viability of erastin-treated H9c2 cells transduced with Ad.ENPP2 and control vector respectively. Cell viability assays revealed that Ad.ENPP2 transduced cells were less sensitive to erastin compared to control vector transduced cells (Fig. 3A). The morphology of ENPP2 trans- duced cells also shown the less ferroptosis changes compared to control vector transduced cells (Fig. 3B). Since erastin-induced ferroptosis is reactive oxygen species (ROS)-dependent, we measured the total cellular ROS generation after erastin treatment in H9c2 cells using FACS. The FACS quantiﬁcation analysis revealed that erastin treatment led to 1.494 ± 0.057-fold increase of ROS levels in control vector transduced cells. ENPP2 overexpression could suppress the erastin-induced ROS generation (Fig. 3C). These data indicate that ENPP2 overexpression protects the car- diomyocytes from erastin-induced ferroptosis. 3.4. ENPP2/LPA signal regulates the expression of ferroptosis- related genes in H9c2 cells ENPP2 is an enzyme that generates the bioactive lipid mediator LPA. Thus, we assayed the LPA level in supernatant of H9c2 cells transduced with Ad.ENPP2. The LPA levels in supernatant of ENPP2 transduced cells increase signiﬁcantly compared to that of control cells (Fig. 4A). To study the roles of ENPP2/LPA in regulation of cardiomyocytes functions, we also checked the expression of LPA receptors by H9c2 cells. Cardiomyocytes express mRNA for four LPA receptor subtypes (LPAR1, LPAR3, LPAR4, LPAR5) as indicated by qRT-PCR analysis (Fig. 4B). The supernatant of ENPP2 transduced cells could signiﬁcantly rescue the H9c2 cells from erastin-induced ferroptosis, suggesting ENPP2/LPA are the important regulators in ferroptosis of H9c2 cells (Fig. 4C). To investigate the mechanisms that ENPP2/LPA inhibits erastin- induced ferroptosis, we detected the protein level of ferroptosis regulator GPX4, ACSL4 and NRF2 in transduced H9c2 cells. GPX4 is a phospholipid hydroperoxidase that protects cells against mem- brane lipid peroxidation in ferroptosis. The protein level of GPX4 was upregulated in ENPP2 transduced H9c2 cells both in the absence and presence of erastin (Fig. 4D). ACSL4 is a biomarker and contributor of ferroptosis and dictates ferroptosis sensitivity by shaping cellular lipid composition. The expression of ACSL4 was remarkably downregulated in ENPP2 transduced cells which treated with erastin (Fig. 4E). NRF2 is also signiﬁcantly down- regulated in ENPP2 transduced (Fig. 4F). Collectively, these ﬁndings conﬁrm that ENPP2/LPA regulates GPX4, ACSL4 and NRF2 protein expression in erastin-induced ferroptosis of H9c2 cells. We further checked the activation of MAPK and AKT signaling in ENPP2 transduced cells treated with erastin. ENPP2 transduction could signiﬁcantly enhance the active P-AKT activation in erastin- treated cells. Whereas, it have slightly effect on activation of MAPK (Fig. 4GeH). It is conﬁrming that ENPP2/LPA signal sup- presses the ferroptosis probably through affecting the AKT pathway in H9c2 cells. 4. Discussion ENPP2/LPA signaling is involved in regulating various types of regulated cell death such as apoptosis, necroptosis and autophagy [19,20]. Ferroptosis was recently reported as a novel mechanism of iron-dependent regulated cell death. Its roles in myocardial ho- meostasis and pathology of heart diseases are not well understood. In this study, we demonstrated that ENPP2/LPA inhibits erastin- induced ferroptosis in cardiomyocytes and may play a novel role in myocardial homeostasis. Several small molecules, such as erastin and RSL3, are reported to induce ferroptosis in both normal and cancer cells [21,22]. We conﬁrmed that erastin-induced growth inhibition is an ideal ferroptosis model for cardiomyocytes. The erastin inhibits the growth of H9c2 cells in a Fe-dependent manner, because antiox- idants Fer-1 could signiﬁcantly rescue it. Whereas the H2O2- induced toxicity of H9c2 cells, most caused through apoptosis and necrosis, could not be rescued by Fer-1 . Furthermore, the erastin-induced ferroptosis