Reeive 5 Aug 16 Aepte De 16 Pulishe 19 Jn 17 3 Chins of type V ollgen regulte rest tumour growth vi glypin-1 Guorui Hung 1, Goxing Ge 1,w, Vlerio Izzi & Dniel S. Greenspn 1 DOI: 1.138/nomms1351 OPEN Periellulr 3(V) ollgen n ffet the funtioning of ells, suh s ipoytes n pnreti ells. Here we show tht 3(V) hins re n unnt prout of norml mmmry gln sl ells, n tht 3(V) ltion in mouse mmmry tumour moel inhiits mmmry tumour progression y reuing the prolifertive potentil of tumour ells. These effets re shown to e primrily ell utonomous, from loss of 3(V) hins normlly proue y tumour ells, in whih they ffet growth y enhning the ility of ell surfe proteoglyn glypin-1 to t s o-reeptor for FG. Thus, mehnism is presente for miroenvironmentl influene on tumour growth. 3(V) hins re proue in oth sl-like n luminl humn rest tumours, n its expression levels re tightly ouple with those of glypin-1 ross rest ner types. Eviene inites 3(V) hins s potentil trgets for inhiiting tumour growth n s mrkers of onogeni trnsformtion. 1 Deprtment of Cell n Regenertive Biology, University of Wisonsin Shool of Meiine n Puli Helth, Mison, Wisonsin 5375, USA. Centre of Exellene in Cell-Extrellulr Mtrix Reserh n Bioenter Oulu, Fulty of Biohemistry n Moleulr Meiine, University of Oulu, Apistie 5, Oulu 91, Finln. w Present ress: Institute of Biohemistry n Cell Biology, Shnghi Institutes for Biologil Sienes, Chinese Aemy of Sienes, Shnghi 31, Chin. Corresponene n requests for mterils shoul e resse to D.S.G. (emil: sgreens@wis.eu). NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 1
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 Collgen V [ol(v)] is low unne firillr ollgen wiely istriute in tissues s 1(V) (V) heterotrimers 1 tht integrte into firils of the unnt ollgen I [ol(i)] n regulte the geometry of resulting ol(i)/ol(v) heterotypi firils. 1(V) (V) heterotrimers lso regulte the tensile strength of ol(i)/ol(v) firils, s muttions in the genes for either the 1(V) or (V) hin n use lssi Ehlers Dnlos synrome 3,, whih is hrterize y frgile onnetive tissues 5. There is thir ol(v) hin, 3(V), whih n e foun in 1(V)(V)3(V) heterotrimers n hs more limite tissue istriution thn o 1(V) (V) heterotrimers 6. Tissues in whih the 3(V) hin hs een etete inlue white ipose tissue (WAT), skeletl musle, n pnreti islets, in whih periellulr 3(V) hins re importnt to proper funtioning of ipoytes, myofires n pnreti ells, respetively 6. 3(V) RNA is t reltively high levels in rest 7. Thus, finings of high 3(V) levels in WAT 6 suggeste tht high 3(V) levels in rest might our in mmmry ft ps. We show here tht 3(V) hins re in mmmry ft ps, ut re lso t prtiulrly high levels in ssoition with, n re proue y, mmmry gln sl ells. Intertions etween epithelil ells n the extrellulr mtrix (ECM) re importnt to rest rinom pthogenesis. Stroml firillr ollgens seem of prtiulr importne, s their ensity helps etermine rest rinom risk, n firils n provie trks long whih metstti epithelil ells migrte 8. Col(V) is speifilly upregulte B1-fol in the esmoplsi ssoite with sirrhous infiltrting utl rinoms 9, suggesting role in rest ner etiology. The importne of ollgenous ECM to rest rinom etiology, the speifi upregultion of ol(v) in esmoplsi, n the high 3(V) levels ssoite with mmmry gln prompte us to ssess possile 3(V) roles in mmmry rinom etiology. Towrs this en, effets of lting the 3(V) gene Col53 on mmmry tumour iology were stuie in the MMTV-PyMT mouse moel, whih repitultes mny proesses oserve in humn rest ner progression n metstsis 1. MMTV-PyMT tumour progression ws mrkely slowe y 3(V) ltion, preominntly ue to tumour ell utonomous effets. Col53 / MMTV-PyMT tumour ells h gretly reue prolifertive potentil, pprently ue to loss of intertions etween 3(V) n the ell surfe proteoglyn glypin-1 (), ffeting ility of the ltter to t s o-reeptor for mitogeni ftors. Results thus provie previously unreognize mehnism for ontrol of tumour progression y ollgenous ECM. 3(V) hins n re shown to e seprtely expresse y sl n luminl ells, respetively, in norml mouse n humn mmmry gln, ut to e oexpresse in luminl n sl-like humn rest tumours, whih my provie gin of utonomy n thus growth vntge to tumour ells. Suh growth vntge my e of prtiulr importne to luminl A tumours, with whih prtiulrly high 3(V) n expression levels re strongly ssoite. Dt showing nti-3(v) ntioies to slow tumour ell growth in vitro n in vivo suggest venues for therpeuti interventions. Results Col53 ltion slows tumour growth in MMTV-PyMT mie. Immunofluoresene foun tht 3(V) hins, lthough etete throughout mmmry ft ps, re t espeilly high levels ssoite with mmmry glns (Fig. 1). In ontrst, nti1(v) n -(V) ntioies showe 1(V) (V) heterotrimers to e evenly istriute etween ft p n glns, suggesting enrihment of only 3(V)-ontining ol(v) within the ltter. Co-loliztion showe high 3(V) levels of mmmry glns to e exlusively ssoite with sl ells (Fig. 1), with no pprent ssoition with luminl ells (Fig. 1). To etermine possile effets of 3(V) ltion on mmmry rinoms, Col53 / n MMTV-PyMT mie were interrosse to otin Col53 / /MMTV-PyMT (/PyMT) n Col53 þ / þ /MMTV-PyMT (/PyMT) progeny (Supplementry Fig. 1). Although signifint ifferene in tumour lteny etween /PyMT n /PyMT mie ws not oserve (Supplementry Fig. 1), /PyMT survivl ws signifintly inrese (Fig. 1, P.1, with survivl in groups ompre using the Log-Rnk test) n, t ny ge, /PyMT tumours were mrkely smller thn /PyMT tumours (Fig. 1e). Thus, tumours grew more slowly n survivl ws enhne in /PyMT mie. However, espite the reue size of /PyMT mmmry tumours, Col53 ltion i not pper to signifintly ffet the extent of lung metstsis (Supplementry Fig. 1 e). Although exlusively ssoite with sl ells in norml mouse mmmry uts (Fig. 1), 3(V) hins were expresse y /PyMT tumour ells (Fig. 1f,g), espite the ft tht MMTV-PyMT tumours hve gene expression profiles hrteristi of luminl type tumours 11. Inee, 3(V)-positive /PyMT tumours were lso positive for luminl mrker K8 (Fig. 1f), n negtive for sl mrker K1 (Fig. 1g). In /PyMT tumour setions, 3(V) o-lolize with K1 only in the sl ells of untrnsforme uts (Fig. 1g). Cell- n non-ell-utonomous 3(V) effets on tumour growth. To etermine the extent to whih erese tumour growth might e ue to stroml, non-tumour ell-utonomous, properties, /PyMT