Analysis of Axin2 expression and function in murine models for pancreatic cancer

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1 DOI /s Cell & Bioscience LETTER TO THE EDITOR Open Access Anlysis of Axin2 expression nd function in murine models for pncretic cncer Dietmr Zechner *, Tim Kroemer, Ann Christin Alert, Mri Schönrogge, Tois Rdecke nd Brigitte Vollmr Astrct Bckground: The involvement of Wnt in crcinogenesis nd progression of pncretic cncer is currently intensely discussed. We evluted ctivtion of the Wnt signling pthwy y using Wnt reporter mouse strin expressing β-glctosidse under the control of the Axin2 promotor during pncretitis induced formtion of precncerous lesions. We lso evluted ctivtion of Wnt signling during interction of pncretic cncer with the tumor strom. Results: Activtion of Wnt signling ws oserved during cinr-to-ductl metplsi fter chronic s well s cute pncretitis. Activtion of Wnt signling ws lso noticed during growth of pncretic cncer in n orthotopic syngeneic pncres cncer model. Activtion of Wnt signling ws, however, not oserved in crcinom ssocited firolsts, ut ws detected in few cell clusters inside the tumor. Genetic ltion of Axin2 significntly reduced ody weight without hving mjor impct on lood glucose concentrtion. However, ltion of Axin2 hd no influence on the oserved β-glctosidse positive cell clusters or on tumor weight. Conclusion: These dt demonstrte tht the Wnt signling pthwy is ctivted during cinr-to-ductl metplsi fter injury to the pncres. However these dt do not support mjor role of Wnt signling or of Axin2 in crcinom ssocited firolsts nd tumor growth. Keywords: Solid-pseudoppillry neoplsms, Acinr cell crcinom, Founder muttions, Cncer immunology, Dietes, Hyperglycemi Der Editor The expression of Axin2 is induced y cnonicl Wnt signling during cncerogenesis ut lso during mny other Wnt-regulted physiologicl nd pthophysiologicl processes [1]. The introduction of the β-glctosidse gene into the locus of Axin2 in mice, therefore, generted reporter strin ( ), which relily expresses β-glctosidse in cells where Wnt signling is ctivted [1]. Axin2 functions s scffold protein tht fcilittes the phosphoryltion nd therey the degrdtion of β-ctenin, which is the key protein necessry for cnonicl Wnt signling [2]. Thus, Axin2 inhiits the cnonicl Wnt signling pthwy in form of negtive feedck loop [1]. In Axin2 lcz/lcz mice, oth Axin2 lleles re *Correspondence: dietmr.zechner@uni rostock.de Institute for Experimentl Surgery, Rostock University Medicl Center, Schillingllee 69, 1857 Rostock, Germny replced y β-glctosidse. This cn result in prolonged Wnt signling in vivo [3]. It is well estlished tht Wnt signling is involved in the cncerogenesis of multiple gstrointestinl crcinoms [4]. In colorectl crcinom 9 % of ll tumors hve muttion in gene coding for regultory components of the cnonicl Wnt signling pthwy such s CTNNB1 (β-ctenin) or denomtous polyposis coli (APC). These muttions result in ctivtion of the cnonicl Wnt signling pthwy [4]. In heptocellulr crcinom 3 to 44 % of tumors contin muttions of CTNNB1 nd 5 to 25 % contin muttions in AXIN1 resulting in ctivtion of Wnt signling [4]. Loss of function muttion of AXIN2 cn lso e found in heptocellulr nd colorectl crcinom [4]. However, it is still controversil how importnt this signling pthwy is during crcinogenesis of pncretic 216 The Author(s). This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pulicdomin/zero/1./) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Pge 2 of 6 cncer [5]. Aout 24 % of cinr cell crcinoms (ACC) hve moleculr ltertions in the cnonicl Wnt signling pthwy. Similrly, solid-pseudoppillry neoplsms (SPNs) in the pncres usully hror muttions in CTNNB1 [5]. The prognosis of this rre neoplsm is excellent nd most ptients re cured y surgicl resection [5]. To the contrry, the prognosis of pncretic ductl denocrcinom (PDA) is very disml. Dependent on the study CTNNB1 muttions hve een identified in none or very few PDAs [6, 7]. Nevertheless, recent pulictions suggest tht ctivtion of cnonicl Wnt signling vi lterntive mechnisms might contriute to the crcinogenesis of PDA [6, 7]. For exmple, muttions of