Pyroptosis at the forefront of anticancer__immunity.pdf

REVIEWOpen AccessPyroptosis at the forefront of anticancerimmunityReid Loveless1,Ryan Bloomquist2and Yong Teng3*AbstractTumor resistance to apoptosis and the immunosuppressive tumor microenvironment are two major contributors topoor therapeutic responses during cancer intervention.Pyroptosis,a lytic and inflammatory programmed cell deathpathway distinct from apoptosis,has subsequently sparked notable interest among cancer researchers for itspotential to be clinically harnessed and to address these problems.Recent evidence indicates that pyroptosisinduction in tumor cells leads to a robust inflammatory response and marked tumor regression.Underlying itsantitumor effect,pyroptosis is mediated by pore-forming gasdermin proteins that facilitate immune cell activationand infiltration through their release of pro-inflammatory cytokines and immunogenic material following cellrupture.Considering its inflammatory nature,however,aberrant pyroptosis may also be implicated in the formationof a tumor supportive microenvironment,as evidenced by the upregulation of gasdermin proteins in certaincancers.In this review,the molecular pathways leading to pyroptosis are introduced,followed by an overview ofthe seemingly entangled links between pyroptosis and cancer.We describe what is known regarding the impact ofpyroptosis on anticancer immunity and give insight into the potential of harnessing pyroptosis as a tool andapplying it to novel or existing anticancer strategies.Keywords:Pyroptosis,Antitumor immunity,Gasdermin,cancer,The immune landscapeBackgroundWhile long evading discovery,the existence and physio-logical significance of programmed cell death(PCD)pathways distinct from apoptosis have garnered increas-ing interest in recent years,in part,due to the highprevalence of apoptosis resistance in tumors 1.Ofthese different forms,pyroptosis,a necrotic and lyticPCD,has distinguished itself from others by its ability toinduce a powerful inflammatory response 2.Similar tonecroptosis,a programmed form of necrosis,pyroptosisis believed to exist principally as a defense against patho-gens by triggering an antimicrobial response through thereleaseofimmunogeniccellularcontent,includingdamage-associated molecular patterns(DAMPs)andinflammatory cytokines 3.Unlike necroptosis,which ismediated by mixed lineage kinase domain-like pseudoki-nase(MLKL)and caspase-independent 4,pyroptosis ismediated by gasdermin(GSDM)family proteins and,like apoptosis,largely caspase-dependent 5.Otherforms of regulated necrosis,such as ferroptosis,havealso recently emerged 610 and are compared along-side necrosis and apoptosis in Table 1.The quest to overcome cancer and its grave globalconsequences has repeatedly led us to face the cheat ofdeath and detection by cancer cells.While still a rela-tively obscured process,pyroptosis represents a poten-tially harnessable and potent means to not only bypassapoptosis resistance but to activate tumor-specific im-munity and/or enhance the effectiveness of existing ther-apies.Here,we discussthe current knowledgeofpyroptosis in the context of anticancer immunity to giveinsight into its potential to fight cancer.The Author(s).2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use,sharing,adaptation,distribution and reproduction in any medium or format,as long as you giveappropriate credit to the original author(s)and the source,provide a link to the Creative Commons licence,and indicate ifchanges were made.The images or other third party material in this article are included in the articles Creative Commonslicence,unless indicated otherwise in a credit line to the material.If material is not included in the articles Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use,you will need to obtainpermission directly from the copyright holder.To view a copy of this licence,visit http:/creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver(http:/creativecommons.org/publicdomain/zero/1.0/)applies to thedata made available in this article,unless otherwise stated in a credit line to the data.*Correspondence:yong.tengemory.edu3Department of Hematology and Medical Oncology,Winship CancerInstitute,Emory University,201 Dowman Dr,Atlanta,GA 30322,USAFull list of author information is available at the end of the articleLoveless et al.Journal of Experimental&Clinical Cancer Research (2021)40:264 https:/doi.org/10.1186/s13046-021-02065-8Table 1 Comparison of select cell death formsInducersKey constituentsCharacteristicsCell releaseImmune featuresApoptosis(PCD)TNF-,FasL,TRAIL,Hypoxia,Irradiation,HeatshockBcl-2 protein family,P53,Caspase-2/3/6/7/8/9/10,HSPsPlasma membrane blebbing,Reduced cellularvolume,Nuclear fragmentation and chromatincondensationIn certain cases:DAMPs(e.g.,dsDNA,HMGB1,ATP,calreticulin)Mostly anti-inflammatory.Pro-inflammatory in cases involving therelease of DAMPsPyroptosis(PCD)DAMPs,PAMPs,MicrobialinfectionGSDM protein