The pseudo-caspase FLIP(L) regulates cell fate following p53 activation Andrea Leesa,1, Alexander J. McIntyrea,1, Nyree T. Crawforda, Fiammetta Falconea, Christopher McCanna, Caitriona Holohana, Gerard P. Quinna, Jamie Z. Robertsa, Tamas Sesslera, Peter F. Gallaghera, Gemma M. A. Gregga, Katherine McAllistera, Kirsty M. McLaughlina, Wendy L. Allena, Laurence J. Eganb, Aideen E. Ryanb,c, Melissa J. Labonte-Wilsona, Philip D. Dunnea, Mark Wappetta, Vicky M. Coylea, Patrick G. Johnstona, Emma M. Kerra, Daniel B. Longleya,2,3, and Simon S. McDadea,2,3 aPatrick G Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast, Northern Ireland BT9 7BL, United Kingdom; bDiscipline of Pharmacology & Therapeutics, Lambe Institute for Translational Research, School of Medicine, College of Medicine, Nursing and Health Sciences, National University of Ireland Galway, Galway, Ireland; and cRegenerative Medicine Institute, College of Medicine, Nursing and Health Sciences, National University of Ireland Galway, Galway, Ireland Edited by Karen H. Vousden, Francis Crick Institute, London, United Kingdom, and approved June 8, 2020 (received for review February 12, 2020) p53 is the most frequently mutated, well-studied tumor-suppressor switch from cell-cycle arrest to cell death induction remain gene, yet the molecular basis of the switch from p53-induced cell- poorly understood, although p53-mediated transcriptional up- cycle arrest to apoptosis remains poorly understood. Using a combi- regulation of PUMA has been suggested to be crucial in a nation of transcriptomics and functional genomics, we unexpectedly number of studies (10, 11). identified a nodal role for the caspase-8 paralog and only human Despite p53/TP53 being the most frequently mutated gene in pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we iden- cancer, ∼50% of all tumors retain wild-type p53 (WT-p53) and tify FLIP(L) as a direct p53 transcriptional target gene that is rapidly typically circumvent or suppress p53’s functions through alter- up-regulated in response to Nutlin-3A, an MDM2 inhibitor that po- native nonmutational mechanisms (12). Reawakening the latent tently activates p53. Genetically or pharmacologically inhibiting ex- tumor suppressive functions in cancers retaining WT-p53 pression of FLIP(L) using siRNA or entinostat (a clinically relevant class- I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer therefore is an attractive clinical concept (12). Targeting the cells in response to Nutlin-3A, which otherwise predominantly in- p53-MDM2 interaction has received significant attention and duced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating che- Significance motherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking p53 is the most frequently mutated, well-studied tumor- activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, suppressor gene, yet the switch from p53-induced cell-cycle this activation was not dependent on receptor engagement by its arrest to apoptosis remains poorly understood. We un- ligand, TRAIL. In the absence of caspase-8, another of its paralogs, expectedly identify a critical role for the only human pseudo- caspase-10 (also transcriptionally up-regulated by p53), induced apo- caspase FLIP(L) in regulating this switch. Genetically or phar- ptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser macologically inhibiting expression of FLIP(L), which we iden- extent than in caspase-8-proficient cells. FLIP(L) depletion also modu- tify as a new p53 transcriptional target, promoted apoptosis lated transcription of canonical p53 target genes, suppressing p53- over cell-cycle arrest in response to p53 activation. Mechanis- induced expression of the cell-cycle regulator p21 and enhancing tically, FLIP(L) inhibited p53-induced apoptosis by blocking p53-induced up-regulation of proapoptotic PUMA. Thus, even in the ligand-independent activation of caspase-8 by the TRAIL-R2/ absence of caspase-8/10, FLIP(L) silencing promoted p53-induced ap- DR5 death receptor. Notably, FLIP(L) also modulated tran- optosis by enhancing PUMA expression. Thus, we report unexpected, scription of canonical p53 target genes: increasing expression therapeutically relevant roles for FLIP(L) in determining cell fate of cell-cycle regulator p21 and suppressing upregulation of following p53 activation. apoptosis-inducing PUMA. Thus, we report unexpected, ther- apeutically exploitable roles for FLIP(L) in regulating the switch p53 | apoptosis | FLIP | TRAIL-R2 | entinostat from p53-induced cell-cycle arrest to apoptosis. he mechanisms through which p53 activation differentially Author contributions: A.L., A.J.M., M.J.L.