(2007) 26, 6356–6360 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION Polyomavirus small T transactivates genes by its ability to provoke the synthesis andthe stabilization of MYC

B Klucky1 and E Wintersberger

Division of Molecular Biology, Department of Medical Biochemistry, MFPL, Medical University of Vienna, Vienna, Austria

DNA tumor viruses are capable of driving quiescent cells polyomaviruses, but it is so far unproven that these into the . In case of , two viral viruses play a causative role in the formation of cancers. , the large andthe small (ST) T are However, polyomaviruses have been extremely helpful responsible for this outcome. ST interacts with the in studies of cell cycle regulation and cell transforma- phosphatase PP2A andwith chaperons of the dnaKtype tion. This includes the discovery of important cellular andleadsto the transactivation of several genes, which regulators such as or the play a role in S-phase induction. One of these is the pRB (Moran, 1993). transcription factor myelocytomatosis (MYC), which by All polyomaviruses code for two major early proteins, itself is an important regulator of growth. Microarray the small (ST) and the large (LT) tumor antigen. analysis has revealedseveral ST-inducedgenes, which are Polyomaviruses can infect a variety of cell types also targets of MYC; hence, ST may induce these genes including differentiated and hence growth arrested cells. via MYC. Experiments shown here are in line with this As they depend on the cellular machinery for replication assumption. MYC-regulatedgenes are inducedby ST at of their DNA, the T antigens meet this prerequisite by later times than MYC anda MYC responsive is interfering with the growth control of the host cell, stimulatedby ST. Regulation of MYC occurs through driving cells into the S-phase of the cell cycle. These signal transduction pathways, which are co-ordinated by reactions may form the basis for the tumorigenic PP2A suggesting that they may be targets of ST. Here, capacity of these viruses. we show that this is the case as important kinases involved The contribution of LT oncogene to S-phase induc- in these pathways appear in the active phosphorylated tion consists of the transactivation of E2F-regulated form in the presence of ST. Inhibition of these kinases genes, which requires interactions of the interferes with MYC induction and inhibition of MYC with the pocket proteins (pRB and its relatives p130 and activity blocks ST-mediated transactivation. p107; Harbour and Dean (2000)) with chaperons such Oncogene (2007) 26, 6356–6360; doi:10.1038/sj.onc.1210458; as HSC70 (Sullivan and Pipas, 2002) and with the published online 16 April 2007 coactivator proteins CBP/p300 (Nemethova et al., 2004). Keywords: polyomavirus; T antigens; MYC; S-phase The main cellular interactors of ST antigen are the induction protein phosphatase PP2A (Yang et al., 1991) and chaperons. PP2A is trimeric, consisting of a catalytic subunit C and two regulatory subunits A and B. B The multiple-stage process of cancer development is subunits constitute a family of proteins whereof driven by genetic and epigenetic events. Cancer can also individual members are assumed to confer substrate be associated with virus infection, for instance, with specificity to the phosphatase (Millward et al., 1999; members of the small DNA tumor virus family. The role Virshup, 2000). ST proteins can replace distinct B of human papillomaviruses in the development of subunits (Chen et al., 2004), which results in an carcinomas of the cervix is well documented and a inactivation of PP2A with respect to specific substrates. contribution of polyomaviridae, Simian virus 40 (SV40) Among these substrates is, for instance, p27Kip1 an or the human polyomaviruses, BKV and JCV, in the inhibitor of the cyclin-dependent kinase cdk2 (Porras etiology of human cancers has frequently been con- et al., 1999; Schu¨ chner and Wintersberger, 1999). ST has sidered (Reiss and Khalili, 2003). Up to 80% of been shown to stimulate signal transduction pathways the human population carries information of human (Sontag et al., 1993; Frost et al., 1994; Howe et al., 1998) and to transactivate genes, for example telomerase, Correspondence:Professor E Wintersberger, Division of Molecular cyclin D1 (Watanabe et al., 1996) and cyclin A (Porras Biology, Department of Medical Biochemistry, Medical University of et al., 1996; Schu¨ chner et al., 2001). Thus, ST Vienna, Dr. Bohrgasse 9, Vienna A-1030, Austria. contributes importantly to S-phase induction. Indeed it E-mail:[email protected] was found that neither LT nor ST by itself can induce an 1Current address:DKFZ, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg D-69221, Germany. S-phase in growth-arrested mouse or human fibroblasts, Received 13 June 2006; revised 26 February 2007; accepted 2 March but that the two proteins together do so quite effectively 2007; published online 16 April 2007 (Ogris et al., 1992; Porras et al., 1999). This result was Polyoma ST transactivates genes through induction of MYC B Klucky and E Wintersberger 6357 substantiated more recently when it was found that of ST-induced MYC, their mRNA levels should increase stable transfection of a combination of oncogenic Ras, later than that of MYC. As shown in Figure 1a, this is telomerase and SV40 LT and ST antigen can transform indeed the case. In Swiss 3T3 mouse fibroblasts, which human cells; LT alone was not sufficient for this conditionally express ST under the direction of the reaction (Yu et al., 2001; Hahn et al., 2002). MMTV promoter (Ogris et al., 1992), ST mRNA is Microarray analysis was carried out to more fully induced already 4 h after addition of dexamethasone to comprehend the capacity of ST and LT proteins to serum-starved cells. MYC mRNA also increases already deregulate the transcriptional program of the host cell 4 h after addition of dexamethasone and reaches a (Klucky et al., 2004). This showed that both proteins maximum level after 20 h. mRNAs for telomerase affect transcription of a surprisingly large number of (TERT) and cyclin D1 increase later, which is in line genes. Interestingly, among the proteins found tran- with the assumption that they are targets of MYC. If scriptionally upregulated by ST was the transcription this were so, one would expect that a reporter gene factor myelocytomatosis (MYC). Intact binding sites for PP2A and chaperons (J domain) were found essential for this reaction. This finding led us to propose that the a S3T3 STwt induction of a variety of genes seen in the microarray 0h 28h 0h 4h 8h 16h 18h 20h 24h 28h Dex study may be a consequence of the upregulation of 30 MYC (Klucky et al., 2004). 25 MYC dimerizes with MAX to bind specifically to a 20 DNA sequence, called E-box, present in promoters of 15 many genes. Thereby MYC controls the expression 10

