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Deregulated β‐catenin induces a p53‐ and ARF‐dependent growth arrest and cooperates with Ras in transformation

Deregulated β‐catenin induces a p53‐ and ARF‐dependent growth arrest and cooperates with Ras in... The EMBO Journal Vol. 20 No. 17 pp. 4912--4922, 2001 Deregulated P-catenin induces a p53- and ARF-dependent growth arrest and cooperates with Ras in transformation and axin/conductin (Behrens et al., 1998; Ikeda et al., Alexander Damalas, Sharon Kahan, 1998). The N-terminally phosphorylated P-catenin then Michael Shtutman, Avri Ben-Ze'ev and 1 becomes a target for recognition by a specific SCF-type E3 Moshe Oren ubiquitin ligase (Hart et al., 1999; Winston et al., 1999; Department of Molecular Cell Biology, Weizmann Institute of Sadot et al., 2000), leading to its polyubiquitylation and Science, Rehovot 76100, Israel subsequent proteasomal degradation. Corresponding author Upon physiological activation of the Wnt-wingless e-mail: [email protected] pathway, P-catenin is stabilized and translocates into the nucleus. There, in complex with members of the TCF/LEF Aberrant activation of -catenin contributes to the family of DNA binding proteins, it serves as a transcrip­ onset of a variety of tumors. We report that a tumor­ tional regulator and induces the expression of cognate derived -catenin mutant induces accumulation and target genes (Behrens et al., 1996; Riese et al., 1997; van activation of the p53 tumor suppressor protein. de Wetering et al., 1997). Aberrant stabilization of Induction is mediated through ARF, an alternative P-catenin, resulting in constitutive transcriptional activity, reading frame product of the INK4A tumor suppres­ is encountered in a variety of cancers (Bienz, 2000; Peifer sor locus, in a manner partially dependent on the and Polakis, 2000; Polakis, 2000; Zhurinsky et al., 2000a). transcription factor E2Fl. In wild-type mouse embryo Stabilization can be achieved either through mutational fibroblasts, mutant P -catenin inhibits cell proliferation inactivation of APC or axin, or through direct mutation of and imposes a senescence-like phenotype. This does P-catenin. The latter mutations usually occur within the not occur in cells lacking either ARF or p53, where N-terminal domain of P-catenin, rendering it refractory to deregulated -catenin actually overrides density­ phosphorylation by GSK-3p (Polakis, 1999). dependent growth inhibition and cooperates with acti­ The c-myc and cyclin Dl genes, encoding important vated Ras in transformation. Thus, the oncogenic positive regulators of cell proliferation, have been iden­ activity of deregulated -catenin is curtailed by con­ tified as transcriptional targets of deregulated P-catenin current activation of the p53 pathway, thereby provid­ (He et al., 1998; Shtutman et al., 1999; Tetsu and ing a protective mechanism against cancer. When the McCormick, 1999). Constitutive activation of those p53 pathway is impaired, deregulated -catenin is free genes may account, at least in part, for the putative to manifest its oncogenic features. This can occur not contribution of P-catenin deregulation to tumorigenesis. only by p53 mutations, but also by ablation of ARF Additional P-catenin target genes such as WISP-I and expression, as observed frequently in early stages of PPARo (He et al., 1998; Xu et al., 2000), may also play a colorectal carcinogenesis. positive role in cancer processes. The oncogenic potential Keywords: ARF/P-catenin/p53/Ras/senescence of deregulated P-catenin has been convincingly demon­ strated in vivo in mouse tumor models (Oshima et al., 1995; Gat et al., 1998; Chan et al., 1999). Introduction The p53 tumor suppressor gene is also subject to frequent mutations in cancer (Levine, 1997). Such muta­ P-catenin is a versatile protein. On the one hand, it is an tions primarily abolish the tumor inhibitory activities of important structural component of the cell adhesion machinery that mediates cell-cell interactions (Kemler, the wild-type (wt) p53 protein. In non-stressed cells, p53 1993; Ben-Ze'ev and Geiger, 1998). On the other hand, as protein levels are usually very low, owing to rapid a pivotal constituent of the Wnt-wingless pathway, proteolytic degradation ofwt p53. Numerous stress signals P-catenin is also intimately involved in intracellular signal lead to activation and accumulation of p53 (Giaccia and transduction (Cox and Peifer, 1998; Willert and Nusse, Kastan, 1998; Prives, 1998; Lohrum and Vousden, 1999; 1998). Oren, 1999; Prives and Hall, 1999; Ashcroft et al., 2000). The levels, subcellular localization and activity of These signals include diverse types of DNA damage and P-catenin are tightly regulated within the cell (Aberle et al., chromosomal aberrations, as well as deregulation of 1997; Bienz, 1999; Clevers, 2000; Hecht et al., 2000; various growth stimulatory signaling pathways. The latter Rosin-Arbesfeld et al., 2000). The free cytosolic pool of type of deregulation may be achieved via aberrant P-catenin is unstable and subject to rapid proteolytic activation of oncogenes such as c-myc and ras, or through degradation, which requires its phosphorylation by the the action of viral oncoproteins such as the products of the glycogen synthase kinase 3P (GSK-3P). Phosphorylation adenovirus ElA region. In several cases, including c-myc, occurs on sites within the N-terminal domain of P-catenin, ras, E2Fl, and adenovirus ElA, induction ofp53 relies to and is mediated through interaction of P-catenin with a a great extent on another tumor suppressor protein, ARF complex including, in addition to GSK-3P, the APC (Bates et al., 1998; de Stanchina et al., 1998; Palmero (adenomatous polyposis coli) tumor suppressor protein et al., 1998; Stott et al., 1998; Zindy et al., 1998). ARF is 4912 © European Molecular Biology Organization ARF-dependent induction of p53 by P-catenin the product of an alternative transcript of the INK4a tumor recombinant retrovirus encoding B-catenin S33Y, a suppressor locus (Quelle et al., 1995; reviewed in constitutively active B-catenin mutant derived from a Sharpless and DePinho, 1999; Sherr and Weber, 2000). CRC patient (Morin et al., 1997). A retrovirus expressing ARF binds to the Mdm2 protein, which plays a key role in only the puromycin resistance gene served as a negative the degradation of p53 through its activity as a p53- control. Infection efficiencies were routinely close to specific E3 ubiquitin ligase (Honda and Yasuda, 1999). 100%, as assessed by puromycin selection of representa­ The binding of ARF to Mdm2 sequesters Mdm2 in the tive infected cultures, and by infection of parallel cultures nucleolus, where it cannot target p53 for ubiquitylation; in with a retrovirus encoding B-galactosidase (data not addition, it directly inhibits the E3 activity of Mdm2 shown). The total levels of B-catenin in the infected (Honda and Yasuda, 1999; Tao and Levine, 1999; Weber MEFs, representing the combination of endogenous wt et al., 1999, 2000b; Lohrum et al., 2000). Hence, induction B-catenin and transduced mutant B-catenin, were only of ARF by deregulated oncogenes inhibits the ubiquityl­ moderately elevated relative to the endogenous wt protein ation and subsequent proteasomal degradation of p53, alone in control MEFs (Figure lA). More importantly, resulting in accumulation of p53 and stimulation of p53- these levels were comparable with those present in cell mediated cellular responses. This is believed to serve as a lines derived from human CRC, where B-catenin is failsafe mechanism that protects against the tumorigenic deregulated owing to either direct B-catenin mutations consequences of oncogene activation (Sherr, 1998). In (SW48 and HCT116) or inactivation of the APC tumor addition, ARF may also have p53-independent anti­ suppressor (SW480). As seen in Figure lB, such levels of proliferative functions (Carnero et al., 2000; Esteller oncogenic B-catenin were already sufficient to elicit a et al., 2000; Weber et al., 2000a). marked increase in the steady state levels of p53. We reported previously that overexpression of B-cate­ Several oncoproteins trigger p53 accumulation through nin leads to accumulation of transcriptionally active p53 in stimulation of ARF expression (Lowe, 1999; Sherr and transfected cells (Damalas et al., 1999). We now show that Weber, 2000). We, therefore, investigated whether ARF activated, tumor-derived mutant B-catenin triggers p53 plays a role in the induction of p53 by tumor-associated accumulation when expressed at levels comparable with B-catenin. Analysis of infected wt MEFs revealed a those present in cells derived from human colorectal dramatic increase in murine p19ARF protein levels in cancer (CRC). The accumulation of p53 is due to induction the presence of B-catenin S33Y (Figure lB). Hence, of ARF expression. Maximal induction of ARF and p53 deregulated B-catenin can effectively enhance ARF requires the presence of the E2Fl transcription factor. In expression. normal primary mouse embryo fibroblasts (MEFs), In p53-null MEFs, basal ARF expression is significantly deregulated B-catenin arrests cell proliferation and im­ elevated (Figure lB), owing to relief of p53-dependent poses a senescence-like phenotype that is dependent on repression of ARF transcription (Robertson and Jones, both ARF and p53 function. Conversely, in ARP-deficient 1998). Nevertheless, B-catenin elicited a further increase or p53-deficient MEFs, the same mutant B-catenin exerts in ARF protein (2.5-fold after normalization for a-tubulin, a growth stimulatory effect, reflected in an ability to over­ Figure lB). Remarkably, B-catenin augmented to a similar come density-dependent growth inhibition. Moreover, extent the transcriptional activity of the murine ARF in the absence of either ARF or wt p53, deregulated promoter (Inoue et al., 1999) in these cells (Figure lC). B-catenin cooperates with oncogenic ras to enhance cell Moreover, activation of this promoter was effectively transformation. abrogated by overexpression of dominant negative TCF4 Our findings support a model where the emergence of (DN-TCF4), known to block the transcriptional activity of deregulated, constitutively active B-catenin is coupled B-catenin (Korinek et al., 1997). These findings imply that with the transcriptional stimulation of ARF expression. deregulated B-catenin can induce ARF expression at the This in tum triggers an anti-proliferative response, transcriptional level. orchestrated largely through p53. Such anti-proliferative The ARF protein induced by B-catenin resides predom­ response is proposed to curb the potential oncogenic inantly in the nucleolus (Figure 1D, a), as confirmed by consequences of deregulated B-catenin, thereby providing double staining for the nucleolar protein nucleophosmin/ a potent mechanism for tumor suppression. Elimination of B23 (Figure 1D, b); this is consistent with earlier reports this inhibitory arm, through inactivation of either p53 or (Weber et al., 1999). ARF, might unveil the full oncogenic potential of Upregulation of p53 by B-catenin was accompanied by deregulated B-catenin and allow it to play a more effective elevated levels of p21, a p53 target gene product role in tumorigenesis. The observed silencing of ARF (Figure lE), confirming that the upregulated p53 is expression in a sizable fraction of colorectal adenomas, functional. Moreover, induction of ARF and p53 by corresponding to early stages of cancer progression, is mutant B-catenin could be observed in the presence of consistent with this model. either low (Figure lE) or high (Figure lF) serum concentrations, although its extent was milder in cells growing in high serum. Results To further investigate whether physiological activation Induction of p53 by oncogenic p-catenin is of B-catenin-mediated signaling can induce ARF expres­ mediated through ARF sion, wt MEFs were treated with LiCl. LiCl is a selective Experimental overexpression of B-catenin can induce p53 inhibitor of GSK-3B; exposure of cells to LiCl results in accumulation (Damalas et al., 1999). To investigate the B-catenin stabilization and augmentation of B-catenin­ molecular mechanism underlying this induction, primary mediated signaling (Woodgett, 1994). As seen in Figure 2, MEFs were employed. wt MEFs were infected with a high concentrations of LiCl elicited a significant increase 4913 A.Damalas et al. in ARF protein levels. As expected, this was accompanied by a proportionate increase in p53 protein; induction of p53 by LiCl has recently been reported also by Mao et al. (2001). The role of ARF in the induction of p53 by P-catenin deregulated 13-catenin was investigated with the aid of ARP-deficient MEFs (Karnijo et al., 1997). As seen in WI B p$J-./· MEI' Figure 3, 13-catenin S33Y was totally incapable of V V ' � n-,- -�-----, augmenting p53 in the absence of ARF, nor could it (7 elevate p21 expression. It should be noted that very extensive 13-catenin overexpression, for example by f-!' .