The multifaceted roles of gasdermins in cancer biology and oncologic therapies

BBA - Re iews on Cance 1876 (2021) 188635 A ailable online 14 Oc obe 2021 0304-419X/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Re iew A icle The mul i ace ed oles o gasde mins in cance biology and oncologic he apies Da id Sa i´ o a , b , * , Jeanne e Ma ínez-Val c , ´ Angela Molina-C espo a , b , Lau a S´ anchez c , Gema Mo eno-Bueno a , b , d , * a Biochemis y Depa men , UAM, & IIBm "Albe o Sols" CSIC-UAM, c/ A zobispo Mo cillo 4, 28029 Mad id, Spain. b Cen o de In es igaci´ on Biom´ edica en Red, ´ a ea de C´ ance (CIBERONC), c/ Melcho Fe n´ andez Almag o 3, 28029 Mad id, Spain. c Zoology, Gene ics and Physical An h opology Depa men , San iago de Compos ela Uni e si y, A da/ Al onso X O Sabio s/n, 27002 Lugo, Spain. d MD Ande son Cance Cen e Founda ion, c/ A u o So ia 270, 28033 Mad id, Spain. ARTICLE INFO Keywo ds: Gasde min Py op osis cance cell dea h cance ea men In lamma ion ABSTRACT The in ol emen o he Gasde min (GSDM) p o ein amily in cance and o he pa hologies is one o he ho es opics in biomedical esea ch. The e a e six GSDMs in humans (GSDMA, B, C, D, GSDME/DFNA5 and PJVK/ DFNB59) and, excep PJVK, hey can igge cell dea h mos ly by py op osis (a o m o ly ic and p o- in lamma o y cell dea h) bu also o he mechanisms. The exac ole o GSDMs in cance is in ica e, since depending on he biological con ex , hese p o eins ha e di e se cell-dea h dependen and independen unc- ions, exhibi ei he p o- umo o an i- umo unc ions, and p omo e ei he sensi iza ion o esis ance o onco- logic ea men s. In his e iew we p o ide a comp ehensi e o e iew on he mul i ace ed oles o he GSDMs in cance , and we c i ically discuss he possibili ies o exploi ing GSDM unc ions as de e minan s o an i-cance ea men and as no el he apeu ic a ge s, wi h special emphasis on inno a i e GSDM-di ec ed nano- he a- pies. Finally, we discuss he issues o be esol ed be o e GSDM-media ed oncologic he apies became a eali y a he clinical le el. 1. The gasde min gene amily The gasde min amily (GSDM) comp ises six genes in humans (GSDMA, B, C, D, GSDME/DFNA5, and PJVK/DFNB59) and en in mice (Gsdma1–3, Gsdmc1–4, Gsdmd, Gsdme, and D nb59) [1–3]. GSDME (A.K.A DFNA5, dea ness au osomal dominan 5) is he mos ancien GSDM gene, being p esen in some in e eb a es like co als [2,4], while GSDMB appea s only in speci ic mammalian species, being he only GSDM membe no p esen in he mouse and a genomes [2]. GSDM usually clus e in speci ic ch o- mosomal egions (17q21 con ains GSDMA and GSDMB; 8q24 GSDMC and GSDMD; Table 1) indica ing ha gene duplica ions occu ed du ing e eb a e e olu ion [2,4]. In ac , GSDMB likely o igina ed om a local duplica ion and DNA-s and in e sion o GSDMA in some mammals [2,4]. The name Gasde min o igina es om he “Gas ic and de mal” exp ession o GSDMA, he i s iden i ied GSDM gene [5]. GSDMs a e gene ally exp essed in he diges i e ac , whe e hey show speci ic exp ession pa - e ns: in he gas o-esophageal epi helium GSDMA is mos ly de ec ed in di e en ia ed cells, GSDMB in he basal laye s, GSDMC in he sup abasal and di e en ia ed egions and GSDMD in he di e en ia ing cells [1,3]. In addi ion, GSDMs show speci ic exp ession pa e ns in mul iple issues/o - gans. Fo example, GSDMA is exp essed in skin, lung, mamma y glands; GSDMB in li e , lung, colon and immune cells; GSDMC in skin and spleen; GSDMD in he in es ine and leukocy es; GSDME in ep oduc i e o gans and ne ous sys em and PJVK in es es and he audi o y ne ous sys em, among o he s [1–4,6,7]. 2. GSDM a he c oss oads be ween py op osis and o he cell dea h mechanisms GSDMs a e cy oplasmic p o eins (a ound 50 KDa) wi h nine Abb e ia ions: CT, C- e minal domain; DAMPs, damaged-associa ed molecula pa e ns; DCT, Deci abine; GZM, G anzyme; KO, Knock-ou ; LDH, Lac a e dehy- d ogenase; LPS, lipopolysaccha ide; NLRP3, NOD-like ecep o p o ein 3; NC, Nanocapsules; NT, N- e minal domain; NP, Nanopa icles; PLK1, Polo like kinase 1; ROS, eac i e oxygen species; SCC, Squamous Cell Ca cinoma; TGF-β, ans o ming g ow h ac o -β; TNF, Tumo nec osis ac o ; TSG, Tumo supp esso gene; WB, Wes e n blo ; WT, Wild- ype. * Co esponding au ho s a : Biochemis y Depa men UAM, & IIBm "Albe o Sols" CSIC-UAM, & CIBERONC E-mail add esses: [email p o ec ed] (D. Sa i´ o), [email p o ec ed] (G. Mo eno-Bueno). Con en s lis s a ailable a ScienceDi ec BBA - Re iews on Cance jou nal homepage: www.else ie .com/loca e/bbacan h ps://doi.o g/10.1016/j.bbcan.2021.188635 Recei ed 30 June 2021; Recei ed in e ised o m 28 Sep embe 2021; Accep ed 10 Oc obe 2021 BBA - Re iews on Cance 1876 (2021) 188635 2 conse ed mo i s (con aining leucine- ich egions) o s ill unknown unc ion, dis ibu ed h oughou he p o ein [3]. GSDMs, wi h he excep ion o PJVK, sha e a mode a ely conse ed N- e minal (NT) and C- e minal (CT) domains ha a e in e connec ed by a cen al hinge e- gion, which is speci ic o each amily membe [2]. Fo many yea s, GSDMs unc ions ha e been unclea un il he disco e y, in 2015, ha GSDMD igge in lammasome-dependen ly ic cell dea h [8–10] by o ming memb ane po es [11–15]. Fu he s udies e ealed ha o he GSDMs exhibi a simila p o ein 3D s uc u e [15–18] and sha e he p o- cell dea h unc ion ha is ac i a ed by a common mechanism [15,19,20]. The common model shows ha cy osolic GSDMs ha e an inac i e con o ma ion in which he NT po e- o ming domain is au o- inhibi ed by binding o speci ic esidues in he hyd ophobic co e o he CT [11–15,19,20]. This olded con o ma ion is main ained by he linke in e domain egion, which is clea ed by speci ic p o eases ac i- a ed a e pa icula s imuli (Fig. 1A). The eleased GSDM NT binds o speci ic acid lipids (e.g., phosphoinosi ides and phospha idylse ine) and inse s in o he cell memb ane in e nal laye , oligome izing and o ming la ge ansmemb ane po es [11–15,19,20] (Fig. 1B). The ups eam pa hways leading o GSDM NT elease a e speci ic o each GSDM [7,19,20]. Rega ding GSDMD, mul iple pa hogen signals (e.g., LPS, lagellin) o cell damage molecula pa e ns “DAMPs” (e.g., eac i e oxygen species “ROS”, dsDNA) ac i a e he mul ip o ein complex in lammasome, which hen p o okes he elease o GSDMD NT domain by he p o-in lamma o y caspases1/4/5/11 [7,19,20]. GSDMD NT po es acili a e he sec e ion o ma u e in lamma o y cy okines (IL1β, IL18) and o he in acellula molecules (i.e., Lac a e dehyd ogenase; LDH), while he in lux o wa e and ions migh be esponsible o cell swelling [7,19,20]. La e , he ac i a ion and oligome iza ion o Ninju in-1 (NINJ1) p oduces he ex ensi e memb ane up u e and cell lysis (Fig. 1B), which esul s in he elease o di e se in acellula con en , including he p o-in lamma o y DAMP HMGB1 [21]). This in lammasome/GSDMD-media ed mechanism is e med py op osis (“ ie y dea h”) and can be di ided in o “canonical” (caspase 1) o “non- canonical” (caspase 4/5/11) depending on he s imuli and caspases in ol ed [7,19,20]. Py op osis occu s mos ly in leukocy es and epi he- lial cells and p o okes an in lamma o y eac ion o he su ounding cells [7,19,20]. Py op osis is mechanis ically and biologically di e en o o he p og ammed cell dea h ypes, such as apop osis, nec op osis o e op osis, and dying cells exhibi dis inc mo phologic ea u es: ex ensi e cell swelling and memb ane ballooning in absence o cell de achmen , in ac nucleus wi h ch oma ic condensa ion, among o he s [22]. Excep