primry tumour ells were introue into the fourth ominl ft ps of Col53 þ / þ () n Col53 / C57BL/6 mie. Survivl n tumour sizes of the two groups were then ompre t vrious time points. Survivl ws mrkely inrese (Fig., P.1, with survivl in groups ompre using the Log-Rnk test) n tumour size mrkely erese (Fig. ) in Col53 /, ompre with, mie injete with /PyMT ells, initing non-ell-utonomous ontriutions from miroenvironments of stroml origin. To ssy for possile tumour ell-utonomous effets of Col53 ltion, /PyMT primry tumour ells were injete into n Col53 / mie. Interestingly, survivl of oth n Col53 / mie injete with /PyMT ells ws gretly prolonge up to B115 ys post injetion (Fig. ) n tumour growth ws gretly elye, with tumours first plple t B ys post injetion (Fig. ). These results re in ontrst to survivl times of or Col53 / mie injete with /PyMT ells, in whih mouse survivl ws only up to B3 or B5 ys post injetion, respetively (Fig. ), n in whih tumours were first plple only B1 ys post injetion (Fig. ). Superimposition of non-ell-utonomous results of Fig., on ell utonomous results (Fig.,, respetively) illustrtes the extents to whih ell-utonomous effets exee non-ell-utonomous effets of Col53 ltion on survivility n tumour growth. In ft, survivl times of versus Col53 / mie injete with /PyMT ells were not signifintly ifferent from eh other (Po.98, with survivl in groups ompre using the Log-Rnk test), further emphsizing the extent to whih ell-utonomous effets exee non-ellutonomous effets. Derese /PyMT tumour ell prolifertive tivity. /PyMT tumours hve mrkely lower lelling inex for prolifertion ntigen Ki-67 thn o /PyMT tumours (Fig. 3,). This is of interest, s it inites impirment of NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 ARTICLE α3v K1-Bsl Overly α3v αsm Atin Perilipin α3v K8-Luminl Overly Type V Survivl rtio 1 8 6 e Tumour volume (mm 3 ) 5,, 3,, 1, 16 18 6 8 Age in weeks 6 8 1 1 1 Weeks fter tumour pperne f α3v K8-Luminl DAPI Overly g α3v K1-Bsl DAPI Overly Figure 1 Altion of 3(V), foun t high levels juxtpose to mmmry uts, yiels erese tumour size n inrese host survivl. () Representtive immunofluoresene stining shows 3(V) hins (top pnels, re); -smooth musle tin (SM tin, top pnels, green), whih mrks utl myoepithelil ells; perilipin (ipoyte mrker); n ol(v) (ottom pnels, green). Blue; DAPI stining. Representtive immunofluoresene stining shows () o-loliztion of 3(V) (re) with mrker K1 (green) in sl ells, n () lk of o-loliztion of 3(V) (green) with mrker K8 (re) in luminl ells, of mmmry uts. Arrowhes n rrows enote luminl n sl ells, respetively. Col53 ltion Kpln Meier plots show signifintly inrese survivl (Po.1, survivl in groups ws ompre using the Log-Rnk test) in /PyMT mie lking funtionl Col53 gene (). (e) Volume of tumour uren is inresingly reue, reltive to /PyMT ontrols, in /PyMT mie t times fter initil tumour pperne. All plple tumours were mesure for volume lultions. All /PyMT mie were kille y 8 weeks fter initil tumour pperne, euse of tumour urens X3, mm 3.(,e) /PyMT n ¼ 3, /PyMT n ¼ 3. Dt re presente s men±s.. P vlues: o.5, o.5. Sttistil nlysis ws vi two-tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. Representtive immunofluoresent stining shows o-loliztion of 3(V) (re) with mrker K8 (green) (f) in the tumour ells of /PyMT tumours, n the lk of o-loliztion with mrker K1 (green) in the sme tumours, exept in sl ells of untrnsforme uts (g). White sle rs, 5 mm. In f, rrowhe n rrow enote luminl n sl ells, respetively, in n untrnsforme ut. In g, n rrow enotes o-loliztion of 3(V) n K1 only in the sl ells of n untrnsforme ut. NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 3
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 Survivl rtio 1 mie mie 8 6 5 3 35 5 Dys fter tumour ell injete Tumour volume (mm 3 ) 5,, 3, mie mie, 1, 1 15 5 3 35 Dys fter tumour ell injete 5 Survivl rtio 1 mie mie 8 mie mie 6 Tumour volume (mm 3 ) 5,, 3,, 1, mie mie mie mie 6 8 1 1 Dys fter tumour ell injete 6 8 1 1 Dys fter tumour ell injete Figure Non-ell-utonomous n ell-utonomous effets of Col53 ltion on survivility n tumour volume. /PyMT primry tumour ells were injete into wil type (-) or Col53 / (-) mie (n ¼ 8 mie per eh group), whih were then hrterize for survivl time () n tumour volume () t time points post tumour ell injetion. /PyMT primry tumour ells were injete into (-) or Col53 / (-) mie, whih were then hrterize for survivl time () n tumour volume () t time points post tumour ell injetion. Injete /PyMT ell urves from, were superimpose on the injete /PyMT ell urves in, to filitte omprison of survivl rtios n tumour volumes. Survivl nlyses were performe using the Kpln Meier metho. For tumour volume urves, t re presente s men±s.. P vlue: o.5. Sttistil nlysis ws vi two-tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. /PyMT tumour ell prolifertion in vivo, n s the Ki-67 lelling inex n e prognosti initor in rest ners 1. This ifferene in Ki-67 lelling inex ws ell-utonomous, s tumours resulting from /PyMT ells injete into C57BL/6 mie h muh lower Ki-67 lelling inies thn i tumours resulting from injetion of /PyMT ells into C57BL/6 mie (Fig. 3,). Consistent with the erese Ki-67 lelling inex, ulture primry tumour ells from /PyMT mie h gretly erese prolifertive tivity ompre with /PyMT ells, when ssye vi mitohonril ehyrogense reution of MTT (Fig. 3e) or 3 H-thymiine inorportion (Fig. 3f) ssys. In ontrst to the mrke ifferene in Ki-67 lelling, no signifint ifferene ws foun in leve spse 3 levels etween /PyMT n /PyMT tumours or ulture tumour ells (Supplementry Fig. ). Thus, while erese prolifertive tivity likely ontriutes to reue /PyMT tumour size, ifferenes in poptoti rtes o not. Altertions in /PyMT tumour ell signlling omponents. For insights into intrinsi ifferenes etween /PyMT n /PyMT tumour ells tht might ffet in vivo ehviours, we ompre levels n tivtion of signlling pthwy omponents importnt to mmmry tumour ehviour. PyMT tivtes Rs 13, whih n signl vi its iret effetor Rf-1 n the mitogen-tivte protein kinse (MAPK) pthwy to rive tumour ell growth 1. We thus ompre omponents of this pthwy in /PyMT n /PyMT tumour ells n tumour tissue smples. Although /PyMT n /PyMT totl Rs levels i not iffer (Fig. 3g,h), levels of tivte Rs-GTP, ple