&--J transient transfection with a CMV-driven expression plasmid, promotes p53 accumulation even in ARF­ , - - - - h•ta.b,illn deficient immortalized NIH-3T3 cells (Damalas et al., 1999). Yet, the data in Figure 3 clearly demonstrates that ARF is essential for induction of active p53 by the more physiological levels of deregulated 13-catenin achieved in the infected cells. Together, these findings imply that deregulated 13-catenin promotes ARF expression, most likely through enhanced transcription, thereby preventing p53 degrad­ ation and allowing accumulation of functional wt p53. Role of E2F1 in the stimulation of the ARF-p53 pathway by deregulated p.catenin Members of the E2F family of transcription factors play a critical role in cell-cycle progression (Nevins, 1998). E2F transcriptional activity is positively regulated by kinase complexes consisting of cyclin D and cdk4 or cdk6 (Sherr, 1996). Hence, increased expression of cyclin D proteins is expected to elevate E2F-mediated transcription. E2Fl, a member of the E2F family, transactivates the ARF promoter (Bates et al., 1998) and was implicated in the induction of ARF expression by several oncoproteins Fi . 1. Induction of p53 and ARP by deregulated �-catenin. (A) Comparison of �-catenin levels in different cell types. Cell extracts were prepared from CRC lines (SW48, HCTI 16, SW480) and from fibroblasts infected with recombinant retroviruses encoding either �-catenin S33Y (wtMEF + �-cat) or puromycin resistance only ( wtMEF). Four micrograms of protein of each sample was subjected to western blot analysis with a monoclonal antibody directed against �-catenin (Sigma). (B) Low passage MEFs, derived from either wt or p53-null animals, were infected with recombinant retroviruses encoding either HA-tagged S33Y tumor-derived mutant �-catenin (�) or puromycin resistance only (v). Cells were trypsinized and replated 48 h later. Cells were re-fed the next day with medium containing 0.1 % serum, and harvested after another 48 h. Twenty micrograms of protein of each sample was subjected to sequential western blot analysis with antibodies directed against p53, p19ARP and a-tubulin as p53 47� � a control for equal loading. (C) Low passage p53-null MEFs, infected p19ARF 20. with either �-catenin (�) or control retrovirus ( v ), were transiently transfected with a firefly luciferase reporter plasmid driven by the murine ARP promoter, either alone or together with a plasmid expressing dominant negative TCF4 (DN-TCF4). Transfections were a-tubulin done in triplicate. Luciferase activity was normalized for Renilla luciferase readings in the same extracts (see Materials and methods). (D) wt MEFs infected with a retrovirus encoding HA-tagged �-catenin � S33Y (a-c) or with pBabe-puro control retrovirus (d-f) were fixed and stained with pl9ARP-specific antiserum (a,d), B23 antiserum to visualize nucleoli (b,e) and DAPI to visualize nuclei (c,f). (E) wt MEFs were infected and processed essentially as in (B), except that blots 21) . J WafI were probed sequentially for p53, p2 and a-tubulin. Identical samples, run in parallel lanes of the same gel, were probed for pl9ARP. (F) wt MEFs were infected and processed as in (B), except -tubulin that the cells were maintained in medium containing I 0% serum throughout the experiment. _ 19All.f ._ _ _ _ � _ _ _ fl ARF-dependent induction of p53 by -catenin LlO(mfl.ll 0 2.5 wt MEF ARl'-/ - v V ll � n .s 1 �----- ---- 20.l>-j �- - -- - -- �f-P 19ARF -- a--l ubulln �p21 20 ,8--i - - Fig. 2. Induction of ARF and p53 by LiCI. Early passage wt MEFs were plated at a density of 2 X 10/10-cm dish. Fourteen hours later, 1- - -- f-.a- wbuJi n LiCI was added to the indicated final concentration, for an additional 48 h. Cells were then harvested and processed as in Figure 1. Fig. 3. ARF is required for accumulation and activation of p53 in response to oncogenic fl-catenin. Early passage MEFs, derived from wt or ARP-deficient (ARP-'-) mice, were infected as in Figure IB . Protein extracts (20 µg total protein/lane) were subjected to western blot (de Stan ch ina et al., 1998). Th e r o le o f E 2 Fl in th e analysis essentially as in Figure IB , except that HA-tag specific upregula ti o n o f ARF and p53 by P- c a tenin was s tudied antibodies were employed to visualize the mutant fl-catenin (HA­ thr o ug h th e use o f E 2 Fl-null MEFs . As seen in Figure 4A, mfl-catenin). indu c ti o n o f bo th ARF and p53 by P- c a tenin S33Y was par tially co mpr o mised in th ese c ells; th e fold indu c ti o n, n o rmalized for a- tubulin, is indi c a ted bel o w th e respe c tive wt A B M_EF Elf'I./- lanes . Never th eless, a residual in c rease in bo th pr o teins V V � � f:I was s till re tained, indi c a ting th a t w h ile E 2 Fl is an c:ydinOl 38.7 - }- HA-mjl-aten ln imp o r tan t media to r o f th e effe c t o f deregula ted P- c a tenin ....... a •la bulin - =i.- p s3 o n p53, i ts r o le is n o t ex c lusive . 47� - -· 1.0 4..0 0.1! 1.6 As expe c ted (Sh tu tman et al., 1999 ; T e tsu and - - - �p19A.RF :zo.a I Mc C o rmi c k, 1999) , infe c ti o n o f MEFs wi th the P- c a tenin l,0 ?.& 1,0 � S33Y re tr o virus led to a subs tan tial in c rease in c y c lin DI - _, .._ ._.. fi-tubuH.n pr o tein (Figure 4B ). Deregula ted P- c a tenin may th us s timula te ARF expressi o n thr o ug h indu c ti o n o f c y c lin Dl, Fig. 4. Involvement of E2Fl in the induction of the ARF-p53 pathway leading to in c reased E 2 Fl-media ted syn th esis o f ARF by fl-catenin. (A) Induction of ARF and p53 in E2Fl-null cells. MEFs mRNA . Th e impa c t o f c y c lin Dl upregula ti o n o n ARF from wt and E2Fl-null mice were infected and analyzed as in Figure 3. expressi o n is, ho wever, mo re co mplex . Spe c ific ally, ARF Band intensities were detemrined by densitometry (NIH-Image). trans c rip ti o n is subje c t to p o si tive regula ti o n by an o th er Numbers below lanes indicate the fold induction of the respective protein by mutant fl-catenin, calculated relative to the parallel control trans c rip ti o n fa c to r, DMPl (In o ue et al., 1999). DMPl virus-infected wt MEF sample, after normalizing for u-tubulin. a c tivi ty, in turn, is in h ibi ted by c y c lin Dl (In o ue and Sh err, (B) Western blot analysis of cyclin DI protein in wt MEFs infected 1998). Th us, c y c lin Dl als o p o ssesses th e p o ten tial to with either control (v) or mutant fl-catenin (Ii) retrovirus. Infection and nega tively regula te ARF expressi o n th r o ug h in h ibi ti o n o f protein analysis were as in Figure IB . DMPI . Th e fa c to rs tha t di c ta te the ne t effe c t o f c y c lin Dl indu c ti o n o n ARF expressi o n s till remain to be elu c ida ted . subs tan tially re tarded rela tive to th eir co un terpar ts in­ Sustained deregulation of JJ- catenin induces a fe c ted wi th th e co n tr o l virus . Unlike w t c ells, MEFs senescence -like phenotype dependent on ARF and la c king ei th er ARF o r p53 co n tinued to pr o lifera te after p infe c ti o n wi th the P- c a tenin S33Y re tr o virus; in nei th er To evalua te th e p h en o typi c co nsequen c es o f sus tained c ell type was th ere any eviden c e for senes c en c e-ass oc ia ted P- c a tenin deregula ti o n, MEFs o f differen t gen o types were mo rp ho l o gi c al al tera ti o ns (Figure 5 ). In fa c t, o verexpres­ infe c ted wi th re tr o viruses en co ding ei th er P- c a tenin S33Y si o n o f P- c a tenin S33Y in th ese c ells exer ted a measurable o r pur o my c in resis tan c e o nly . Infe c ted c ul tures were p o si tive effe c t. Af ter several days o f c ul tiva ti o n, fo c al areas o f h ig h - c ell densi ty co uld be dis c erned in ARF-null trypsinized, and an iden ti c al number o f c ells fr o m ea ch c ul ture were re-pla ted a t l o w densi ty . Mi c r o s co pi c exam­c ul tures . Wh ile co n tr o l ARF-null MEFs pr o lifera ted to ina ti o n o f th e vari o us c ul tures, several days la ter, revealed sa tura ti o n densi ty and th en s to pped, c ul tures o verexpres­ th a t w t MEFs infe c ted wi th th e co n tr o l re tr o virus rea ch ed a sing P- c a tenin S33Y failed to s to p a t th a t p o in t and h ig h c ell densi ty (Figure 5 ). In s triking co n tras t, w t MEFs co n tinued to pr o lifera te avidly (Figure 6B). Th us, in th e infe c ted wi th P- c a tenin S33Y remained rela tively sparse; absen c e o f ARF, deregula ted P- c a tenin c an augmen t th e mo re o ver, many o f th e c ells were flat and remarkably abili ty o f c ells to o ver co me densi ty-dependen t gr o w th spread o u t, ex h ibi ting a senes c en c e-like mo rp ho l o gy . Th e in h ibi ti o n, a fea ture th a t may fa c ili ta te tum o r pr o gressi o n . experimen t s ho wn in Figure 5 was perf o rmed in th e Th e effe c t o f P- c a tenin S33Y o n p53-null MEFs was presen c e o f 10% serum . Essen tially similar resul ts were s o mew h a t mo re co mpli c a ted . Cells infe c ted wi th th e o btained in l o w (0. 1 % ) serum, ex c ep t th a t th e mo rp ho ­ co n tr o l virus grew to a h ig h densi ty, bu t th en pr o lifera ti o n l o gi c al ch anges in P- c a tenin-infe c ted w t MEFs were mo re c eased and c ell numbers a c tually wen t do wn (Figure 6C ). drama ti c , w h ile all o th er c ul tures gradually c eased pr o lif­ Th a t la tter dr o p was ab o lis h ed by o verexpressi o n o f era ti o n wi tho u t displaying senes c en c e-like fea tures (da ta P- c a tenin S33Y, sugges ting th a t under tho se co ndi ti o ns n o t s ho wn ). P- c a tenin may pr o mo te c ell survival . Gr o w th ra te analysis (Figure 6A ) co nfirmed tha t th e Th e dependen c e o f th e senes c en c e-like p h en o type o n pr o lifera ti o n o f MEFs o verexpressing P- c a tenin S33Y was p53 was fur th er co nfirmed by th e use o f a re co mbinan t 4915 control A.Damalas et al. f}-catenin wt MEF p53-/- Fi . 5. Overexpression of mutant �-catenin elicits a senescence-like phenotype dependent on ARF and p53. Early passage MEFs, derived from wt, ARF-null or p53-null mice, were infected with control retrovirus or retrovirus encoding HA-tagged �-catenin S33Y. Cells were trypsinized and replated 48 h later, and after an additional day fresh medium containing 10% serum was added. Cultures were maintained in the same medium for 6 days. Phase contrast photographs were taken at a magnification of IO OX. A B ARF-/- C wt p53-/- 10 18 35 .;;- i 16 i 0 :;:: ';;! -f '£ 12 ., .,, 20 .,, 10 § 8 ::l 15 z z z 6 2 4 ol ol ol u u 5 7 11 1 3 5 7 9 11 1 3 5 7 9 11 Days Days Days Fi . 