o PJVK, he NT o o he GSDMs can p oduce ly ic cell dea h [15,19,20] wi h he elease o LDH and o he molecules. The e- o e, py op osis was enamed as “Gasde min-media ed p og ammed cell dea h” [23], bu accumula ing e idences p o ed ha GSDMs can o ches a e o he cell dea h p ocesses and addi ional dea h-independen unc ions. Indeed, each GSDM is ac i a ed by speci ic p o eases in a cell con ex -dependen way (Fig. 1A) ha could lead o di e en biological consequences: GSDMD NT is eleased by in lamma o y caspases du ing canonical and non-canonical py op osis [9,10], whe eas Ye sinia in ec- ion [24,25] o RIPK3 signaling [26] p o okes caspase-8-media ed GSDMD ac i a ion. Ca hepsin-G ac i a es GSDMD du ing NOD-like e- cep o p o ein 3 (NLRP3)-s imula ed py op osis in mac ophages and neu ophils [27], bu neu ophil elas ase (NE) clea age in neu ophils could esul in py op osis [28] o NETosis [29]. O no e, GSDMD po es can elease cy okines in leukocy es in absence o cell dea h [30], and GSDMD-cell dea h can be inhibi ed by he Endosomal so ing complexes equi ed o anspo (ESCRT-III) mechanism [31]. GSDME is ac i a ed by apop o ic caspase-3, in esponse o mul iple s imuli [32,33], leading o cell lysis conside ed by some au ho s as “nec osis seconda y o apop osis” [32,34], while GSDME p ocessing ia kille cell-de i ed G anzyme-B (GZMB) induces py op o ic and immunogenic cell dea h in umo cells [35]. Su p isingly, e en caspase-8 (a e Ye sinia in ec- ion) and caspases 4/11 can ini ia e signaling pa hways leading o GSDME clea age and cell dea h [36]. Immunocy e- eleased G anzyme-A (GZMA) clea age o GSDMB can p oduce ei he ly ic cell dea h in cance cells [37] o selec i e killing o Shigella in acellula bac e ia bu no he in ec ed cells [38]. Mo eo e , unp ocessed GSDMB enhances caspase-4/ GSDMD non-canonical py op osis [39]. Finally, bo h TNF- α +hypoxia [40] and α -Ke oglu a a e [41] induce Caspase-8 clea age o GSDMC bu in di e en esidues (Fig. 1A). GSDMC NT p o okes py op osis in cance cells bu i could esul in ch onic umo nec osis [40]. The p o eases ac i a ing human and mouse GSDMA and PJVK p o eins a e s ill un- known. No ably, cell-dea h unc ion can be inhibi ed by p o ease p o- cessing wi hin he GSDM NT domain, like GSDMD clea age by caspases- 3/7 [42] o en e o i us 3C p o ease [43]. Likewise, di e se caspases (1/ 3/6/7) can clea e and inac i a e he GSDMB NT [16,39], con adic ing he wo k by Panganiban and collabo a o s, which sugges ed ha caspase-1 clea age o GSDMB linke induced py op osis du ing as hma [44]. O no e, GSDM-media ed cell dea h is mo e complex han me ely o ming cell memb ane po es, since some GSDM NT can also a ge in acellula o ganelle (mi ochond ia, neu ophil g anules and possibly he nucleus) [4,7] (Fig. 1C). In pa icula , ac i a ed GSDMs (GSDMA/ A3/D/E) igge mi ochond ial damage [45–48], and con e sely, al e ed mi ochond ial unc ion induces GSDMD/E clea age [49]. In ac , ime lapse mic oscopy e eals ha du ing py op osis mi ochond ial damage p ecedes cell lysis [45,46]. Mi ochond ia dys unc ion can occu in di e se ways. A e caspase-3/GSDME clea age, GSDME po es pe - meabilize he mi ochond ial memb ane leading o downs eam apop- osome ac i a ion [49]. Thus, GSDME ac i a ion cause a posi i e eedback loop enhancing mi ochond ial apop osis and/o py op osis. By con as , cons i u i ely ac i e mu an GSDMA3 p o eins could cause mi ochond ial damage by wo mechanisms: binding o he mi ochon- d ial chape one T ap1, which p omo es oxida i e s ess and loss o he mi ochond ia memb ane po en ial igge ing apop osis-independen cell dea h [47], o h ough s imula ion o p o cell-dea h au ophagy [48]. Consis en wi h he GSDM mi ochond ial a ge ing, GSDM NTs exhibi s ong binding a ini y o ca diolipin, a lipid en iched in he in e nal mi ochond ial memb anes [15,50]. Taken oge he , hese e idences p o ed ha GSDMs coo dina e an ex ensi e and complex c oss- alk be ween di e se cell dea h pa hways [22,34], bu he p ecise unc ions o each GSDM in physiology and disease has only s a ed o eme ge. The molecula mechanisms egula ing in lammasome-dependen and independen py op osis and he implica ion o GSDMs in physio- logical p ocesses (e.g., esponse o in ec ious agen s) and pa hologies has been ex ensi ely e ised elsewhe e [7,19,20]. As examples, GSDM p o- cell dea h unc ions a e in ol ed in mul iple in lamma o y pa hologies such as sepsis, au oimmune encephalomyeli is (GSDMD), as hma and in lamma o y bowel diseases (GSDMB), among o he s. Addi ionally, a e GSDM mu a ions (Fig. 1) p o oke pa hogenic pheno ypes in mice (Gsdma3 mu a ions ha impede CT p o ein inhibi o y unc ion igge skin in lamma ion and hai loss, among o he de ec s) and humans (GSDME and PJVK unca ing mu a ions p oduce he edi a y dea ness diso de s) [7,19,20]. In his e iew we p o ide a comp ehensi e o e iew on he mul i- ace ed oles o he GSDM amily in cance , co e ing no only hei p o- cell dea h ac i i ies in pa icula umo con ex s, bu also o he unc ions ha e en ually could lead o umo p og ession. Mo eo e , we will ocus on he implica ion o GSDMs in media ing cance esponse o ea men and he ele ance o GSDMs as no el he apeu ic a ge s o clinical oncology managemen , wi h special emphasis on no el GSDM-di ec ed nano- he apies. 3. An i- umo and p o- umo e ec s o GSDMs The p o-cell dea h ac i i y o GSDMs could lead o he idea ha hese p o eins play mainly an an icance unc ion. Ye , GSDM genes a e o en loca ed in genomic egions equen ly ampli ied in cance s (Table 1), D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 3 Table 1 Exp ession and unc ional oles o GSDMs in human cance s. Cance ype (sugges ed unc ion*) Exp ession in umo s (me hod) & e ec s in un ea ed cance cells. Re Human GSDMA (GSDM, GSDM1) [17q21.1] Mouse Gsdma1/2/3 [11D] B eas , gas ic & o a ian (an i- umo ) No exp ession was de ec ed in any o he 24 cance cell lines (No he n blo ), e en hough GSDMA gene was ampli ied in 4/4 (100%) b eas and 2/8 (25%) gas ic HER2 cell lines. [52] Esophageal & Gas ic (an i- umo ) Unde ec able exp ession in 58/60 (97%) umo s and 17/21 (81%) cell lines (RT-PCR). O e exp ession dec eases colony o ma ion in MKN28 cells. [1] Gas ic (an i- umo ) Unde ec able exp ession in 11/18 (61%) umo s and 8/11 (73%) cell lines (RT-PCR). GSDMA is es o ed by inhibi ion o p omo e hype me hyla ion in 6/8 (75%) lines. O e exp ession in cell lines dec eases cell g ow h and induces cell dea h. [51] O a ian (p o- umo ) G ea e mean exp ession in umo s (n =379) compa ed o no mal issue (n =88) (mRNA DBs). Highe GSDMA cance exp ession associa es wi h poo su i al. [53] Human GSDMB (GSDML, PRO2521) [17q21.1] No p esen in mouse Bladde (p o- umo ) La ge mean exp ession in umo s compa ed o no mal issue (n =19) (mRNA DBs). GSDMB and USP24 p o ein exp ession a e posi i ely co ela ed in bladde cance s (n =80; IHC). GSDMB silencing in T24 and 5637 cells dampens p oli e a ion, mig a ion and in asion and educes in i o umo g ow h o T24 xenog a s. GSDMB con ols glycolysis and in i o cance g ow h ia STAT3 signaling. [59] B eas (p o- umo ) G ea e mean exp ession in umo s compa ed o no mal issue (RT-PCR, n =18). Highe mRNA umo exp ession associa es wi h poo p ognosis (n =1628; mRNA DBs). I s o e -exp ession in MCF7 cells induces cell mo ili y, in asion, gela in deg ada ion in i o & in i o umo g ow h and me as asis (mos ly iso o m 2). GSDMB silencing educes mig a ion and in