CD44 glycoprotein in cancer: A molecular conundrum hampering clinical applications

Aze edoe al. Clin P o eom (2018) 15:22 h ps://doi.o g/10.1186/s12014-018-9198-9 LETTER TO THE EDITOR CD44 glycop o ein incance : amolecula conund um hampe ing clinical applica ions Ri a Aze edo1,2, C is iana Gai ei o1,2,3, And eia Peixo o1,2,4, Ma a Rel as‑San os1, Luís Lima1,4, Lúcio La a San os1,2,5,6 and José Alexand e Fe ei a1,2,4,5,7,8* Abs ac CD44 is a hea ily glycosyla ed memb ane ecep o playing a key ole in cell adhesion, signal ansduc ion and cy oskele on emodelling. I is also one o he mos s udied glycop o eins in cance , equen ly explo ed o s em cell iden i ica ion, and associa ed wi h chemo esis ance and me as asis. Howe e , CD44 is a gene al designa ion o a la ge amily o splicing a ian s exhibi ing di e en deg ees o glycosyla ion and, po en ially, unc ionally dis inc oles. Mo eo e , s uc u al di e si y associa ed wi h ambiguous nomencla u e has delayed clinical de elopmen s. He ein, we a emp o comp ehensi ely add ess hese aspec s and sys ema ize CD44 nomencla u e, se ing miles ones o bioma ke disco e y. In addi ion, we suppo ha CD44 may be an impo an sou ce o cance neoan igens, mos likely esul ing om al e ed splicing and/o glycosyla ion. The disco e y o po en ially a ge able CD44 (glyco)iso o ms will equi e he combina ion o glycomics wi h p o eogenomics app oaches, explo ing cus omized p o ein sequence da abases gene a ed using genomics and ansc ip omics. Ne e heless, he necessa y high‑ h oughpu analy ical and bioin o ma ics ools a e now a ailable o add ess CD44 ole in heal h and disease. Keywo ds: CD44, Glycosyla ion, Cance bioma ke s, Nomencla u e, CD44 iso o ms © The Au ho (s) 2018. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/ publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. The ansmemb ane glycop o ein ecep o CD44 plays a key ole in cell adhesion o he ex acellula ma ix and in e ac s wi h g ow h ac o s and se e al ex acellula ligands, including hyalu onic acid, collagen, os eopon in and many me allop o einases o d i e signal ansduc- ion and cy oskele on ea angemen s [1]. By in e ac - ing wi h co- ac o s and adap o p o eins, CD44 has been u he implica ed in lymphocy e homing, haema opoie- sis, cell mig a ion and adhesion, umou in asion and me as asis [1]. Se e al iso o ms o CD44 can be gene - a ed h ough he inse ion o al e na i e exons a he a iable egion in a p ocess egula ed a bo h issue and cellula le els. While ubiqui ously exp essed in heal hy adul and oe al issues, he molecula plas ici y o al e - na i ely spliced CD44 accoun s o di e si ied unc ional oles. Howe e , he in ica e co ela ion be ween CD44 iso o ms and unde lying biological unc ions is ye o be ully disclosed. I has been long desc ibed ha malignan ans o ma ion and p og ession a e accompanied by a de egula ion o CD44 splicing mechanisms, comp ehen- si ely add essed in ecen e iews [2, 3]. The e en s lead- ing o CD44 iso o m molecula emodelling ha e di ec implica ions in se e al cance hallma ks and appea o a y acco ding o he ype o lesion, suppo ing he exis ence o disease-speci ic molecula inge p in s and po en ially a ge able bioma ke s [4]. No su p isingly, CD44 has been a ho opic in cance esea ch, equen ly associa ed wi h mo e agg essi e pheno ypes and widely explo ed o cance s em-cell iden i ica ion [5]. Pa icula ocus has been se on na owing CD44 sc eening o i s cance -associa ed iso o ms