of ining Rf-1, were mrkely higher in /PyMT thn in /PyMT ell n tumour extrts (Fig. 3h), s were tivte phospho-rf-1 levels (Fig. 3g). Similrly, tivte phospho-erk levels were mrkely higher in /PyMT thn in /PyMT smples (Fig. 3g), onsistent with inrese MAPK signlling ownstrem of tivte Rs-GTP n Rf-1 (ref. 15). Interestingly, phosphorylte FGF reeptor (p-fgfr) levels were lso higher in /PyMT thn in /PyMT smples, onsistent with less signlling y mitogeni ftors in the sene of 3(V) hins. All of these ifferenes etween /PyMT n /PyMT smples were signifint (Supplementry Fig. 3). Thus, lower MAPK signlling orreltes with the intrinsi reue growth properties of /PyMT ells n tumours. As nonil Wnt signlling levels n ffet mmmry tumour ehviour vi effets on sl stem ell n luminl progenitor popultions 16, we ompre /PyMT n /PyMT tumour extrts for -tenin levels, ut foun no ifferenes (Fig. 3g). /PyMT tumour ells hve elye ell yle progression. The Rs/Rf/Erk se, whih hs reue tivity in /PyMT ells (ove), normlly trnsmits mitogeni signls from ell surfe reeptors (for exmple, FGFRs) to trnsription ftors tht ontrol ell yle progression 17. To otin further insights into the nture of the erese prolifertive tivity of /PyMT tumour ells, omprtive ell yle nlyses were performe on /PyMT n /PyMT primry tumour ells. Flow ytometri DNA profiles (Fig.,) showe tht, in norml growth meium, mrkely lower proportion of /PyMT thn NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 ARTICLE Ki67 Overly # of Ki67-positive ells 15 1 5 # of Ki67-positive ells 1 5 e Growth urve 1 1 1 8 6 1 3 Dys 5 f 3 H-Thymiine inorportion (1, CPM/1 5 ell) 15 1 5 g β Ctenin p-rf-1 Rf-1 Cells Tumours Gln 1 K h GTP-Rs Totl Rs Cells Tumours Gln K K Rs p-erk1/ Erk1/ p-fgfr 1 K FGFR 1 K α Tuulin 5 K Figure 3 /PyMT tumour ells hve reue ell-utonomous prolifertive tivity n Rs/Rf/ERK signling ompre with ontrols. Representtive Ki-67 immunohistohemil stining (white sle rs, 5 mm) (,) n quntifition of stining (,) re shown for /PyMT n /PyMT tumour setions (,), n for setions of tumours resulting from injetion of /PyMT () or,pymt ells into or Col53 / mie (,). Quntifition ws of six fiels per tumour on setions from three ifferent /PyMT n three ifferent /PyMT tumours, n from three ifferent tumours eh from injetion of /PyMT or,pymt ells into or Col53 / mie (- n -, respetively). (e) Cell prolifertion levels of primry /PyMT n /PyMT tumour ells were ssesse vi olorimetri MTT ssy. Vlues on the orinte xis represent OD t 57 nm, given n ritrry vlue of 1 t y, n then refleting fol hnges on susequent ys. (f) 3 H-thymiine inorportion is ompre for primry /PyMT n /PyMT tumour ells. All ell prolifertion ssys were performe in triplite. Dt re presente s men±s.. P vlues: o.1, o.5. Sttistil nlysis ws vi two-tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. (g) Representtive immunolots re shown of lystes of /PyMT () n /PyMT () primry tumour ells n tumours, n of norml C57BL/6 () or Col53 / mmmry gln tissue. Blots were proe with ntioies to -tenin, phospho-rf-1 (p-rf-1), Rf-1, Rs, phospho-erk1/ (p-erk1/), Erk, phospho-fgfr, FGFR or -tuulin, the ltter s loing ontrol. (h) Lystes were sujete to pull-owns with Rf-1 RBD omin to otin tivte Rs (GTP-Rs), whih ws then etete y immunolot, using ntioy to Rs. An immunolot isplying totl Rs levels in the sme smples is lso shown. Immunolots were repete three times, from three inepenent tumour or ell lystes. Representtive lots re shown. Quntifition of results for signifine of ifferenes is shown in Supplementry Fig. 3. NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 5
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 Tumours Cells of ells of ells 5 3 1 3 6 9 1 15 Chnnels (FL3-AFL3-Are PI) 16 1 8 Dip G1 Dip G Dip s Dip G1 Dip G Dip s Cell yle ells (%) 5 3 1 G1 G/M S CDK6 CDK Cylin D Cylin E Cylin B Cylin A α-tuulin 5 K 5 K 5 K 5 K 8 1 16 Chnnels (FL3-AFL3-Are PI) e Dip G1 Dip G Dip s 1 8 6 8 1 16 Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Dip G1 Dip G Dip s 1 9 6 3 8 1 16 G1 phse : 6 h, h Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Dip G1 Dip G Dip s 1 9 6 3 8 1 16 1 9 6 6 1 36 8 7 Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Dip G1 Dip G Dip s 15 1 5 8 1 16 1 8 6 3 6 9 1 15 1 9 6 3 3 6 9 1 15 1 8 6 16 1 8 8 1 16 6 5 3 1 3 6 9 1 15 16 1 8 3 6 9 1 15 3 1 6 8 6 8 1 16 6 8 1 1 9 6 3 8 1 16 1 9 6 3 3 6 9 1 15 1 9 6 1 8 6 8 1 16 8 6 3 6 9 1 15 1 8 6 1 8 6 3 6 9 1 15 16 1 8 1 9 6 3 6 9 1 15 5 3 1 8 1 16 9 1 36 8 G/M phse : h, 9 h 1 9 6 3 1 3 3 3 8 1 16 3 6 9 1 15 8 1 16 8 1 16 3 6 9 1 15 3 6 9 1 15 Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) Chnnels (FL3-AFL3-Are PI) 3 6 9 1 15 Chnnels (FL3-AFL3-Are PI) f p-h3 Ser1 Histone H3 GAPDH Tumours Cells h p-h3 Ser1 Histone H3 3 5 6 7 8 9 1 11 1 13 GAPDH g p-h3 Ser1 -positive ells (%) 5 3 1 Col53 / 3 5 6 7 8 9 1 11 1 13 15 p-h3 Ser1 Histone H3 GAPDH Figure Cell yle elys in /PyMT tumours n primry tumour ells. () DNA profiles re shown of /PyMT n /PyMT ells grown in norml growth meium, then stine with propiium ioie n nlyse y flow ytometry. () Quntittion is shown for the DNA profiles of. DNA profiles were performe on triplite smples. Immunolotting is shown for ylins n ylin regultors (). DNA profiles re shown of ells sorte y FACS into GM () n S (e) popultions, n then relese into norml mei, hrveste t inite time points (in hours) n nlyse y flow ytometry. (f) Immunolotting ws one for totl histone H3, n ntioy to phospho-serine 1 of histone H3 etete p-h3 ser1 levels in /PyMT () n /PyMT () tumour extrts n primry tumour ells. (g) Quntifition is shown for perent p-h3 ser1 -positive ells etete y immunofluoresent stining of /PyMT n /PyMT tumour setions. Quntifition ws of six fiels per tumour on setions from three ifferent /PyMT n three ifferent /PyMT tumours. Dt re presente s men±s.. P vlue: o.5. Sttistil nlysis ws vi two-tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. (h) Cells were synhronize t the G1/S orer vi oule thymiine lok, relese, n then hrveste t inite time points (in hours), n nlyse y immunolot for totl histone H3 n p-h3 Ser1 levels. GAPDH in immunolots ws loing ontrol. Immunolots were repete three times, from three inepenent tumour or ell lystes. Representtive lots re shown. Quntifition of results for