asion o HCC1954 cells. [56] HER2 B eas (p o- umo ) O e exp essed (80/212; 66%) (IHC) and gene-ampli ied (FISH) (73/123; 59%) in HER2-posi i e umo s. Highe le els (n =212; IHC & n =2096; mRNA DBs) associa e wi h me as asis, poo p ognosis and ea men elapse. [55] HER2 B eas (p o- umo ) GSDMB exp ession p omo es mig a ion in HCC1954, SK-BR-3, and BT474 cells. Enhances lung me as asis in MDA-MB-231 cells. [61] Ce ical (p o- umo ) Mo e equen ly exp essed in umo s (16/21; 76%) han co esponding non-neoplas ic a eas (8/21; 38%) (IHC). I s silencing educes cell g ow h in Hela cells. [57] Ce ical (unce ain) Equal exp ession equency in umo s (23/27; 85%) han co esponding non-neoplas ic a eas (24/27; 89%) (IHC). [37] Colo ec al (unce ain) Simila exp ession equency in umo s (166/230; 72%) han co esponding non-neoplas ic a eas (154/230; 67%) (IHC). [37] Esophageal (an i- umo ) Less equen exp ession in umo s (44/80; 55%) han co esponding non-neoplas ic a eas (72/80; 90%) (IHC). [37] Esophageal & Gas ic (p o- umo ) Exp essed in 47/60 (78%) umo s and 21/21 (100%) cell lines (RT-PCR). Co-ampli ied wi h HER2 in 2/8 (25%) gas ic cance s. I s o e exp ession in MKN28 cells does no a ec colony o ma ion. [1] Gas ic (an i- umo ) Less equen exp ession in umo s (34/75; 45%) han co esponding non-neoplas ic a eas (66/75; 88%) (IHC). [37] Gas ic (p o- umo ) Mo e equen ly exp essed in umo s (44/52; 85%) han no mal issues (29/82; 35%) (RT-PCR). S onge exp ession in umo cells compa ed o no mal cells (ISH, n =5). [58] Gas ic, CRC & HCC (unce ain) Simila mean exp ession be ween umo s (gas ic n =21; HCC n =15; colon n =9) han co esponding non-neoplas ic a eas (RT-PCR). [6] HCC (unce ain) Simila exp ession equency in umo s (17/21; 81%) han co esponding non-neoplas ic a eas (21/21; 100%) (IHC). I s silencing does no a ec cell g ow h in HepG2 cells. [57] Lung (p o- umo ) Highe mean exp ession in lung adenoca cinomas han no mal issue (n =515; mRNA DBs). [54] Mul iple cell line ypes GSDMB p o ein endogenous exp ession de ec ed only in 18/54 (33%) cell lines (WB). I s exp ession could be induced by IFN-Ɣ in 11 cell lines (WB). GZMA induces py op osis killing o GSDMB-exp essing cell lines. [37] OSCC (p o- umo ) Highe mean exp ession in lymph-node me as a ic cance s s non-me as a ic (n =53; mic oa ays and RT-PCR) [60] Panc ea ic (unce ain) Simila exp ession equency in umo s (41/77; 53%) han co esponding non-neoplas ic a eas (45/77; 45%) (IHC). [37] Human GSDMC (MLZE) [8q24.21] Mouse Gsdmc1–4 [15D1] B eas (p o- umo ) Highe le els associa e wi h wo se o e all su i al (n =626; IHC). [40] Colo ec al (p o- umo ) Highe mean exp ession in umo s s adjacen no mal issue (n =44; RT-PCR). GSDMC p o ein de ec ed only in umo s, no in no mal issue (n =44; IHC). GSDMC was silenced (DLD-1 and LoVo cells) o o e -exp essed (SW480 and WiD cells). GSDMC up egula ion p omo ed in i o p oli e a ion, ancho age-independen g ow h and in i o umo igenesis. [68] Esophageal & Gas ic (an i- umo ) Exp essed in 34/60 (57%) o umo s and 16/21 (76%) cell lines (RT-PCR). I s o e exp ession educes sligh ly colony o ma ion in MKN28 cells. [1] (con inued on nex page) D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 4 Table 1 (con inued) Cance ype (sugges ed unc ion*) Exp ession in umo s (me hod) & e ec s in un ea ed cance cells. Re Lung (p o- umo ) G ea e mean exp ession in lung adenoca cinomas s no mal issue (n =515; mRNA DBs). Highe GSDMC exp ession associa es wi h poo p ognosis, me as asis and adio- esis ance (mRNA DBs). Hype me hyla ion associa es wi h gene silencing. [54] Melanoma (p o- umo ) Exp essed in 8/26 (30%) melanomas bu no in ne i (n =5). Mo e equen ly exp essed in me as a ic (6/11; 55%) han non-me as a ic (2/15; 13%) melanomas (IHC). [67] Human GSDMD (GSDMDC1, DFNA5L, DF5L, FKSG10) [8q24.3] Mouse Gsdmd [15D3-E1] ESCC (unce ain) Highe mean exp ession in umo s han in no mal issues (n =148; mRNA DBs and n =30; IHC). [133] Esophageal & Gas ic (an i- umo ) Exp essed in 38/60 (63%) o umo s and 21/21 (100%) cell lines (RT-PCR). I s o e exp ession s ongly educes colony o ma ion in MKN28 cells. [1] Gas ic (an i- umo ) S ong exp ession mo e equen in no mal issue (41/61; 67%) han in umo s (27/102; 27%) (IHC). mRNA le els highe in no mal s ma ched umo issue (n =39; RT-PCR). GSDMD exp ession in BGC823 cells educes in i o p oli e a ion and in i o cance g ow h. [69] Lung (p o- umo ) Highe exp ession (IHC sco e; n =168) in adenoca cinomas and SCCs han no mal issue. O e exp ession associa es wi h inc eased umo size, s age and lowe su i al in adenoca cinomas. GSDMD silencing in PC9, H1703 and H1975 cells educes p oli e a ion, p omo es apop osis and a enua es in i o umo g ow h. [70] Os eo-sa coma (p o- umo ) Exp essed in 20/41 (49%) umo s and none o non-neoplas ic a eas (IHC). G ea e mean p o ein exp ession in umo s han ma ched no mal issues (n =61; WB). Highe le els associa e wi h me as asis, esis ance o chemo he apy, and poo su i al. [134] Sali a y ACC (p o- umo ) Mo e equen ly exp essed in ACC (33/33; 100%), han adenomas (23/29; 79%) & no mal issues (24/33; 73%) (IHC). S ong IHC exp ession only seen in ACC. O e -exp ession in ACC-LM and ACC-83 cells enhances in i o in asion. [71] Human GSDME (DFNA5) [7p15.3] Mouse D na5 [6B2.3] B eas (an i- umo ) Lowe mean exp ession in umo s s no mal samples (n =1142; mRNA DBs). Reduced GSDME in umo s does no associa e wi h su i al. G ea e GSDME le els in ER-nega i e and lobula ca cinomas. Highe p omo e hype me hyla ion in umo s. GSDME gene body me hyla ion associa es wi h educed o e all su i al. [79] B eas (an i- umo ) Reduced mean exp ession in cance s no mal samples (n =2509; mRNA DBs). Highe umo GSDME does no associa e wi h su i al bu co ela e wi h immune- ela ed genes. No associa ion be ween p omo e me hyla ion and gene exp ession. [96] B eas (an i- umo ) Dec eased mean exp ession in cance s s no mal samples (n =10; RT-PCR). Highe me hyla ion in umo s. GSDME exp ession can be ac i a ed wi h deme hyla ing agen s. GSDME silencing inc eases colony o ma ion, p oli e a ion and in asion o MDA-MB-231 cells. [75] B eas and colon (an i- umo ) Lowe mean exp ession in b eas (n =1097) and colon (n =286) cance s s no mal samples (mRNA DBs). GSDME supp esses in i o umo g ow h and p omo es an i- umo immuni y o mu ine cells (EMT6, CT26, 4 T1 & B16–10) in immunocompe en mice. [35] Colo ec al (an i- umo ) Lowe exp ession in cance s s no mal issue (n =5) (RT-PCR). Highe hype me hyla ion in umo s (65%) han no mal issue (3%). GSDME in HCT116 cells educes cell g ow h and colony o ma ion. [73] Colo ec al (unce ain) Using Gsdme KO mice, no clea e ec s we e seen in wo expe imen al models o in es inal cance : he chemical induc ion by azoxyme hane “AOM” o c ossing wi h he Apc1638N/+s ain. [82] ESCC (an i- umo ) G ea e exp ession (IHC sco e) in cance s compa ed o no mal issue (IHC). Inc eased GSDME in SCCs (n =104; IHC) associa es wi h enhanced he apy esponse and be e p ognosis. [116] Gas ic (an i- umo ) F equen p omo e hype me hyla ion in umo s (53%). GSDME exp ession up egula ed by deme hyla ing agen s in gas ic cell lines. GSDME o e -exp ession educes colony o ma ion and augmen s apop osis in NUGC3 cells. [74] GBM (unce ain) Highe mean exp ession in umo s han in no mal b ain (mRNA DBs), bu GSDME le els do no associa e wi h pa ien s’ su i al. [89] Lung (unce ain) Exp essed in 59% o cance s, simila