MYC mRNA 5 of genes involved in proliferation, differentiation and Fold Induction apoptosis (Levens, 2003; Pelengaris and Khan, 2003; 0 Patel et al., 2004). We found concurrencies by compar- 7 ing data of the microarray analysis of cells expressing ST 6 with the results of -wide investigations of MYC- 5 regulated genes. For instance, like MYC, PyST upregu- 4 3 lates several ribosomal proteins, heat shock proteins, 2 nucleolin, cyclins and telomerase (Klucky et al., 2004). 1 Fold Induction Induction of all of these presumptive MYC targets was Cyclin D1 mRNA 0 found to depend on an interaction of ST with PP2A and chaperons. If these genes were indeed secondary targets 20 16 12 Figure 1 PyST induction of MYC target genes depends on an intact PP2A and chaperon binding site. (a) Time course of 8

induction of mRNA for cyclin D1, TERT and ST. Swiss 3T3 TERT mRNA 4 fibroblast and derived cell lines, conditionally expressing PyST wild Fold Induction 0 type or mutants under the control of the Dexamethasone-inducible MMTV promoter, was grown as described (Ogris et al., 1992). 30 Growth arrest and further treatment of cells, isolation of RNA and 25 quantitative PCR were carried out as described previously (Klucky et al., 2004), using Light Cycler Fast Start DNA Master SYBR 20 Green I Kit as recommended by the supplier (Roche, Applied 15 Science Corp.). Primer sequences:MYC forward:5 0-GCCCGCG 10

0 0 ST mRNA CCCAGTGAGGATA-3 , reverse:5 -GCGGCGGCGGTGAGG 5 TC-30; mTERT forward:5 0-TACCAACCCCATCAGGCAAAT Fold Induction -30, reverse:5 0-TGGGCTGGTGTTCAAGGCATC-30, cyclin D1 0 forward:5 0-CTGTGCGCCCTCCGTATCTTA-30, reverse:5 0- 0 0 GGCGGCCAGGTTCCACTTGAG-3 ; ST forward:5 -CA GG b ST max CATATAAGCAGCAGTC-30; reverse:5 0-CATCTCGGGTTGG TGTTC-30; b-actin forward:5 0-TGGCACCACACCTTCTACA MYC ATGAG-30; reverse:5 0 CAAGAAGGAGGCTGGAAAAGAG-30. E-box Luc b-Actin was used for normalization of the data. (b) ST activates myc-responsive elements. The pMYC-TA-LUC vector (Clontech, Palo Alto, CA, USA), was used to measure the stimulation of transcription from a MYC-responsive promoter by ST. The MYC- 8 luciferase vector was co-transfected with a pCIneo mammalian 6 expression vector, containing ST wild type or mutants defective in binding PP2A (PP2AÀ) or chaperons (STJÀ), into REF 52 cells. 4 After serum starvation of the cells for 48 h, expression of luciferase 2 was measured. CMV b-galactosidase was co-transfected to control for transfection efficiency. ST wild type, but not the mutants, 0 responsive element