6. Deregulated �-catenin inhibits cell proliferation in an ARF- and p53-dependent manner. Cultures of wt, ARF-null and p53-null MEFs were infected with control retrovirus (circles) or retrovirus encoding HA-tagged �-catenin S33Y (squares) and processed as in Figure 5. Infected cells were replated in 10-cm dishes, at a seeding density of 2 X 10 cells/dish. Triplicate cultures were counted at the indicated number of days after replating. The SE is indicated. retrovirus encoding a temperature sensitive (ts) p53 activated p53 alone (control + p53Val135, 32C). In mutant, p53Val135 (de Rozieres et al., 2000). ps3-t­ contrast, avid proliferation with no morphological MEFs were first infected with the P-catenin S33Y alterations, despite the presence of P-catenin, was retrovirus, and then re-infected with p53Val135. When seen at 37.5 C where the ts p53 assumes a mutant the infected cultures were shifted to 32C, resulting in conformation devoid of wt p53 activity. It is note­ activation of the ts p53 (Michalovitz et al., 1990), the worthy that under those conditions, allowing for cells assumed a typical senescence-like morphology relatively high levels of p53 even in the absence of (Figure 7 A). This did not occur in the presence of deregulated P-catenin, p53 levels increased only mildly 4916 13 ARF-dependent induction of p53 by -catenin �-catenin+p53Val135 control +p53Val135 32 C 37°C tJ -- p53 �---� •tubulin -I Fig. 7. wt p53 activity is required for induction of a senescence-like phenotype by deregulated 13 -catenin. Early passage p53 - MEFs were infected with control retrovirus or retrovirus encoding HA-tagged 13 -catenin S33Y. Forty-eight hours later cells were trypsinized, replated and subjected to a second round of infection with a retrovirus encoding the ts p53 mutant p53Val135. After 48 h fresh medium containing 10% serum was added, and ° ° some of the cultures were transferred to 32 C while the others were left at 37.5 C. (A) Phase contrast photographs taken 6 days after plating (magnification = lOO X) . (B) Western blot analysis of p53 protein levels in cultures maintained at 32 C and extracted 4 days after replating. in response to excess -catenin S33Y (Figure 7B). This contrast, Ras Val 12 elicited many transformed foci in raises the possibility that tumor-associated -catenin can ARF-null cells (Figure 8), as reported earlier (Kamijo regulate not only the levels of p53, but also its specific et al., 1997). Importantly, while -catenin alone did biochemical activity. not give rise to a significant number of foci, the P P In conclusion, whereas deregulated -catenin exerts combination of -catenin and activated Ras resulted in potent anti-proliferative effects and elicits a senes­ extensive transformation; foci were more numerous and cence-like phenotype in the presence of wt p53 and substantially thicker than with Ras alone (Figure 8). ARF, inactivation of either ARF or p53 alleviates Similarly, -catenin S33Y also augmented Ras-medi­ ated focus formation in p53-null MEFs, although those anti-proliferative effects and reveals the potential growth-promoting features of deregulated P -catenin. overall numbers of foci were lower than in ARF-null MEFs (data not shown). Thus, constitutively activated Ras can cooperate with Tumor-associated p.catenin and activated Ras deregulated P -catenin to transform MEFs; this oncogenic cooperate in transformation in the absence of ARF activity of mutant P -catenin is, however, unleashed only or p53 upon inactivation of the p53 pathway. To further explore whether deregulated P -catenin can contribute to oncogenesis, we asked whether P -catenin S33Y cooperates with constitutively active mutant Ras Regulation of ARF by P-catenin in human in the transformation of cultured cells. This combin­ CRC-derived cells ation was chosen because deregulation of P -catenin is The oncogenic activity of P -catenin has been extensively frequently followed by Ras mutations during colorectal studied in CRC cell lines, representing a type of cancer carcinogenesis (Kinzler and Vogelstein, 1996). MEFs where deregulated -catenin plays a pivotal role in of various genotypes were sequentially infected with tumorigenesis. It was, therefore, important to determine P P retroviruses expressing -catenin S33Y and the tumor­ whether deregulated -catenin can upregulate ARF expres­ associated mutant H-RasVall2. In wt MEFs, no trans­ sion in CRC cells. SW480 cells, in which -catenin is formed foci were detectable upon infection with either deregulated owing to APC gene mutation, were transiently Ras alone or Ras plus -catenin (data not shown). In transfected with either DN-TCF4 or axin, both of which are 4917 A.Damalas et al. consequence of mutations in the P-catenin gene itself, or owing to lesions in genes that control the ubiquitylation and degradation of cytosolic P-catenin, such as the APC tumor suppressor (Kinzler and V ogelstein, 1996) and axin (Satoh et al., 2000). In many cases, best exemplified by CRC, accumulation of transcriptionally active P-catenin is �-ca tenin later followed by p53 mutations that inactivate the tumor suppressor functions of p53 (Kinzler and Vogelstein, 1996). We show here that deregulated P-catenin can activate the p53 pathway and trigger a p53-mediated anti­ proliferative response, which in primary fibroblasts takes the form of a growth inhibition associated with senes­ Ras cence-like morphological features. Moreover, inactivation of the p53 pathway uncovers the oncogenic properties of deregulated P-catenin, measured by its ability to cooperate with mutant Ras in the transformation of primary MEFs. These observations may explain, at least in part, the �-caten in selective pressure to inactivate the p53 pathway in tumors involving excessive P-catenin-dependent signaling. Ras Induction of p53 by deregulated P-catenin is strictly dependent on the ARF tumor suppressor. Deregulated P-catenin stimulates ARF expression and augments tran­ scription from the ARF promoter in CRC-derived cells as Fi g . 8. Oncogenic �-catenin cooperates with Ras in transformation of 5 well as in fibroblasts. This is completely abrogated by ARF-null MEFs. Early passage ARP-I- MEFs (1. 5 X 10 cells/ 10 -cm dish) were infected with either control retrovirus or retrovirus encoding negative dominant TCF, strongly implicating the tran­ �-ca tenin S33Y. Two days later the cultures were trypsinized and scriptional activity of P-catenin in the induction of ARF replated again at 1. 5 X I 0 cells/I 0-cm dish, and subjected to a second and hence of p53. Of note, the P-catenin S33Y mutant is round of infection with a retrovirus encoding the Va ll2 mutant H-Ras. derived from a human tumor (Morin et al., 1997), and Cultures were fixed 8-9 days later and stained with Giemsa stain. possesses enhanced transcriptional activity (Zhurinsky Triplicate dishes are shown. Where only a single oncogene is indicated, the cultures were actually also subj ected to a second infection with et al., 2000b). control retrovirus to maintain the infection history of each culture The ability of P-catenin to activate p53 through equal. upregulation of ARF adds it to a growing list of oncoproteins and growth regulatory proteins that can exert a similar effect (Bates et al., 1998; de Stanchina et al., potent negative regulators of P-catenin signaling (Behrens 1998; Palmero et al., 1998; Radfar et al., 1998; Zindy et al., 1998; Ikeda et al., 1998; Clevers, 2000). In a et al., 1998; Eischen et al., 1999; Ries et al., 2000). considerable minority of SW480 cells, ARF protein can Moreover, it explains the ability of overexpressed easily be detected either in the nucleolus or in a mixed P-catenin to protect p53 against Mdm2-mediated protea­ nucleolar/nucleoplasmic pattern (see Figure 9A). The somal degradation (Damalas et al., 1999). Conceivably, overall fraction of ARF positive SW480 cells was found the induced ARF protein binds to Mdm2, sequesters it in to be -15-20% (Figure 9B). It is noteworthy that the nucleolus, and blocks the p53-specific E3 ubiquitin constitutive ARF expression is not expected to exert ligase activity of Mdm2 (Honda and Yasuda, 1999). marked anti-proliferative effects in SW480 cells, which Indeed, the ARF protein accumulated in response to carry mutant p53 (Rodrigues et al., 1990). In striking P-catenin deregulation is largely nucleolar (Figures ID contrast, overexpression of either DN-TCF4 [Figure 9A, and 9A). a-c; putative positive cells were identified by cotransfec­ Our findings predict that the oncogenic action of tion with green fluorescent protein (GFP)] or axin P-catenin will be facilitated in vivo not only by loss of (Figure 9A, d-f) reduced ARF staining to levels not wt p53 expression, but also by ablation of ARF expression. significantly above background (see also Figure 9B). This is indeed the case, at least in CRC. While Hence, as in MEFs, constitutive P-catenin signaling drives homozygous deletions of the INK4a/ARF locus are not ARF expression also in these CRC cells. Moreover, associated with CRC (Cairns et al., 1995), many primary analysis of the transcriptional activity of the ARF promoter CRC tumors and cell lines exhibit complete or partial loss in SW480 cells revealed a substantial inhibition by either of ARF expression attributable to hypermethylation of the DN-TCF or axin (Figure 9C). Taken together, these results ARF promoter (Esteller et al., 2000). Importantly, silen­ argue strongly that deregulated P-catenin can tum on ARF cing of the ARF gene occurs early in colorectal transcription in colorectal epithelial cells, thereby trigger­ carcinogenesis, and is seen at equally high frequency ing the p53 pathway and placing a selective pressure for the already in adenomas (Esteller et al., 2000). We propose subsequent inactivation of this protective pathway. that ARF silencing enables emerging colorectal tumors to attenuate the activation of the p53 pathway by deregulated P-catenin, thereby averting the consequent restrictions on Discussion cell proliferation (Figure 10). The later emergence of Accumulation of deregulated P-catenin is observed in a mutations in the p53 gene itself may partly serve to enable variety of human cancers. This occurs either as a progression of tumors that have maintained ARF 491 8 ARF-dependent induction of p53 by jl-catenin DN-TCF4+GFP Axin CII CII _e. .:!l 15 + 10 V DN-TCF4 axin "'+ �� Fi g . 