en isaging he necessa y sen- si i i y and speci ici y o clinical applica ions. Howe e , he lack o p o ocols o i s ull iso o m disc imina ion a he p o ein le el, and he exis ence o many unc ion- ally dis inc iso o ms poses a majo d awback. Cu en ly, hese hu dles can only be pa ially ci cum en ed by a - ge ing CD44 ansc ip s using a ian -speci ic p obes o Open Access Clinical P o eomics *Co espondence: jose.a. [email p o ec ed]‑saude.p 1 Expe imen al Pa hology and The apeu ics G oup, Po uguese Ins i u e o Oncology, Rua D . An ónio Be na dino de Almeida, 4200‑072 Po o, Po ugal Full lis o au ho in o ma ion is a ailable a he end o he a icle Page 2 o 5 Aze edoe al. Clin P o eom (2018) 15:22 by eme ging RNAseq app oaches. Analy ical di icul ies a e agg a a ed by as pos - ansla ional modi ica ions, wi h emphasis on he e y high glycosyla ion densi y o a iable egions. Mo eo e , glycans o en p esen a non- empla ed and con ex -dependen na u e, wi h se e al glyco o ms coexis ing o he same p o ein on a gi en biological milieu, u he inc easing CD44 molecula and unc ional di e si y (Table1). This poses a majo chal- lenge o iden i ica ion by con en ional immunoassays as well as high- h oughpu p o eomics, which has delayed he de ini ion o CD44 iso o ms in heal h and disease. The absence o nomencla u e s anda diza ion also eme ges as a key issue, making in e -s udy compa isons and clinical ansla ion almos impossible. Showcasing some examples, CD44H and CD44E e minologies a ise om he i s obse a ions in hema opoie ic and epi- helial cells; gp116 and gp85 dis inguish glyco o ms by molecula weigh s (116 and 85kDa, espec i ely); CD44 hema opoie ic cell E-/L-selec in ligand (HCELL) e e s o CD44 iso o ms exp essed in hema opoie ic and cance cells showing ele a ed sialo ucosyla ed glycans con en and high a ini y o E-/L-selec in ligands [6, 7]. All hese designa ions ail o p o ide clea insigh s on he molecu- la na u e o he iso o ms. No wi hs anding, some s udies adop a nomencla u e based on comme cial names o he used monoclonal an ibodies, highligh ing he a ge ed a iable exon, being CD44 3, CD44 6, CD44 9 amongs he mos associa ed wi h cance , including chemo esis - ance and p ognosis [8, 9]. Mo eo e , hese s udies o en dis ega d ha he analysis o a speci ic a iable exon can esul in he de ec ion o all iso o ms con aining i ins ead o one pa icula p o ein [10]. In addi ion, mos a iable egions may be signi ican ly hinde ed by dense glycosyla ion, biasing de ec ion. On he o he hand, epo s explo ing he ansc ip ome ha e eme ged as a powe ul ool o de e mining CD44 iso o ms di e si y; howe e , ew ha e p o ided he necessa y alida ion a he p o ein le el due o abo e men ioned analy ical di i- cul ies. Ano he impo an sou ce o nomencla u e ambi- gui y esul s om he di ec ansla ion o esul s om Mus musculus s udies o Homo sapiens dis ega ding ha mice CD44 gene p esen s an ex a a iable exon ( 1) no p esen in humans. Fu he con ibu ing o ambi alence, UniP o and NCBI p o ein da abases adop di e en des- igna ions o he same iso o m (illus a ed in Table1). Al oge he hese aspec s impac nega i ely on ou unde - s anding o he biological and clinical ele ance o CD44 iso o ms in cance and o he diseases, u ging nomencla- u e s anda diza ion. Facing hese challenges, we ha e conduc ed a com- p ehensi e in silico analysis o CD44 iso o ms h ough NCBI and UniP o da abases, using BLAST and TMHMM Se e 2.0 ools. The human CD44 gene is loca