Alpha signaling for cancer treatment Breaking the of tnf signaling for cancer treatments p sasi1, 2, x yan2, 3, h enderling1, 3, d park2, hy gilbert1, 2, c curry2, c coleman2, l hlatky1, 3, g qin4, r kishore4 and d a goukassian1, 2, 3received 15 july 2011;Revised 4 november 2011;Accepted 6 november 2011advance online publication 12 december 2011 Top of pageabstracttumor necrosis factoralpha(Tnf binds to two distinct receptors, tnfr1 and tnfr2 tnf is implicated in the processes of tumor groWth, survival, differentiation, invasion, metastases, secretion of cytokines and proangiogenic factors.We have shown that tnfr2 signaling promotes ischemiainduced angiogenesis via modulation of several angiogenic groWth factors.We hypothesized that tnfr2 may promote tumor groWth and angiogenesis.GroWth of mouse lewis lung carcinoma(Llc1)And mouse melanoma b16 cell was evaluated in wIld type(Wt), p75 knocKout(Ko)And double p55ko mouse tumor xenograft models.Compared with wt and p55ko mice, growth of tumors in p75ko mice was significantly decreased(Twofold)In both llc and b16 tumors.Tumor growth inhibition was correlated with decreases in vascular endothelial growth factor(Vegf)Expression and capIllary density, as well as bone marrowderived endothelial progenitor cells incorporation into the functional capIllary network, and an increase in apoptotic cells in llc xenografts.Gene array analysis of tumor tissues showed a decrease in gene expression in pathways that promote tumor angiogenesis and cell survival.Blocking p75 by shorthairpin rna in cultured llcs led to increases in tnfmediated apoptosis, as well as decreases in the constitutive and tnfmediated expression of angiogenic growth factors(Vegf, hgf, plgf), and SDF1 receptor CXCR4.In summary, p75 is essential for tumor angiogenesis and survival in highly vascularized murine lung tumor xenografts.Blocking p75 expression may lead to tumor regression.The effect of tnf on angiogenesis, however, is controversial(Fraterschroder et al., 1987;Leibovich et al., 1987;Sato et al., 1987).In cell culture, the effect of tnf on angiogenesis depends on the concentration and the duration of the treatment(Fraterschroder et al., 1987;Leibovich et al., 1987;Sato et al., 1987;Fajardo et al., 1992;Slowik et al., 1993).In endothelial cells(Ecs), TNF increases the expression of angiogenic factors, such as vascular endothelial growth factor(Vegf), basic fibroblast growth factor(Bfgf)And interleukin(Il)8(Yoshida et al., 1997).In addition, impaired tnf signaling in ecs was associated with enhanced apoptotic response in cutaneous microvasculature in adult tissue(Fajardo et al., 1992). Tnf exerts its function via binding to p55(Tnfr1)And p75(Tnfr2). Both p55And p75bind to TNF receptorassociated factor 1 and 2 (Traf1 and traf2) (Rothe et al., 1994;Sethi et al., 2008), thus providing a mechanism for shared activity between the two receptors. On the other hand, the cytoplasmic domains of p55And p75are significantly different and these receptors are reported to trigger divergent signaling pathways upon interaction with TNF (Jacobsen et al., 1994;Rothe et al., 1994;Barbara et al., 1996;Sethi et al., 2008).The intracellular signaling involving p55 has been well deciphered.Studies have shown that p55 activates both apoptosis and cell survival signaling pathway, and the proapoptotic effects of p55 are related to the death domain on its cytoplasmic part of the receptor(Jacobsen et al., 1994;Barbara et al., 1996;Bhardwaj and aggarwal, 2003).Inversely, p55, via activating nf increases the expression of genes that in turn suppress tnfmediated apoptosis(Jacobsen et al., 1994;Barbara et al., 1996;Bhardwaj and aggarwal, 2003).Compared with p55, few signaling pathways and functions of p75 have been elucidated, especially in the context of tumor angiogenesis(Fraterschroder et al., 1987;Slowik et al., 1993).Unlike p55, the cytoplasmic domain of p75 lacks the death domain(Mukhopadhyay et al., 2001).Studies have shown that in ecs, bmx(Endothelial tyrosine kinase)Binds to p75 constitutively.Tnf stimulation induces reciprocal phosphorylation between p75bound bmx and vegfr2, which leads to the activation of the akt pathway and in turn results in ec migration and angiogenesis(Zhang et al., 2003).Studies