9. ARP is induced by constitutive Jl-catenin signaling in SW480 CRC cells. (A) SW480 cells were plated at a density of 5 X 10 cells/well in a 6-well plate. Two days later, cultures were transiently transfected with either myc-tagged axin (5 µg) (a--c) or a combination of GFP (50 ng) plus DN-TCF4 (5 µg) (d-f). Twenty-four hours later cells were fixed. Endogenous pl4ARP (a,c) was visualized by staining with polyclonal anti-p14ARP antibodies. Cells positive for transfected axin were visualized by staining with a monoclonal antibody directed against the myc tag (b ). Putative DN-TCF4 transfectants were identified by GFP fluorescence (e). DAPI staining was employed to visualize nuclei (c,f). (B) Quantitative analysis of the experiment shown in (A). The left and right panels depict the data for SW480 cultures transfected with either axin or DN-TCF+GFP, respectively. (-)and (+) relate to the cell subpopulations staining negative or positive for the transfected protein (myc-tagged axin and GFP, respectively; GFP is expected to mark DN-TCF positive cells). In each case, the number relates to the percentage of cells within the given subpopulation where prominent nucleolar ARP staining was easily discemable. (C) SW480 cells were transiently transfected with a luciferase reporter driven by the mouse p19ARP promoter plus either control vector (v) or plasmids expressing DN-TCF4 or axin. Transfections were done in triplicate. Luciferase activity was normalized for Renilla luciferase readings in the same extracts (see Materials and methods). expression. Alternatively, p53 mutations may provide In many human tumors, including those driven by additional features that are not enabled by the mere loss of constitutive �-catenin signaling, activating Ras mutations ARF expression. Such mutations may eliminate the ability are also observed; CRC is a good example (Kinzler and of tumor cells to undergo p53 activation through ARF­ Vogelstein, 1996). As shown here, Ras can indeed independent pathways, in response to conditions such as cooperate with tumor-associated mutant �-catenin in the DNA damage, chromosomal aberrations or hypoxia (Oren, transformation of cultured cells. Of note, this oncogenic 1999; Ashcroft et al., 2000; Sherr and Weber, 2000). activity of �-catenin is not revealed as long as the p53 Moreover, p53 mutations may also contribute directly to pathway is intact. tumorigenesis through dominant gain of function activities Recent findings suggest that the impact of Ras acti­ (Michalovitz et al., 1991; Dittmer et al., 1993). Indeed, vation on p53 is largely dependent on the ARF status (Ries while ARF promoter hypermethylation may be more et al., 2000). When ARF is non-functional, Ras leads primarily to p53-independent induction of Mdm2 expres­ frequent in CRC tumors retaining wt p53, it also occurs in a significant number of tumors carrying p53 mutations sion, which in tum enhances p53 degradation and in­ (Esteller et al., 2000). capacitates p53-mediated anti-proliferative signaling. i'x..1 "',t,, A.Damalas et al. In conclusion, our findings imply that the oncogenic Normal A B Epithelium [3 activity of deregulated -catenin places a selective pres­ sure for the inactivation of the p53 pathway in emerging deregulated B-catenin B-catenin activation tumors. In some cases, the pressure is effectively allevi­ ( APC mutations ) ated early on by ARF silencing. In other cases, the tumor cells can apparently cope with this problem without turning off ARF expression, either because the extent of p53 activation is relatively mild and tolerable, or because ARF ARF sil ncing other genetic alterations render the cells more refractory to some of the inhibitory effects of p53. In both types of cases, however, further progression down the road to (p53) malignancy often seems to entail the buildup of additional (p53) pressure on the p53 pathway, eventually resulting in direct p53 mutational inactivation of the p53 gene itself. Proliferation Adenoma Materials and methods Growth Inhibition Cells Additional alterations p53 -null mice (Jacks et al., 19 94) were obtained from Jackson Laboratorie s. Mouse MEFs were prepared from day 13.5 embr yos. MEFs derived from E2Fl knockout mice and ARF knockout mice were p53 mutations obtained from L. Yamasaki and C. Sherr, respectively. HC T1 16 , SW48 and SW480 cells were obtained from ATCC. MEFs were maintained in Dulbec co 's modified Eagl e's medium (DMEM) plus 10% fetal calf I Carcinoma I serum (FCS; Sigma), supplemented with non-es sential amino acids and B-mercaptoethanol. HCT116 and SW48 cells were maintained in Fig. 10. Schematic model depicting the relationship between McCoy 's SA medium plus 10% FC S. SW480 cells were maintained in deregulated B-ca tenin and the ARF-p53 pathway in MEFs and during DMEM plus 10% FCS. carcinogenesis. In MEFs, ablation of ARF prevents the activation of p53 by deregulated B-ca tenin and spares the cells from p53 -mediated Retroviral infection growth inhibition (this study). In carcinogenesis, it is proposed that High titer retroviral stocks were produced by transfecting retroviral ARF silencing by promoter hypermethylation or by other mechanisms constructs (pBabe-puro, pBabe-HA-B-ca tenin S33Y, pBabe-Ras Va ll2, enables emerging tumor cells to benefit from the oncogenic activities of or pBabep53Val 13 5) into 293T cells (2 X 10 /1 0-cm dish), by the deregulated B-ca tenin while avoiding the inhibitory consequences of calcium phosphate co-precipitation method, together with the ecotropic p53 activation. Additional genetic alterat ions, occurring at later stages packaging vector pSV-\jrE-MLV, providing ecotropic packaging helper of carcinogenesis, eventually generate a selective pressure for mutation fu nction. Virus-con taining culture supematants were collected 24-72 h of the p53 gene and lead to full malignancy. See Discussion for further post-transfection, at 6 h interval s, and pooled together. Frozen low details. passage MEFs were thawed and plated at a density of 2 X 10 /1 0-cm dish. Cells were infected 14 h later with filtered supematants in the presence of polybrene (8 µg/ ml; Sigma). Fresh supematants were added three times, at 4 h interval s. Forty-eight hours post-infection, cells were trypsinized Conversely, when ARF function is retained, deregulated and replated; 24 h later, fresh medium containing 10% FCS was added to Ras has the opposite outcome: ARF is induced and Mdm2 the cells. Whenever indicated, cells were replenished with medium function is blocked, resulting in accumulation of active containing 0. 1 % FC S. Cells were analyzed at the times indicated in the p53. One might, therefore, predict that the strongest respective figure legends. selective pressure for elimination of wt p53 would be lmmunoblot and immunostaining analysis exerted in those tumors that experience sequential Cells were washed with ice-cold phosphate-buffered saline (PBS) and [3 constitutive activation of -catenin and Ras on a back­ lyzed in NP-40 buffer (50 mM Tris pH 8, 15 0 mM NaCl, 5 mM EDTA, ground of sustained ARF expression. This prediction 0.5% NP-40) containing protease inhibitors (phenylmethylsulfonyl remains to be proven. fluoride and aprotinin) . After 15 min on ice, with intermittent vigorous vortexing, lysates were cleared by centrifugation at 4 °C. Samples [3 Deregulation of -catenin is primarily associated with corresponding to 20 µg total protein (Bio-Rad protein assay) were epithelial tumors. While we show that CRC-derived resolved on 12.5 or 15% SDS-polyacrylarnide gels and transferred to epithelial cells induce ARF expression in response to the nitrocellulose membranes (BA83, Schleicher & Schnell). Membranes [3 transcriptional activity of deregulated -catenin (Figure 9), were probed with polyclonal antibodies specific for mouse p53 (CMS, wan Novocastra), p19 ARF (gift of R.DePinho), mouse p21 (gift of most of the work in the present study was performed with C.S chneider), cyclin Dl (gift of T. Hunter) and Ha-tag (YI 1; Santa Cruz). fibro blasts . It is, therefore, important to point out that For the analysis of B-catenin levels in various cell lines (Figure IA), only [3 deregulation of -catenin is also implicated in the 4 µg total protein was loaded on each lane, and blots were probed with a development of desmoid fibroma, a tumor resulting from monoclonal antibody specific for B-ca tenin (Sigma). Western blots were developed with the ECL kit (Amersham) . Immunofluorescence micro­ excessive proliferation of fibroblasts, which arises fre­ scopy was performed as described (Simcha et al., 19 98). The myc tag was quently in individuals with germline mutations in the APC detected with the aid of the 9E 10 monoclonal antibody (gift of G.Evan). gene. A recent study employing APC-deficient Min mice revealed that loss of p53 is critical for the development of Luciferase assays p53-n ull MEFs (20 000 cells/well, in a 6-well dish) were transfected with such tumors (Halberg et al., 2000) . The ability of a combination of plasmids encoding firefly luciferase under control of the [3 deregulated -catenin to induce a vigorous p53-dependent mouse ARF promoter (2.6 kb genomic DNA fragment, gift of C.S h err) anti-proliferative response offers a particularly appealing and Renilla luciferase under the cytomegalovirus (CMV) promoter ( 40 ng explanation for the need to dispose of p53 function in order of each plasmid), with or without a dominant negative DN-TCF4 for such fibroblastic tumors to arise. (Korinek et al. , 19 97) expression plasmid (200 ng). p53 -null MEFs, rather 4920 P ARF-dependent ind ucti on of p53 b -cat enin than wt MEFs, were used in order to avoid activation of the endogenous Zhurinsky,J., Geiger,B. and Oren,M. (1999) Excess P-catenin p53 protein by the transfection procedure, potentially leading to p53- promotes accumulation of transcriptionally active p53. EMB O J. , mediated repression of ARF promoter activity. Cells were harvested 48 h 18, 3054--3063. later. de Rozieres,S., Maya,R., Oren,M. and Lozano,G. (2000) The loss of For luciferase assays in SW480 cells, cells were plated at 5 X 10 cells/ mdm2 induces p53-mediated apoptosis. Oncogene, 19, 1691-1697. well in a 6-well dish. Two days later, the cells were transfected with a de Stanchina,E. et al. (1998) ElA signaling to p53 involves the combination of plasmids encoding firefly luciferase under control of the p19(ARF) tumor suppressor. Genes Dev. , 12, 2434--2442. mouse ARF promoter (1 µg) and Renilla luciferase under the CMV Dittmer,D., Pati,S., Zambetti,G., Chu,S., Teresky,A.K., Moore,M., promoter (50 ng), with or without either axin (Shtutman et al., 19 99; 4 µg) Finlay,C. and Levine,A.J. (1993) Gain of function mutations in p53. or dominant negative DN-TCF4 (4 µg).Cells were harvested 24 h later. Nature Genet. , 4, 42-46. Luciferase assays were performed with a commercial double luciferase Eischen,C.M., Weber,J.D., Roussel,M.F., Sherr,C.J. and Cleveland,J.L. kit (Promega), employing a TD-20e luminometer (Turner Design) as (1999) Disruption of the ARF-Mdm2-p53 tumor suppressor pathway described (Damalas et al., 1999). Values of luciferase activity driven by in Myc-induced lymphomagenesis. Genes Dev. , 13: 2658-2669. the ARF promoter were normalized for Renilla luciferase readings in the Esteller,M., Tortola,S., Toyota,M., Capella,G., Peinado,M.A., Baylin, same extracts. S.B. and Herman,J.G. (2000) Hypermethylation-associated inacti­ vation of pl4(ARF) is independent of pl6(INK4a) methylation and Transformation assays p53 mutational status. Cancer Res. , 60, 129-133. Transformation assays were performed essentially as described Gat,U., DasGupta,R., Degenstein,L. and Fuchs,E. (1998) De novo hair (Michalovitz et al., 1990), employing low passage MEFs in conjunction follicle morphogenesis and hair tumors in mice expressing a truncated with retroviral infection. Cultures were maintained in culture for about P-catenin in skin. Cell, 95, 605-614. 2 weeks and then washed twice with PBS and fixed with methanol for Giaccia,A.J. and Kastan,M.B. (1998) The complexity of p53 modulation: 5 min. Fixed cultures were stained with Giemsa stain. emerging patterns from divergent signals. Genes Dev. , 12, 2973-2983. Halberg,R.B., Katzung,D.S., Hoff,P.D., Moser,A.R., Cole,C.E., Lubet,R.A., Donehower,L.A., Jacoby,R.F. and Dove,W.F. (2000) Tumorigenesis in the multiple intestinal neoplasia mouse: redundancy Acknowledgements of negative regulators and specificity of modifiers. Proc. Natl Acad. We are indebted to B.Geiger for many inspiring discussions and Sci. USA , 97, 3461-3466. continued support. We thank C.Sherr and M.Roussel for ARF-null Hart,M. et al. 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(1998) The alternative product from the human CDKN2A http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Deregulated β‐catenin induces a p53‐ and ARF‐dependent growth arrest and cooperates with Ras in transformation

