Phosphorylated p70s6k in noninvasive low grade urothelial carcinoma of the bladder: correlation with tumor recurrence

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1 (2014) 16, AJA, SIMM & SJTU. All rights reserved X Mle Helth Open Access ORIGINAL ARTICLE Phosphorylted p70s6k in noninvsive low grde urothelil crcinom of the ldder: correltion with tumor recurrence Soon J Kim 1, Jung Hoon Kim 2, Hui Seok Jung 2, Te Jin Lee 3, Kyung Mee Lee 2, In Ho Chng 2 We investigted whether inhiiting phosphorylted p70s6k (p p70s6k) suppresses the prolifertion nd growth of noninvsive low grde urothelil crcinom (LG URC) in vitro nd whether p p70s6k cn serve s predictive iomrker for the recurrence of noninvsive LG URC of the ldder in ptients. We constructed tissue microrry (TMA) for 95 LG URC nd 35 enign urothelium smples nd performed immunohistochemicl stining for p p70s6k nd p 4E BP1. A Cox regression model ws used to investigte the predictive fctors for recurrence of LG URC. We investigted the dose dependent ntiprolifertive effect of rpmycin, its ntiprolifertive effect nd the growth inhiition effect of p70s6k sirna trnsfection in RT4 nd 253J cell lines. The pt1 stged group (P < 0.05; hzrd rtio (HR), 2.415) nd the high p p70s6k stining group (P < 0.05; HR, 2.249) were independent fctors for predicting recurrence. Rpmycin inhiited RT4 nd 253J cell prolifertion in dose dependent mnner (r = 0.850, P < in RT4 cells; r = 0.835, P < in 253J cells). RT4 nd 253J cell prolifertion nd growth were inhiited y the trnsfection of p70s6k sirna nd rpmycin, respectively (P < 0.05). Trnsfection of p70s6k sirna resulted in inhiitory effects on cell prolifertion nd growth tht were similr to those of rpmycin. Our results suggest tht inhiiting p70s6k phosphoryltion is importnt to prevent recurrence nd tht p70s6k phosphoryltion cn e used s moleculr iomrker to predict recurrence of certin LG URC of the ldder. (2014) 16, ; doi: / X ; pulished online: 11 Mrch 2014 Keywords: ldder; cncer; p70s6k INTRODUCTION Approximtely 70% 80% cses of urothelil crcinom (URC) of the ldder present s non muscle invsive tumors (pt nd pt1). Such non muscle invsive URC is treted with trnsurethrl resection of ldder nd intrvesicl instilltion of cillus Clmette Guerin, with fvorle prognosis nd 5 yer survivl rte of pproximtely 90%. 1 However, recurrence is oserved in 50% 90% of cses nd 10% 20% of these progresses to more invsive URC. 1 Therefore, the outcomes of ptients with non muscle invsive URC is relted to recurrence nd progression nd precise prediction of recurrence nd progression is lrgely dependent on URC pthologic grde. URC is clssified into four ctegories: ppillom, ppillry urothelil neoplsm of low mlignnt potentil, low grde URC (LG URC) nd high grde URC (HG URC). Despite tretment with trnsurethrl resection of ldder nd pproprite therpy, the recurrence rte for non muscle invsive HG URC is >80% nd >50% of recurrent cses progress to more invsive form. The rte of invsion, progression or metstsis is low in non muscle invsive LG URC treted with trnsurethrl resection of ldder nd pproprite therpy, lthough recurrence is oserved in pproximtely 50% of cses. 2 However, the mechnism of non muscle invsive LG URC recurrence is not yet known. Lortory methods tht predict the risk of recurrence nd progression include exmintion of proliferting cell nucler ntigens, the rs gene, nd p21, p53 nd R expression. Such fctors re significntly expressed mostly in HG URC or invsive URC, ut not in LG URC; therefore, identifying high risk fctors tht predict disese recurrence is very importnt. 3 The phosphtidylinositol 3 kinse (PI3K) pthwy ctivtes numer of signling molecules nd the Akt/mmmlin trget of the rpmycin (mtor) pthwy is of prticulr interest ecuse of its role in inhiiting poptosis nd promoting cell prolifertion. 