from our laboratory have shown that p75 signaling promotes ischemiainduced angiogenesis via modulation of several angiogenic growth factors in mice(Goukassian et al., 2007). We hypothesize that p75 signaling may be essential for the angiogenic and survival processes in tumor tissue and blocking p75 signaling may affect tumor cell survival.We also investigated p75mediated effects on p38(Mapk)Activation in bone marrow(Bm)Derived endothelial progenitor cells(Epcs).Bmderived epcs are known to be critical regulators of the angiogenic switch in progression of micrometastasis to lethal macrometastasis(Gao et al., 2008;Rafii and lyden, 2008), and tumorderived TNF signaling had been linked in vivo to differentiation of myeloid progenitors cells to myeloid(Li et al., 2009).In this study, we identified the role of functional p75 signaling in bmderived epcs in vitro and in the context of tumor angiogenesis and tumor growth in vivo. ToPof pAGErEsultstnfr2 ko miCroEnvironmEnt DoEs not support lEwis lunG CArCinomA(LlC)AnD B16 mElAnomA tumor GrowthAftEr 21 DAys of inoCulAtion, tumor(LlC)VolumEs wErE lArGEr in wt AnD DouBlEko(DBlko)MiCE CompARed with p75ko miCE(SupplEmEntAry fiGurE s1).A similAr Growth wAs oBsErvED in All thrEE GEnotypEs BEtwEEn DAy 0(Figure 1a).CompARed with DAy 7, tumors in wt AnD DBlko miCE BECAmE 370(PAnD 1030(PlArGEr on DAys 14 AnD 21, rEspECtivEly, whErEAs in p75ko miCE, tumors BECAmE only 180(PAnD 420(PlArGEr on DAys 14 AnD 21, rEspECtivEly.KinEtiCs of tumor Growth AnD tumor volumEs in DBlko miCE wErE CompArABlE with wt miCE with no stAtistiCAl DiffErEnCE At Any timE point.CompARed with wt AnD DBlko miCE, tumor Growth in p75ko miCE wAs ReduCED By 30(Pon DAy 14 AnD 50(Pon DAy 21(Figure 1a).ThEsE rEsults suGGEst thAt thE ABsEnCE of siGnAlinG throuGh p75, But not Both tnf rECEptors(For ExAmplE, DBlko)In tumor tissuE miCroEnvironmEnt inhiBits tumor Growth. (A)GraphiC rEprEsEntation ofLlctumor volumEs in wt, p75ko anD DBlko miCE. (B)QuantifiCation of tnf immunostaininG(Red)In tumors anD normal skin of wt anD p75ko miCE shown as pErCEnt of mEan pixEl intEnsity usinG nih imaGE j proGram(HErE anD ElsEwhErE). (C)Tnf protEin rElEasE(PG measuRed in tumor homoGenatesfrom wt anD p75ko micE(P(D)Quantification of vEGf immunostaininG(Red)In tumors from wt and p75ko micE shown as pErcEnt of mEan pixEl intEnsity(PWT vs p75KO). (E)VeGf protein release(PG measuRed in tumor homoGenates(PWT vs p75KO). (F representative imaGes(RiGht panel)And quantification of f, cd31(Red)Immunofluorescence, and(G)Quantification of cd31(Cells in the wholetumor tissue from wt vs p75ko mice using fasc analysis, when wt is set as 100 note, compared with fasc analysis, tumor associated cd31(Cells were twice as higher in immunofluorescent studies, suggesting a significant heterogeneity in tumor vascular network and a superior quantitative nature of the facs analysis of tumorassociated cd31(Cells.Tumor growth kinetics revealed that between days 0 tumors grew similarly in wt vs p75 mice.Compared with day 7, melanoma tumors in wt mice became 1195 larger(Twelvefold)On day 14, whereas at the same time period tumor growth was inhibited in p75ko mice by more than two times(Pon DAy 14 (Only 474 increase between day 7 vs day 14).These results indicate that in the absence of the signaling via tnfr2 at least in the tumor tissue microenvironment, melanoma growth can be inhibited 2.5fold. NotE thAt in thEsE ExpErimEnts B16Cells wErE unAltERed (Socalled type and the tumor growth inhibition was predominantly dependent on unsupportive host tissue in p75ko mice.Llc(Figure 1a)And b16 melanoma xenograft studies(Supplementary figure s2)Taken together suggest that this may be a common response of vascularized tumors of different origin, at least in part, due to decreased tumor angiogenesis. Tnf expression is similar in the tumor tissue from wt and p75ko miceimmunostaining showed that the intensity of tnf staining in tumor or normal tissue(2 distant from tumor)Was not different between wt and p75ko mice(Figure 1b and supplementary figures s3a and b).However, compared with normal skin(Supplementary figure s3b), TNF expression was ten and ninefold(P higher in tumor tissue from wt and p75ko mice, respectively.Enzymelinked immunosorbent assay(Elisa)Of