family,Caspase-1/3/4/5/8/11,InflammasomesPlasma membrane rupture and pore formation,Cytoplasmic swelling,Chromatin condensationIntracellular content,DAMPs(e.g.,IL-18,IL-1,dsDNA,ATP,HMGB1)Pro-inflammatoryNecroptosis(PCD)TNF-,TRAIL,Fas ligand,Microbial infectionMLKL,RIPK1/3(Necrosome),TRADDPlasma membrane rupture,Cytoplasmic andorganelle swelling,Moderate chromatincondensationIntracellular content,DAMPs(e.g.,IL-1,IL-33,IL-6,HSPs)Mostly pro-inflammatory.Anti-inflammatory in certain casesFerroptosis(PCD)ROS from ironaccumulation and lipidperoxidationGPX4,System XC,GSH,ACSL4No plasma membrane blebbing or rupture,Small mitochondria with ruptured outermembrane,Normal nucleusDAMPs(e.g.,HMGB1,dsDNA),lipid oxidization products(e.g.,4-HNE,LTB4)Pro-inflammatoryNecrosis(Accidental)Microbial infection,Toxins,Trauma,Ischemia,Thermal stressUnspecificPlasma membrane rupture,Cytoplasmic andorganelle swelling,Random DNA degradationIntracellular content,DAMPs(e.g.,IL-1,IL-33,dsDNA,ATP,HMGB1)Pro-inflammatoryACSL4,Acyl-CoA synthetase long-chain family member 4;Bcl-2,B-cell lymphoma 2;DAMPs,danger-associated molecular patterns;dsDNA,double-stranded DNA;GPX4,glutathione peroxidase 4;GSDM,gasdermin;GSH,glutathione;HMGB1,high-mobility group box protein 1;HSPs,heat shock proteins;IL,interleukin;LTB4,Leukotriene B4;MLKL,mixed lineage kinase domain-like protein;PAMPs,pathogen-associated molecularpatterns;PCD,programmed cell death;RIPK1/3,receptor-interacting serine/threonine-protein kinase 1/3;ROS,reactive oxygen species;TNF-,tumor necrosis factor-alpha;TRADD,TNFR-associated death protein;TRAIL,TNF-related apoptosis-inducing ligand;XC,cysteine/glutamate transporter receptor;4-HNE,4-HydroxynonenalLoveless et al.Journal of Experimental&Clinical Cancer Research (2021)40:264 Page 2 of 14Pyroptosis at a glancePyroptosis was first described in the 1990s in macro-phages infected with S.enterica serovar Typhimurium(S.Typhimurium)11 and S.flexneri 12.Although ori-ginally thought to be a process of apoptosis,furtherstudy revealed that this bacteria-induced cell death washeavily dependent on caspase-1 13,a caspase that isnot involved in apoptosis execution(i.e.,caspase-3).Shortly afterward in 2001,this PCD was coined pyropto-sis,or“fiery falling”,to describe the release of pro-inflammatory signals by the dying cells.Pyroptotic cellsshare several features with apoptotic cells,such as chro-matin condensation and DNA fragmentation,but aredistinguishable by their intact nucleus,pore formation,cell swelling,and osmotic lysis(Table 1)14.Generally,pyroptotic cell rupture is achieved through the caspase-mediated activation of pore-forming GSDM proteins fol-lowing the binding of DAMPs or pathogen-associatedmolecular patterns(PAMPs)15.These same caspasesmay also directly or indirectly contribute to the matur-ation of pro-inflammatory cytokines that,alongsideDAMPs,initiate or perpetuate an inflammatory responsewhen released.Although serving an important protective role inpathogen resolution,pyroptosis has been implicated as acomplicating factor in several human diseases,such ascardiovascular disease 16,neurodegenerative disease17,and HIV/AIDS 18.Metabolic disorders like dia-betes may also be promoted by pyroptosis throughchronic inflammation and the production of insulin-interfering cytokines 19.In cancer,the role of pyropto-sis appears to be double-edged.On one side,pyroptosiscan rapidly lead to tumor regression and,on the other,itcan facilitate the development of the tumor microenvir-onment.Hence,cancer cells may either suppress or in-cite pyroptosis to support their progression dependingon the context.Molecular mechanisms of pyroptosisAlthough the number of known pyroptosis pathways islikely to increase in the future,there are currently twoprincipal and several alternative pathways that have beenelucidated to date(Fig.1).In the principal pathways,pyroptosis is induced by GSDMD and involves inflam-matory caspase-1(canonical pathway)or caspase-4/5(ormouse caspase-11)(non-canonical pathway).Of the al-ternativepathways,themostwidelyregardedisGSDME-inducedpyroptosisthroughcaspase-35,though different pathways involving other GSDM familymembers and caspases or granzymes have also been re-ported.Structurally,GSDMA,GSDMB,GSDMC,GSDMD,and GSDME are all comprised of an N-terminalpore-forming domain and a C-terminal regulatory do-main that are joined by a linker region 20.Undernormal conditions,the linker region allows the C-terminal domain to fold over the top of the N-terminaldomain and functionally inhibit its lethal activity.Cleav-age at the linker site by caspases or granzymes,however,relinquishes this auto-inhibitory structure and leads tothe translocation of the N-terminal domain fragmentinto the plasma and mitochondrial membranes.Oncebound,the N-terminal domain oligomerizes and forms-barrel transmembrane pores that facilitate the secre-tion of pro-inflammatory content,like interleukin(IL)-1 and IL-18,and cause cell lysis through