signifine of ifferenes for n f is shown in Supplementry Fig.. 6 NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 ARTICLE /PyMT ells re in S phse, n mrkely lrger proportions re in G1 n G/M, onsistent with erese /PyMT prolifertive tivity. As ylin synthesis n onsequent ylin-epenent kinse (CDK) tivtion re key to regulting ell yle progression, levels of oth were ssye. Lower ylin D n A levels, ut similr ylin B n E levels, were foun in /PyMT ompre with /PyMT ells (Fig. ). Lower ylin D levels re onsistent with lower /PyMT Rs/Rf/Erk signlling levels (ove), s suh signlling is thought to ffet ell yle progression y inuing ylin D expression erly in G1 (ref. 17). Lower ylin A levels my result from the lower proportion of /PyMT ells in S phse, in whih ylin A umultes 18. Also onsistent with lower /PyMT prolifertive tivity were lowere levels of CDK6 n CDK, whih in n re stilize/tivte y ylin D 19, (Fig. ). All these ifferenes etween /PyMT n /PyMT smples were signifint (Supplementry Fig. ). Lower ylin D-CDK/6 tivity levels inite y the results suggest prtil lokge of /PyMT ells in G1, ontriuting to lowere prolifertive tivity. To further ssess ell yle ifferenes, /PyMT n /PyMT tumour ells were fluoresene-tivte ell sorting (FACS) sorte into G1-, S- n G/M-phse ells, whih were then relese into growth meium n hrveste t inite time points (Fig.,e, n Supplementry Fig. f). By 6 h post sorting, smll numer of G/M-sorte /PyMT ells h trverse G1 n were in S-phse (Fig. ), showing G/G1 urtion to er6 h. In ontrst, for G/M-sorte /PyMT ells, S-phse ells were not lerly etete until h fter relese into growth meium (Fig. ), initing /PyMT G/G1 urtion to eb h, n thus lokge in G/G1. To guge ifferenes in the urtion of S n G/M in /PyMT n /PyMT ells, S- (Fig. e) n G1- (Supplementry Fig. f) sorte ells relese into growth meium were nlyse. For S-sorte ells, /PyMT G1-phse ells first pper fter h, wheres /PyMT G1-phse ells pper, in smll numers, fter 9 h, onsistent with ely in G/M (Fig. e). Anlysis of G1-sorte /PyMT n /PyMT ells foun no ifferenes in S-phse urtion (Supplementry Fig. f). Results (summrize in Supplementry Tle 1) thus show the /PyMT ell yle to e onsierly elye, with loks in G1 n G/M. As histone H3 Ser1 phosphoryltion is ruil for hromosome onenstion uring mitosis 1, n thus ell yle progression, it is of interest tht /PyMT tumours n tumour ells h mrkely higher p-h3 Ser1 levels (Fig. f n Supplementry Fig. e), n tht /PyMT tumours h mrkely higher proportion of p-h3 Ser1 -positive ells, thn i /PyMT ounterprts (Fig. g). H3 ser1 phosphoryltion, whih egins t prophse, must e remove upon metphse/nphse trnsition for hromosoml eonenstion to our t the en of mitosis 1. A oule thymiine lok, to synhronize ells t the G1/S orer, followe y nlysis of timing for p-h3 ser1 pperne/ ispperne upon relese of ells from the lok, showe highest p-h3 ser1 levels eginning in oth /PyMT n /PyMT ells B7 h fter G1/S orer lok relese (Fig. h). This showe no ifferene in time neee for /PyMT n /PyMT ells to mke the S/G trnsition, or trnsverse S efore H3 ser1 phosphoryltion. However, high p-h3 ser1 levels persiste only 3 h in /PyMT ells, ut t lest 6 h in /PyMT ells (Fig. h), onsistent with elye ephosphoryltion. Thus the /PyMT G/M lok eviene in Fig. e is shown y p-h3 ser1 t (Fig. h) to e ue to prolonge mitosis. In sum, t re onsistent with loks in G1 n mitosis of /PyMT ells, onsierly elying the ell yle n orrelting with lowere prolifertive tivity. 3(V) hins intert with ell surfes vi. Importnt to unerstning how 3(V) hins ffet tumour ell ehviour is etermining the ell surfe moieties with whih they intert. It hs een reporte tht (V) ollgen hins, whih re similr to, if not orthologous to, 3(V) hins, ffet Shwnn ell funtion vi intertions with ell surfe heprn sulfte proteoglyn (HSPG) (ref. ). As is over-expresse in rest n other ners, in whih it ppers to regulte the Rs/Erk pthwy n moulte mitogeni responses to HSPG-ining growth ftors 3 8, we teste for possile 3(V)- intertions in /PyMT tumours. Immunofluoresene showe to e present in oth /PyMT n /PyMT tumours, n to o-lolize with 3(V) in the former (Fig. 5). Moreover, 3(V) immunopreipittion from /PyMT tumour extrts o-preipitte (Fig. 5) n, onversely, immunopreipittion of o-preipitte 3(V) hins (Fig. 5). Thus, 3(V) hins o-lolize with n re oun to in /PyMT tumours. Proing the immunopreipittes of Fig. 5, with ntioies to the 1(V) hin of ol(v) showe 1(V) hins to hve o-preipitte with 3(V) hins n (Fig. 5,), onsistent with the proility tht tumour 3(V) hins in in the ontext of 1(V)(V)3(V) heterotrimers. Immunopreipittes were lso proe with ntioy to the 1(XI) hin of ol(xi) s 1) 1(XI) is very similr in struture to 1(V) n ple of sustituting for 1(V) in ining other ol(v) hins 9 31, suh tht it ws possile tht 3(V) hins might our in 1(XI)-ontining heterotrimers; n ) 1(XI) is mrker tht is upregulte in, n my ply role in filitting vrious ners, inluing rest ner 3. However, 1(XI) hins were not etete in 3(V) or immunopreipittes from /PyMT tumour lystes (Fig. 5,). We previously suggeste tht 3(V) might intert with ell surfe moieties vi highly hrge sequenes in its N-terminl non-triple helil omin (NTD) 6. In pull-owns of reominnt proteins, 3(V)-NTD immunopreipittion ws suffiient to o-preipitte (Fig. 5), n vie vers (Fig. 5e). Thus 3(V) NTD sequenes re suffiient to in. ffets stility/eposition of 3(V)-ontining ECM.To etermine the extent to whih rogtion of ining might ffet ssoition of 3(V) hins with /PyMT tumour ells, the ltter were infete with previously esrie 33 enovirl vetor (A-Sh-1) for shrna knokown. Interestingly, upon knokown, 3(V) hins were no longer reily etetle y immunofluoresene (Fig. 5f), n were foun y immunolotting to e mrkely erese (Fig. 5g,h). In ontrst, 1(V) levels were not ffete y knokown (Fig. 5g), suggesting tht levels of ol(v) ontining 3(V) hins, ut not ol(v) lking 3(V) hins (for exmple, 1(V) (V) heterotrimers), re ffete y intertions. 3(V) moultes ility to ffet prolifertion. /PyMT tumour ells infete with A-Sh-1 lso h mrkely erese prolifertion (Fig. 6), onsistent with reporte ilities to moulte mitogeni responses n stimulte prolifertion 3,3. This mrke erese in prolifertive tivity ws ompnie y signifintly erese Rs signlling n FGFR phosphoryltion (Fig. 6, n Supplementry Fig. 5), similr to tht resulting from 3(V) knokout (Fig. 3g,h), n thus onsistent with the possiility tht n 3(V) intert to ffet ell prolifertion vi the sme pthwy. Infetion with A-Sh-1 of /PyMT tumour ells, whih lrey hve reue prolifertive potentil (ove), further reue prolifertion (Fig. 6), lthough the ifferene NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 7