tumor homogenates confirmed that tnf levels were similar in tumor tissue from wt vs p75ko mice(1010 vs 976 pg P(Figure 1c). Vegf expression and capillary density are decreased, and apoptosis is increased in tumors from p75ko micecompared with wt tumors, the intensity of vegf immunostaining in tumor tissue from p75ko mice was decreased more than 50(P(Figure 1d and supplementary figure s4a).Accordingly, elisa of tumor homogenates http://www.teamskyuser.co.uk/beats-by-dr-dre-pro.html showed 40(Pdecrease in VEGF levels in the tumor tissue from p75KO mice (Figure 1e).Cd31 immunostaining was significantly decreased in tumors from p75ko mice(80 decrease, PFigure 1f and Supplementary Figure S4B).Fluorescenceactivated cell sorting analysis revealed a 41(Pdecrease in CD31 (Cell in the wholetumor tissue from p75ko vs wt mice(Figure 1g). Terminal transferase dUTPnick end labeling (Tunel)Staining was increased 40(Pin tumor tissue from p75KO mice (Figure 1h and supplementary figure s4c). DeCreAseD tumor Angiogenesis in the periphery oF the tumor in p75ko miCeAs shown in Figures 2A AnD B, Apoptosis(As meAsuReD By Tunel)In tumors From p75ko miCe were inCreAseD(Figure 2B, leFt imAge AnD triple overlAy).This wAs AssoCiAteD with A signiFiCAnt DeCreAse in CApillAry network in tumor But not peritumorAl tissue(Figure 2B, miDDle imAge AnD triple overlAy), inDiCAting suBstAntiAl DeFiCienCy in tumor Angiogenesis in the tumormiCroenvironment interFACe in p75KO miCe.These results suggest thAt wt host tissue supports tumor Angiogenesis AnD survivAl, whereAs Both BiologiCAl proCesses Are CompromiseD in p75kos miCe.Apoptosis AnD tumor Angiogenesis wAs Also evAluAteD At the interFACe oF tumor tissue By triple stAining with terminAl trAnsFerAse Dutp niCk enD lABeling(Tunel), CD31AnD Topro3.The peritumorAl AnD tumor AreA wAs iDentiFieD By h stAining oF ADjACent seCtions(Not shown).RepresentAtive imAges oF tripleimmunostAineD tumors(PAnel on the FAr right)In the periphery oF tumor tissue For tunel(Green), CD31(ReD)AnD Topro3(Blue)In wt(A)AnD p75ko(B). (A, B)Tunel staining(Top panel)In wttumors. (B)Insets in peritumoral(Top)AnD tumors(Bottom)In p75kos show Double positive(Tunel staining, arrowheaDs), inDiCating apoptosis oF p75KO ECs. (C)Representative images oF bmDeriveD(GFP Cells reCruiteD into WTAnD p75kotumor tissue (Arrows). (D)QuantiFication oF bmderived gFp(Cells recruitment into the tumor tissue.Small arrowheads indicate bmderived gFp(Cells that are not incorporated into Functional vessels(Green)In p75ko tumors. (F)Quantification of bmderived epcs incorporation into the functional capillary network. Full figure and legend(523K) Incorporation ofBmderived epcsinto tumor vasculature is decreased in tumors from p75ko beats by dre online store uk micethe recruitment of bmderived cells was not different in chimeric(Syngeneic green fluorescent protein(Gfp)Bm transplanted)Wt vs p75ko mice(Figures 2c and d, green fluorescence and arrows).Bmderived epcs(Figures 2e and f, yellow fluorescence and arrows)Were incorporated into functional tumor vessels(Gfp cells also positive for bs1lectinrhodamine perfusion in tumors of wt mice, whereas this was significantly decreased(45 P in tumors of p75KO mice(Figures 2e and f, yellow vs green this finding suggests that incorporation of bmderived epc into functional vascular network within the tumor tissue requires, at least in part, the presence of tnf receptor p75 in tumor stroma and on epc themselves. AngiogeniC, prosurvival and antiapoptotiC gene expression is deCreased in tumors from p75ko miCeCombined data from angiogeniC, apoptotiC and CanCer pathways miCroarrays(Total of 360 genes tested)Revealed that 29 genes were signifiCantly regulated in tumors from p75ko vs wt miCe(28 genes were downregulated and only one, BCl2l1, was upregulated).Strikingly, 17 out of 28 downregulated genes are involved in regulation of angiogenesis(For example, vegf, hgf, Pgf, il1b, p38 mapk14, CxCl2 and others).Compared with tumors from wt miCe, tumors drebtshdphones from p75ko miCe showed two to fivefold deCrease in the expression of angiogeniC growth faCtors, Cytokines and Chemokines(Figures 3a the expression of antiapoptotiC(Bag 1, 3, 4, bnip3, dad1 and faim)And bCl2 family(Bnip3l and bok)Genes