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Springer Journals
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Copyright © European Molecular Biology Organization 2001
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10.1093/emboj/20.17.4912
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The EMBO Journal Vol. 20 No. 17 pp. 4912--4922, 2001 Deregulated P-catenin induces a p53- and ARF-dependent growth arrest and cooperates with Ras in transformation and axin/conductin (Behrens et al., 1998; Ikeda et al., Alexander Damalas, Sharon Kahan, 1998). The N-terminally phosphorylated P-catenin then Michael Shtutman, Avri Ben-Ze'ev and 1 becomes a target for recognition by a specific SCF-type E3 Moshe Oren ubiquitin ligase (Hart et al., 1999; Winston et al., 1999; Department of Molecular Cell Biology, Weizmann Institute of Sadot et al., 2000), leading to its polyubiquitylation and Science, Rehovot 76100, Israel subsequent proteasomal degradation. Corresponding author Upon physiological activation of the Wnt-wingless e-mail: [email protected] pathway, P-catenin is stabilized and translocates into the nucleus. There, in complex with members of the TCF/LEF Aberrant activation of -catenin contributes to the family of DNA binding proteins, it serves as a transcrip­ onset of a variety of tumors. We report that a tumor­ tional regulator and induces the expression of cognate derived -catenin mutant induces accumulation and target genes (Behrens et al., 1996; Riese et al., 1997; van activation of the p53 tumor suppressor protein. de Wetering et al., 1997). Aberrant stabilization of Induction is mediated through ARF, an alternative P-catenin, resulting in constitutive transcriptional activity, reading frame product of the INK4A tumor suppres­ is encountered in a variety of cancers (Bienz, 2000; Peifer sor locus, in a manner partially dependent on the and Polakis, 2000; Polakis, 2000; Zhurinsky et al., 2000a). transcription factor E2Fl. In wild-type mouse embryo Stabilization can be achieved either through mutational fibroblasts, mutant P -catenin inhibits cell proliferation inactivation of APC or axin, or through direct mutation of and imposes a senescence-like phenotype. This does P-catenin. The latter mutations usually occur within the not occur in cells lacking either ARF or p53, where N-terminal domain of P-catenin, rendering it refractory to deregulated -catenin actually overrides density­ phosphorylation by GSK-3p (Polakis, 1999). dependent growth inhibition and cooperates with acti­ The c-myc and cyclin Dl genes, encoding important vated Ras in transformation. Thus, the oncogenic positive regulators of cell proliferation, have been iden­ activity of deregulated -catenin is curtailed by con­ tified as transcriptional targets of deregulated P-catenin current activation of the p53 pathway, thereby provid­ (He et al., 1998; Shtutman et al., 1999; Tetsu and ing a protective mechanism against cancer. When the McCormick, 1999). Constitutive activation of those p53 pathway is impaired, deregulated -catenin is free genes may account, at least in part, for the putative to manifest its oncogenic features. This can occur not contribution of P-catenin deregulation to tumorigenesis. only by p53 mutations, but also by ablation of ARF Additional P-catenin target genes such as WISP-I and expression, as observed frequently in early stages of PPARo (He et al., 1998; Xu et al., 2000), may also play a colorectal carcinogenesis. positive role in cancer processes. The oncogenic potential Keywords: ARF/P-catenin/p53/Ras/senescence of deregulated P-catenin has been convincingly demon­ strated in vivo in mouse tumor models (Oshima et al., 1995; Gat et al., 1998; Chan et al., 1999). Introduction The p53 tumor suppressor gene is also subject to frequent mutations in cancer (Levine, 1997). Such muta­ P-catenin is a versatile protein. On the one hand, it is an tions primarily abolish the tumor inhibitory activities of important structural component of the cell adhesion machinery that mediates cell-cell interactions (Kemler, the wild-type (wt) p53 protein. In non-stressed cells, p53 1993; Ben-Ze'ev and Geiger, 1998). On the other hand, as protein levels are usually very low, owing to rapid a pivotal constituent of the Wnt-wingless pathway, proteolytic degradation ofwt p53. Numerous stress signals P-catenin is also intimately involved in intracellular signal lead to activation and accumulation of p53 (Giaccia and transduction (Cox and Peifer, 1998; Willert and Nusse, Kastan, 1998; Prives, 1998; Lohrum and Vousden, 1999; 1998). Oren, 1999; Prives and Hall, 1999; Ashcroft et al., 2000). The levels, subcellular localization and activity of These signals include diverse types of DNA damage and P-catenin are tightly regulated within the cell (Aberle et al., chromosomal aberrations, as well as deregulation of 1997; Bienz, 1999; Clevers, 2000; Hecht et al., 2000; various growth stimulatory signaling pathways. The latter Rosin-Arbesfeld et al., 2000). The free cytosolic pool of type of deregulation may be achieved via aberrant P-catenin is unstable and subject to rapid proteolytic activation of oncogenes such as c-myc and ras, or through degradation, which requires its phosphorylation by the the action of viral oncoproteins such as the products of the glycogen synthase kinase 3P (GSK-3P). Phosphorylation adenovirus ElA region. In several cases, including c-myc, occurs on sites within the N-terminal domain of P-catenin, ras, E2Fl, and adenovirus ElA, induction ofp53 relies to and is mediated through interaction of P-catenin with a a great extent on another tumor suppressor protein, ARF complex including, in addition to GSK-3P, the APC (Bates et al., 1998; de Stanchina et al., 1998; Palmero (adenomatous polyposis coli) tumor suppressor protein et al., 1998; Stott et al., 1998; Zindy et al., 1998). ARF is 4912 © European Molecular Biology Organization ARF-dependent induction of p53 by P-catenin the product of an alternative transcript of the INK4a tumor recombinant retrovirus encoding B-catenin S33Y, a suppressor locus (Quelle et al., 1995; reviewed in constitutively active B-catenin mutant derived from a Sharpless and DePinho, 1999; Sherr and Weber, 2000). CRC patient (Morin et al., 1997). A retrovirus expressing ARF binds to the Mdm2 protein, which plays a key role in only the puromycin resistance gene served as a negative the degradation of p53 through its activity as a p53- control. Infection efficiencies were routinely close to specific E3 ubiquitin ligase (Honda and Yasuda, 1999). 100%, as assessed by puromycin selection of representa­ The binding of ARF to Mdm2 sequesters Mdm2 in the tive infected cultures, and by infection of parallel cultures nucleolus, where it cannot target p53 for ubiquitylation; in with a retrovirus encoding B-galactosidase (data not addition, it directly inhibits the E3 activity of Mdm2 shown). The total levels of B-catenin in the infected (Honda and Yasuda, 1999; Tao and Levine, 1999; Weber MEFs, representing the combination of endogenous wt et al., 1999, 2000b; Lohrum et al., 2000). Hence, induction B-catenin and transduced mutant B-catenin, were only of ARF by deregulated oncogenes inhibits the ubiquityl­ moderately elevated relative to the endogenous wt protein ation and subsequent proteasomal degradation of p53, alone in control MEFs (Figure lA). More importantly, resulting in accumulation of p53 and stimulation of p53- these levels were comparable with those present in cell mediated cellular responses. This is believed to serve as a lines derived from human CRC, where B-catenin is failsafe mechanism that protects against the tumorigenic deregulated owing to either direct B-catenin mutations consequences of oncogene activation (Sherr, 1998). In (SW48 and HCT116) or inactivation of the APC tumor addition, ARF may also have p53-independent anti­ suppressor (SW480). As seen in Figure lB, such levels of proliferative functions (Carnero et al., 2000; Esteller oncogenic B-catenin were already sufficient to elicit a et al., 2000; Weber et al., 2000a). marked increase in the steady state levels of p53. We reported previously that overexpression of B-cate­ Several oncoproteins trigger p53 accumulation through nin leads to accumulation of transcriptionally active p53 in stimulation of ARF expression (Lowe, 1999; Sherr and transfected cells (Damalas et al., 1999). We now show that Weber, 2000). We, therefore, investigated whether ARF activated, tumor-derived mutant B-catenin triggers p53 plays a role in the induction of p53 by tumor-associated accumulation when expressed at levels comparable with B-catenin. Analysis of infected wt MEFs revealed a those present in cells derived from human colorectal dramatic increase in murine p19ARF protein levels in cancer (CRC). The accumulation of p53 is due to induction the presence of B-catenin S33Y (Figure lB). Hence, of ARF expression. Maximal induction of ARF and p53 deregulated B-catenin can effectively enhance ARF requires the presence of the E2Fl transcription factor. In expression. normal primary mouse embryo fibroblasts (MEFs), In p53-null MEFs, basal ARF expression is significantly deregulated B-catenin arrests cell proliferation and im­ elevated (Figure lB), owing to relief of p53-dependent poses a senescence-like phenotype that is dependent on repression of ARF transcription (Robertson and Jones, both ARF and p53 function. Conversely, in ARP-deficient 1998). Nevertheless, B-catenin elicited a further increase or p53-deficient MEFs, the same mutant B-catenin exerts in ARF protein (2.5-fold after normalization for a-tubulin, a growth stimulatory effect, reflected in an ability to over­ Figure lB). Remarkably, B-catenin augmented to a similar come density-dependent growth inhibition. Moreover, extent the transcriptional activity of the murine ARF in the absence of either ARF or wt p53, deregulated promoter (Inoue et al., 1999) in these cells (Figure lC). B-catenin cooperates with oncogenic ras to enhance cell Moreover, activation of this promoter was effectively transformation. abrogated by overexpression of dominant negative TCF4 Our findings support a model where the emergence of (DN-TCF4), known to block the transcriptional activity of deregulated, constitutively active B-catenin is coupled B-catenin (Korinek et al., 1997). These findings imply that with the transcriptional stimulation of ARF expression. deregulated B-catenin can induce ARF expression at the This in tum triggers an anti-proliferative response, transcriptional level. orchestrated largely through p53. Such anti-proliferative The ARF protein induced by B-catenin resides predom­ response is proposed to curb the potential oncogenic inantly in the nucleolus (Figure 1D, a), as confirmed by consequences of deregulated B-catenin, thereby providing double staining for the nucleolar protein nucleophosmin/ a potent mechanism for tumor suppression. Elimination of B23 (Figure 1D, b); this is consistent with earlier reports this inhibitory arm, through inactivation of either p53 or (Weber et al., 1999). ARF, might unveil the full oncogenic potential of Upregulation of p53 by B-catenin was accompanied by deregulated B-catenin and allow it to play a more effective elevated levels of p21, a p53 target gene product role in tumorigenesis. The observed silencing of ARF (Figure lE), confirming that the upregulated p53 is expression in a sizable fraction of colorectal adenomas, functional. Moreover, induction of ARF and p53 by corresponding to early stages of cancer progression, is mutant B-catenin could be observed in the presence of consistent with this model. either low (Figure lE) or high (Figure lF) serum concentrations, although its extent was milder in cells growing in high serum. Results To further investigate whether physiological activation Induction of p53 by oncogenic p-catenin is of B-catenin-mediated signaling can induce ARF expres­ mediated through ARF sion, wt MEFs were treated with LiCl. LiCl is a selective Experimental overexpression of B-catenin can induce p53 inhibitor of GSK-3B; exposure of cells to LiCl results in accumulation (Damalas et al., 1999). To investigate the B-catenin stabilization and augmentation of B-catenin­ molecular mechanism underlying this induction, primary mediated signaling (Woodgett, 1994). As seen in Figure 2, MEFs were employed. wt MEFs were infected with a high concentrations of LiCl elicited a significant increase 4913 A.Damalas et al. in ARF protein levels. As expected, this was accompanied by a proportionate increase in p53 protein; induction of p53 by LiCl has recently been reported also by Mao et al. (2001). The role of ARF in the induction of p53 by P-catenin deregulated 13-catenin was investigated with the aid of ARP-deficient MEFs (Karnijo et al., 1997). As seen in WI B p$J-./· MEI' Figure 3, 13-catenin S33Y was totally incapable of V V ' � n-,- -�-----, augmenting p53 in the absence of ARF, nor could it (7 elevate p21 expression. It should be noted that very extensive 13-catenin overexpression, for example by f-!' .