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 in prolifertive tivity etween A-Sh-1 n ontrol vetorinfete /PyMT ells ws not s gret s the ifferene in prolifertive tivity etween A-Sh-1 n ontrol vetor-infete /PyMT ells. Thus, ontriutes to prolifertive tivity of tumour ells in the presene n sene of the 3(V) hin, lthough this ility is reue in the sene of 3(V). α3(v) DAPI Overly μm μm μm μm IP: α α3(v) IgG Cell extrts Pre Post Pre Post α3(v) IP: α- Cell extrts Pre Post Pre Post IgG α3(v) α1(v) α1(v) α1(xi) α1(xi) _ 1 K e _ 1 K IP: α α 3(V) IP: α- f S-1 Sh-1 α3(v)/dapi g α3(v) α1(v) Lm α1 -S-1 -Sh-1 -S-1 5 K h Fol reltive to 1. 1..8.6... Figure 5 o-lolizes with n ins 3(V) hins in /PyMT tumours. () Representtive immunofluoresent stining for (re) n 3(V) hins (green) shows o-loliztion in /PyMT () tumours n the sene of 3(V) stining in /PyMT () tumours. Blue stining is DAPI. Overly pnels show res of n 3(V) o-loliztion (yellow). Immunopreiptitions (IP) from /PyMT () or /PyMT () tumour extrts were performe using nti-3(v) () or nti- () ntioies. Immunopreipittes were then nlyse y immunolot using ntioies for, or for 3(V) or 1(V) hins (,). In,, immunolots re lso shown of mounts of 3(V) n present in tumour extrts efore (Pre) n fter (Post) immunopreipittions of the ognte proteins performe from suh extrts, to emonstrte input levels of eh protein, n to provie sense of how muh of the ville proteins were immunopreipittte. In,e, reominnt n 3(V)-NTD sequenes were o-inute, followe y immunopreipittion with nti-3(v)-ntd () or nti- ntioies (e), n then immunolotting with nti-, nti-3(v)-ntd n nti-1(v) ntioies. (f) Representtive immunofluoresent stining for 3(V) n stining for DAPI were performe on /PyMT primry tumour ells infete with enovirl vetor A-Sh-1 (Sh-1) for shrna knokown 33, or with srmle ontrol vetor A-S-1 (S-1). (g) Immunolots of ECM ssoite with /PyMT primry tumour ells infete with A-Sh-1 or A-S-1, show lowere 3(V) levels upon knokown. knokown h no effet on 1(V) levels. Lminin 1 hin ws stine s loing ontrol. Immunolotting of /PyMT ells infete with A-S-1 (-S-1) serve s ontrol for levels of n 1(V) in the sene of 3(V) hins. (h) Quntifition of three immunolots from three seprte experiments shows no signifint ifferene in levels etween -S-1 n -S-1 ells. White sle rs, 5 mm. 8 NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 ARTICLE Growth urve 1 1 8 6 -S-1 -Sh-1 1 3 Hours 5 6 7 p-rf-1 Rf-1 p-erk Erk p-fgfr FGFR α-tuulin S-1 Sh-1 1 K 1 K 5 K 7 6 5 3 1 -S-1 -Sh-1 GTP-Rs Totl Rs S-1 Sh-1 5 K 5 K Growth urve 1 3 5 6 Hours 7 Figure 6 Aility of to ontriute to the prolifertive tivity of mmmry tumour ells is reue in the sene of the 3(V) ollgen hin. Growth rtes, mesure vi MTTssy, were ompre for /PyMT () n /PyMT () primry tumour ells infete with enovirl vetor A-Sh-1, for knokown of, or with srmle ontrol vetor A-S-1. All ell prolifertion ssys were performe in triplite. Dt re presente s men±s.. P vlues: o.5, o.1, o.5. Sttistil nlysis ws vi two-tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. () Immunolots re shown of lystes of /PyMT primry tumour ells infete with S-1 or Sh-1 vetor, n of lystes of /PyMT () ells. Blots were proe with ntioies to phospho-rf-1 (p-rf-1), Rf-1, phospho-erk1/ (p-erk1/), Erk, phospho-fgfr (p-fgfr), FGFR, or -tuulin, the ltter s loing ontrol. () Lystes were sujete to pull-owns with Rf-1 RBD omin to otin tivte Rs (GTP-Rs), whih ws then etete y immunolot, using ntioy to Rs. Immunolots were repete three times, from three inepenent tumour or ell lystes. Representtive lots re shown. Quntifition of results for signifine of ifferenes for, is shown in Supplementry Fig. 5. Effets of 3(V) ltion on ell growth ftor intertions. n ffet ell prolifertion s o-reeptor for HSPG-ining mitogeni growth ftors, ut oes not ffet mitogeni responses to non-hspg-ining growth ftors 3,7. Thus we etermine whether effets of 3(V) ltion on prolifertion n Rs/Rf/Erk signlling might operte, t lest in prt, vi ffets on ility of to t s o-reeptor for HSPG-ining growth ftors. Towrs this en, /PyMT n /PyMT tumour ells were grown in 1% fetl ovine serum (FBS)-mei in the presene/sene of high onentrtions of HSPG-ining growth ftor FG or non-hspg-ining growth ftor EGF. Interestingly, perhps euse EGF levels in FBS were suffiient for mximl signlling, e EGF h no isernle effet on /PyMT or /PyMT ell growth (Fig. 7,). In ontrst, e FG h lrge effet in inresing /PyMT ell prolifertion (Fig. 7), ut reltively smll effet on /PyMT ell prolifertion (Fig. 7). Thus, wheres FG onentrtions provie y 1% FBS-growth meium seeme suffiient to inue pek signlling/prolifertion in /PyMT ells, ility of FG-ugmentttion to inrese /PyMT prolifertion suggests tht FG signlling ws not t pek levels in 1% FBS-growth meium. Therefore, FG signlling seems less effiient in /PyMT thn in /PyMT ells, n /PyMT tumour ells pper to hve efiits in HSPG-epenent, ut not HSPG-inepenent, mitogeni signlling. Exogenously e FGF1, whih hs less ffinity for HSPGs thn oes FG (ref. 35), h no effet on /PyMT tumour ell prolifertion (Fig. 7,). Interestingly, susequent to shrna knokown, ugmenttion of 1% FBS-mei with high levels of FG no longer inrese /PyMT ell prolifertion (Fig. 7). Thus, the ifferene in sensitivity to FG etween /PyMT n /PyMT tumour ells, use y 3(V) ltion, is -epenent. To further investigte how 3(V) n might intert to ffet FG signlling, ELISAs were performe to quntittively exmine FG ining to soli-phse, 3(V)-NTD, or þ 3(V)-NTD omine. FG oun either or 3(V)-NTD seprtely, n oun the two omine with somewht inrese ffinity (Fig. 7), with lulte K D vlues of 56, 36 n 1 nm, respetively. In omplementry ELISAs, oun to soli-phse FG or 3(V)-NTD, n oun soli-phse FG þ 3(V)-NTD omine with somewht inrese ffinity (Fig. 7e), with lulte K D vlues of 57, 3 n 9 nm, respetively. In thir set of ELISAs of 3(V)-NTD oun to soli-phse or FG, n oun þ FG omine with somewht inrese ffinity (Fig. 7f), with lulte K D vlues of 33, 35 n 16 nm, respetively. Clulte K D vlues of B57 nm for FG- ining in the ssys of Fig. 7,e ws similr in mgnitue to previous estimtes for FG ining to heprin 36, while ffinity of FG-3(V)-NTD ining (lulte K D vlues of B35 nm) in the ssys of Fig. 6,f ws it higher. Interestingly, eh protein oun mixture of the other two with higher ffinity thn it oun to either lone (Fig. 7 f). Consistent with ELISA results, immunopreipittion of FG, 3(V)-NTD or o-preipitte eh of the other NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 9