RNF43, which cn regulte Wnt signling, were detected in 6 1 % of PDAs [6, 7]. Only few pulictions exist tht suggest tht Wnt signling might lso contriute to tumor strom interction [8, 9]. This interction cn e sed on the expression of distinct Wnts in cncer ssocited firolsts, which promotes tumor progression [8]. Alterntively, expression of Wnts in crcinom cells cn induce Wnt signling in the desmoplstic rection, which indirectly promotes tumor ggressiveness [9]. Thus, the purpose of this study ws to evlute the ctivtion of the Wnt signling pthwy in precncerous lesions such s tuulr complexes fter cinr-to-ductl metplsi (ADM) nd during the tumor strom interction of fully estlished PDA. Wnt signling in tuulr complexes The formtion of tuulr complexes ws induced y repetitive cerulein dministrtion from dy 22 to dy 4 of the experimentl prdigm (Fig. 1). Since previous pulictions demonstrted detrimentl influence of streptozotocin () induced hyperglycemi on the progression of chronic pncretitis [1], the pncres of hyperglycemic mice with chronic pncretitis () ws compred to the pncres of normoglycemic mice with chronic pncretitis () nd hyperglycemic mice, which hd no pncretitis (). A relile induction of hyperglycemi in treted mice (: 21.5/ , : 2.3/ ) compred to control mice (: 5.7/ ; medin/interqurtile rnge in mmol/l on dy 22) ws noticed. In mice without pncretitis no β-glctosidse stined tuulr complexes were oserved, wheres strong β-glctosidse stining ws oserved in tuulr complexes during chronic pncretitis (Fig. 1; Tle 1). No β-glctosidse stining ws oserved in the pncres of hyperglycemic Axin2 +/+ mice during chronic pncretitis indicting tht β-glctosidse stining is dependent on the knock in llele of β-glctosidse into the Axin2 locus (Fig. 1). Thus, β-glctosidse stining in tuulr complexes is not cused y cellulr senescence. Since only few tuulr complexes could e oserved during chronic pncretitis, we lso evluted the formtion of precncerous lesions fter cute pncretitis. Acute pncretitis ws induced on dy in normoglycemic () or hyperglycemic mice () nd the pncres ws compred to normoglycemic () or hyperglycemic mice () without pncretitis (Fig. 2). A strong nd relile induction of hyperglycemi in treted mice (: 21.1/ ; : 22.1/ ) compred to control mice (: 5.5/ ; : 5.6/ ; medin/interqurtile rnge in mmol/l) ws noticed. Tuulr complexes with strong β-glctosidse stining were not oserved in the pncres of shm or treted mice, ut in the pncres of normoglycemic s well s hyperglycemic Axin2 +/ lcz mice during cute pncretitis (Fig. 2; Tle 2). No β-glctosidse stining ws oserved in the pncres of hyperglycemic Axin2 +/+ mice fter cute pncretitis (Fig. 2). Thus, Wnt signling is ctivted in ssumed precncerous lesions such s tuulr complexes y pncretitis, which is well defined risk fctor for the development of pncretic cncer. This oserved ctivtion of cnonicl Wnt signling upon tissue dmge is consistent with previous pulictions. For exmple, it ws suggested tht the oserved increse in β-ctenin mrna level [11] or β-ctenin protein concentrtion [12] fter tissue dmge in the pncres indictes ctivtion of Wnt signling. In ddition, tissue dmge induced the expression of Axin2 s demonstrted y quntittive PCR [12] nd the ctivtion of Wnt signling in ductl cells ws lso oserved fter prtil pncretic duct ligtion [13]. The following three hypotheses cn e defined when descriing the oserved ctivtion of Wnt signling during ADM in respect to cncerogenesis: () Activtion of Wnt signling hs no effect on cncerogenesis, ut is concomitnt phenomenon when cells re stuck in dedifferentition, () Wnt signling inhiits cncerogenesis, (c) Wnt signling promotes cncerogenesis. Possily, the function of Wnt signling is completely different during cncerogenesis leding to PDA or other sutypes of pncretic cncer. In respect to cncerogenesis of PDA most dt support the first or second hypothesis nd disgree with the third hypothesis. For exmple, ctivtion of Wnt signling does not cooperte with oncogenic Rs to induce PDA formtion in mice [12]. Moreover, only few muttions hve een found in humn PDAs tht ctivte Wnt signling suggesting tht these muttions re not founder muttions of the crcinom [6, 7].