le els han no mal issues (IHC). Exp essed in 95% o cell lines (WB). GSDME does no a ec umo g ow h o human NCI-H3122 and HCC827 xenog a s. [78] Mul iple cell lines (an i- umo ) P o ein de ec ed in 30/57 (53%) cell lines, s ong exp ession in 11/57 (19%) (WB). Gsdme KO mice do no exhibi de elopmen al de ec s. [33] Melanoma (an i- umo ) GSDME silencing in B16-O a cells inc eases in i o umo g ow h. [49] Human PJVK (Pej akin, DFNB59) [2q31.2] Mouse D nb59 [2C3] O a ian (an i- umo ) Lowe mean exp ession in umo s (n =379) compa ed o no mal issue (mRNA DBs). Lowe PJVK cance exp ession associa ed wi h poo su i al. [53] Table ows a e o de ed alphabe ically by GSDM gene name (aliases indica ed in pa en heses and gene locus in squa e b acke s) and hen by umo ypes. * The o e all e ec (p o en o in e ed) on umo biology (excluding e ec s on cance ea men ) acco ding o s udy esul s. Abb e ia ions: ACC: Adenoid Cys ic Ca cinoma; CRC: Colo ec al; ESCC: Esophageal Squamous Cell Ca cinoma; GBM: Glioblas oma Mul i o me; IHC: Immunohis ochemis y; ISH: in si u hyb idiza ion; mRNA DBs: Da a Bases o mRNA exp ession (mic oa ays o RNAseq); SCC: Squamous Cell Ca cinoma; WB: Wes e n Blo . D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 5 such as he 17q12–21 egion (GSDMA and GSDMB), ha con ains he HER2/ERBB2 oncogene, o he 8q24 egion (GSDMC and GSDMD), close o MYC oncogene. Mo eo e , GSDMs a e ei he up egula ed o down- egula ed (mRNA and/o p o ein) in a wide ange o cance ypes compa ed o hei espec i e no mal issues (Table 1). In ac , accumu- la ing e idences indica e ha GSDMs play complex oles in cance biology, possibly ha ing ei he p o- umo o an i- umo unc ions depending on he cellula con ex (summa ized in Table 1). The in ol emen o GSDMs in oncologic ea men esponse will be p e- sen ed in sec ion 4. GSDMA was ini ially desc ibed as equen ly los in gas o- esophageal cance s [1,51] and b eas cance cells [52], e en in HER2- posi i e cell lines wi h GSDMA gene co-ampli ica ion [52]. GSDMA exp ession could be es o ed by me hyla ion inhibi o s [51]. In gas ic cance cells, GSDMA up egula ion, media ed by TGF-β ( ans o ming g ow h ac o -β) and he ansc ip ion ac o LMO1 (LIM domain only 1), p omo ed an apop o ic signal leading o educed cell g ow h [51]. These da a poin o a po en ial umo -supp esso gene (TSG) ole o GSDMA. Con a ily, in exp ession da abases GSDMA mRNA was up e- gula ed in o a ian ca cinomas e sus no mal issue, and highe GSDMA le els associa ed wi h wo se su i al [53]. Likewise, GSDMA mRNA is up egula ed in some lung cance da ase s [54] and b eas umo s [55,56], bu he ele ance o hese obse a ions is unce ain. In ac , despi e being he i s GSDM gene iden i ied [5], he mechanism o clea age/ac i a ion and he p ecise unc ional e ec s o GSDMA in cance emains unclea . Unlike o he GSDMs, GSDMB does no consis en ly educe cell g ow h o gas ic cance cells o o he cell models [1,56,57]. Mo eo e , con a y o i s neighbo gene GSDMA, GSDMB is equen ly exp essed (mRNA and/o p o ein) in se e al human umo ypes and cance cell lines, including gas ic, hepa ic, and b eas , among o he s (Table 1). None heless, compa ed o no mal issues, GSDMB le els a e ei he up egula ed [1,54–59], down egula ed [37] o unchanged [6,37,57] depending on he umo ype and he me hods used o measu e i s exp ession (Table 1). In e ms o mRNA, GSDMB o e -exp ession asso- cia es wi h ad anced/me as a ic disease in o al SCC [60] and gas ic umo s [58], sho e disease ee and me as asis ee su i al in b eas cance [56], and o e all su i al in only 3 o 33 cance ypes es ed by Zhou and colleagues [37]. Howe e , since GSDMB mRNA is de ec ed in a ious no mal cell ypes [7] he ue GSDMB exp ession in umo s should be assessed by immunohis ochemis y. Using a alida ed GSDMB an ibody and a speci ic FISH p obe, He gue a-Redondo and colleagues [55] demons a ed ha GSDMB is o e -exp essed/gene-ampli ied in >60% o HER2 b eas cance s. Simila ly, in gas o-esophageal cance s GSDMB and HER2 co-ampli ica ion is a common inding [1]. Impo - an ly, in HER2 b eas ca cinomas, GSDMB p o ein up egula ion asso- cia ed signi ican ly wi h a ious ad e se clinical pa ame e s: disease p og ession, elapse and esponse a e neoadju an he apy, as well as dis an me as ases and lymph node posi i i y in he adju an se ing, independen ly o ho monal ecep o s s a us [55]. Mo eo e , GSDMB exp ession media es mul iple p o- umo unc ions in b eas neoplasias: inc eases in i o cell mig a ion, possibly by modula ing he Rac GTPa- ses, in asion and gela in deg ada ion [56], s imula es in i o me as a ic dissemina ion [56,61], and educes sensi i i y o an i-HER2 he apy. Ac ually, all hese p o- umo ac i i ies could be dec eased wi h a no el an i-GSDMB nano he apy [61], hus demons a ing ha GSDMB plays a key ole in he biology and clinical beha io o HER2 b eas ca cinomas. Simila ly, in bladde cance cells GSDMB p omo es p oli e a ion, Fig. 1. GSDM po e- o ming unc ions. A: Regula ion o GSDM unc ion by clea age o mu a ion. Schema ic ep esen a ion o GSDM egions (NT, N- e minal domain; CT, C- e minal; and linke in e domain) showing he known p o eases and espec i e clea age si es (in pa en heses, he clea age si es o mu ine p o eins a e indica ed in g ey le e s). The clea age e en s inducing po e- o ming ac i i y a e shown in ed lines, and inhibi o y p ocessing in black. Gain-o – unc ion mu a ions in he CT o GSDMA3 and GSDME a e indica ed by as e isks. B: Main s eps in he o ma ion o GSDM memb ane po es. The e a e di e ences in he s uc u e and size o GSDM po es. GSDMA3 o m po es con aining a ound 27 monome s and wi h an in e nal po e diame e o 16–18 nm; GSDMD, 33 monome s and up o 21 nm in e nal diame e . C: Summa y o mechanisms o GSDM ac i a ion and biological e ec s. Casp, Caspase; Ca G Ca hepsin G; GZM, G anzyme; NE, Neu ophil Elas ase. (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he web e sion o his a icle.) D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 6 mig a ion and in asion [59]. These e idences and he p e ious obse - a ions in gas ic ca cinomas [1,58] indica e ha GSDMB could ac like an oncogene. Howe e , he in i o e ec o GSDMB on umo igenesis and p og ession migh depend on he cance models, since i p omo es umo g ow h in MCF7 b eas [56] and T24 bladde cance xenog a s [59] bu no o he human [61] and mu ine [37] cance models (Table 1). A his poin , i is impo an o highligh he exis ence o mul iple GSDMB ansc ip ional a ian s [6,57] and a leas ou dis inc ans- la ed p o ein iso o ms (ENSEMBL:ENSG00000073605). Though, o unknown easons he 411 aa p o ein sequence -Q8TAX9-, which was pa ially c ys alized [16], and is ega ded as “canonical” in all da a- bases, does no ma ch any o he ansc ip ional a ian s. The ou ansla ed iso o ms, which di e on he al e na i e usage o exons 6–7, can play di e en unc ions in cance [56] and in lamma o y diseases [44,62]. The iso o m di e en ial exp ession was epo ed in no mal and cance issues [6,57,58,63–65] and could be egula ed by speci ic genomic elemen s [58,64,65] and SNPs [44,63,66]. Howe e , he e is sca ce in o ma ion on he p ecise unc ions o each ansla ed a ian in no mal and pa hological con ex s. In his ega d, He gue a-Redondo e al. epo ed ha in MCF7 b eas cance cells bo h GSDMB iso o m 1 (GSDMB-1; lacks exon 6) and 2 (GSDMB-2; he sho es iso o m, lacking exons 6–7) enhanced mig a ion and in asion in i o, while only GSDMB2 p