&--J transient transfection with a CMV-driven expression plasmid, promotes p53 accumulation even in ARF­ , - - - - h•ta.b,illn deficient immortalized NIH-3T3 cells (Damalas et al., 1999). Yet, the data in Figure 3 clearly demonstrates that ARF is essential for induction of active p53 by the more physiological levels of deregulated 13-catenin achieved in the infected cells. Together, these findings imply that deregulated 13-catenin promotes ARF expression, most likely through enhanced transcription, thereby preventing p53 degrad­ ation and allowing accumulation of functional wt p53. Role of E2F1 in the stimulation of the ARF-p53 pathway by deregulated p.catenin Members of the E2F family of transcription factors play a critical role in cell-cycle progression (Nevins, 1998). E2F transcriptional activity is positively regulated by kinase complexes consisting of cyclin D and cdk4 or cdk6 (Sherr, 1996). Hence, increased expression of cyclin D proteins is expected to elevate E2F-mediated transcription. E2Fl, a member of the E2F family, transactivates the ARF promoter (Bates et al., 1998) and was implicated in the induction of ARF expression by several oncoproteins Fi . 1. Induction of p53 and ARP by deregulated �-catenin. (A) Comparison of �-catenin levels in different cell types. Cell extracts were prepared from CRC lines (SW48, HCTI 16, SW480) and from fibroblasts infected with recombinant retroviruses encoding either �-catenin S33Y (wtMEF + �-cat) or puromycin resistance only ( wtMEF). Four micrograms of protein of each sample was subjected to western blot analysis with a monoclonal antibody directed against �-catenin (Sigma). (B) Low passage MEFs, derived from either wt or p53-null animals, were infected with recombinant retroviruses encoding either HA-tagged S33Y tumor-derived mutant �-catenin (�) or puromycin resistance only (v). Cells were trypsinized and replated 48 h later. Cells were re-fed the next day with medium containing 0.1 % serum, and harvested after another 48 h. Twenty micrograms of protein of each sample was subjected to sequential western blot analysis with antibodies directed against p53, p19ARP and a-tubulin as p53 47� � a control for equal loading. (C) Low passage p53-null MEFs, infected p19ARF 20. with either �-catenin (�) or control retrovirus ( v ), were transiently transfected with a firefly luciferase reporter plasmid driven by the murine ARP promoter, either alone or together with a plasmid expressing dominant negative TCF4 (DN-TCF4). Transfections were a-tubulin done in triplicate. Luciferase activity was normalized for Renilla luciferase readings in the same extracts (see Materials and methods). (D) wt MEFs infected with a retrovirus encoding HA-tagged �-catenin � S33Y (a-c) or with pBabe-puro control retrovirus (d-f) were fixed and stained with pl9ARP-specific antiserum (a,d), B23 antiserum to visualize nucleoli (b,e) and DAPI to visualize nuclei (c,f). (E) wt MEFs were infected and processed essentially as in (B), except that blots 21) . J WafI were probed sequentially for p53, p2 and a-tubulin. Identical samples, run in parallel lanes of the same gel, were probed for pl9ARP. (F) wt MEFs were infected and processed as in (B), except -tubulin that the cells were maintained in medium containing I 0% serum throughout the experiment. _ 19All.f ._ _ _ _ � _ _ _ fl ARF-dependent induction of p53 by -catenin LlO(mfl.ll 0 2.5 wt MEF ARl'-/ - v V ll � n .s 1 �----- ---- 20.l>-j �- - -- - -- �f-P 19ARF -- a--l ubulln �p21 20 ,8--i - - Fig. 2. Induction of ARF and p53 by LiCI. Early passage wt MEFs were plated at a density of 2 X 10/10-cm dish. Fourteen hours later, 1- - -- f-.a- wbuJi n LiCI was added to the indicated final concentration, for an additional 48 h. Cells were then harvested and processed as in Figure 1. Fig. 3. ARF is required for accumulation and activation of p53 in response to oncogenic fl-catenin. Early passage MEFs, derived from wt or ARP-deficient (ARP-'-) mice, were infected as in Figure IB . Protein extracts (20 µg total protein/lane) were subjected to western blot (de Stan ch ina et al., 1998). Th e r o le o f E 2 Fl in th e analysis essentially as in Figure IB , except that HA-tag specific upregula ti o n o f ARF and p53 by P- c a tenin was s tudied antibodies were employed to visualize the mutant fl-catenin (HA­ thr o ug h th e use o f E 2 Fl-null MEFs . As seen in Figure 4A, mfl-catenin). indu c ti o n o f bo th ARF and p53 by P- c a tenin S33Y was par tially co mpr o mised in th ese c ells; th e fold indu c ti o n, n o rmalized for a- tubulin, is indi c a ted bel o w th e respe c tive wt A B M_EF Elf'I./- lanes . Never th eless, a residual in c rease in bo th pr o teins V V � � f:I was s till re tained, indi c a ting th a t w h ile E 2 Fl is an c:ydinOl 38.7 - }- HA-mjl-aten ln imp o r tan t media to r o f th e effe c t o f deregula ted P- c a tenin ....... a •la bulin - =i.- p s3 o n p53, i ts r o le is n o t ex c lusive . 47� - -· 1.0 4..0 0.1! 1.6 As expe c ted (Sh tu tman et al., 1999 ; T e tsu and - - - �p19A.RF :zo.a I Mc C o rmi c k, 1999) , infe c ti o n o f MEFs wi th the P- c a tenin l,0 ?.& 1,0 � S33Y re tr o virus led to a subs tan tial in c rease in c y c lin DI - _, .._ ._.. fi-tubuH.n pr o tein (Figure 4B ). Deregula ted P- c a tenin may th us s timula te ARF expressi o n thr o ug h indu c ti o n o f c y c lin Dl, Fig. 4. Involvement of E2Fl in the induction of the ARF-p53 pathway leading to in c reased E 2 Fl-media ted syn th esis o f ARF by fl-catenin. (A) Induction of ARF and p53 in E2Fl-null cells. MEFs mRNA . Th e impa c t o f c y c lin Dl upregula ti o n o n ARF from wt and E2Fl-null mice were infected and analyzed as in Figure 3. expressi o n is, ho wever, mo re co mplex . Spe c ific ally, ARF Band intensities were detemrined by densitometry (NIH-Image). trans c rip ti o n is subje c t to p o si tive regula ti o n by an o th er Numbers below lanes indicate the fold induction of the respective protein by mutant fl-catenin, calculated relative to the parallel control trans c rip ti o n fa c to r, DMPl (In o ue et al., 1999). DMPl virus-infected wt MEF sample, after normalizing for u-tubulin. a c tivi ty, in turn, is in h ibi ted by c y c lin Dl (In o ue and Sh err, (B) Western blot analysis of cyclin DI protein in wt MEFs infected 1998). Th us, c y c lin Dl als o p o ssesses th e p o ten tial to with either control (v) or mutant fl-catenin (Ii) retrovirus. Infection and nega tively regula te ARF expressi o n th r o ug h in h ibi ti o n o f protein analysis were as in Figure IB . DMPI . Th e fa c to rs tha t di c ta te the ne t effe c t o f c y c lin Dl indu c ti o n o n ARF expressi o n s till remain to be elu c ida ted . subs tan tially re tarded rela tive to th eir co un terpar ts in­ Sustained deregulation of JJ- catenin induces a fe c ted wi th th e co n tr o l virus . Unlike w t c ells, MEFs senescence -like phenotype dependent on ARF and la c king ei th er ARF o r p53 co n tinued to pr o lifera te after p infe c ti o n wi th the P- c a tenin S33Y re tr o virus; in nei th er To evalua te th e p h en o typi c co nsequen c es o f sus tained c ell type was th ere any eviden c e for senes c en c e-ass oc ia ted P- c a tenin deregula ti o n, MEFs o f differen t gen o types were mo rp ho l o gi c al al tera ti o ns (Figure 5 ). In fa c t, o verexpres­ infe c ted wi th re tr o viruses en co ding ei th er P- c a tenin S33Y si o n o f P- c a tenin S33Y in th ese c ells exer ted a measurable o r pur o my c in resis tan c e o nly . Infe c ted c ul tures were p o si tive effe c t. Af ter several days o f c ul tiva ti o n, fo c al areas o f h ig h - c ell densi ty co uld be dis c erned in ARF-null trypsinized, and an iden ti c al number o f c ells fr o m ea ch c ul ture were re-pla ted a t l o w densi ty . Mi c r o s co pi c exam­c ul tures . Wh ile co n tr o l ARF-null MEFs pr o lifera ted to ina ti o n o f th e vari o us c ul tures, several days la ter, revealed sa tura ti o n densi ty and th en s to pped, c ul tures o verexpres­ th a t w t MEFs infe c ted wi th th e co n tr o l re tr o virus rea ch ed a sing P- c a tenin S33Y failed to s to p a t th a t p o in t and h ig h c ell densi ty (Figure 5 ). In s triking co n tras t, w t MEFs co n tinued to pr o lifera te avidly (Figure 6B). Th us, in th e infe c ted wi th P- c a tenin S33Y remained rela tively sparse; absen c e o f ARF, deregula ted P- c a tenin c an augmen t th e mo re o ver, many o f th e c ells were flat and remarkably abili ty o f c ells to o ver co me densi ty-dependen t gr o w th spread o u t, ex h ibi ting a senes c en c e-like mo rp ho l o gy . Th e in h ibi ti o n, a fea ture th a t may fa c ili ta te tum o r pr o gressi o n . experimen t s ho wn in Figure 5 was perf o rmed in th e Th e effe c t o f P- c a tenin S33Y o n p53-null MEFs was presen c e o f 10% serum . Essen tially similar resul ts were s o mew h a t mo re co mpli c a ted . Cells infe c ted wi th th e o btained in l o w (0. 1 % ) serum, ex c ep t th a t th e mo rp ho ­ co n tr o l virus grew to a h ig h densi ty, bu t th en pr o lifera ti o n l o gi c al ch anges in P- c a tenin-infe c ted w t MEFs were mo re c eased and c ell numbers a c tually wen t do wn (Figure 6C ). drama ti c , w h ile all o th er c ul tures gradually c eased pr o lif­ Th a t la tter dr o p was ab o lis h ed by o verexpressi o n o f era ti o n wi tho u t displaying senes c en c e-like fea tures (da ta P- c a tenin S33Y, sugges ting th a t under tho se co ndi ti o ns n o t s ho wn ). P- c a tenin may pr o mo te c ell survival . Gr o w th ra te analysis (Figure 6A ) co nfirmed tha t th e Th e dependen c e o f th e senes c en c e-like p h en o type o n pr o lifera ti o n o f MEFs o verexpressing P- c a tenin S33Y was p53 was fur th er co nfirmed by th e use o f a re co mbinan t 4915 control A.Damalas et al. f}-catenin wt MEF p53-/- Fi . 5. Overexpression of mutant �-catenin elicits a senescence-like phenotype dependent on ARF and p53. Early passage MEFs, derived from wt, ARF-null or p53-null mice, were infected with control retrovirus or retrovirus encoding HA-tagged �-catenin S33Y. Cells were trypsinized and replated 48 h later, and after an additional day fresh medium containing 10% serum was added. Cultures were maintained in the same medium for 6 days. Phase contrast photographs were taken at a magnification of IO OX. A B ARF-/- C wt p53-/- 10 18 35 .;;- i 16 i 0 :;:: ';;! -f '£ 12 ., .,, 20 .,, 10 § 8 ::l 15 z z z 6 2 4 ol ol ol u u 5 7 11 1 3 5 7 9 11 1 3 5 7 9 11 Days Days Days Fi . 