transactivates the transcription factor MYC and in turn mediates FoldI nduction of myc- mock STwt PP2A- STJ- strong expression of the reporter luciferase gene. Equal expression of ST was verified by Western blot analysis using an antibody α PyST against the N-terminus of ST.

Oncogene Polyoma ST transactivates genes through induction of MYC B Klucky and E Wintersberger 6358 under an artificial promoter containing a MYC-respon- STwt PP2A- STJ- sive E-box should be transactivated by ST. Figure 1b - + - + - + shows that this is the case. As expected, activation of α PyST this promoter depends on intact binding sites for PP2A α phospho-AKT and chaperons. Deregulated expression of MYC is found in many α AKT human tumors (Nesbit et al., 1999) and multiple signal α phospho-Tor transduction pathways are implicated in the activation α of MYC, among these are the RAS effector pathways, actin PI3K/AKT and MAPK. Recently, two kinases were found to be crucial in the activation, the stabilization b α pMEK1/2 and eventually the inactivation of MYC (Sears et al., α MEK1/2 2000, Yeh et al., 2004). Phosphorylated and hence active α extracellular-signal-regulated kinase (ERK) phosphory- pERK1/2 lates MYC at serine 62, which results in an activation α ERK1/2 and a stabilization of the transcription factor. GSK3b, c which in its phosphorylated form is inactive, can α E cadherin phosphorylate MYC on threonine 58 only when the RAS pathway is shut off. This leads to MYC degrada- α cadherin11 tion by the ubiquitine/proteasome pathway. Removal of α ß-catenin the phosphate from serine 62 in a doubly phosphory- lated MYC (serine 62 and threonine 58) by PP2A α MYC resulting in a threonine 58 monophosphorylated species α actin of the transcription factor is a prerequisite for degrada- tion (Yeh et al., 2004). d STwt In growth-arrested cells ST-induced upregulation of 0 4 8 12 24 28h Dex particular steps of these pathways may render them independent of external growth signals. Consequently, α MYC we have analysed several kinases with respect to their α status in the presence and absence of actin ST protein. Our data strongly indicate that ST activates the AKT pathway by altering the function of PP2A, Figure 2 Deregulation of signal transduction pathways by ST. leading to the appearance of phosphorylated forms of Swiss 3T3 cells conditionally expressing ST were grown and serum- AKT and TOR (Figure 2a). PI3K was shown to be starved as in Figure 1. After induction of ST for 28 h whole- cell extracts were prepared as described (Klucky et al., 2004) activated by the SV40 ST, leading to PKC and mitogen (a) Phosphorylation of AKT and TOR was determined using activated protein kinase (MEK) activation (Sontag antibodies specific for Ser 473 phosphorylated AKT (Cell Signal- et al., 1993) and cDNA microarray analysis showed ling Technology Inc., Beverly, MA, USA, #9271) and for the that polyomavirus ST increases mRNA levels of the p85 phosphorylated downstream kinase, target of rapamycin (TOR, Cell Signalling #2971). Total amount of AKT protein was not regulatory subunit of PI3-kinase (Klucky et al., 2004). affected by the viral antigens as shown using an antibody against ST can furthermore be shown to cause the emergence of unphosphorylated AKT (Cell Signalling #9270). Determination of phosphorylated and hence activated versions of MEK b-actin with anti-actin antibody AC-74 (SIGMA-Aldriche, St and ERK kinases in serum-starved cells (Figure 2b). By Louis, MO, USA) was used to ensure equal loading of protein. (b) giving rise to an accumulation of phosphorylated ERK, Phosphorylation of MEK and ERK proteins. Western blots were probed with phospho-MEK1/2 antibody (Cell Signalling #9121) ST specifically creates exactly those conditions, which and phospho ERK antibody (Cell signalling #9106) respectively. result in an activation and stabilization of MYC. The Total amount of these kinases was determined using phospho- negatively active kinase, GSK3b, remains inactive independent antibodies (Cell Signalling #9122 and #9102). (c) because the RAS pathways will be active as long as ST Alteration of b-catenin and cadherins by ST. Western blot analysis indicates downregulation of cadherin 11 and E-cadherin while inactivates PP2A. Since PP2A is also required for the b-catenin is induced in the presence of PyST. MYC protein was removal of phosphate from serine 62 of MYC, ST will detected in STwt cells, but not in the mutants. The antibodies used inhibit this reaction as well and thus will keep MYC in were, E cadherin:BD Bioscience (Palo Alto, CA, USA), #610181; stable and active form. In fact, as shown in Figure 2d, b-catenin:BD Bioscience #610153; cadherin-11:(sc-6463) and MYC protein increases with time in cells expressing ST MYC (sc-764) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Equal loading was determined using antibody against and accumulates further even when MYC mRNA starts b-actin. (d) Increase of MYC protein with time of induction of ST. already to decline (see Figure 1a), which is in accord The antibodies used in these Western blots were as in (c). with MYC stabilization. Taken together, ST, by its capacity to activate RAS pathways, is competent to specific kinase inhibitors. Under these conditions neither create those conditions, which activate and stabilize MYC nor its target genes, cyclin D1 and TERT, are MYC induced by ST (Figure 3b). On the other hand, if MYC Support for this conclusion comes from experiments is induced by ST in the presence of a MYC inhibitor, shown in Figure 3a, where the ST-induced conversion of which competes with the transcription factor for bind- the kinases to their phosphorylated form is inhibited by ing to the E box in MYC-regulated promoters, no