-W., P.D.D., V.M.C., P.G.J., E.M.K., D.B.L., and S.S.M. designed research; A.L., A.J.M., N.T.C., F.F., C.M., C.H., J.Z.R., T.S., P.F.G., Tregulates cell survival (by initiating cell-cycle arrest and DNA G.M.A.G., K.M., K.M.M., and E.M.K. performed research; G.P.Q., L.J.E., A.E.R., and M.W. damage repair) and cell death (by promoting apoptosis) remain contributed new reagents/analytic tools; A.L., A.J.M., N.T.C., F.F., C.H., J.Z.R., T.S., P.F.G., enigmatic (1). Canonically, phosphorylation of the p53 N ter- G.M.A.G., K.M., W.L.A., E.M.K., and S.S.M. analyzed data; and A.L., M.J.L.-W., P.D.D., minus blocks its interaction with the E3 ubiquitin ligase Mouse D.B.L., and S.S.M. wrote the paper. Double Minute 2 (MDM2) (2) reducing ubiquitination of the C The authors declare no competing interest. terminus and stabilizing p53. Stabilized p53 directly activates This article is a PNAS Direct Submission. target genes to induce cell-cycle arrest mediated by the cyclin- Published under the PNAS license. dependent kinase inhibitor p21/CDKN1A and other effectors (3, Data deposition: The raw data and RNA-Seq analyses discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI) Gene Ex- 4). In the event of sustained stress and/or irreparable DNA pression Omnibus (GEO) database and linked sequence read archive files, which are ac- damage, sequential posttranslational modification of p53 is cessible at accession no. GSE113682. thought to direct p53 activity toward proapoptotic target genes, 1A.L. and A.J.M. contributed equally to this work. including those encoding BAX, PUMA, and NOXA (5, 6) and 2D.B.L., and S.S.M. contributed equally to this work. the death receptor TRAIL receptor 2 (TRAIL-R2 also known as 3To whom correspondence may be addressed. Email: [email protected] or S.McDade@ death receptor-5, DR5) (7). We and others have contributed to qub.ac.uk. progress in identifying direct and indirect p53 target genes using This article contains supporting information online at https://www.pnas.org/lookup/suppl/ chromatin immunoprecipitation sequencing (ChIP-Seq) (8) doi:10.1073/pnas.2001520117/-/DCSupplemental. (summarized in ref. 9); however, the mechanisms underlying the First published July 13, 2020. 17808–17819 | PNAS | July 28, 2020 | vol. 117 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.2001520117 Downloaded by guest on September 25, 2021 has led to the development of the selective small molecule in- Fig. S1G). In the extrinsic pathway, crosstalk between caspase-8 hibitors of MDM2 (MDM2i) such as Nutlin-3A (13). While and the mitochondria is mediated by the BH3 protein BID, highly effective at stabilizing p53 (12), such agents promote which when cleaved by caspase-8 to its truncated form (tBID) variable cellular fates depending on cell type in all but MDM2- translocates to the mitochondria and promotes mitochondrial amplified cells, in which they induce cell death (14). The ob- outer membrane permeabilization (MOMP). In line with this, servation that potent p53 stabilization elicited by MDM2i is of- the mechanism of apoptosis induced by Nutlin-3A/siFLIP(L) was ten insufficient to induce cell death suggests the existence of significantly attenuated, although not completely prevented, in fundamental mechanisms that regulate the cell death-inducing BID knockout cells (Fig. 1 J and K). By comparison, the effects effects of stabilized p53. Strategies targeting such p53-mediated of the direct extrinsic apoptosis pathway activator isoleucine cell death inhibitory mechanisms have potential to not only en- zipper-TRAIL (IZ-TRAIL) were completely abrogated by BID hance MDM2i-induced cell death, but also to augment the ef- deletion, confirming this model as apoptotically type-2 (Fig. 1 J ficacy of p53-induced cell death induced by radiotherapy and and K). DNA damaging chemotherapies. In this study, we unexpectedly identify a critical role for the FLIP(L) Is a Direct, Acetylation-Dependent p53-Target Gene. From the apoptosis regulator and only human pseudo-caspase FLIP(L) Western blot analyses in Fig. 1, it was apparent that Nutlin-3A (Fas-associated death domain [FADD]-like interleukin-1β-con- treatment up-regulated expression FLIP(L) in p53-WT CRC verting enzyme inhibitory protein) in regulating the switch from cells. Reanalysis of previous ChIP-Seq data from our group in p53-induced cell-cycle arrest to apoptosis. Importantly, this bi- human foreskin keratinocytes treated with cisplatin (8) or pub- ology is therapeutically exploitable using class-I HDAC inhibi- licly available ChIP-Seq data from p53-WT MCF7 breast cancer tors, which prevent p53-mediated transcriptional up-regulation cells treated with Nutlin-3A (22) identified direct, treatment- of FLIP(L) leading to synergistic inductions of apoptosis when induced binding of p53 to the CFLAR/FLIP promoter (SI Ap- these agents are combined with Nutlin-3A or DNA damaging pendix, Fig. S2A), and we confirmed this in HCT116 cells fol- chemotherapy. lowing treatment with Nutlin-3A (Fig. 2A).