4 mtor controls protein synthesis y phosphorylting downstrem sustrtes, including p70s6 kinse (p70s6k) nd eukryotic initition fctor (elf) 4E inding protein 1 (4E BP1). 5 The p70s6k protein phosphoryltes the 40S riosoml protein S6 nd is proposed to ply crucil role in the trnsltion of 5 terminl oligopyrimidine trct mrnas. 6 Phosphoryltion y mtor 4E BP1 disrupts its inding to elf4e, protein tht inds the 5 cp structure of mrna. The relesed elf4e llows the formtion of functionl trnsltion initition complex, therey llowing trnsltion. 7 We previously reported tht ctivting the mtor pthwy, such s phosphorylting p70s6k, is relted to high recurrence nd disese progression in non muscle invsive URC of the ldder. 8 Severl studies hve reveled tht the PI3K/AKT/mTOR pthwy is 1 Deprtment of Convergence Medicine nd Phrmceuticl Biosciences, Chung Ang University Grdute School, Seoul, 2 Deprtment of Urology nd 3 Deprtment of Pthology, Chung Ang University College of Medicine, Seoul, Kore. Correspondence: Dr. IH Chng (cucih@cu.c.kr) Received: 28 Ferury 2013; Revised: 02 July 2013; Accepted: 30 August 2013

2 612 lrgely responsile for promoting cell growth in non muscle invsive LG URC of the ldder. 9,10 Therefore, we investigted whether inhiiting phosphorylted p70s6k suppresses the prolifertion nd growth of non muscle invsive LG URC in vitro models nd whether phosphorylted p70s6k is predictive iomrker for recurrence nd progression of noninvsive LG URC of the ldder in ptient model. MATERIALS AND METHODS Humn noninvsive LG URC tissue microrry (TMA) construction nd immunohistochemistry After otining institutionl review ord pprovl, we retrieved 95 LG URC ldder specimens collected t Chung Ang University Hospitl etween 1989 nd Prffin locks were ville in 95 cses nd TMAs were constructed using mnul rry device (TMA set, Lro, Seoul, Kore) for the 95 LG URC smples nd 35 enign urothelium smples, s descried previously. 8 All clinicopthologicl ptient dt were retrieved from electronic medicl records nd included demogrphics, pthologicl stge, tumor size, tumor multiplicity nd concurrent crcinom in situ. Follow up dt on postopertive intrvesicl cillus Clmette Guerin tretment, disese recurrence nd progression were otined vi review of ptient medicl records nd letters of inquiry to ptients. Immunohistochemistry sections were incuted with the primry ntiody (nti p p70s6k (Ser371): diluted 1:100 nd nti p 4E BP1: diluted 1:400), followed y peroxidse conjugted secondry ntiody. Smples were ssessed in lind mnner y one pthologist with no knowledge of the ptients clinicopthologicl dt. We modified stndrd H score to evlute immunohistochemicl positivity. The stndrd H score (scle of 0 300) ws clculted sed on the product of the percentge of cells showing cytoplsmic leling (0 100) multiplied y the intensity of leling (1 = wek; 2 = moderte nd 3 = strong). 11 And then finl H score ws generted y clculting the verge of three tissue smples. A cutoff ws used for the expression of ech mrker sed on the medin tumor H score (H score 10 nd 93 for p p70s6k nd p 4E BP1, respectively). We defined high expression of mrker s the cutoff nd higher nd low expression s none or less thn the cutoff. Cell cultures nd regents The humn non muscle invsive LG URC cell lines (RT4 nd 253J) were purchsed from the Americn Type Culture Collection (Mnsss, VA, USA). Rpmycin ws purchsed from Sigm (St Louis, MO, USA) nd the following ntiodies were purchsed from Cell Signling Technologies (Beverly, MA, USA): mtor, phosphorylted mtor (p mtor, Ser 2448), p70 S6 kinse, phosphorylted p70 S6 kinse (p p70 S6 kinse, Ser371) nd ctin. Western lot nlysis Cells were wshed with ice cold phosphte uffered sline nd trypsinized nd lysis uffer (Intron, Seoul, Kore) ws dded. The lystes were stored t 20 C until nlysis. The mount of protein ws quntified y the Brdford ssy (Bio Rd, Hercules, CA, USA). Equl mounts of protein were loded onto Redygels (4% 20% Tris HCL; Bio Rd) nd