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 Growth urve 16 Con 1 FGF1 FG EGF 1 1 8 6 1 3 Hours Growth urve Growth urve -S-1 -S-1FG 1 16 Con -Sh-1 1 1 FGF1 1 -Sh-1-FG 8 FG 1 6 EGF 8 6 5 6 7 1 3 5 6 7 1 3 5 6 7 Hours Hours e Bining (%) :(.5:.5) 5 3 3 1 1 Bining (%) FG FG:(.5:.5) f Bining (%) 3 5 15 1 5 FG :FG(.5:.5) 1 3 FG (nm) 5 6 1 3 5 6 1 3 5 6 (nm) (nm) g FG FG _ IP: α-fg _ IP: α α3(v) IP: α- _ 1 K 5 K h α-fg α-α3(v) α 3(V) FG FGF1 i FG Lm α1 -S-1 ECM -Sh-1 5 K Figure 7 3(V)::FG intertions moulte FG signlling in tumour ells. /PyMT () n /PyMT () ells were trete with FGF1, FG, or EGF n fol inreses in ell numers t given times, reltive to numers t time, were etermine using MTT. () /PyMT ells infete with A-Sh-1 (Sh-1) for knokown, or srmle vetor A-S-1, were grown in the presene/sene of FG. () Seril onentrtions of FG were inute in wells ote with, 3(V)-NTD or oth. Plotte vlues re perent of O.D. otine from 5 nm FG ining to 3(V)-NTD-ote wells. Seril onentrtions of (e) or 3(V)-NTD (f) were inute in wells ote with FG, 3(V)-NTD or oth (e) or with FG, or oth (f). Plotte vlues re perent of O.D. otine from inution of 5 nm ining to FG-ote wells (e) or 5 nm 3(V)-NTD ining to -ote wells (f). Vlues in f re the men±s.. of three experiments. (g) FG, 3(V)-NTD n were o-inute, then immunopreipitte with ntioies to FG, 3(V) or, followe y immunolotting with the sme ntioies. (h) Immunopreipittions from /PyMT n /PyMT tumour extrts employe ntioies to FG n 3(V), followe y immunolotting with the sme ntioies. (i) /PyMT or /PyMT tumour ells infete with A-Sh-1 or srmle vetor A-S-1 were ethe from ulture ishes, followe y immunolotting of ECM remining on ishes to etet oun FG. Lminin 1 immunolotting ws loing ontrol. All ell prolifertion ssys n ELISAs were performe in triplite. Dt re presente s men±s.. P vlues: o.5, o.1, o.5. Sttistil nlysis ws vi -tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. two proteins; n, in most ses, eh protein ppere to in more strongly to eh of the other two in the presene of the thir (Fig. 7g). Results thus support the potentil for formtion of FG:3(V): triprtite omplexes n suggest some level of oopertive ining. Consistent with inility of FGF1 to ffet /PyMT ell prolifertion (Fig. 7,), FGF1 ining to 3(V)-NTD or ws not etete vi immunopreipittion (Fig. 7h). In ition to the ove in vitro intertions with reominnt proteins, immunopreipittion of enogenous FG from /PyMT tumour extrts o-preipitte enogenous 3(V) hins n vie vers (Fig. 7h). Thus, 3(V) n FG re oun together, iretly or iniretly, in vivo. Consistent with this possiility, 3(V)-free ECM from /PyMT tumour ells (Fig. 7i) n ECM of /PyMT tumour ells in whih h een knoke own (Fig. 7i) oth ontine less FG thn i ontrol /PyMT tumour ell ECM. These results re, together, onsistent with the possiility tht 3(V)-ontining ECM n t together to onentrte FG t the ell-ecm interfe. Exogenous 3(V)-NTD inues /PyMT tumour ell growth. Beuse of our hypothesis tht 3(V) hins ffet tumour ell growth properties vi NTD sequenes, we etermine whether exogenously e reominnt 3(V)-NTD sequenes might ffet /PyMT tumour ell growth. Interestingly, e 3(V)-NTD inrese ulture /PyMT tumour ell prolifertion (Fig. 8), ompnie y inrese levels of ylin D, CDK6, MAPK tivity, n FGFR phosphoryltion; n erese p-h3 ser1 levels (Fig. 8; quntifition in Supplementry Fig. 6). Thus, tretment of ells with exogenous 3(V)-NTD is suffiient to resue some portion of 3(V) funtion. 1 NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 ARTICLE BSA NTD Growth urve 16 1 1 1 8 6 BSA 6 Hours 8 Cylin D CDK6 p-h3 Ser1 Histone H3 α-tuulin BSA NTD 5 K p-erk Erk GTP-Rs Totl Rs p-fgfr FGFR K K 1 K 1 K Growth urve 3 5 15 1 5 IgG α-eα3(v) α-mα3(v) 6 Hours 8 Cylin D CDK6 p-h3 Ser1 Histone H3 -Tuulin α-α3(v) IgG E M 5 K p-erk Erk GTP-Rs Totl Rs p-fgfr FGFR α-α3(v) IgG E M K K 1 K 1 K e Volume of tumour (mm 3 ), IgG α-α3(v) 3,, 1, 1 3 5 6 Dys fter injetion f COL α3(v) PARP PARP VAR α-α3(v) HS GPCI HS GPCI Extrellulr mtrix Cytoplsmi mtrix Rs Rf1 Ativte FGFR Lowere tivtion of FGFR P Erk P Cytoplsm VAR Nuleus Erk P 18 P P 1 CDK CDK/6 Cylin E Cylin D COL G1 S Figure 8 Effets of tretment with reominnt 3(V)-NTD sequenes n nti-3(v)-ntd ntioies on in vitro n in vivo tumour ell growth. Culture /PyMT ells were trete with reominnt 3(V)-NTD sequenes prepre in mmmlin ells, followe y monitoring of growth y MTT ssy (), or y immunolotting for ylin D, CDKs n 6, p-h3 Ser1, phospho-erk1/ (p-erk1/), Erk1/, phospho-fgfr (p-fgfr), n Rs; n for tivte GTP-Rs pulle-own from lyste with Rf-1 RBD omin (). Culture /PyMT ells were trete with ntioies rise ginst reominnt 3(V)-NTD sequenes prepre in mmmlin ells (M) or E. oli (E), followe y monitoring of growth y MTT ssy (), or y immunolotting for ylin D, CDKs n 6 n p-h3 Ser1, phospho-erk1/ (p-erk1/), Erk1/, phospho-fgfr (p-fgfr), n Rs; n for tivte GTP-Rs pulle-own from lyste with Rf-1 RBD omin (). Immunolotting for -tuulin ws loing ontrol. (e) tumour volumes were mesure in C57BL/6 mie injete with /PyMT ells n then, 7 ys lter, with nti-3(v)-ntd (M) ntioies or with non-immune ontrol IgG. Cell prolifertion ssys were performe in triplite. Mie injete with 3(V)-NTD ntioy or IgG were n ¼ 8 per group. Dt re presente s men±s.. P vlues: o.5, o.1, o.5. Sttistil nlysis ws vi two-tile Stuent s t-test, with ifferenes onsiere signifint t Po.5. Immunolots were repete three times, from three inepenent ell lystes. Representtive lots re shown. Quntifition of results for signifine of ifferenes for, is shown in Supplementry Fig. 6. (f) A moel in whih the ii PARP n si vrile (VAR) 3(V) suomins in si FG () n ii heprn sulfte (HS) GAG hins, respetively. Vi these intertions 3(V) my t s linker tht enhnes the effiieny of signlling y HSPG-ining growth ftors, whih in PyMT ells t vi Rs to tivte MAPK signlling n the ell yle. Anti-3(V) ntioies inhiit tumour ell growth. We next ssesse whether tretment with nti-3(v)-ntd ntioies might ffet /PyMT tumour ell growth. Suh tretment ws suffiient to inhiit /PyMT tumour ell prolifertion in ulture (Fig. 8), with onomitntly reue ylin D, CDK 6, MAPK tivity, n FGFR phosphoryltion levels; n somewht inrese p-h3 ser1 levels (Fig. 8; quntifition in Supplementry Fig. 6). To etermine whether nti-3(v)-ntd ntioies oul hve in vivo effets, 5 1 5 /PyMT ells were injete into 1-week-ol mie, followe 7 ys lter y injetion with mg of nti-3(v)-ntd ntioies or ontrol IgG twie weekly. The result ws signifint ely of tumour growth NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 11