6. Deregulated �-catenin inhibits cell proliferation in an ARF- and p53-dependent manner. Cultures of wt, ARF-null and p53-null MEFs were infected with control retrovirus (circles) or retrovirus encoding HA-tagged �-catenin S33Y (squares) and processed as in Figure 5. Infected cells were replated in 10-cm dishes, at a seeding density of 2 X 10 cells/dish. Triplicate cultures were counted at the indicated number of days after replating. The SE is indicated. retrovirus encoding a temperature sensitive (ts) p53 activated p53 alone (control + p53Val135, 32C). In mutant, p53Val135 (de Rozieres et al., 2000). ps3-t­ contrast, avid proliferation with no morphological MEFs were first infected with the P-catenin S33Y alterations, despite the presence of P-catenin, was retrovirus, and then re-infected with p53Val135. When seen at 37.5 C where the ts p53 assumes a mutant the infected cultures were shifted to 32C, resulting in conformation devoid of wt p53 activity. It is note­ activation of the ts p53 (Michalovitz et al., 1990), the worthy that under those conditions, allowing for cells assumed a typical senescence-like morphology relatively high levels of p53 even in the absence of (Figure 7 A). This did not occur in the presence of deregulated P-catenin, p53 levels increased only mildly 4916 13 ARF-dependent induction of p53 by -catenin �-catenin+p53Val135 control +p53Val135 32 C 37°C tJ -- p53 �---� •tubulin -I Fig. 7. wt p53 activity is required for induction of a senescence-like phenotype by deregulated 13 -catenin. Early passage p53 - MEFs were infected with control retrovirus or retrovirus encoding HA-tagged 13 -catenin S33Y. Forty-eight hours later cells were trypsinized, replated and subjected to a second round of infection with a retrovirus encoding the ts p53 mutant p53Val135. After 48 h fresh medium containing 10% serum was added, and ° ° some of the cultures were transferred to 32 C while the others were left at 37.5 C. (A) Phase contrast photographs taken 6 days after plating (magnification = lOO X) . (B) Western blot analysis of p53 protein levels in cultures maintained at 32 C and extracted 4 days after replating. in response to excess -catenin S33Y (Figure 7B). This contrast, Ras Val 12 elicited many transformed foci in raises the possibility that tumor-associated -catenin can ARF-null cells (Figure 8), as reported earlier (Kamijo regulate not only the levels of p53, but also its specific et al., 1997). Importantly, while -catenin alone did biochemical activity. not give rise to a significant number of foci, the P P In conclusion, whereas deregulated -catenin exerts combination of -catenin and activated Ras resulted in potent anti-proliferative effects and elicits a senes­ extensive transformation; foci were more numerous and cence-like phenotype in the presence of wt p53 and substantially thicker than with Ras alone (Figure 8). ARF, inactivation of either ARF or p53 alleviates Similarly, -catenin S33Y also augmented Ras-medi­ ated focus formation in p53-null MEFs, although those anti-proliferative effects and reveals the potential growth-promoting features of deregulated P -catenin. overall numbers of foci were lower than in ARF-null MEFs (data not shown). Thus, constitutively activated Ras can cooperate with Tumor-associated p.catenin and activated Ras deregulated P -catenin to transform MEFs; this oncogenic cooperate in transformation in the absence of ARF activity of mutant P -catenin is, however, unleashed only or p53 upon inactivation of the p53 pathway. To further explore whether deregulated P -catenin can contribute to oncogenesis, we asked whether P -catenin S33Y cooperates with constitutively active mutant Ras Regulation of ARF by P-catenin in human in the transformation of cultured cells. This combin­ CRC-derived cells ation was chosen because deregulation of P -catenin is The oncogenic activity of P -catenin has been extensively frequently followed by Ras mutations during colorectal studied in CRC cell lines, representing a type of cancer carcinogenesis (Kinzler and Vogelstein, 1996). MEFs where deregulated -catenin plays a pivotal role in of various genotypes were sequentially infected with tumorigenesis. It was, therefore, important to determine P P retroviruses expressing -catenin S33Y and the tumor­ whether deregulated -catenin can upregulate ARF expres­ associated mutant H-RasVall2. In wt MEFs, no trans­ sion in CRC cells. SW480 cells, in which -catenin is formed foci were detectable upon infection with either deregulated owing to APC gene mutation, were transiently Ras alone or Ras plus -catenin (data not shown). In transfected with either DN-TCF4 or axin, both of which are 4917 A.Damalas et al. consequence of mutations in the P-catenin gene itself, or owing to lesions in genes that control the ubiquitylation and degradation of cytosolic P-catenin, such as the APC tumor suppressor (Kinzler and V ogelstein, 1996) and axin (Satoh et al., 2000). In many cases, best exemplified by CRC, accumulation of transcriptionally active P-catenin is �-ca tenin later followed by p53 mutations that inactivate the tumor suppressor functions of p53 (Kinzler and Vogelstein, 1996). We show here that deregulated P-catenin can activate the p53 pathway and trigger a p53-mediated anti­ proliferative response, which in primary fibroblasts takes the form of a growth inhibition associated with senes­ Ras cence-like morphological features. Moreover, inactivation of the p53 pathway uncovers the oncogenic properties of deregulated P-catenin, measured by its ability to cooperate with mutant Ras in the transformation of primary MEFs. These observations may explain, at least in part, the �-caten in selective pressure to inactivate the p53 pathway in tumors involving excessive P-catenin-dependent signaling. Ras Induction of p53 by deregulated P-catenin is strictly dependent on the ARF tumor suppressor. Deregulated P-catenin stimulates ARF expression and augments tran­ scription from the ARF promoter in CRC-derived cells as Fi g . 8. Oncogenic �-catenin cooperates with Ras in transformation of 5 well as in fibroblasts. This is completely abrogated by ARF-null MEFs. Early passage ARP-I- MEFs (1. 5 X 10 cells/ 10 -cm dish) were infected with either control retrovirus or retrovirus encoding negative dominant TCF, strongly implicating the tran­ �-ca tenin S33Y. Two days later the cultures were trypsinized and scriptional activity of P-catenin in the induction of ARF replated again at 1. 5 X I 0 cells/I 0-cm dish, and subjected to a second and hence of p53. Of note, the P-catenin S33Y mutant is round of infection with a retrovirus encoding the Va ll2 mutant H-Ras. derived from a human tumor (Morin et al., 1997), and Cultures were fixed 8-9 days later and stained with Giemsa stain. possesses enhanced transcriptional activity (Zhurinsky Triplicate dishes are shown. Where only a single oncogene is indicated, the cultures were actually also subj ected to a second infection with et al., 2000b). control retrovirus to maintain the infection history of each culture The ability of P-catenin to activate p53 through equal. upregulation of ARF adds it to a growing list of oncoproteins and growth regulatory proteins that can exert a similar effect (Bates et al., 1998; de Stanchina et al., potent negative regulators of P-catenin signaling (Behrens 1998; Palmero et al., 1998; Radfar et al., 1998; Zindy et al., 1998; Ikeda et al., 1998; Clevers, 2000). In a et al., 1998; Eischen et al., 1999; Ries et al., 2000). considerable minority of SW480 cells, ARF protein can Moreover, it explains the ability of overexpressed easily be detected either in the nucleolus or in a mixed P-catenin to protect p53 against Mdm2-mediated protea­ nucleolar/nucleoplasmic pattern (see Figure 9A). The somal degradation (Damalas et al., 1999). Conceivably, overall fraction of ARF positive SW480 cells was found the induced ARF protein binds to Mdm2, sequesters it in to be -15-20% (Figure 9B). It is noteworthy that the nucleolus, and blocks the p53-specific E3 ubiquitin constitutive ARF expression is not expected to exert ligase activity of Mdm2 (Honda and Yasuda, 1999). marked anti-proliferative effects in SW480 cells, which Indeed, the ARF protein accumulated in response to carry mutant p53 (Rodrigues et al., 1990). In striking P-catenin deregulation is largely nucleolar (Figures ID contrast, overexpression of either DN-TCF4 [Figure 9A, and 9A). a-c; putative positive cells were identified by cotransfec­ Our findings predict that the oncogenic action of tion with green fluorescent protein (GFP)] or axin P-catenin will be facilitated in vivo not only by loss of (Figure 9A, d-f) reduced ARF staining to levels not wt p53 expression, but also by ablation of ARF expression. significantly above background (see also Figure 9B). This is indeed the case, at least in CRC. While Hence, as in MEFs, constitutive P-catenin signaling drives homozygous deletions of the INK4a/ARF locus are not ARF expression also in these CRC cells. Moreover, associated with CRC (Cairns et al., 1995), many primary analysis of the transcriptional activity of the ARF promoter CRC tumors and cell lines exhibit complete or partial loss in SW480 cells revealed a substantial inhibition by either of ARF expression attributable to hypermethylation of the DN-TCF or axin (Figure 9C). Taken together, these results ARF promoter (Esteller et al., 2000). Importantly, silen­ argue strongly that deregulated P-catenin can tum on ARF cing of the ARF gene occurs early in colorectal transcription in colorectal epithelial cells, thereby trigger­ carcinogenesis, and is seen at equally high frequency ing the p53 pathway and placing a selective pressure for the already in adenomas (Esteller et al., 2000). We propose subsequent inactivation of this protective pathway. that ARF silencing enables emerging colorectal tumors to attenuate the activation of the p53 pathway by deregulated P-catenin, thereby averting the consequent restrictions on Discussion cell proliferation (Figure 10). The later emergence of Accumulation of deregulated P-catenin is observed in a mutations in the p53 gene itself may partly serve to enable variety of human cancers. This occurs either as a progression of tumors that have maintained ARF 491 8 ARF-dependent induction of p53 by jl-catenin DN-TCF4+GFP Axin CII CII _e. .:!l 15 + 10 V DN-TCF4 axin "'+ �� Fi g . 