electrophoresis ws performed ccording to the mnufcturer s instructions. Proteins were lotted onto polyvinylidene fluoride (PVDF) memrnes (Invitrogen, Crlsd, CA, USA) nd incuted for 1 h t room temperture in 5% skim milk for locking. Blots were incuted with primry ntiodies overnight t 4 C nd with horserdish peroxidse conjugted secondry ntiodies for 1 h t room temperture. The memrnes were developed using enhnced chemiluminescence. sirna, plsmid DNA construct nd trnsfection sirna oligonucleotides ginst p70s6k (sirna I/II) were designed y nd purchsed from Cell Signling Technologies (Beverly, MA, USA). Trnsient trnsfections into RT4 nd 253J cells were performed using Lipofectmine 2000 regent (Invitrogen) ccording to the mnufcturer s instructions. Cells were hrvested t 48 h for susequent experiments. A plsmid contining the coding sequence of Homo spiens p70s6k (GenBnk: NM ) ws prepred y cloning into the pcdna3.1 vector (Invitrogen). The coding region of the p70s6k gene ws PCR mplified from pcmv SPORT6. Next, the p70s6k frgment nd the pcdna3.1 vector (Invitrogen) were digested with the EcoRI nd Xhol restriction enzymes nd ligted y infusion technology to uild the pcdna3.1 p70s6k plsmid. Competent Escherichi coli DH5 cells were trnsformed nd positive clones were identified y PCR. The PCR products were digested nd sequenced. After sequencing, positive clones were selected to grow nd propgte. The pcdna3.1 p70s6k plsmid ws purified nd trnsfected into RT4 nd 253J cells using Lipofectmine 2000 in Opti miniml essentil medium (Invitrogen) ccording to the mnufcturer s protocol. RT4 nd 253J cells were grown in six well pltes until they reched 85% 90% confluence. One microliter of Lipofectmine 2000 regent nd 0.4 µg pcdna3.1 p70s6k ws used to trnsfect ech well of cells in the sence of serum. The cells were hrvested for Western lot nlysis 24 h fter trnsfection. Cell prolifertion ssy Cell prolifertion ws determined using EZ Cytoz viility ssy kit (Dil l, Seoul, Kore) ccording to the mnufcturer s instructions. This ssy is sed on the clevge of tetrzolium slt to wter solule formzn y the succinte tetrzolium reductse system. Briefly, RT4 nd 253J cells were seeded in 96 well pltes t density of cells per well. After 24 h in culture, the cells were treted with 100 nmol l 1 p70s6k sirna, 0.5 µg pcdna3.1 p70s6k plsmid nd 10 µmol l 1 rpmycin for 48 h. The cell prolifertion ssy ws performed 1, 2 or 3 dys fter tretment. The medium ws replced with fresh medium (200 µl) nd 10 µl EZ cytox regent ws dded to ech well. The cell culture ws continued for 1 h nd the culture medium ws then removed to wells of new plte. Opticl density ws quntified t wvelength of 450 nm. Crystl violet growth ssy RT4 nd 253J cells were seeded in 24 well pltes t density of cells per well. After 24 h in culture, the cells were treted with 100 nmol l 1 p70s6k sirna, 0.5 µg pcdna3.1 p70s6k plsmid nd 10 µmol l 1 rpmycin for 24 h. Growth inhiition ws determined using the crystl violet method. Briefly, fter 2 dys of tretment, the cells were fixed in 5% glutrldehyde in phosphte uffered sline, rinsed with distilled wter nd dried completely. The cells were incuted in 1:1 volume mixture of 200 mmol l 1 3 (cyclohexylmino) 1 propnesulfnoic cid, ph = 9.5 nd 0.2% crystl violet t 25 C for 30 min nd then wshed nd dried. Crystl violet stin ws susequently dissolved in 10% cetic cid (Sigm, MO, USA) nd the sornce ws red t 560 nm following ckground sutrction. Cell growth ws determined nd photogrphed using Zeiss Axiovert 200M cell live microscope. Sttisticl nlysis Sttisticl Pckge for Socil Sciences softwre pckge version 14.0 (SPSS Inc, Chicgo, IL, USA) ws used for ll sttisticl nlyses. The reltionships etween ordinry vriles were investigted using two tiled Student s t test nd nlysis of vrince. Spermn s