omo ed in i o umo g ow h and me as a ic dissemina ion in immunode icien mice [56]. Opposing i s p o- umo unc ions, GSDMB can, unde speci ic s im- uli, exhibi py op o ic ac i i y in cance cells [37,61], and hus an an i umo e ec . Speci ically, in a con ex o an ac i a ed an i umo immune esponse, he lymphocy e-de i ed G anzyme A (GZMA) can clea e GSDMB wi hin cance cells (Fig. 1A,C), ac i a ing caspase- independen py op osis and p omo ing umo clea ance [37]. Con- as ing wi h o he s udies (Table 1), Zhou and cols [37] epo ed a lowe GSDMB immunohis ochemical s aining in gas o-esophageal u- mo s compa ed o no mal issues, bu no o he cance ypes (Table 1), sugges ing ha GSDMB could ac as umo supp esso in hese neo- plasias. Un o una ely, his s udy did no e alua e he associa ion o GSDMB s aining wi h clinicopa hological a iables o disease su i al, hus he clinical ele ance o hese obse a ions emains unclea . GSDMC was ini ially iden i ied as a ma ke o melanoma p og es- sion, being pa icula ly o e -exp essed in me as a ic melanoma cells [67]. Fu he s udies epo ed opposing esul s ega ding i s exp ession and unc ional e ec s in umo s. On one hand, Saeki and cols ound GSDMC exp ession in 50% o gas o-esophageal umo s and cell lines bu was mos ly los in di use- ype gas ic ca cinomas. In gas ic cance cells, GSDMC exp ession educed cell p oli e a ion, sugges ing a possible umo supp esso unc ion [1]. On he o he hand, GSDMC up egula ion in lung [54] and b eas [40] umo s associa es wi h ad e se clinical pa ame e s (Table 1), and inc eases p oli e a ion along wi h in i o umo g ow h in colo ec al cance cells [68], sugges ing a p o- umo ole. Ac i a ion o GSDMC clea age by d ugs o me aboli es ( α -KG) [41] induces py op osis in cance cells (Fig. 1; Table 2), bu can also p o oke ch onic nec osis [40]. In he la e case, unde hypoxia nuclea PD-L1 ac i a es GSDMC ansc ip ion in b eas cance cells. In pa allel, ac i a ed mac ophages sec e e TNF- α esul ing in caspase-8 ac i a ion wi hin cance cells. The coo dina ed up egula ion o GSDMC and caspase-8 induces GSMDC clea age, causes py op osis and subsequen nec osis in b eas cance cells [40]. Pa adoxically, ch onic umo nec osis uels cance p og ession, and he e o e pa ien s wi h GSDMC-o e exp essing b eas cance s show poo su i al [40]. This s udy challenges he idea ha GSDM-media ed cance cell dea h can ha e bene icial e ec s on cance p og ession. Whe eas GSDMD ole in leukocy e py op osis is well known, i s implica ion in cance is deba able. Depending on he s udy, bo h GSDMD up egula ion and down egula ion (mRNA and/o p o ein) ha e been desc ibed in umo s, whe e GSDMD o e exp ession associa es wi h ei he good o un a o able p ognosis, as well as an i umo o p o- umo e ec s (Table 1). Fo ins ance, GSDMD exp ession diminishes p oli e a ion in gas ic cance cells, ei he ia cell dea h induc ion [1] o by egula ing cell cycle- ela ed p o eins [69], bu i inc eases lung cance g ow h in i o [70] and in asion o sali a y gland adenoca ci- nomas [71]. Among he GSDMs, GSDME exhibi s he mos consis en an i- umo unc ions in he li e a u e and is commonly conside ed as a po en ial TSG o se e al easons. Fi s , GSDME exp ession in cance models (un ea ed cells) inhibi s cell g ow h and/o p o okes cell dea h [72,73]. Second, di e se an i-oncologic ea men s induce GSDME clea age and enhance cance dea h (Table 2; discussed in sec ion 4). Thi d, GSDME is usually exp essed in no mal issues bu is equen ly down egula ed in di e se cance ypes, being his al e a ion some imes associa ed wi h inc eased malignancy o poo p ognosis (Table 1). Fou h, p omo e hype me hyla ion (a common mechanism o silencing TSGs) o GSDME is commonly obse ed in cance s [72–77]. Besides, GSDME loss-o - unc ion mu a ions occu in a small p opo ion o umo s, as an al e na i e mechanism o me hyla ion o inac i a ing GSDME- media ed cance cell dea h [35]. Finally, he umo supp esso ac i i y o GSDME is media ed by i s clea age by kille -cell GZMB, esul ing in caspase-independen py op osis o cance cells and enhancemen o an i- umo immuni y [35]. Despi e hese e idences, and he consis en e ec o GSDME on umo sensi iza ion o cance he apies (Table 2), i is s ill ques ionable i GSDME pe se gene ally ac s as a bona ide TSG, and whe he he e alua ion o umo GSDME p o ein le els ha e a eliable p ognos ic u ili y (Table 1). Fo ins ance, GSDME p o ein up egula ion was obse ed in he majo i y in lung umo s and cell lines, and GSDME clea age (py op o ically ac i e) was de ec ed e en in un ea ed speci- mens [78]. Besides, GSDME p omo e hype me hyla ion does no uni- e sally co ela e wi h gene silencing in di e se cance da ase s (Table 1) [77]. In e es ingly, he gene body o GSDME is equen ly hypome hyla ed in cance compa ed o no mal issues, and in ac , he pa icula pa e ns o me hyla ion in he GSDME gene p omo e and body egions, a he han GSDME exp ession, can be used as a powe ul bioma ke o pan-cance de ec ion [79–81]. Apa om human sam- ples, s udies wi h in i o animal models epo ed seemingly con adic- o y esul s ega ding GSDME ole in umo de elopmen and p og ession. In xenog a cance models, GSDME does no a ec umo g ow h o human lung cance cells [78], bu educes umo g ow h o mu ine melanoma [49], b eas , and colon cell lines [35]. Mo eo e , compa ing Gsdme Knock-ou (KO) mice wi h wild ype (WT) animals in wo expe imen al models o in es inal cance , no clea e ec s on ca ci- nogenesis, umo di e en ia ion and p og ession we e e idenced, hough an inc eased umo al in lamma ion was obse ed in WT mice [82]. By con as , Gsdme KO mice exhibi educed umo igenesis in in lamma ion–media ed coli is-associa ed colo ec al cance models [83], sugges ing ha GSDME-media ed py op osis and in lamma ion play a ole in cance ini ia ion. None heless, hese con lic ing da a could be pa ly explained conside ing new indings demons a ing ha GSDME umo supp essi e e ec depends mos ly on he subsequen umo in lamma ion and immunogenic ac i a ion (see sec ion 6). Finally, hi he o, he implica ion o PVJK is la gely unknown. Recen ly, using o a ian ca cinoma exp ession da abases, PJVK was ound down egula ed in umo s compa ed wi h no mal samples, and lowe PJVK mRNA le els in cance s associa ed wi h poo su i al a es [53]. 4. GSDMs modula e umo esponse o an i-cance ea men s E ading apop osis is a cance hallma k ha can p o ide esis ance o an i-cance he apies. In his scena io, ac i a ing py op osis o o he cell dea h mechanisms could lead o umo eg ession. GSDM-media ed cell dea h, which can p oceed in caspase-dependen and independen ways (Fig. 1), occu s in esponse o mul iple cell-damaging s imuli and an i- cance ea men s (chemo he apy, a ge ed d ugs and immuno- he apy) (Table 2). These da a would b ing o he idea ha GSDM- D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 7 Table 2 E ec o GSDMs on an i-cance he apy esponse. Tumo s /cell lines T ea men E ec on cance cells Re GSDMB HER2 B eas umo s, PDXs & cell lines T as uzumab Highe le els associa e wi h poo ea men esponse (adju an o neoadju an con ex s) in human umo s. T as uzumab esis ance associa es wi h GSDMB up egula ion in human PDXs. GSDMB exp ession p omo es d ug su i al in SK-BR-3 and HCC1954 cells. [55] HER2 B eas cell lines T as uzumab GSDMB up egula ion inc eases as uzumab su i al in HCC1954, SK-BR-3, and BT474 cells. This e ec can be pa ially blocked wi h an i-GSDMB nano he apy. [61] Colon & Melanoma cell lines An i-PD1 Exogenous GSDMB o e -exp ession in mu ine CT26 and B16-F10 cell line xenog a s sensi izes o an i-PD1 ea men in i o. [37] GSDMC B eas cell line Mul iple chemo d ugs Mos d ugs up egula e GSDMC bu only he an ibio ic- ype induce caspase-8/GSDMC py op osis in MDA-MB-231 cells. [40] Mul iple cell lines DM- α -KG Induces ROS and ac i a ion o DR6/caspase-8 axis leading o GSDMC py op osis in mul iple human and mouse cance cell lines in i o. DM- α -KG educes in i o umo g ow h (Hela and B16 xenog a s) and me as asis (B16) and his e ec depends on he p esence o GSDMC and DR6. [41] GSDMD AML p ima y cul u es and lines Val-bo oP o (DPP8/9 inhibi o ) Ac i a es CARD8/caspase-1/GSDMD-media ed py op osis and e icien ly kills AML p ima y cells and 12/17 AML cell lines in i o. Hal s umo p og ession o one PDX and MV4;11 cell line xenog a s in i o. [86] NPC cell lines Taxol Caspase-1 clea es GSDMD and ac i a es py op osis in i o in HNE-2, 5-8F cell lines. GSDMD silencing does no a ec umo g ow h in i o in un ea ed 5-8F xenog a s. GSDMD sensi izes 5-8F xenog a s o axol ea men . [135] Esophageal SCC cell lines Me o min Me o min up egula es miR-497, which in u n down egula es PELP1. PELP1 educ ion inc eases GSDMD py op osis in KYSE510 and KYSE140 cells. [133] O a ian cell lines Alpha-NETA Induces caspase-4 and GSDMD up egula ion and cell dea h in Ho8910PM cells. GSDMD silencing pa ly inc eases cell su i al o he compound. [136] GSDME (TN) B eas cance cell lines Te a‑a senic hexoxide Induces mi ochond ial ROS-media ed caspase-3/GSDME clea age and py op osis in mouse EO771, 4T1 and human Hs578T, MDA-MB-231 cance cells bu no in non- umo igenic cell lines (MCF10A, NMuMG). GSDME silencing educes d ug-induced cell dea h. [125] (TN) B eas cell lines Ce uximab miR-155-5p an agomi up egula es GSDME and swi ches apop osis o py op osis a e ce uximab ea men in EGF-o e exp essing MDA-MB-231 and MDA-MB-468 cells. [112] Colon cell lines Lobapla in A e lobapla in, caspase-3 clea es GSDME and p omo es py op osis ia ROS/JNK/bax mi ochond ial signaling pa hway in HT-29 and HCT116 cells. GSDME silencing swi ched om py op osis o apop osis bu did no a ec in i o umo g ow h upon lobapla in ea men . [90] Colon cell lines TNF +CHX o na i oclax These d ugs induce BAK/BAX/Caspase-3 ac i a ion and GSDME clea age and py op osis in HCT116 cells. GSDME silencing educes cell dea h o ea men . [121] Colon cell lines Ionizing adia ion (IR) IR induces GSDME-py op osis ia he LncRNA NEAT1 and miR488 in HCT116 cells. [113] Gas ic cell lines 5-FU S imula es Caspase-3 and GSDME clea age and py op osis in SGC-7901 and MKN-45 cells. GSDME knock ou swi ches py op osis o apop osis. [137] GBM cell lines Galangin (na u al la onoid) Simul aneously induces au ophagy plus caspase-3/GSDME clea age and py op osis in U87MG and U251 cells. GSDME silencing swi ches py op osis o apop osis. Inhibi ion o au ophagy plus Galangin inc eases cell dea h in i o in U87MG xenog a s. [89] Esophageal SCC cell lines Cispla in and BI2536 The Plk1 inhibi o BI2536 combined wi h cispla in p o okes caspase-3/GSDME clea age and py op osis in nine SCC cell lines in i o. The d ug combina ion se e ely educes in i o umo g ow h o KYSE150 umo xenog a s. [116] Head & Neck cell lines T ip olide T ip olide ac i a es BAD/BAX-caspase 3-GSDME py op osis by ep essing mi ochond ial associa ed hexokinase-II and p o oking ROS in HK1 and FaDu cells. Combining T ip olide wi h e as in (SLC7A11 inhibi o ) hal s in i o umo g ow h in HK1 xenog a s. [88] Li e cell lines Mil i one Induces ROS and mi ochond ial damage leading o caspase-3/GSDME clea age and py op osis in HepG2 and Hepa1–6 cells in i o and cell dea h in i o using Hepa1–6 umo xenog a s. GSDME silencing swi ches py op osis o apop osis. [126] Lung cell lines and p ima y human umo s T ame inib, E lo inib o Ce i inib GSDME sensi izes o T ame inib, e lo inib and ce i inib a ge ed he apies in i o and in i o using mul iple cell lines. GSDME knock ou swi ches py op osis o apop osis bu does no a ec umo g ow h when un ea ed. GSDME py op osis was obse ed in pa ien s ea ed p ima y umo s. [78] Lung cell line Cispla in o pacli axel Cispla in p o okes a s onge ac i a ion o caspase-3/GSDME clea age and py op osis han pacli axel in A549 cells. GSDME knockdown inhibi s cispla in- bu no pacli axel-induced pyop osis in i o. [138] Lung & neu oblas oma cell lines Dasa inib Dasa inib up egula es GSDMD and GSDME p o ein le els and p o okes caspase-3/GSDME clea age and py op osis in A549 and SH-SY5Y cells in i o. [139] Melanoma Cell lines Doxo ubicin S imula es au ophagy plus caspase-3/GSDME clea age and py op osis in SK-MEL-5, SK-MEL-28, and A-375 cells. Inhibi ion o eEF-2K dec eases au ophagy and up egula es sensi i i y o doxo ubicin and py op osis in i o. [140] Melanoma cell line I on plus ROS ac i a o s I on up egula es ROS which in u n ac i a es Tom20/bax/caspase-3 and GSDME py op osis in A375 cells. GSDME silencing bu no GSDMD p e en s I on/CCCP py op osis in i o. I on supplemen a ion plus sul asalazine inhibi s xenog a umo g ow h and me as asis h ough GSDME-py op osis induc ion. [124] Melanoma cell lines BRAF and MEK inhibi o s BRAFi+MEKi ac i a e caspase-3 / GSDME clea age and py op osis in i o and in i o in di e se melanoma models. GSDME-py op osis s imula es immune an i- umo esponse. [85] Mul iple cell line ypes Mul iple chemo d ugs Chemo he apy d ugs p o oke caspase-3/GSDME clea age and py op osis in SH-SY5Y, Mewo, Hela, NCI-H522 and EMT6 cance cells bu also in no mal ke a inocy es, placen al epi helial cells and smoo h muscle cells. Gsdme KO mice display educed in i o oxici y o no mal issues o Cispla in, 5-FU o bleomycin ea men . [33] AML: Acu e myeloid leukemia; DM- α -KG: Dime hyl- α -Ke oglu a a e; GBM: Glioblas oma; NPC: nasopha yngeal ca cinoma; SCC: Squamous Cell Ca cinoma; TN: T iple Nega i e B eas Cance . D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 8 exp essing cance cells may be gene ally mo e sensi i e o oncologic he apeu ic challenge, bu he eali y is much mo e complex. Fi s , he p ecise e ec o GSDMs on cell dea h depends on he bal- ance o di e en s imuli, signaling pa hways, molecula al e a ions, and cellula con ex s. In ac , he co-ac i a ion o speci ic signaling pa hways migh be equi ed o GSDM py op osis. Fo ins ance, in HepG2 cells GSDME enhances e oposide cell dea h only in he p esence o WT p53 [84] and GSDMC is up egula ed by di e se chemo he apy d ugs in MDA- MB-231 cells, bu only he an ibio ic- ype (doxo ubicin/epi ubicin) induce caspase-8/GSDMC-dependen py op osis [40]. Second, he GSDMs media e an ex ensi e c oss alk among di e se cell dea h mech- anisms, and hese could coope a e o compe e in a biological con ex - dependen way. Fo example, in lammasome- igge ed caspases (1/4/ 5/11) ac i a e GSDMD py op osis bu he apop o ic caspases 3/7 in- hibi s py op osis in monocy es [42]. Thi d, umo cells could exhibi in insic o acqui ed esis ance o py op osis. While loss o unc ion mu a ions in human GSDM genes ha e been epo ed only o GSDME so a [35], al e a ions in ups eam signaling pa hways o caspases could also occu . Acco dingly, GSDME-posi i e BRAF V600E/K melanomas esis an o BRAF+MEK inhibi o s exhibi weak induc ion o he p o- apop o ic p o eins BIM-EL and BMF, and hus educed caspase-3 ac i - i y [85]. Simila ly, 5/17 AML lines we e in insically esis