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 in ntioy-trete mie, with lesser effet fter tumour volume inrese pst size threshol (Fig. 8e). 3(V) sl n luminl loliztion in humn rest. As n 3(V) o-lolize n n intert to enhne growth in rest ner ells (Figs 5 n 7), it ws relevnt to etermine reltive istriutions of the two proteins in norml mmmry ut. As in mouse (Fig. 1), 3(V) hins were ssoite with K1-positive sl ells n not with K8-positive luminl ells in norml humn mmmry glns (Supplementry Fig. 7). However, the inverse ws true for, whih ws ssoite with K8-positive luminl ells, n not with K1-positive sl ells of norml humn (Fig. 9) n mouse (Supplementry Fig. 8) mmmry glns. Thus, in norml mmmry gln, 3(V)- intertions woul require ollortive sl n luminl ell intertion. 3(V) n o-lolize in humn tumours. Similr to its ssoition with norml K1-positive sl ells, 3(V) hins were ssoite with the sl-like ells of humn triple negtive sl-like rest tumours (Fig. 9). However, s in K8-positive luminl-like 11 tumours of MMTV-PyMT mie (for exmple, Fig. 1f), 3(V) hins were lso ssoite with humn luminl A tumour ells (Fig. 9). In oth humn sl-like (Fig. 9) n luminl A (Fig. 9) tumours, s in luminl-like 11 MMTV-PyMT tumours (Fig. 5), 3(V) hins o-lolize with. Interestingly, signl seeme onsistently higher in luminl A thn in sl-like tumours (for exmple, Fig. 9,). Correltion of 3(V)/ expression in rest ner types. To etermine the extent to whih 3(V) n expression levels might orrelte with humn rest tumour phenotypes, we querie the Cner Genome Atls Brest Cner (TCGA_BRCA) tset 37. Interestingly, high expression levels for oth the 3(V) gene COL5A3 (Fig. 1) n the gene (Fig. 1) re strongly ssoite with luminl A rest tumours, with expression level istriution of oth genes in humn rest ner in the orer luminl Aluminl Bsl-like (Po.1). In ft, ssoition n liner regression nlyses emonstrte tight orreltion etween 3(V) n expression levels in these humn rest ner types (Fig. 1), onsistent with the onlusion of meningful funtionl intertions etween the two proteins. Consistent with the strong ssoition of high 3(V) n expression with luminl tumours, high expression of the two proteins lso ssoites strongly with oestrogen reeptor-positive (ER þ ) n progesterone reeptor-positive (PR þ ) tumours, ut not with humn epierml growth reeptor -positive (HER þ ) tumours (Fig. 1,e). Disussion ECM is key omponent of miroenvironments tht ffet ell ifferentition, funtion, trnsformtion, tumour growth n metstti potentil 38. We previously showe the 3(V) hin to mrkely ffet speilize funtions of ertin ell types, inluing ipoytes n pnreti ells 6. Here, we emonstrte 3(V) to ffet mmmry tumour ell growth in the MMTV-PyMT moel, n provie moleulr mehnism for 3(V)-ell intertions. Others hve previously shown mmmry tumour metststi potentil to e ffete y ltertions to the ensity, thikness, orienttion n rosslinking of ol(i) firils 8,38. However, lthough 1(V) (V) heterotrimers, the most ommonly ourring ol(v) form, inorporte into n ffet physil properties of interstitil ol(i) firils, 3(V) hins hve periellulr istriution unhrteristi of ol(i), n n e synthesize y ells tht o not synthesize ol(i), resulting in 3(V)-ontining, ol(i)-free ECM 6. In ft, effets of 3(V) ltion on tumour growth re shown here to e preominntly tumour ell-utonomous, ue to loss of 3(V) hins normlly proue y tumour ells themselves, with only reltively minor non-ell-utonomous effets, presumly ontriute y strom. This is in ontrst to the non-ell-utonomous effets on mmmry tumour metstsis y ol(i) firils, exlusively ontriute y strom, onsistent with the possiility tht 3(V) hins ply roles in tumour progression issimilr to those of ol(i). Known ell surfe ollgen reeptors re the 1-integrins n DDRs (Disoiin Domin Reeptors), LAIR-1 (Leukoyte- Assoite Immunogloulin-Like reptor-1), n GPVI (Glyoprotein VI), ll of whih in Gly-X-Y repets of ollgen triple helies 1. In prtiulr, mehnotrnsution y 1-integrins ppers responsile for hnges to tumour ell ehviour in response to hnge ol(i) firil physil properties 39,. Here, 3(V) hins re shown to intert with mmmry tumour ells vi ifferent type of reeptor,, ell surfe HSPG over-expresse in rest n other ners 3,,7. is lso involve in moulting mitogeni responses to HSPG-ining growth ftors n signlling vi the Rs/Erk pthwy 3 5,7,8 in ner ells, n in stimulting ell yle progression in vrious ell types 3. Tht 3(V)- intertions re funtionlly importnt is supporte y the oservtions tht 3(V) ltion results in reue mmmry tumour ell prolifertion, Rs/Erk signlling, n ell yle progression just those properties stimulte y. Interestingly, knokown resulte in mrkely reue 3(V) levels in mmmry tumour ell-ssoite ECM. Thus, reiprol intertions my our in rest rinom, in whih tumour ell surfe ontriutes to stiliztion n/or eposition of 3(V)-ontining ECM, while 3(V)-ontining ECM enhnes tumour ell growth y engging ell surfe, in fst-forwr proess tht my ffet rinom outomes. One wy in whih ffets ell prolifertion is s o-reeptor for HSPG-ining mitogeni growth ftors 3,7. Thus, our finings tht /PyMT tumour ells re efiient in prolifertive responses to HSPG-epenent (FG), ut not HSPG-inepenent (EGF) growth ftors re onsistent with the possiility tht 3(V) hins i o-reeptor funtion. Moreover, the fining tht shrna knokown of removes ility of FG to resue /PyMT prolifertion to /PyMT levels (Fig. 7), inites the ifferene in sensitivity to FG signlling etween /PyMT n /PyMT tumour ells to epen on. ELISAs n