exhibited morphologically distinct and disparate from H2O2-induced cell death. Ferroptosis is mediated through the redoxactive metal Fe and ROS generation. We further conﬁrmed that erastin-induced the ROS increase in erastin-treated H9c2 cells. All these observations suggested that erastin-induced ferroptosis is an ideal ferroptosis model of cardiomyocytes. Given the important roles of ENPP2/LPA signals in regulating the cardiomyocyte functions, we further checked the effect of ENPP2/ LPA on H9c2 cells ferroptosis and its mechanisms. Adenovirus mediated ENPP2 overexpression slightly affects the proliferation and migration of H9c2 cells, but signiﬁcantly inhibits the erastin- induced ferroptosis. ENPP2 overexpression also inhibits the erastin-induced ROS generation. Morphologically changes in ENPP2 transduced cells show resistance to erastin. These observa- tions suggested ENPP2/LPA plays important roles in regulating ferroptosis of cardiomyocytes. The mechanisms that ENPP2/LPA signal in suppressing ferrop- tosis were further investigated. H9c2 cardiomyocytes express the LPA receptor, LPAR1, LPAR3, LPAR4 and LPAR5. Adenovirus medi- ated ENPP2 overexpression leads to increase LPA level in cell su- pernatants. And the supernatants from H9c2 ENPP2 transduced cells exhibit suppressive effect on erastin-induced ferroptosis. It is suggested that autocrine ENPP2/LPA might play a central role in regulation cardiomyocytes ferroptosis. Several genes such as GPX4, ACSL4 and NRF2 have recently been identiﬁed to regulate ferrop- tosis by directly or indirectly targeting iron metabolism and lipid peroxidation. GPX4 is an antioxidant enzyme which prevents the iron-mediated reactions of peroxides and induces ferroptotic cell death . ENPP2 overexpression causes upregulation of GPX4 in H9c2 cells. The tumor suppressor p53 has been demonstrated to promote ferroptosis via a transcription-dependent mechanism . NRF2 is a major target of ARF in p53-independent tumor suppression and considered as an executive molecule in ferroptosis . NRF2 inhibition reverses the resistance of cancer cells to artesunate-induced ferroptosis [26,27]. It is indicated that NRF2 is as major regulator in ferroptosis. ACSL4 acts as an essential component for ferroptosis execution [28,29]. In erastin-induced ferroptosis of H9c2 cells, both NRF2 and ACSL4 are increased, whereas ENPP2 overexpression reduces their expression in erastin- treated H9c2 cells. The a6b4 integrin has been reported to protect adherent epithelial and carcinoma cells from ferroptosis induced by erastin . It also had been reported to promote expression of ENPP2 autocrine and cooperates with LPA to activate multiple signals such as Rho and Rac small GTPases [30,31]. The collabora- tion with integrins may contribute to the inhibitory effect of ENPP2/ LPA on H9c2 cell ferroptosis. In summary, our studies have identiﬁed that ENPP2/LPA signal protects cardiomyocytes against ferroptosis through regulating ferroptosis related gene GPX4, ACSL4 and NRF2 and modulating cell survival signals. These ﬁndings demonstrated that ENPP2/LPA signal pathway plays an important role in regulation of ferroptosis of cardiomyocytes. he effect of supernatant of H9c2 cells transduced with Ad.ENPP2 or control vector on the viability of erastin-treated H9c2 cells. (DeH) H9c2 cells infected with 20 MOI of Ad.ENPP2 or Ad.Null. 24 h later, cells were treatment with erastin (5 mM). Cells were incubated for another 24 h, and then, the GPX4, ACSL4, NRF2, P-AKT, P-MAPK was detected by Western bloting. All the data were repeated at least three times and are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ad.Null group. Conﬂicts of interest statement None declared. Acknowledgements This work was supported by grants from National Natural Sci- ence Foundation of China (No.81470320, No.31571231, No.81573086) and Qinghai-Utah Joint Research Key Lab for High Altitude Medicine (No.2014-ZJ-Y39). References  H. Akazawa, Mechanisms of cardiovascular homeostasis and pathophysiologyefrom gene expression, signal transduction to cellular communication, Circ. J. 79 (12) (2015) 2529e2536.  