ed on he sho a m o ch omosome 11 [GRCh38. p7, NC_000011.10 (35138870-35232402)] and i s p e- cu so mRNA consis s o 19 exons. Namely, exons 1–16 encode he ex acellula domain, exon 17 encodes he highly conse ed ansmemb ane domain, and exons 18 and 19 encode he cy oplasmic domain. In silico analysis in NCBI has p edic ed o e wen y-one possible mRNA ansc ip s de i ed om he al e na i e splicing o exons 6–14 and 18, ele en o which we e also ound in UniP o . Howe e , only eigh ha e been expe imen ally con i med (de ailed in Table1 and Fig.1), speci ically six iso o ms wi h a iable ex acellula domain ex ensions, one iso- o m wi h a unca ed cy oplasmic ail, and one iso o m unca ed a he ex acellula domain. He e we a emp o s anda dize he nomencla u e o he abo e desc ibed iso o ms h ough a logical e minology. Following p e- exis ing designa ions, we p opose ha human CD44 iso- o ms o igina ed by al e na i e splicing o he a iable egion should highligh he included exons. Con e sely, Table 1 P oposed CD44 nomencla u e o expe imen ally obse ed iso o ms, i s co espondence wi hUniP o andNCBI da abases andp edic ed N- andO-glycosyla ion si es a N-glycosyla ion si es p edic ed using Ne NGlyc se e 1.0 (h p://www.cbs.d u.dk/se i ces/Ne NG lyc/) b O-glycosyla ion si es p edic ed using Ne OGlyc se e 4.0 (h p://www.cbs.d u.dk/se i ces/Ne OG lyc/) P oposed nomencla u e UniP o NCBI P edic ed glycosyla ion si es N-glycosyla ionaO-glycosyla ionbTo al CD44 2‑10 Iso o m 1 Iso o m 1 8 146 154 CD44 3‑10 Iso o m 4 Iso o m 2 8 133 141 CD44 8‑10 Iso o m 10 Iso o m 3 7 79 86 CD44 10 Iso o m 11 Iso o m 6 7 56 63 CD44s Iso o m 12 Iso o m 4 6 32 38 CD44s Iso o m 15 Iso o m 8 6 32 38 CD44s‑exon 15 Iso o m 18 Iso o m 7 6 23 29 CD44soluble Iso o m 19 Iso o m 5 2 3 5 Page 3 o 5 Aze edoe al. Clin P o eom (2018) 15:22 iso o ms lacking he a iable egion should p esen a nominal designa ion. Figu e 1 cons i u es a schema ic ep esen a ion o he expe imen ally de e mined human CD44 iso o ms, and he p oposed nomencla u e is desc ibed below: •CD44 2-10 The canonical CD44 iso o m includes a pep ide sequence encoded by exons 6–14, while splicing ou exon 18. This iso o m has a p edic ed molecula weigh o 82 kDa bu p esen s ex en- si e glycosyla ion, he eby a ising o app oxima ely 250kDa o highe . •CD44 3-10 Also known as epican, his iso o m esul s om he e en ion o exons 7–14 ( 3– 10) and splicing ou o exon 18. I s unmodi ied o m has 77kDa, esul ing in an up o 200kDa glycop o ein a e pos - ansla ional modi ica ions. •CD44 8-10 Also known as CD44E o CD44R1, his iso o m is o igina ed h ough e en ion o exons 12–14 ( 8– 10) and splicing ou o exon 18. I has Fig. 1 Schema ic ep esen a ion o expe imen ally con i med human CD44 p e‑mRNA and espec i e iso o ms. Blue illed boxes ep esen cons an egion exons, while whi e illed boxes ep esen exons o he a iable egion p esen in he designa ed CD44 iso o m. Da k blue illed boxes wi h educed box size ep esen unca ed exons om he cons an egion. The blue line ep esen s missing exon(s). Exon 18, illed black, con ains an ea ly 3’UTR and only makes pa o CD44s iso o m Page 4 o 5 Aze edoe al. Clin P o eom (2018) 15:22 53kDa in i s unal e ed o m, while eaching 130kDa in i s glycosyla ed o m. •CD44 10 Also known as gp116 o CD44R2, his iso- o m e ains he a iable exon 14 ( 10), while splicing ou exon 18. The unglycosyla ed o m has app oxi- ma ely 47kDa whe eas he glycosyla ed o ms ha e been obse ed a 