Pge 3 of 6 dy 1 2 3 4 5 22 24 26 29 31 33 36 38 4 dy 1 2 3 4 5 22 23 28 3x 8x 3x 8x Axin2 +/+ Fig. 1 Chrcteriztion of the niml model for chronic pncretitis.

cerulein (5 µg/kg) on the indicted dys; the control group () ws treted with the pproprite vehicle solution insted of.

Brs 5 μm In respect to cncerogenesis of SPNs or ACCs most dt disgree with the first nd second hypothesis nd support the third hypothesis.

Thus, permnent ctivtion of Axin2 +/+ Fig. 2 Chrcteriztion of the niml model for cute pncretitis.

3 Pge 3 of 6 dy dy x 8x 3x 8x Axin2 +/+ Fig. 1 Chrcteriztion of the niml model for chronic pncretitis. Hyperglycemi ws induced in two cohorts (, ) y ip injection of 5 mg/kg streptozotocin on dy 1 5 nd chronic pncretitis ws then induced in two cohorts (, ) y dministrtion of three ip injections of cerulein (5 µg/kg) on the indicted dys; the control group () ws treted with the pproprite vehicle solution insted of. β-glctosidse stining in tuulr complexes of the pncres in normoglycemic nd hyperglycemic Axin2 +/ lcz mice ws oserved fter pncretitis, wheres no β-glctosidse stining ws oserved in Axin2 +/+ mice. Brs 5 μm In respect to cncerogenesis of SPNs or ACCs most dt disgree with the first nd second hypothesis nd support the third hypothesis. For exmple, ctivtion of Wnt signling in mice results in the formtion of tumors resemling humn SPNs [14], wheres ctivtion of Wnt signling in p53 deficient mice results in the formtion of tumors resemling humn ACC [15]. Moreover, humn SPNs usully hror muttions in CTNNB1 tht ctivte Wnt signling nd out 24 % of ACCs in ptients show moleculr chnges in key proteins of the cnonicl Wnt signling pthwy [5]. Thus, permnent ctivtion of Axin2 +/+ Fig. 2 Chrcteriztion of the niml model for cute pncretitis. Hyperglycemi ws induced in two cohorts (, ) y ip injection of 5 mg/kg streptozotocin on dy 1 5 nd chronic pncretitis ws then induced in two cohorts (, ) y dministrtion of eight ip injections of cerulein (5 µg/kg) on dy 22 nd 23; the control groups (, ) were treted with the pproprite vehicle solutions. β-glctosidse stining in tuulr complexes of the pncres in normoglycemic nd hyperglycemic mice ws oserved fter pncretitis, wheres no β-glctosidse stining ws oserved in Axin2 +/+ mice. Brs 5 μm cnonicl Wnt signling my promote cncerogenesis of SPNs nd ACCs, ut not of PDA. Wnt signling during tumor strom interction In order to evlute, if Wnt signling cn e oserved in cncer ssocited firolsts, 666PDA cells were injected into the pncres of C57BL6-Tg ACTB-eGFP1Os/J s well s mice (Fig. 3). Crcinoms induced desmoplstic rection, which ws chrcterized y GFP + Tle 1 Quntifiction of β-glctosidse + tuulr complexes during chronic pncretitis on dy 4 Numer of β-glctosidse + tuulr complexes Numer of tuulr complexes oserved Numer of mice nlyzed

Pge 4 of 6 Tle 2 Quntifiction of β-glctosidse + tuulr complexes fter cute pncretitis on dy 28 Numer of β-glctosidse + tuulr complexes Numer of tuulr complexes oserved Numer of mice nlyzed 3 5 6 8 5 5

666PDA cells were injected on dy into the pncres of C57BL6-Tg ACTB-eGFP1Os/J, Axin2 +/+, or Axin2 lcz/lcz mice nd the tumors were nlyzed on dy 2 or 21.