3 613 correltion coefficients were clculted to test the reltionships mong prmeters. Recurrence free survivl curves were estimted using the Kpln Meier method nd ny differences in the survivl curves were compred using log rnk tests. A Cox regression model ws used to investigte predictive fctors for the recurrence of LG URC in multivrite nlysis. A P < 0.05 ws considered significnt. RESULTS p p70s6k nd p 4E BP1 expression in humn LG URC TMA The medin ge of dignosis for the ptient cohort ws 64 (29 85) yers, with mle: femle rtio of 6.3:1 nd medin follow up period of 84 months (rnge, months). Recurrence ws oserved in 37 ptients (39.3%). No reltionship ws oserved etween p 4E BP1 H scores nd clinicopthologicl vriles (ge, sex, pthologicl stge, tumor size, tumor multiplicity nd concurrent crcinom in situ), ut the p p70s6k H scores were higher for older ge (15.6 ± 24.0 vs 27.0 ± 25.5, P < 0.05) nd concurrent crcinom in situ (16.1 ± 22.5 vs 52.2 ± 30.5) (Tle 1). As shown in Figure 1, p p70s6k nd p 4E BP1 were expressed in the cytoplsm of LG URC of the ldder nd in enign epithelium nd p p70s6k stining ws less intense in low grde superficil umilicl cord cell tissue compred to tht in the enign urothelium. However, p 4E BP1 stining ws greter in LG URC tissue. The men p p70s6k H score ws lower in the LG URC cohort thn in the enign cohort (27.4 ± 4.6 vs ± 16.0, P < 0.05), wheres the men p 4E BP1 score ws higher in the LG URC cohort thn in the enign cohort (117.8 ± 7.9 vs 61.2 ± 14.1, P < 0.05) (Figure 1). The medin tumor H scores were 10 nd 93 for p p70s6k nd p 4E BP1, respectively. We investigted the correltion etween p p70s6k nd p 4E BP1 expression nd recurrence rtes. In the Kpln Meier survivl nlysis, LG URC, with high p p70s6k immunohistochemicl stining, exhiited higher recurrence (P < 0.05, Tle 1: Clinicopthologicl chrcteristics of the 95 ptients with LG URC nd summry of p p70s6k nd p 4E BP1 expression (men H score), ctegorized y clinicopthologicl prmeters Clinicl nd pthologic fetures Age (yer) Sex No. of ptients (%) p 4E BP1 H score (men ± s.d.) P vlue p p70s6k H score (men ± s.d.) P vlue <70 69 (72.6) ± ± 24.0 < (27.4) 98.5 ± ± 25.5 Mle 82 (86.3) ± ± Femle 13 (13.7) 86.5 ± ± 16.1 T stge T 90 (94.7) ± ± T1 5 (5.3) ± ± 26.9 Numer of tumors 1 67 (70.5) ± ± (29.5) ± ± 23.8 Tumor dimeters <3 cm 51 (53.7) ± ± cm 44 (46.3) ± ± 30.5 Concurrent CIS No 88 (92.6) ± ± 22.5 <0.05 Yes 7 (7.4) ± ± 30.5 CIS: crcinom in situ; LG URC: low grde urothelil cell crcinom; p 4E BP1: phosphorylted 4E BP1; p p70s6k: phosphorylted 4E BP1 log rnk test), lthough p 4E BP1 sttus ws not relted to recurrence (Figure 1c nd 1d). Tle 2 shows the results of univrite nd multivrite nlyses using the Cox regression model for recurrence free survivl. T1 stging group (P < 0.05; hzrd rtio (HR), 2.145) nd high p p70s6k stining group (P < 0.05; HR, 2.249) were independent fctors predicting recurrence. Therefore, we decided not to investigte the 4E BP 1 pthwy in vitro ecuse of the lck of correltion etween p 4E BP 1 expression nd recurrence in vivo. Western lot nlysis for mtor pthwy protein expression in the URC cell lines We nlyzed the expression nd ctivtion sttus of the mtor nd p70s6k proteins in the RT4 nd 253J cell lines. As shown in (Figure 2), mtor nd p70s6k were expressed in oth URC cells nd mtor nd p70s6k were phosphorylted in RT4 nd 253J cells. Thus, oth low grde non muscle invsive URC cell lines exhiited constitutively ctivted mtor/p70s6k signling. Dose dependent ntiprolifertive effect of rpmycin in RT4 nd 253J cells RT4 nd 253J cells were treted with rpmycin (1 pmol l 1, 10 pmol l 1, 100 pmol l 1, 1 nmol l 1, 10 nmol l 1, 100 nmol l 1, 1 µmol l 1 nd 10 µmol l 1 ) for 48 h. The cell prolifertion ssy ws performed 1, 2 or 3 dys fter tretment. Ech of the concentrtions ove ws considered s one treted group nd there ws no rpmycin in the control group. Rpmycin hd demonstrted inhiitory effects on RT4 nd 253J cell prolifertion in dose dependent mnner