120kDa. •CD44s Also known as CD44H o gp85, he s anda d o m o CD44 splices ou all a iable exons and exon 18. O iginally wi h 39 kDa, he subsequen pos - ansla ional addi ion o N-linked and O-linked oli- gosaccha ides gi es ise o a 85–90kDa glycop o ein. •CD44s Also known as sho - ail o ail-less, is a 32kDa iso o m splicing ou he a iable egion and exon 19, while e aining exon 18. Impo an ly, exon 18 con ains a s op codon ha o igina es a unca ed cy oplasmic ail, consequen ly leading o he loss o in acellula p o ein domains and signalling mo i s necessa y o in e ac ion wi h cy oskele al compo- nen s. •CD44s-exon15 A CD44s homolog o 37kDa lacking he pep ide sequence encoded by exon 15. •CD44sol This CD44 soluble iso o m only e ains exons 1–4, while p esen ing unca ed o ms o he exons 3 and 4. The modi ica ion o he wo la e exons leads o a smalle ex acellula domain as well as o he loss o ansmemb ane and cy oplasmic domains. This 16kDa iso o m is o en shed o bodily luids h ough ma ix me allop o ease ac i i y. In summa y, he wide a ay o s uc u ally simila CD44 iso o ms, associa ed wi h dense glycosyla ion and inad e en lack o nomencla u e consensus has posed a signi ican challenge o in e ing on CD44 ole in can- ce . These aspec s ha e biased many p e ious conclu- sions, p o ided se e al con lic ing da a, and signi ican ly delayed clinical de elopmen . Mos s udies dis ega d CD44 glycosyla ed domains, which some imes mo e han double he molecula weigh o he iso o ms, decisi ely modula ing biophysical, biochemical and unc ional p ope ies o he ecep o (p edic ed glycosyla ion si es de ailed in Table1). Glycosyla ion also aises a emen- dous challenge o CD44 mapping based on con en ional p o eomics, u ging he in oduc ion o glycan- a ge ed app oaches. As such, mo e comp ehensi e s a egies will ce ainly equi e he in eg a ion o glycomics/gly- cop o eomics wi h eme ging p o eogenomics, explo - ing cus omized p o ein sequence da abases gene a ed using genomics and ansc ip omics. This app oach is also expec ed o pinpoin ele an cance neoan igens o d i ing a ge ed he apeu ics and immuno he apy de elopmen . Ne e heless, we augmen ha he neces- sa y echnologies a e now a ailable o add essing CD44 molecula and unc ional di e si y in heal h and disease, ul ima ely p o iding a ge able bioma ke s o oncology. Abb e ia ions CD44: clus e o di e en ia ion 44; HCELL: hema opoie ic cell E‑/L‑selec in ligand; RNAseq: RNA sequencing; mRNA: messenge ibonucleic acid. Au ho s’ con ibu ions RA, CG and JAF: de eloped he concep ; RA and AP: e ised he li e a u e and pe o med bioin o ma ics analysis; RA, CG, AP and JAF: w o e he manusc ip ; MRS, LL and LLS: e ised he manusc ip . All au ho s ead and app o ed he inal manusc ip . Au ho de ails 1 Expe imen al Pa hology and The apeu ics G oup, Po uguese Ins i u e o Oncology, Rua D . An ónio Be na dino de Almeida, 4200‑072 Po o, Po u‑ gal. 2 Ins i u e o Biomedical Sciences Abel Salaza , Uni e si y o Po o, Po o, Po ugal. 3 P og am o Immunology and Immuno he apy, Cen e o Applied Medical Resea ch (CIMA), Pamplona, Spain. 4 Ins i u o de In es igação e Ino ação em Saúde (I3S), Uni e sidade do Po o, Po o, Po ugal. 5 Po o Comp ehensi e Cance Cen e (P.ccc), Po o, Po ugal. 6 Uni e si y Fe nando Pessoa, Po o, Po ugal. 7 Glycobiology in Cance , Ins i u e o Molecula Pa hol‑ ogy and Immunology o he Uni e si y o Po o (IPATIMUP), Po o, Po ugal. 8 In e na ional Ibe ian Nano echnology Labo a o y (INL), B aga, Po ugal. Acknowledgemen s No applicable. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. A ailabili y o da a and ma e ials Da a sha ing no applicable o his a icle as no da ase s we e gene a ed o analysed du ing he cu en s udy. Consen o publica ion All au ho s p o ide ull consen o publica ion. E hics app o al and consen o pa icipa e The e a e no e hics issues associa ed o his publica ion. Funding The au ho s wish o acknowledge he Po uguese Founda ion o Science and Technology (FCT) o he human esou ces G an s: Ph.D. G an s SFRH/ BD/105355/2014 (RA), SFRH/BD/111242/2015 (AP), SFRH/BD/127327/2016 (CG); Pos doc o al G an s SFRH/BPD/101827/2014 (LL) and SFRH/ BPD/111048/2015 (JAF). FCT is co‑ inanced by Eu opean Social Fund (ESF) unde Human Po en ial Ope a ion P og amme (POPH) om Na ional S a egic Re e ence F amewo k (NSRF). The au ho s also acknowledge FCT he unding o CI‑IPOP esea ch uni (PEs ‑OE/SAU/UI0776/201), he Po uguese Oncology Ins i u e o Po o Resea ch Cen e (CI‑IPOP‑29‑2014; CI‑IPOP‑58‑2015) and Ph.D. P og ams in Biomedicine and Pa hology and Molecula Pa hology o ICBAS‑Uni e si y o Po o. Publishe ’s No e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub‑ lished maps and ins i u ional a ilia ions. Recei ed: 23 May 2018 Accep ed: 23 June 2018 Re e ences 1. Senbanjo LT, Chellaiah MA. CD44: a mul i unc ional cell su ace adhesion ecep o is a egula o o p og ession and me as asis o cance cells. F on Cell De Biol. 2017;5:18. Page 5 o 5 Aze edoe al. Clin P o eom (2018) 15:22 • as , con enien online submission • ho ough pee e iew by expe ienced esea che s in you ield • apid publica ion on accep ance • suppo o esea ch da a, including la ge and complex da a ypes • gold Open Access which os e s wide collabo a ion and inc eased ci a ions maximum isibili y o you esea ch: o e 100M websi e iews pe yea • A BMC, esea ch is always in p og ess. Lea n mo e biomedcen al.com/submissions Ready o submi you esea ch ? Choose BMC and bene i om: 2. Pon a H, She man L, He lich PA. CD44: om adhesion molecules o signalling egula o s. Na Re Mol Cell Biol. 2003;4:33–45. 3. P ochazka L, Tesa ik R, Tu anek J. Regula ion o al e na i e splicing o CD44 in cance . Cell Signal. 2014;26:2234–9. 4. Olsson E, Hone h G, Bendahl PO, Saal LH, G u be ge ‑Saal S, Ringne M, Vallon‑Ch is e sson J, Jonsson G, Holm K, Lo g en K, e al. CD44 iso o ms a e he e ogeneously exp essed in b eas cance and co ela e wi h umo sub ypes and cance s em cell ma ke s. BMC Cance . 2011;11:418. 5. Mo a h I, Ha mann TN, O ian‑Rousseau V. CD44: mo e han a me e s em cell ma ke . In J Biochem Cell Biol. 2016;81:166–73. 6. Jacobs PP, Sacks ein R. CD44 and HCELL: p e en ing hema ogenous me as asis a s ep 1. FEBS Le . 2011;585:3148–58. 7. Jiang H, Pe e son RS, Wang W, Ba nik E, Knudson CB, Knudson W. A equi emen o he CD44 cy oplasmic domain o hyalu onan binding, pe icellula ma ix assembly, and ecep o ‑media ed endocy osis in COS‑7 cells. J Biol Chem. 2002;277:10531–8. 8. Thapa R, Wilson GD. The impo ance o CD44 as a s em cell bioma ke and he apeu ic a ge in cance . S em Cells In . 2016;2016:2087204. 9. Nagano O, Okazaki S, Saya H. Redox egula ion in s em‑like cance cells by CD44 a ian iso o ms. Oncogene. 2013;32:5191–8. 10. E b U, Megap che AP, Gu X, Buchle MW, Zolle M. CD44 s anda d and CD44 10 iso o m exp ession on leukemia cells dis inc ly in luences niche embedding o hema opoie ic s em cells. J Hema ol Oncol. 2014;7:29.