4 Pge 4 of 6 Tle 2 Quntifiction of β-glctosidse + tuulr complexes fter cute pncretitis on dy 28 Numer of β-glctosidse + tuulr complexes Numer of tuulr complexes oserved Numer of mice nlyzed dy 666PDA 2/21 Fig. 3 Chrcteriztion of the niml model for studying desmoplsi. 666PDA cells were injected on dy into the pncres of C57BL6-Tg ACTB-eGFP1Os/J, Axin2 +/+, or Axin2 lcz/lcz mice nd the tumors were nlyzed on dy 2 or 21. Desmoplstic rection visulized y nti-gfp immunohistochemistry in C57BL6-Tg ACTB-eGFP1Os/J mouse, which uiquitously expresses GFP, shows strong GFP expression in firolst like cells surrounding the crcinom (rrow), nd few GFP expressing cells within the crcinom (rrowhed). c Chrcteristic imges of tumors in mice showing no β-glctosidse stining in firolst like cells surrounding the crcinom (rrow), ut few clusters of β-glctosidse + cells within the crcinom (rrowhed). Brs 5 µm C57BL6-Tg ACTB-eGFP1Os/J c firolst-like cells surrounding the crcinom nd few GFP + cells inside the crcinom (Fig. 3). In ll Axin2 +/ lcz mice firolst like cells surrounding the crcinom did not express β-glctosidse (Fig. 3c). β-glctosidse positive cells were however noticed in out 28 % of tumors in ring like clusters within the tumor (Fig. 3c). In order to evlute if these cells re dependent on functionl Axin2 gene, we injected 666PDA cells into the pncres of Axin2 lcz/lcz mice. These chrcteristic clusters of β-glctosidse positive cells were lso oserved in Axin2 lcz/lcz mice (Additionl file 1). Thus Axin2 is not necessry for the formtion of these β-glctosidse positive cell clusters. Since Wnt signling hs een reported to e importnt t vrious stges of T cell development [16], these cells might e immune cells migrting into the crcinom. However, these dt do not support the hypothesis tht Wnt signling is ctivted in crcinom ssocited firolsts. In order to evlute if Axin2 influences the growth of pncretic cncer we compred the tumor weight in Axin2 lcz/lcz to the tumor weight in or Axin2 +/+ mice. Loss of Axin2 function significntly reduced the ody weight without hving mjor influence on the lood glucose concentrtion (Fig. 4, ). We did not oserve mjor difference in the ody weight ( : 29.8/ ; Axin2 +/+ : 29.7/ medin/interqurtile rnge in g) or in lood glucose concentrtion ( : 8.2/ ; Axin2 +/+ : 8.8/ medin/interqurtile rnge in mmol/l) etween mice lcking one or none llele of Axin2. A lood cell count did not revel mjor differences in the numer of leukocytes (Axin2 +/+ : 4.2/ ; Axin2 +/ lcz : 6./ ; Axin2 lcz/lcz : 6.9/ ), lymphocytes (Axin2 +/+ : 2.5/1.9 4.; : 3.9/ ; Axin2L lcz/lcz : 3.8/ ), or monocytes plus neutrophil grnulocytes (Axin2 +/+ : 1.7/1.3 2.; Axin2 +/ lcz : 2.1/ ; Axin2L lcz/lcz : 3.2/ medin/

5 Pge 5 of 6 5 * 12 c 4 ody weight [g] lood glucose [mmol/l] tumor weight [mg] Fig. 4 Anlysis of ody weight, lood glucose nd tumor weight in or Axin2 lcz/lcz mice. Axin2 lcz/lcz mice hve significntly decresed ody weight. No mjor influence of Axin2 genotype on lood glucose concentrtion ws oserved. c No significnt influence of Axin2 genotype on tumor weight could e demonstrted. Significnt difference: *P =.6 interqurtile rnge in 1 9 cells/l) dependent on the mouse genotype. We did lso not oserve n ovious influence of Axin2 function on the tumor weight when compring tumors grown in to tumors grown in Axin lcz/lcz (Fig. 4c). We did lso not oserve mjor difference in tumor weight ( : 166/86-215; Axin2 +/+ : 283/ medin/interqurtile rnge in mg) etween mice lcking one or none llele of Axin2. Thus, no mjor function of Wnt signling on the growth of pncretic cncer could e oserved in this syngeneic orthotopic niml model. Additionl file Additionl file 1. In the Supplementl Mteril Section results from etglctosidse stinings, lood glucose concentrtions, ody weights, tumor weights, s well s numer of leucocytes, lymphocytes nd monocytes plus gnulocytes re presented. Arevitions ACC: cinr cell crcinom; ADM: cinr-to-ductl metplsi; BrdU: 5-romo- 2 -deoxyuridine; H/E: hemtoxylin/eosin; PDA: pncretic ductl denocrcinom; SPN: solid-pseudoppillry neoplsm. Authors contriutions DZ crried out the study design, the interprettion of dt, the cell injections into mice, histochemicl evlutions, nd drfted the mnuscript. All other uthors prticipted in the study (TK: Figs. 1, 2; Tles 1, 2; ACA: Figs. 3c, nd 4; MS: Fig. 4, TR: Fig. 3), crried out the nlysis nd interprettion of dt, nd revised the mnuscript. BV mde sustntil contriutions to the design of the study nd revision of the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements We thnk Berit Blendow, Dorothe Frenz, Ev Loreer-Rehfeldt, nd Mren Nerowski (Institute for Experimentl Surgery, Rostock University Medicl Center) for excellent technicl ssistnce. The Axin2 tm1wm mouse strin ws generous gift from Prof. Wlter Birchmeier (Berlin, Germny). Funding cknowledgement: Supported y the Forschungsförderung der Medizinischen Fkultät der Rostocker Universität (FORUN) (project 88917). Competing interests The uthors declre tht they hve no competing interests. Avilility of dt All dt generted or nlyzed during this study re included in this pulished rticle nd its Additionl file 1. Ethics pprovl All experiments on mice were executed in ccordnce with Germn legisltion nd the EU-directive 21/63/EU nd were pproved y the Lndesmt für Lndwirtschft, Leensmittelsicherheit und Fischerei Mecklenurg-Vorpommern. Received: 11 July 216 Accepted: 14 July 216 References 1. Lustig B, Jerchow B, Schs M, Weiler S, Pietsch T, Krsten U, et l. Negtive feedck loop of Wnt signling through upregultion of conductin/xin2 in colorectl nd liver tumors. Mol Cell Biol. 22;22(4):