t 3 dys fter tretment. Cell prolifertion ws correlted with rpmycin concentrtion nd the Spermn s correltion vlues for these two prmeters were r = 0.850, P < for RT4 nd r = 0.835, P < for 253J cells (Figure 2). Tle 2: Results of univrite nd multivrite nlyses of clinicopthologicl vriles nd the expression levels of p p70s6k nd p 4E BP1 in reltion to recurrence free survivl in 95 ptients with LG URC Vriles Univrite nlysis Multivrite nlysis Age (yer) Sex HR (95% CI) P vlue HR (95% CI) P vlue ( ) ( ) Femle ( ) ( ) T stge T ( ) < ( ) <0.05 No. of tumors ( ) ( ) Tumor dimeter CIS 3 cm ( ) ( ) Yes ( ) ( ) Previous BCG Yes ( ) ( ) p 4E BP1 High ( ) ( ) p p70s6k High ( ) < ( ) <0.05 BCG: cillus clmette guerin; CI: confidence intervl; CIS: crcinom in situ; HR: hzrd rtio; LG URC: low grde urothelil cell crcinom; p 4E BP1: phosphorylted 4E BP1; p p70s6k: phosphorylted 4E BP1

4 Phosphorylted P70S6K in urothelil crcinom 614 c d Figure 1: Expression of phosphorylted-p70s6k (p-p70s6k) nd p-4e-bp1 in ldder tissue. () Immunohistochemicl stining of p-p70s6k nd p-4ebp1 in prffin-emedded sections of enign urothelium nd low-grde urothelil crcinom (LG-URC) tissues. P-p70S6K stining (red) ws lower in LG-URC tissues thn in enign urothelil tissues, ut p-4e-bp1 stining (red) ws higher in LG-URC tissues. () Grph displying the comprisons etween the H-scores of p-p70s6k nd p-4e-bp1 with respect to enign nd LG-URC tissues. The men H-scores for p-4e-bp1 were significntly higher in LG-URC tissues nd the men H-scores for p-p70s6k were significntly lower (*P < 0.05). (c) LG-URC with high p-p70s6k immunohistochemicl stining exhiited higher risk of recurrence (P < 0.05). (d) LG-URC with p-4e-bp1 sttus ws not relted to recurrence in LG-URC. (P = 0.314). Scle rs = 100 mm. The cell prolifertion inhiition effect of p70s6k sirna trnsfection nd rpmycin in RT4 nd 253J cells Figure 3 shows tht p70s6k sirna medited the downregultion of p70s6k nd its phosphor ylted counterprt nd tht Figure 2: Western lot nlysis for mtor pthwy protein expression in LG-URC cell lines nd the ntiprolifertive effect of rpmycin in RT4 nd 253J cells. () Cell extrcts from four URC cell lines (RT4 nd 263J) were sujected to immunolotting using ntiodies directed ginst mtor, p-mtor, p70s6k nd p-p70s6k. Protein loding ws normlized using n ntiody recognizing -ctin. RT4 nd 253J cells expressed phosphorylted mtor nd p70s6k proteins. Blots representtive of three seprte experiments re shown. () Incuting RT4 nd 263J cells with incresing doses of rpmycin demonstrted dose-dependent reduction in cell prolifertion t 3 dys fter tretment (r = , P < in RT4 nd r = , P < in 253J cells). pcdna3.1 p70s6k medited the upregultion of p70s6k nd its phosphorylted counterprt in RT4 nd 253J cells, lthough the downregultory effect of p70s6k sirna ws not solute. Rpmycin inhiited p70s6k phosphoryltion in RT4 nd 253J cells. RT4 nd 253J cells were treted with 100 nmol l 1 p70s6k sirna, 100 nmol l 1 pcdna3.1 p70s6k nd 10 nmol l 1 rpmycin for 48 h (Figure 3). RT4 nd 253J cell prolifertion ws inhiited y the trnsfection of p70s6k sirna, respectively (P < 0.05), s compred with tht in the negtive control t 48 nd 72 h fter trnsfection. Rpmycin tretment resulted in reduced cell prolifertion (P < 0.05). The p70s6k sirna trnsfection showed inhiitory effects similr to those of rpmycin. pcdna3.1 p70s6k tretment tended to result in higher cell prolifertion t 48 h thn tht in the control nd mock groups, lthough it ws not significnt in RT4 or 253J cells. The cell growth inhiition effect of p70s6k sirna trnsfection nd rpmycin in RT4 nd 253J cells In the crystl violet growth ssy, RT4 nd 253J cell growth ws inhiited y the trnsfection of p70s6k sirna nd rpmycin, respectively (P < 0.05), s compred with tht in the negtive control. The p70s6k sirna trnsfection showed inhiitory effects similr to those of rpmycin (Figure 