were deCreased 2 in tumors from p75ko miCe Compared with wt miCe(Figures 4a furthermore, absenCe of p75 in the host tissue had little effeCts on the expression of genes in CanCer pathways(Supplementary figures s5a in agreement with the gene arrays, quantitative realtime pCr(Qrt results showed that as in gene arrays the expression of ang1, ang2, hif1 and mmp2 was not modified in the llC tumors from wt vs p75ko miCe;Whereas CxCl2 expression was deCreased in tumors from p75ko vs wt miCe(Supplementary figures s6a in summary, miCroarray results suggest that signaling through tnfr in miCroenvironment has an important role in supporting angiogenesis and Cell survival in llC tumors.These results also suggest that inhibition of tumor growth in p75ko miCe is, predominantly, a ConsequenCe of unsupportive tumor miCroenvironment. (For the results of b16 mouse melanoma angiogeniC gene array analysis, see supplementary figure 7). (A, b)Representative angiogenesis pathways miCroarray of tumors from wt and p75ko miCe at 5 exposure and the loading Controls(Bottom, small insets)At 5 exposure time.Dottedline CirCles indiCate gene expression deCrease in p75ko vs the same gene in wt(Solidline CirCles). (C)FunCtional grouping of genes in angiogenesis pathways miCroarray with the fold Changes(Dottedline CirCles, deCreased)In p75ko vs wt tumors.In p75ko tumor tissue deCreased gene expression was observed in growth faCtors, Cytokines and Chemokines, suCh as vasCular endothelial growth faCtor(Vegfa and b), plaCental growth faCtor(Pgf), Chemokine(CxC motif)Ligands(CxCl1, CxCl2, CxCl10), interleukins(Il1b, il12a and il18), pleiotrophin(Ptn, known as, heparinbinding growth faCtor 8), signal transduCtion and transCription faCtors MAPK14(Known as p38), prostaglandinendoperoxide synthase 1(Ptgs1), adhesion moleCules and proteases, suCh as Collagen, type XVIII, alpha 1(Col18a1), neuropilin 1(Nrp1), angiopoietinlike 4(Angptl4)And matrix metalloproteinase 9(Mmp 9).The densitometry values of hsp90ab1 and bas2C genes were used as internal Control.An arbitrary Cutoff was set at 1.6(Up or down).We assigned an arbitrary Cutoff thresholds for fold Changes at 1.6(Here and elsewhere). (A, b)Representative apoptosis pathways miCroarray of tumors from wt and p75ko miCe at 5 exposure and the loading Controls(Bottom, small insets)At 5 exposure time. (C)Functional grouping of genes in apoptosis microarray with the fold changes of the same gene(Solidline vs dottedline circles, decreased)In wt vs p75ko. Full figure and legend(119K) Tnfr2 is required for the ActivAtion of p38 mApk in Bmderived epcsin cultured Bmderived epcs oBtAined from wt mice And in cultured llcs, tnf treAtment ActivAted p38 mApk in A timedependent mAnner(Figures 5A And B And e And f), whereAs in EPCs oBtAined from p75KO mice, TNFmediAted p38 ActivAtion wAs significAntly weAker(0.4fold, P Figures 5c These results suggest thAt(A)Signaling via tnf receptor p75 is necessary for proper p38 activation in Bmderived epcs and(B)That p38 is constitutively activate in llcs and there is significant tnfmediated p38 activation in llcs. (A, c, e)Representative images of phosphop38(Pp38)And totalp38(Tp38)Western blot analysis in wt and p75ko epcs and unaltered llcs. (B, d, f)Quantification p38 protein level and phosphorylation using densitometric analysis of pp38 band intensity after adjusting for actin(Not shown)And tp38 band intensity.Band intensity at time 0 for each cell type was set at 100 and percent change over 60 was calculated.Results represent data from three independent experiments.We generated p75 receptor kd llcs(P75kd using shorthairpin rna technology(Supplementary materials and methods).Qrt data of percent inhibition of p75 receptor in stably transfected p75kd is presented in Supplementary figure s8.P75kd or llcs(Plasmid combinations 1 and 1 showed 74 or 90 inhibition of p75 receptor expression, respectively(Supplementary figure s8).Compared with control, scrambled plasmidtransfected llcs, tnf treatment induced 56 and 90 apoptotic cells(Cells in subg0 population) (Riccardi and nicoletti, 2006)In p75kd(1 and p75KD(1 respectively(Figure 6a).Increase in apoptotic cells was in direct correlation with percent inhibition of p75 receptor(Figure 6a and supplementary figure s8).