an o Val- bo oP o agen due o he lack o p o-caspase-1, which was equi ed o GSDMD-media ed py op osis [86]. Apa om hese conside a ions, he gene al pic u e shows ha he GSDMs a e mainly in ol ed in swi ching apop osis o ly ic and in lam- ma o y cell dea h p ocesses (py op osis and nec osis, bu no nec op osis o e op osis) [87] a e cance ea men (Table 2). GSDME is he main de e minan o his apop osis- o-py op osis swi ch in mul iple in i o and in i o models ea ed no only wi h a my iad o an i-cance agen s (Table 2) bu also wi h speci ic na u al an i umo p oduc s, such as he epoxide ip olide [88] o he la onol galangin [89]. In clinical speci- mens, GSDME-py op osis was also e idenced (measu ing se um LDH concen a ions) in lung cance pa ien s a e chemo- o EGFR inhibi o - based ea men s [78]. None heless, in many s udies i is unclea i his apop osis-py op osis swi ch ansla es in o a bigge cance killing. Fo example, in lobapla in- ea ed colo ec al cance cells GSDME silencing did no a ec umo esponse (g ow h a e) in i o and in i o [90], and TRAIL ea men esul ed in simila Hela cell dea h quan i ies, i e- spec i e o GSDME le els [35]. Like GSDME, GSDMD ac s as a he apy sensi ize o media es apop osis-py op osis swi ch in pa icula cance cell ypes and oncologic ea men s (Table 2), hus, hese GSDMs could be impo an modula o s o cance he apy esponse. Un o una ely, many o he s udies lis ed in Table 2 we e no pe o med in clinical human specimens, so he impac o GSDM-media ed py op osis on cance pa ien su i al is s ill o be e i ied. Con a y o o he GSDMs, GSDMB up egula ion has been associa ed wi h he apy esis ance. Speci ically, in HER2 b eas ca cinomas GSDMB ende s cance cells mo e esis an o he an i-HER2 agen s as uzumab and lapa inib, bu no o axol chemo he apy [55,61], likely in a py op osis independen mechanism. Fo his eason, GSDMB signi i- can ly associa es wi h wo se b eas cance pa ien s’ p ognosis in bo h adju an and neoadju an an i-HER2 ea men s [55]. Summa izing, he inal e ec o GSDMs on umo ea men esponse depends on mul iple ac o s, including he co-ac i a ion o speci ic signaling pa hways, he d ug used, cellula con ex and, as discussed la e , he e ec on he umo mic oen i onmen . 5. Ta ge ed ac i a ion o GSDM cy o oxici y in umo cells as no el he apeu ic op ions Di e se oncologic ea men s induce GSDM-media ed cell dea h, hus sugges ing ha a ge ed app oaches speci ically aimed a ac i a ing o GSDM cy o oxici y in umo s can be exploi ed as no el he apeu ic op ions (Fig. 2). To his end, p omising he apeu ic e ec s has been ecen ly ob ained using wo gene al ypes o app oaches: a) Ta ge ac i a ion o he in insic GSDM p o-cell dea h unc ion in cance cells h ough nanomedicines; b) Deli e ing GSDM cy o oxic pep ides/ exp ession cons uc s in o umo s. Wi hin he i s ype o app oaches, h ee s udies a ge ed GSDME- py op osis wi h nanoca ie s ha combined agen s o induce GSDME ansc ip ion (such as he DNA me hyla ion inhibi o deci abine, DCT) wi h an i-cance d ugs (Fig. 2A). Fan and colleagues [91] used umo - a ge ing nanoliposomes loaded wi h cispla in (LipoDDP) and DCT o igge GSDME-py op osis in 4T1 b eas xenog a s. The in a enous adminis a ion o LipoDDP-DCT up egula ed GSDME exp ession (by inhibi ing i s gene p omo e hype me hyla ion), induced caspase-3/ GSDME clea age and he subsequen py op osis led o a educed umo g ow h and me as a ic sp ead in i o. Simila ly, Zhao and col- labo a o s [92] designed biomime ic nanopa icles (BNP), by using b eas cance memb anes on o a poly-lac ic-co-glycolic acid (PLGA) co e, ha we e loaded wi h he pho osensi ize indocyanine g een (ICG) and DCT. In 4T1 umo xenog a s he “BNP” nanomedicine was ac i- a ed by low-dose pho o-ac i a ion (local hype he mia) and induced GSDME-dependen py op osis by he combina ion o wo e ec s: On one hand, ICG-media ed punc u e o cance cell memb anes p omp ed a sha p inc ease in cy oplasmic Ca 2+ , p o oking cy och ome c elease and caspase-3 ac i a ion. On he o he hand, DCT elease up- egula ed GSDME ansc ip ion. Impo an ly, his “ac i a able” he apeu ic agen educed signi ican ly he size o p ima y umo and me as asis by ac i- a ing a s ong sys emic an i umo immune esponse. Finally, Hu e al. [93] designed a nano-d ug deli e y sys em based on mPEG-PLGA-PLL copolyme ha was loaded wi h a senic ioxide (As 2 O 3 ) as he apeu- ic agen o hepa ocellula ca cinoma (HCC) ea men . In a umo adminis a ion o hese nanopa icles (As 2 O 3 -NPs) esul ed in caspase- 3/GSDME clea age and py op osis o Huh7 xenog a ed umo cells. Mo eo e , As 2 O 3 educed he exp ession o DNA me hyl ans e ases (Dnm 1, 3a and 3b) which modula e he ansc ip ional le els o GSDMD o GSDME in a cell line-dependen way. In e ms o p e-clinical e icacy, compa ed o ee As 2 O 3 , As 2 O 3 -NPs g ea ly inhibi ed umo g ow h and showed no ob ious sys emic oxici y. Ins ead o using d ugs/compounds a ec ing GSDM exp ession and/ o ac i a ion, Molina-C espo and collabo a o s [61] c ea ed he i s nano he apy a ge ing di ec ly a GSDM p o ein wi h a he apeu ic an ibody (Fig. 2B). Speci ically, he nanomedicine ( e med AbGB-NC) combined a he apeu ic an i-GSDMB monoclonal an ibody (AbGB) wi h biocompa ible nanocapsules (NCs) unc ionalized wi h hyalu onic acid (HA). Since GSDMB up egula ion p omo es mul iple p o- umo e ec s in HER2 b eas ca cinomas [55,56], he au ho s nex alida ed he he apeu ic e ec o his nanomedicine in di e en HER2/GSDMB+ b eas cance models. The esul s demons a ed ha he in acellula eleased AbGB, bu no an i ele an an ibody, was able o impac signi ican ly on umo beha io in di e se ways: a) dec easing in i o cell mig a ion; b) inc easing he sensi i i y o as uzumab an i-HER2 he apy; c) educing umo g ow h in i o o o ho opic b eas cance xenog a s; and d) limi ing lung me as asis coloniza ion in i o, wi h no ob ious unwan ed cy o oxic e ec s. A he mechanis ic le el, in i o es s indica ed ha he binding o he AbGB o GSDMB (wi hin he CT egion and close o he hinge egion) could a ec he p o ein con igu- a ion hus enhancing he in e ac ion o GSDMB p o ein wi h sul a ides. This in e ac ion would subsequen ly dec ease cell mig a ion, and impo an ly may elease he in insic p o-cell dea h ac i i y o GSDMB. In ag eemen wi h his, he AbGB-NCs, bu no he con ol ea men s, inc eased he cell dea h a e (TUNEL assay, no speci ic es o py op- osis) o cance cells in i o, and speci ically o GSDMB-o e exp essing umo s (Fig. 2B). O e all, his s udy p o ed ha GSDMB is a no el he apeu ic a ge o agg essi e HER2/GSDMB+b eas cance s, and indica ed, o he i s ime, ha GSDMB cy o oxic e ec and o he unc ions in cance can be modula ed di ec ly wi h in acellula an ibodies. Whe eas he abo e nano he apies ely on he endogenous exp ession D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 9 o he GSDMs by he umo cells, o he he apeu ic app oaches ha e been designed o ex insically deli e ing GSDM cy o oxic cons uc s in o cance cells. As a po en ial ea men o schwannoma umo s Ahmed and collabo a o s [94] gene a ed an adeno-associa ed se o ype- 1 i us (AAV1)-based ec o ha , unde he con ol o a Schwann cell- speci ic p omo e (P0), exp esses he py op o ic NT egion o GSDMD ( esidues 1–276). The in a umo al injec ion in o in a-scia ic ne e o he AAV1-P0-GSDMD-NT in human and syngeneic mouse cells xenog a schwannoma models educed umo bu den by inc easing cell dea h and educing cell p oli e a ion. Mo eo e , his ea men alle ia