6 Pge 6 of 6 2. Behrens J, Jerchow BA, Würtele M, Grimm J, Asrnd C, Wirtz R, et l. Functionl interction of n xin homolog, conductin, with et-ctenin, APC, nd GSK3et. Science. 1998;28(5363): Yu HM, Jerchow B, Sheu TJ, Liu B, Costntini F, Puzs JE, et l. The role of Axin2 in clvril morphogenesis nd crniosynostosis. Development. 25;132(8): White BD, Chien AJ, Dwson DW. Dysregultion of Wnt/β-ctenin signling in gstrointestinl cncers. Gstroenterology. 212;142(2): Shi C, Dniels JA, Hrun RH. Moleculr chrcteriztion of pncretic neoplsms. Adv Ant Pthol. 28;15(4): Witkiewicz AK, McMilln EA, Blji U, Bek G, Lin WC, Mnsour J, et l. Whole-exome sequencing of pncretic cncer defines genetic diversity nd therpeutic trgets. Nt Commun. 215;6: Wddell N, Pjic M, Ptch AM, Chng DK, Ksshn KS, Biley P, et l. Whole genomes redefine the muttionl lndscpe of pncretic cncer. Nture. 215;518(754): Fu L, Zhng C, Zhng LY, Dong SS, Lu LH, Chen J, et l. Wnt2 secreted y tumour firolsts promotes tumour progression in oesophgel cncer y ctivtion of the Wnt/β-ctenin signlling pthwy. Gut. 211;6(12): Avgustinov A, Irvni M, Roertson D, Ferns A, Go Q, Klingeil P, et l. Tumour cell-derived Wnt7 recruits nd ctivtes firolsts to promote tumour ggressiveness. Nt Commun. 216;7: Zechner D, Knpp N, Borowski A, Rdecke T, Genz B, Vollmr B. Dietes increses pncretic firosis during chronic inflmmtion. Exp Biol Med (Mywood). 214;239(6): Jensen JN, Cmeron E, Gry MV, Strkey TW, Ginni R, Jensen J. Recpitultion of elements of emryonic development in dult mouse pncretic regenertion. Gstroenterology. 25;128(3): Morris JP 4th, Cno DA, Sekine S, Wng SC, Herok M. Bet-ctenin locks Krs-dependent reprogrmming of cini into pncretic cncer precursor lesions in mice. J Clin Invest. 21;12(2): Huch M, Bonfnti P, Boj SF, Sto T, Loomns CJ, vn de Wetering M, et l. Unlimited in vitro expnsion of dult i-potent pncres progenitors through the Lgr5/R-spondin xis. EMBO J. 213;32(2): Heiser PW, Cno DA, Lndsmn L, Kim GE, Kench JG, Klimstr DS, et l. Stiliztion of et-ctenin induces pncres tumor formtion. Gstroenterology. 28;135(4): Clrke AR, Cummings MC, Hrrison DJ. Interction etween murine germline muttions in p53 nd APC predisposes to pncretic neoplsi ut not to incresed intestinl mlignncy. Oncogene. 1995;11(9): Gttinoni L, Ji Y, Restifo NP. Wnt/et-ctenin signling in T-cell immunity nd cncer immunotherpy. Clin Cncer Res. 21;16(19): Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t

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