4). pcdna3.1 p70s6k tended to induce cell growth s compred with tht in the control nd mock groups, lthough it ws not significnt in RT4 nd 253J cells. The morphologicl chnges were in concordnce with the tretment effects on cell growth (Figure 4). DISCUSSION URC of the ldder is unique epithelil tumor in terms of the two divergent pthwys of tumorigenesis, which correspond to phenotypiclly distinct; non muscle invsive, LG URC nd muscle invsive, HG URC. 12 LG URC frequently exhiits ctivting muttions in the HRAS gene nd the firolst growth fctor receptor 3 gene, wheres most HG URC contins inctivted p53 nd/or deletion of the retinolstom gene.3 Despite the two distinct moleculr pthwys of progression, suset of LG URC progress to more ggressive, higher grde nd muscle invsive disese, usully fter course of multiple recurrences. Therefore, identifying iomrkers tht would help in the recurrence of LG URC is n re of ctive reserch.

5 615 Figure 3: The p70s6k sirna nd pcdna3.1-p70s6k trnsfection nd the cell prolifertion inhiitory effect of p70s6k sirna trnsfection nd rpmycin in RT4 nd 253J cells. () Western lot nlysis of p70s6k nd p-p70s6k protein expression fter p70s6k sirna nd pcdna3.1-p70s6k trnsfection into RT4 nd 253J cells. Cells trnsfected with p70s6k sirnas nd/or pcdna3.1-p70s6k were nlyzed y immunolotting for the indicted proteins nd rpmycin fter 48 h. () Cell prolifertion ssys for RT4 nd 253J treted with p70s6k sirnas nd/or pcdna3.1-p70s6k nd rpmycin. p70s6k sirna nd rpmycin significntly (P < 0.05) inhiited cell prolifertion s compred with the negtive control series t 48 nd 72 h fter exposure. In our study, high p p70s6k expression, s shown y the immunohistochemicl stining, predicted recurrence for LG URC treted y trnsurethrl resection, demonstrting tht the mtor signling pthwy thorough p p70s6k is involved in LG URC recurrence. Using p p70s6k enled us to identify suset of ptients with LG URC who hd n incresed risk for recurrence. These results were similr to those of our previous study in non muscle invsive ldder cncer cohort, in which the high expression of p p70s6k ws predictive fctor for recurrence of erly rest cncer nd lung cncer. 8,13,14 Sun et l., 15 reported tht high expression of p S6, sustrte Figure 4: The growth-inhiitory effect of p70s6k sirna trnsfection nd rpmycin in RT4 nd 253J cells. () RT4 nd 253J cell growth ws inhiited y the trnsfection of p70s6k sirna nd rpmycin, respectively (P < 0.05), s compred with tht in the negtive control. The trnsfection of p70s6k sirna tended to show inhiitory effects similr to those of rpmycin. () Morphologicl chnges were in ccordnce with the tretment effects on cell growth in RT4 nd 253J cells. Scle rs = 100 mm. of p p70s6k, predicts the progression of non muscle invsive URC of the ldder treted y trnsurethrl resection in immunohistochemicl stining of 266 humn UC smples, nd tht the unfvorle prognostic findings of p p70s6k overexpression hve een reported in other solid tumors. 13,16 We demonstrted tht p 4E BP1 nd p70s6k reveled diverse pttern regrding their ssocition with tumor recurrence. This finding suggests tht p 4E BP1 nd p70s6k re not ctivted eqully in the

6 616 pthwy. Incresed p 4E BP1 expression hs een ssocited with worse prognosis in few types of neoplsms. 17,18 By contrst, p 4E BP1 seems to e less relile thn other mrkers s n independent, recurrence predictive fctor for LG URC. We filed to demonstrte n ssocition etween the sttus of the mrker nd recurrence in oth univrite nd multivrite nlyses. We demonstrted tht the URC cell line, line derived from the LG URC of the ldder, expressed the p mtor nd p p70s6k proteins nd tht rpmycin inhiited RT4 nd 253J cell prolifertion dose dependently. These results re similr to those of previous preclinicl studies tht hve investigted the role of the mtor signling pthwy in URC Indeed, rpmycin nd its derivtive RAD001 (everlimus) inhiit URC cell prolifertion nd induce G0 G1 cell cycle rrest without poptosis in URC cell lines Previous studies hve focused on invsive URC cell lines, wheres we focused on LG URC cell lines (RT4 nd 253J). Although considerle numer of cell models exist for dvnced URC, the mjority of URC re dignosed s low grde, non muscle invsive tumors. 