immunopreipittions with reominnt proteins emonstrte tht n 3(V)-NTD n in eh other, n tht eh n in FG. Thus, t were onsistent with possile 3(V)::FG triprtite omplex formtion, n suggeste some level of oopertive ining. Moreover, immunopreipittions from tumour extrts n nlysis of FG mounts ssoite with the ECM of /PyMT, /PyMT, n knokown tumour ells supporte the onlusion tht the three proteins intert in vivo. Unlike the NTDs of other firillr ollgen hins, the 3(V)-NTD is not proteolytilly trimme, prompting our previous suggestion tht 3(V) might iretly intert with ell surfes vi these highly hrge sequenes 6. Within the 3(V)-NTD is n ii (pi ¼.) PARP suomin n si (pi ¼ 1.3) vrile suomin 7. Thus, hrge intertions etween the PARP suomin n si FG (pie1), n etween the vrile suomin n negtively hrge 1 NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 ARTICLE K8-luminl Overly 5 μm 5 μm K1-Bsl Overly Norml α3v Overly K1-Bsl Overly 5 μm 5 μm Bsl-like α3v Overly K8-luminl Overly Luminl A α3v Overly Figure 9 3(V) n re ssoite with sl n luminl ells, respetively, in norml rest, ut o-lolize in humn tumours. () Representtive immunofluoresene o-loliztion of (green) with mrker K8 (re) in luminl ells, ut not with mrker K1 (re) or with 3(V) (re) in sl ells, in norml humn mmmry uts. Arrowhes n rrows enote luminl n sl ells, respetively. (,) Representtive ololiztion of (green) with sl mrker K1 (re) n with 3(V) (re) in humn triple negtive, sl-like tumours (), n with K8-luminl mrker (re) n 3(V) (re) in humn luminl A tumours (). Note tht ppere to e onsistently lower in sl-like thn in luminl tumours. White sle rs, 5 mm. NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions 13
NATURE COMMUNICATIONS DOI: 1.138/nomms1351 COL5A3 expression (pnn normlize) 6 (N=98) (N=31) Bsl-like Luminl A (N=135) Luminl B expression (pnn normlize) (N=98) (N=31) (N=17) Bsl-like Luminl A Luminl B COL5A3 expression (pnn normlize) 1 5 Spermn R:.3991 P <.1 6 6 5 expression (pnn normlize) COL5A3 expression (pnn normlize) 6 6 ER neg ns e 6 ns (N=179) (N=55) (N=65) (N=61) (N=5) (N=11) 6 (N=179) (N=55) (N=65) ER pos PR neg PR pos HER neg HER pos expression (pnn normlize) ER neg (N=61) ER pos (N=5) PR neg PR pos HER neg (N=11) HER pos Figure 1 Tight orreltion of 3(V) n expression in humn tumour types. Expression of COL5A3 (,) n (,e) (gene expression, Pnn normlize vlues) in the TCGA Brest Cner ohort 37, oring to (,) histotype/pam 5 lssifition n (,e) hormone/growth ftor reeptor sttus. Note mrkely higher COL5A3 n expression in luminl-a type tumours in respet to sl-like n luminl-b types (,), n ssoition with ER n PR positivity, ut not to HER expression in,e. All t in,,,e re reporte s men (ross mrk)±s.. The mein of eh group is lso inite y ontinuous line within eh oxplot. The numer of ptients in eh group is inite t the ottom of the orresponing oxplot. () Assoition/liner regression nlysis emonstrtes tight orreltion etween COL5A3 n expression in luminl n sl-like tumour types (n ¼ 56 ptients, Spermn R, Po.1 n R squre,17). Po.5, Po.1, Po.1, s from the Kruskl Wllis test followe y Dunn s post ho test. GAG hins oul e strightforwr mehnism filitting 3(V)::FG omplex formtion (Fig. 8f). In support of this possiility we foun tht, unlike FG, effets of the ii FGF1 (pie6) on tumour ell prolifertion were not ffete y 3(V) ltion (Fig. 7) n tht FGF1 i not in 3(V)-NTD or in immunopreipittions. Thus, lesser FGF1 ffinity for HSPGs 35 n for the 3(V)-NTD together likely ontriute to reue potentil for 3(V)::FGF1 omplex formtion. Exogenously e 3(V)-NTD sequenes signifintly enhne growth of ulture /PyMT tumour ells, initing tht 3(V)-NTDs neen t e prt of full-length 3(V) hin or n integrl prt of the ECM to enhne prolifertive ehviour. Thus, future stuies employing moifie reominnt NTD sequenes e to ell ultures, to lolize 3(V)-NTD resiues involve in effets on tumour ell growth, my further insights into mehnisms involve in 3(V) ollgen s effets on ell growth, n further possile effetive therpeuti interventions esigne to isrupt tumour ell:3(v) intertions. In regr to therpeuti interventions, the fining tht polylonl nti-3(v)-ntd ntioies inhiit mmmry tumour ell growth in ulture n mmmry tumour growth in vivo n e extene y generting high-ffinity monolonl ntioies irete ginst NTD epitopes, to enhne effetiveness in slowing tumour growth. Suh prelinil stuies my e pplie in vrious moels of rest n other ner types. In norml mmmry gln, 3(V) hins re proue y sl n not luminl ells, wheres is proue y luminl n not sl ells. Thus, 3(V)- intertions woul require ollortive intertion of sl n luminl ells. In ontrst, 3(V) n o-lolize in mouse /PyMT, n humn luminl n sl-like, tumours. These results re reminisent of previous exmples in whih growth, ifferentition, n funtion in norml rest result from intertions etween proteins seprtely proue y sl n luminl ells, wheres the sme proteins re proue y single ells in rest ner, proviing gins of utonomy suh tht prrine proesses eome utorine n growth vntge is otine. We speulte tht 3(V)- o-expression my e n erly pttion tht provies growth vntge to trnsforming luminl epithelil preursor ells, from whih oth sl-like n luminl tumours re thought to originte 3. 3(V) n expression levels re tightly ssoite in humn rest ner types, onsistent with possile funtionlly meningful intertions, with istriutions of expression levels of oth proteins in the orer luminl Aluminl Bsl-like. We speulte tht high 3(V)/ expression levels ssoite with humn luminl A tumours my reflet prtiulr importne of suh expression to the growth properties of suh tumours. Tht growth of MMTV-PyMT tumours, whih hve luminl tumour gene expression pttern tht overlps those of humn luminl tumours 11, is reue upon 3(V) loss, is onsistent with the onlusion tht nomlous expression of the normlly sl ell-speifi 3(V) provies growth vntge to luminl tumour ells. In ontrst, the lesser 3(V)/ levels ssoite with sl-like tumours suggest less reline on n 3(V)- funtionl xis, n more reline on ifferent, tumour type-speifi pttions for growth vntge. As 3(V) is expresse in trnsforme, ut not norml luminl ells, it is potentil mrker for neoplsti luminl ells. Aility of nti-3(v) ntioies to slow /PyMT tumour growth suggests 1 NATURE COMMUNICATIONS 8:1351 DOI: 1.138/nomms1351 www.nture.om/ntureommunitions