osmotic bar-rier disruption 21.In the subsequent sections,a sum-mary of the steps involved in each of the pathwaysleading to pyroptosis is provided.Canonical inflammasome pathwayIn the canonical inflammasome pathway to pyroptosis,recognition of DAMPs(e.g.,fibrinogen,heat shock pro-teins,DNA)and/or PAMPs(e.g.,flagellin,glycans,lipo-polysaccharides(LPSs)by pattern recognition receptors(PRRs)leads to the activation of respective cytosolic sig-naling complexes called inflammasomes,which are typ-ically comprised of a sensor protein,adaptor,andeffector caspase 22.Although a variety of PRRs,likeNOD-likereceptors(NLRs)andtoll-likereceptors(TLRs),are involved in this process,only a subset ofthese is known to be able to directly assemble inflamma-somes and activate the cysteine protease caspase-1 23.Specifically,the PRRs/inflammasome sensors in this sub-setincludeNLRfamilypyrindomain-containing(NLRP)1,NLRP3,NLRP4,absentinmelanoma2(AIM2),and Pyrin.Following their activation,the major-ity of these sensors interact with the adaptor proteinapoptosis-associatedspeck-likeproteincontainingCARD(ASC),which activates caspase-1 through pro-caspase-1 recruitment and cleavage.In addition to free-ing and activating the lethal N-terminal domain ofGSDMD(GSDMD-N),caspase-1 also matures pro-IL-1and pro-IL-18 into IL-1 and IL-18,which are releasedthrough the necrotic membrane pores formed by GSDMD-N 24.Non-canonical inflammasome pathwayIn contrast to the canonical inflammasome pathway,thenon-canonical inflammasome pathway is independent ofcaspase-1 and instead reliant on caspase-4 and-5 inhumans and caspase-11 in mice 25.The activation ofthese caspases occurs through the direct binding of LPSto respective pro-caspases and bypasses the need forinflammasome sensors.Originating from gram-negativebacteria,cytoplasmic delivery of LPS may occur throughinfection or membrane vesicles.Although these caspasesdo not activate IL-1 and IL-18 directly,their triggeringof pyroptosis through GSDMD cleavage leads to anLoveless et al.Journal of Experimental&Clinical Cancer Research (2021)40:264 Page 3 of 14efflux of potassium ions that activates the NLRP3inflammasome and upregulates the action of caspase-126.Alternative pathwaysIt was revealed that in certain contexts,such as chemo-therapy or targeted cancer therapy,a pathway fromapoptosis to pyroptosis can be induced through caspase-3 5.Although principally associated with apoptosisexecution and morphological changes,caspases-3 canmediate pyroptosis through the cleavage of GSDME,which similarly leads to GSDME-N pore formation andmembrane permeabilization.When GSDME levels arehigh,pyroptosis is rapidly prompted following caspase-3activation,but when GSDME levels are low,apoptosis isprompted instead 5.Considering that most of theFig.1 Schematic of pyroptosis signaling pathways.The canonical inflammasome pathway to pyroptosis is induced by various stimuli and resultsin caspase-1 activation,while the non-canonical pathway is induced by LPS and results in caspase-4/5 activation.Both activated caspase-1 andcaspase-4/5 cleave autoinhibited GSDMD at its linker region to free the N-terminal domain of GSDMD(GSDMD-N)from its repressor C-terminaldomain(GSDMD-C).GSDMD-N then translocates to the plasma membrane and undergoes oligomerization and pore formation,which causes anincrease in osmotic pressure and eventually cell lysis.Pore formation also facilitates the release of intracellular content and the inflammatorycytokines IL-18 and IL-1 following their activation by caspase-1.Through alternative pathways,GSDMD may also be cleaved by caspase-8,similarto GSDME,which can additionally be cleaved by caspase-3 and granzyme B.Aside,GSDMD-N and GSDMB-N can also respectively activate NLRP3or caspase-4.In the other alternative pathways,GSDMB is cleaved by caspase-1 or granzyme A,while GSDMC is cleaved by caspase-8 andtranscriptionally upregulated under hypoxia through pSTAT3 interaction with programmed death-ligand 1.The mechanisms of GSDMA-mediatedpyroptosis have yet to be elucidated.AIM2,absent in melanoma 2;DAMPs,danger-associated molecular patterns;FADD,Fas-associated deathdomain protein;GSDMA/B/C/D/E,gasdermin A/B/C/D/E;IL,interleukin;LPS,lipopolysaccharides;NLRP1/3/4,NLR family pyrin domain-containing1/3/4;PAMPs,pathogen-associated molecular patterns;RIPK1,receptor-interacting serine/threonine-protein kinase 1;pSTAT3,phospho-signaltransducer and activator of transcription 3;TAK1(also known MAP 3K7),transforming growth factor beta-activated kinase 1Loveless et al.Journal of Experimental&Clinical Cancer Research (2021)40:264 Page 4 of 14proteases involved in pyroptosis can also mediate apop-tosis when their respective GSDM protein is absent 27,28,it is suggestive that the balance between pyroptosisand apoptosis is largely dependent on GSDM proteinlevels.This notion requires further evidence,however,asit is contradicted by studies challenging the role ofGSDME in pyroptosis 29,30.Several