9. ARP is induced by constitutive Jl-catenin signaling in SW480 CRC cells. (A) SW480 cells were plated at a density of 5 X 10 cells/well in a 6-well plate. Two days later, cultures were transiently transfected with either myc-tagged axin (5 µg) (a--c) or a combination of GFP (50 ng) plus DN-TCF4 (5 µg) (d-f). Twenty-four hours later cells were fixed. Endogenous pl4ARP (a,c) was visualized by staining with polyclonal anti-p14ARP antibodies. Cells positive for transfected axin were visualized by staining with a monoclonal antibody directed against the myc tag (b ). Putative DN-TCF4 transfectants were identified by GFP fluorescence (e). DAPI staining was employed to visualize nuclei (c,f). (B) Quantitative analysis of the experiment shown in (A). The left and right panels depict the data for SW480 cultures transfected with either axin or DN-TCF+GFP, respectively. (-)and (+) relate to the cell subpopulations staining negative or positive for the transfected protein (myc-tagged axin and GFP, respectively; GFP is expected to mark DN-TCF positive cells). In each case, the number relates to the percentage of cells within the given subpopulation where prominent nucleolar ARP staining was easily discemable. (C) SW480 cells were transiently transfected with a luciferase reporter driven by the mouse p19ARP promoter plus either control vector (v) or plasmids expressing DN-TCF4 or axin. Transfections were done in triplicate. Luciferase activity was normalized for Renilla luciferase readings in the same extracts (see Materials and methods). expression. Alternatively, p53 mutations may provide In many human tumors, including those driven by additional features that are not enabled by the mere loss of constitutive �-catenin signaling, activating Ras mutations ARF expression. Such mutations may eliminate the ability are also observed; CRC is a good example (Kinzler and of tumor cells to undergo p53 activation through ARF­ Vogelstein, 1996). As shown here, Ras can indeed independent pathways, in response to conditions such as cooperate with tumor-associated mutant �-catenin in the DNA damage, chromosomal aberrations or hypoxia (Oren, transformation of cultured cells. Of note, this oncogenic 1999; Ashcroft et al., 2000; Sherr and Weber, 2000). activity of �-catenin is not revealed as long as the p53 Moreover, p53 mutations may also contribute directly to pathway is intact. tumorigenesis through dominant gain of function activities Recent findings suggest that the impact of Ras acti­ (Michalovitz et al., 1991; Dittmer et al., 1993). Indeed, vation on p53 is largely dependent on the ARF status (Ries while ARF promoter hypermethylation may be more et al., 2000). When ARF is non-functional, Ras leads primarily to p53-independent induction of Mdm2 expres­ frequent in CRC tumors retaining wt p53, it also occurs in a significant number of tumors carrying p53 mutations sion, which in tum enhances p53 degradation and in­ (Esteller et al., 2000). capacitates p53-mediated anti-proliferative signaling. i'x..1 "',t,, A.Damalas et al. In conclusion, our findings imply that the oncogenic Normal A B Epithelium [3 activity of deregulated -catenin places a selective pres­ sure for the inactivation of the p53 pathway in emerging deregulated B-catenin B-catenin activation tumors. In some cases, the pressure is effectively allevi­ ( APC mutations ) ated early on by ARF silencing. In other cases, the tumor cells can apparently cope with this problem without turning off ARF expression, either because the extent of p53 activation is relatively mild and tolerable, or because ARF ARF sil ncing other genetic alterations render the cells more refractory to some of the inhibitory effects of p53. In both types of cases, however, further progression down the road to (p53) malignancy often seems to entail the buildup of additional (p53) pressure on the p53 pathway, eventually resulting in direct p53 mutational inactivation of the p53 gene itself. Proliferation Adenoma Materials and methods Growth Inhibition Cells Additional alterations p53 -null mice (Jacks et al., 19 94) were obtained from Jackson Laboratorie s. Mouse MEFs were prepared from day 13.5 embr yos. MEFs derived from E2Fl knockout mice and ARF knockout mice were p53 mutations obtained from L. Yamasaki and C. Sherr, respectively. HC T1 16 , SW48 and SW480 cells were obtained from ATCC. MEFs were maintained in Dulbec co 's modified Eagl e's medium (DMEM) plus 10% fetal calf I Carcinoma I serum (FCS; Sigma), supplemented with non-es sential amino acids and B-mercaptoethanol. HCT116 and SW48 cells were maintained in Fig. 10. Schematic model depicting the relationship between McCoy 's SA medium plus 10% FC S. SW480 cells were maintained in deregulated B-ca tenin and the ARF-p53 pathway in MEFs and during DMEM plus 10% FCS. carcinogenesis. In MEFs, ablation of ARF prevents the activation of p53 by deregulated B-ca tenin and spares the cells from p53 -mediated Retroviral infection growth inhibition (this study). In carcinogenesis, it is proposed that High titer retroviral stocks were produced by transfecting retroviral ARF silencing by promoter hypermethylation or by other mechanisms constructs (pBabe-puro, pBabe-HA-B-ca tenin S33Y, pBabe-Ras Va ll2, enables emerging tumor cells to benefit from the oncogenic activities of or pBabep53Val 13 5) into 293T cells (2 X 10 /1 0-cm dish), by the deregulated B-ca tenin while avoiding the inhibitory consequences of calcium phosphate co-precipitation method, together with the ecotropic p53 activation. Additional genetic alterat ions, occurring at later stages packaging vector pSV-\jrE-MLV, providing ecotropic packaging helper of carcinogenesis, eventually generate a selective pressure for mutation fu nction. Virus-con taining culture supematants were collected 24-72 h of the p53 gene and lead to full malignancy. See Discussion for further post-transfection, at 6 h interval s, and pooled together. Frozen low details. passage MEFs were thawed and plated at a density of 2 X 10 /1 0-cm dish. Cells were infected 14 h later with filtered supematants in the presence of polybrene (8 µg/ ml; Sigma). Fresh supematants were added three times, at 4 h interval s. Forty-eight hours post-infection, cells were trypsinized Conversely, when ARF function is retained, deregulated and replated; 24 h later, fresh medium containing 10% FCS was added to Ras has the opposite outcome: ARF is induced and Mdm2 the cells. Whenever indicated, cells were replenished with medium function is blocked, resulting in accumulation of active containing 0. 1 % FC S. Cells were analyzed at the times indicated in the p53. One might, therefore, predict that the strongest respective figure legends. selective pressure for elimination of wt p53 would be lmmunoblot and immunostaining analysis exerted in those tumors that experience sequential Cells were washed with ice-cold phosphate-buffered saline (PBS) and [3 constitutive activation of -catenin and Ras on a back­ lyzed in NP-40 buffer (50 mM Tris pH 8, 15 0 mM NaCl, 5 mM EDTA, ground of sustained ARF expression. This prediction 0.5% NP-40) containing protease inhibitors (phenylmethylsulfonyl remains to be proven. fluoride and aprotinin) . After 15 min on ice, with intermittent vigorous vortexing, lysates were cleared by centrifugation at 4 °C. Samples [3 Deregulation of -catenin is primarily associated with corresponding to 20 µg total protein (Bio-Rad protein assay) were epithelial tumors. While we show that CRC-derived resolved on 12.5 or 15% SDS-polyacrylarnide gels and transferred to epithelial cells induce ARF expression in response to the nitrocellulose membranes (BA83, Schleicher & Schnell). Membranes [3 transcriptional activity of deregulated -catenin (Figure 9), were probed with polyclonal antibodies specific for mouse p53 (CMS, wan Novocastra), p19 ARF (gift of R.DePinho), mouse p21 (gift of most of the work in the present study was performed with C.S chneider), cyclin Dl (gift of T. Hunter) and Ha-tag (YI 1; Santa Cruz). fibro blasts . It is, therefore, important to point out that For the analysis of B-catenin levels in various cell lines (Figure IA), only [3 deregulation of -catenin is also implicated in the 4 µg total protein was loaded on each lane, and blots were probed with a development of desmoid fibroma, a tumor resulting from monoclonal antibody specific for B-ca tenin (Sigma). Western blots were developed with the ECL kit (Amersham) . Immunofluorescence micro­ excessive proliferation of fibroblasts, which arises fre­ scopy was performed as described (Simcha et al., 19 98). The myc tag was quently in individuals with germline mutations in the APC detected with the aid of the 9E 10 monoclonal antibody (gift of G.Evan). gene. A recent study employing APC-deficient Min mice revealed that loss of p53 is critical for the development of Luciferase assays p53-n ull MEFs (20 000 cells/well, in a 6-well dish) were transfected with such tumors (Halberg et al., 2000) . The ability of a combination of plasmids encoding firefly luciferase under control of the [3 deregulated -catenin to induce a vigorous p53-dependent mouse ARF promoter (2.6 kb genomic DNA fragment, gift of C.S h err) anti-proliferative response offers a particularly appealing and Renilla luciferase under the cytomegalovirus (CMV) promoter ( 40 ng explanation for the need to dispose of p53 function in order of each plasmid), with or without a dominant negative DN-TCF4 for such fibroblastic tumors to arise. (Korinek et al. , 19 97) expression plasmid (200 ng). p53 -null MEFs, rather 4920 P ARF-dependent ind ucti on of p53 b -cat enin than wt MEFs, were used in order to avoid activation of the endogenous Zhurinsky,J., Geiger,B. and Oren,M. (1999) Excess P-catenin p53 protein by the transfection procedure, potentially leading to p53- promotes accumulation of transcriptionally active p53. EMB O J. , mediated repression of ARF promoter activity. Cells were harvested 48 h 18, 3054--3063. later. de Rozieres,S., Maya,R., Oren,M. and Lozano,G. (2000) The loss of For luciferase assays in SW480 cells, cells were plated at 5 X 10 cells/ mdm2 induces p53-mediated apoptosis. Oncogene, 19, 1691-1697. well in a 6-well dish. Two days later, the cells were transfected with a de Stanchina,E. et al. (1998) ElA signaling to p53 involves the combination of plasmids encoding firefly luciferase under control of the p19(ARF) tumor suppressor. Genes Dev. , 12, 2434--2442. mouse ARF promoter (1 µg) and Renilla luciferase under the CMV Dittmer,D., Pati,S., Zambetti,G., Chu,S., Teresky,A.K., Moore,M., promoter (50 ng), with or without either axin (Shtutman et al., 19 99; 4 µg) Finlay,C. and Levine,A.J. (1993) Gain of function mutations in p53. or dominant negative DN-TCF4 (4 µg).Cells were harvested 24 h later. Nature Genet. , 4, 42-46. Luciferase assays were performed with a commercial double luciferase Eischen,C.M., Weber,J.D., Roussel,M.F., Sherr,C.J. and Cleveland,J.L. kit (Promega), employing a TD-20e luminometer (Turner Design) as (1999) Disruption of the ARF-Mdm2-p53 tumor suppressor pathway described (Damalas et al., 1999). Values of luciferase activity driven by in Myc-induced lymphomagenesis. Genes Dev. , 13: 2658-2669. the ARF promoter were normalized for Renilla luciferase readings in the Esteller,M., Tortola,S., Toyota,M., Capella,G., Peinado,M.A., Baylin, same extracts. S.B. and Herman,J.G. (2000) Hypermethylation-associated inacti­ vation of pl4(ARF) is independent of pl6(INK4a) methylation and Transformation assays p53 mutational status. Cancer Res. , 60, 129-133. Transformation assays were performed essentially as described Gat,U., DasGupta,R., Degenstein,L. and Fuchs,E. (1998) De novo hair (Michalovitz et al., 1990), employing low passage MEFs in conjunction follicle morphogenesis and hair tumors in mice expressing a truncated with retroviral infection. Cultures were maintained in culture for about P-catenin in skin. Cell, 95, 605-614. 2 weeks and then washed twice with PBS and fixed with methanol for Giaccia,A.J. and Kastan,M.B. (1998) The complexity of p53 modulation: 5 min. Fixed cultures were stained with Giemsa stain. emerging patterns from divergent signals. Genes Dev. , 12, 2973-2983. Halberg,R.B., Katzung,D.S., Hoff,P.D., Moser,A.R., Cole,C.E., Lubet,R.A., Donehower,L.A., Jacoby,R.F. and Dove,W.F. (2000) Tumorigenesis in the multiple intestinal neoplasia mouse: redundancy Acknowledgements of negative regulators and specificity of modifiers. Proc. Natl Acad. We are indebted to B.Geiger for many inspiring discussions and Sci. USA , 97, 3461-3466. continued support. We thank C.Sherr and M.Roussel for ARF-null Hart,M. et al. 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(1998) The alternative product from the human CDKN2A

Journal

The EMBO Journal – Springer Journals

Published: Sep 3, 2001

Keywords: ARF; β‐catenin; p53; Ras; senescence

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