ed umo - associa ed pain while causing no e iden neu ologic oxici y. Recen ly, Wang e al. [95] de ised a e y cle e app oach by which he elease o he py op o ic GSDMA3 p o ein occu s speci ically wi hin umo cells (Fig. 2C). Speci ically, he au ho s designed a complex nano- bioo hogonal sys em based on wo in e ac ing componen s. As i s componen , he mouse GSDMA3 clea ed p o ein (NT plus CT domain) was conjuga ed ia a ie hylsilyl (TES) e he linke o gold nanopa icles (NP) o gene a e he NP-GSDMA3, a biocompa ible nanosys em ha accumula ed mos ly in o umo issue o 4T1 cells xenog a s. As second componen , he au ho s used he umo -imaging p obe phenylalanine i uo obo a e (Phe-BF 3 ), which was aken up speci ically cance cells. Phe-BF3 desilya ed he silyl e he bond o NP-GSDMA3 esul ing in he in acellula elease o a py op o ic ac i e GSDMA3 p o ein and he killing o 4T1 umo cells in i o. A e h ee ounds o injec ion wi h Phe-BF3 plus NP-GSDMA3 he umo bu den educed eno mously, while he adminis a ion o Phe-BF 3 o NP-GSDMA3 alone, o a loss-o - unc ion GSDMA3-NP did no ha e any e ec . No ob ious cy o oxic e ec s we e obse ed in o he o gans in mice ea ed Phe-BF 3 plus NP- GSDMA3 [95]. Rema kably, some o hese GSDM- a ge ed nano he apies achie ed umo eg ession h ough he ac i a ion o a po en an icance in lam- ma o y eac ion (Fig. 2) (discussed below), hus indica ing ha py op osis-inducing nano he apies a e p omising app oaches ha could hal umo p og ession simul aneously by a acking umo cells and enhancing immune esponse. 6. GSDM-media ed cell dea h: e ec s on he umo mic oen i onmen and he cance esponse o immuno he apy GSDM py op osis and he immune esponse a e engaged in a com- plex bidi ec ional c oss alk. On one hand, cance cell lysis and he subsequen elease o DAMPs and cy okines p omo es an immunos i- mula o y umo mic oen i onmen ha could lead o cance ejec ion [87]. On he o he hand, cy o oxic T cells and NK cells can di ec ly igge py op osis by clea ing GSDME o GSDMB in cance cells ia pe o in-media ed elease o GZMB and GZMA, espec i ely [35,37]. The e o e, py op osis and ac i a ed immunocy es could gene a e a posi i e eedback loop in an icance immuni y (Fig. 2). GSDME-media ed py op osis gene ally associa es wi h a s ong im- mune an i umo esponse [87]. GSDME up egula ion in human cance s co ela e posi i ely wi h immune in il a ion [96], and i s o e - exp ession in mu ine b eas cance and melanoma xenog a s in immunocompe en mice signi ican ly augmen ed he numbe o umo in il a ing lymphocy es (TILs), mos ly CD8+T and NK cells. Ac i a ed TIL cy o oxici y and inc eased phagocy osis by umo -associa ed mac- ophages p o oked umo e adica ion [35]. Mo eo e , a e he apeu ic challenge by BRAF–MEK inhibi o s GSDME-media ed py op osis in mu ine melanoma models inc eased T cell in il a ion and imp o ed ea men esponses in i o [85]. Consis en wi h he bene icial e ec o GSDME py op osis in cance ea men , as commen ed be o e, he GSDME a ge ed nano he apies LipoDDP-DCT and BNP induced a s ong immune eac ion, including he elease o cy okines and p o- in lamma o y molecules, ec ui men and ac i a ion o cy o oxic T cells, and dend i ic cell ma u a ion, ha al oge he p o oked umo e adica ion [91,92], Alike GSDME nano he apies, he e icacy o he NP- GSDMA3 +Phe-BF3 nanomedicine depends on he immune an i- umo eac ion. In ac , he py op o ic killing o a small ac ion o umo cells (20%) was su icien o ac i a e, ia IL1-β sec e ion, a po en an i- umo e ec (T cells, NK and mac ophages) esul ing in he elimina ion o he bulk umo [95] (Fig. 2C). Rema kably, he GSDME umo supp esso unc ion in bo h un- ea ed and he apy-challenged umo s seem o ely on he p esence o ac i a ed cy o oxic cells. Thus, deple ion o NK and CD8+T cells o knocking-ou pe o in e e ses GSDME e ec s on umo g ow h o un- ea ed cance s [35] and deple ion o CD4+/CD8+T-cells signi ican ly dampens he esponse o xenog a ed melanoma cells o BRAF–MEK inhibi o s. Consequen ly, immunocompe en (e.g., Balb/c) and no immunode icien (nu/nu) mouse s ains should be used o un eil he ull e ec s o GSDME on cance . In ac , di e ences in mouse s ains may explain in pa he con lic ing esul s ob ained wi h GSDME-posi i e cance xenog a s (discussed in sec ion 2). O no e, while in i o GSDME ac i a ion in cance cells occu s ia caspase-3 ( esul ing in nec osis seconda y o apop osis), in i o clea age by kille -cell GZMA p o okes immunogenic cell dea h (Fig. 2), a o m o cell dea h ha is su icien o ac i a e an adap i e immune esponse [35,87]. Ac ually, he “ accine” inocula ion o immunocompe en mice wi h GSDME-o e exp essing cance cells p oduces he e icien killing o he seconda y umo implan a ion. Suppo ing his idea, GSDME- media ed py op osis igge s an immune esponse agains esidual cance , since Gsdme-silenced mu ine melanomas ecu ed mo e equen ly han Gsdme-exp essing ones a e he in e up ion o BRAF- MEK inhibi o s he apy [85]. In opposi ion o he in insic umo supp ession media ed by GSDME, GSDMB p omo es di e se p o- umo e ec s in b eas cance [55,56], and i s exogenous exp ession in mu ine colon and melanoma cells does no a ec umo g ow h in immunocompe en mice [37]. In ac , o expose he po en ial GSDMB an i umo e ec , he s imula ion o he an i umo immune esponse by PD-1 immune checkpoin inhibi o s is equi ed o e icien GZMA/GSDMB-media ed umo py op osis and eg ession [37] (Fig. 2B). This shows ha igge ing GSDMB cy o oxici y wi hin umo s may equi e addi ional signals om he umo mic oen- i onmen in o de o enhance immune ecogni ion and cance killing. In his sense, GSDMB exp ession in cance cells is up egula ed by cy o- kines (IFNs and TNF- α ) p oduced by TILs [37], and his may con ibu e o main aining a posi i e eedback loop o py op osis (Fig. 2B). Mo e- o e , his wo k indica es ha py op osis and immuno he apy could syne gize o p oduce a p o ec i e immune eac ion. Suppo ing his idea, Wang e al. 2020 p o ed ha he NP-GSDMA3 +Phe-BF 3 he apy sensi ized 4T1 umo s o in i o an i-PD-1 ea men [95] (Fig. 2C). The e o e, py op osis-induced in lamma ion, ia GSDM agonis s, can coope a e wi h immune checkpoin inhibi o s, hus leading o an imp o ed e ec i eness o cance immuno he apy. Based on hese da a, he cu en hypo hesis is ha GSDM-media ed py op osis could con e immunologically “cold” o “ho ” umo s, and likely espond be e o immune check-poin inhibi o s. 7. Fine unning o GSDM-media ed cell dea h o oncologic ea men : emaining ques ions Al hough he da a desc ibed abo e e eal a c ucial ole o GSDMs as no el he apeu ic a ge s and as key de e minan s o chemo he apeu ic and immuno he apy ea men s he e a e s ill a numbe o ques ions o be esol ed be o e GSDM-media ed oncologic he apies became a eali y a he clinical le el. 7.1. Con olling he speci ici y, in ensi y and iming o GSDM-media ed cance cell dea h Cu en ly, p edic ing he inal e ec o py op osis in he apy ou come is in ica e. In o de o exploi success ully GSDM-py op osis in cance he apy, he e a e h ee key pa ame e s ha need o be inely con olled (speci ici y, iming and in ensi y o cance lysis and D. Sa i´ o e al. BBA - Re iews on Cance 1876 (2021) 188635 16 [73] M.S. Kim, X. Chang, K. Yamashi a, J.K. Nagpal, J.H. Baek, G. Wu, B. T ink, E. A. Ra o i ski, Abe an p omo e me hyla ion and umo supp essi e ac i i y o he DFNA5 gene in colo ec al ca cinoma, Oncogene 27 (2008) 3624–3634. [74] K. 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