1 Recent genetic nlyses hve identified RT4 nd 253J cells s cliniclly relevnt URC cell lines, representtive of low grde, non muscle invsive chrcteristics. 22 Moreover, the RT4 nd 253J gene expression profile correltes with long term ptient survivorship, hllmrk of LG URC. 22,23 We reveled tht p70s6k sirna suppressed RT4 nd 253J cell prolifertion nd growth nd tht the efficcy of p70s6k sirna ws similr to tht of rpmycin. These findings suggest tht effectively inhiiting p70s6k y mtor sirna, p70s6k sirna nd rpmycin inhiited URC cell prolifertion nd growth. Moreover, we showed incresed cell prolifertion nd growth y trnsient trnsfection using pcdna3.1 p70s6k, indicting tht the phosphoryltion of p70s6k, which is downstrem of the mtor pthwy, is essentil for the prolifertion nd growth of LG URC in vitro. These results correlted with our results from the LG URC TMA immunohistochemistry in vivo. Therefore, recurrence of LG URC cn e prevented y inhiiting p70s6k phosphoryltion nd p p70s6k my e iomrker to predict the recurrence of LG URC. There re severl limittions of our study. First, phos4e BP1 expression ws higher nd p p70s6k expression ws lower in the ldder cncer cohort thn in the enign cohort. However, we did not show these results in vitro ecuse norml urothelil cell line could not e otined. Schultz et l. 24 reported lower p p70s6k expression in the ldder cncer cohort compred with enign urothelium in n immunohistochemicl study of 144 ptients with ldder cncer who hd undergone rdicl cystectomy. They suggested tht these findings were consistent with downregultion of the mtor downstrem effect. However, in our study, the ldder cncer cohort exhiited higher expression of p 4E BP1 tht tht in enign urothelium, indicting tht the mtor pthwy ws consistently processed; ut tht the pthwy ws shifted from p70s6k phosphoryltion to 4E BP1 phosphoryltion. Therefore, we hypothesize tht high phos4e BP1 expression my e relted to the tumorigenesis of ldder cncer. 4E BP1 is protein tht inds to elf 4E nd plys criticl role in the regultion of gene expression. When 4E BP1 is phosphorylted, elf 4E is relesed nd the initition complex my e formed to promote trnsltion. 25 Severl studies hve implicted components of the protein initition synthesis pprtus in crcinogenesis. For exmple, elf 4E hs een reveled to induce mlignnt trnsformtion when overexpressed in mmmlin cells. 26 Further study is required to evlute these mechnisms. Finlly, rigorous vlidtion process must precede the incorportion of novel moleculr iomrkers into clinicl mngement. Therefore, we need further prospective investigtions using lrger numer of ptients. Despite severl limittions, this is one of the few preclinicl nd clinicl studies indicting tht inhiiting p70s6k phosphoryltion in the LG URC is importnt in preventing recurrence. Our results suggest tht phosphorylted p70s6k my e useful s moleculr iomrker to predict recurrence in certin LG URCs of the ldder. AUTHOR CONTRIBUTIONS IHC nd JHK concevied nd designed the experiments. SJK nd KML performed the experiments. SJK, HSJ nd IHC nlyzed the dt. TJL contriuted regents/mterils/nlysis tools. SJK nd IHC wrote the pper. COMPETING INTERESTS The uthors declre no competing finncil interests. ACKNOWLEDGMENTS This study ws supported y the Deprtment of Convergence Medicine nd Phrmceuticl Biosciences Reserch Scholrship Grnts, Chung Ang University in REFERENCES 1 Pshos CL, Bottemn MF, Lskin BL, Redelli A. Bldder cncer: epidemiology, dignosis, nd mngement. 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