S. Lavandero, et al., Autophagy in cardiovascular biology, J. Clin. Invest. 125 (1) (2015) 55e64.  B. Lu, et al., The role of ferroptosis in cancer development and treatment response, Front. Pharmacol. 8 (2017) 992.  Fan, Z., et al., Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis. 2017. 6(8): p. e371.  Brown, C.W. and J.J. Amante, The alpha6beta4 integrin promotes resistance to ferroptosis. 2017. 216(12): p. 4287e4297.  L.C. Chang, et al., Heme oxygenase-1 mediates BAY 11-7085 induced ferrop- tosis, Canc. Lett. 416 (2018) 124e137.  I. Ingold, et al., Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis, Cell 172 (3) (2018) 409e422 e21.  A. Tarangelo, et al., p53 suppresses metabolic stress-induced ferroptosis in cancer cells, Cell Rep. 22 (3) (2018) 569e575.  B.R. Stockwell, et al., Ferroptosis: a regulated cell death nexus linking meta- bolism, redox biology, and disease, Cell 171 (2) (2017) 273e285.  S.J. Guiney, et al., Ferroptosis and cell death mechanisms in Parkinson's dis- ease, Neurochem. Int. 104 (2017) 34e48.  Y.A. Hannun, L.M. Obeid, Sphingolipids and their metabolism in physiology and disease, Nat. Rev. Mol. Cell Biol. 19 (3) (2018) 175e191.  N.J. Pyne, S. Pyne, Sphingosine 1-phosphate receptor 1 signaling in mammalian cells, Molecules 22 (3) (2017).  X. Tang, M.G. Benesch, D.N. Brindley, Lipid phosphate phosphatases and their roles in mammalian physiology and pathology, J. Lipid Res. 56 (11) (2015) 2048e2060.  Y.C. Yung, N.C. Stoddard, J. Chun, LPA receptor signaling: pharmacology, physiology, and pathophysiology, J. Lipid Res. 55 (7) (2014) 1192e1214.  A. Perrakis, W.H. Moolenaar, Autotaxin: structure-function and signaling, J. Lipid Res. 55 (6) (2014) 1010e1018.  R. Hilal-Dandan, et al., Lysophosphatidic acid induces hypertrophy of neonatal cardiac myocytes via activation of Gi and Rho, J. Mol. Cell. Cardiol. 36 (4) (2004) 481e493.  T. Pulinilkunnil, et al., Lysophosphatidic acid-mediated augmentation of car- diomyocyte lipoprotein lipase involves actin cytoskeleton reorganization, Am. J. Physiol. Heart Circ. Physiol. 288 (6) (2005) H2802eH2810.  A. Abdel-Latif, et al., Lysophospholipids in coronary artery and chronic ischemic heart disease, Curr. Opin. Lipidol. 26 (5) (2015) 432e437.  G.O. Latunde-Dada, Ferroptosis: role of lipid peroxidation, iron and ferriti- nophagy, Biochim. Biophys. Acta 1861 (8) (2017) 1893e1900.  W.S. Yang, B.R. Stockwell, Ferroptosis: death by lipid peroxidation, Trends Cell Biol. 26 (3) (2016) 165e176.  J. Dachert, et al., RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death, Oncotarget 7 (39) (2016) 63779e63792.  Shintoku, R., et al., Lipoxygenase-mediated generation of lipid peroxides en- hances ferroptosis induced by erastin and RSL3. 2017. 108(11): p. 2187e2194.  Y. Li, et al., Corin protects H2O2-induced apoptosis through PI3K/AKT and NF- kappaB pathway in cardiomyocytes, Biomed. Pharmacother. 97 (2018) 594e599.  V.S. Viswanathan, et al., Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway, Nature 547 (7664) (2017) 453e457.  Y. Xie, et al., The tumor suppressor p53 limits ferroptosis by blocking DPP4 activity, Cell Rep. 20 (7) (2017) 1692e1704.  D. Chen, et al., NRF2 is a major target of ARF in p53-independent tumor suppression, Mol. Cell. 68 (1) (2017) 224e232 e4.  J.L. Roh, et al., Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis, Redox Biol. 11 (2017) 254e262.  Doll, S., et al., ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. 2017. 13(1): p. 91e98.  H. Yuan, et al., Identiﬁcation of ACSL4 as a biomarker and contributor of ferroptosis, Biochem. Biophys. Res. Commun. 478 (3) (2016) 1338e1343.  K.L. O'Connor, M. Chen, L.N. Towers, Integrin alpha6beta4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation, Am. J. Physiol. Cell Physiol. 302 (3) (2012) C605eC614.  M. Chen, K.L. O'Connor, Integrin alpha6beta4 promotes expression of Erastin auto- taxin/ENPP2 autocrine motility factor in breast carcinoma cells, Oncogene 24 (32) (2005) 5125e5130.