Oncogene Polyoma ST transactivates genes through induction of MYC B Klucky and E Wintersberger 6359 a STwt transactivation of the two target genes, cyclin D and --+-MEK Inhibitor TERT, can be observed (Figure 3b), which underlines ---+PI3K/AKT Inhibitor the conclusion that upregulation of these genes by ST indeed occurs via MYC. -+++Dexamethasone Finally, ST interferes with WNT signalling by α phospho-AKT upregulating the major key regulator of the pathway, b-catenin (Figure 2c), which itself is involved in MYC α AKT induction. Polyoma ST decreases cadherin 11 and E cadherin mRNA (Klucky et al., 2004) and protein α pMEK1/2 (Figure 2c) and may thereby prevent cadherin/ b-catenin complex formation, resulting in an increase in b-catenin α MEK1/2 in the cytoplasm (Figure 2c). Owing to the upregulation of RAS signal transduction pathways by ST, GSK3b, α MYC the kinase, which within the APC complex is responsible for phosphorylating and thereby destabilizing b-catenin, α PyST is inactive. b-Catenin thus remains stable and is transported into the nucleus where it binds to T-cell α actin factor (TCF) and activates transcription of many genes including MYC (Barker et al., 2000). In accor- b STwt dance with this proposed sequence of events, ST can be shown to strongly induce the expression of --+-- MEK Inhibitor a transgene under a TCF-responsive promoter ---+- AKT Inhibitor (Figure 3c), which proves the involvement of b-catenin ----+ MYC Inhibitor in ST-mediated production of MYC. Altogether the O 2 Dexamethasone

-++++ H outcome is a strong activation of the expression and a stabilization of MYC. MYC An independent study also came to the conclusion that MYC is a major target of ST protein. In this work, Cyclin D1 the role of phosphorylation and dephosphorylation of MYC was studied and PP2A was found to play an TERT important role in the switch from stabilization to degradation of MYC (Yeh et al., 2004, see also the PyST above discussion). As expected, inhibition of PP2A by ST resulted in continuously high and active MYC. Even HPRT more interesting is that a mutated version of MYC, which escapes regulated destruction, can replace ST in c 35 the transformation system consisting of mutated Ras, 30 telomerase and SV40 LT and ST proteins (Yeh et al., 2004). This provides strong support for the conclusion 25 that the induction of MYC is a central function of ST in TCFwt 20 transformation and S-phase induction. By addressing TCFmut 15 PP2A, ST interferes with the function of a central regulator of signal transduction and cell cycle control. It 10 is interesting to note that in contrast to ST, LT does not TCF Fold Induction 5 upregulate MYC in arrested cells (Klucky et al., 2004). 0 Thus, LT and ST deal with two different transcription STwt STPP2A- STJ- systems leading to S-phase induction, which, at least in part, explains the requirement of both viral proteins Figure 3 MYC induction by ST depends on MEK and AKT in this process. activation. (a) Kinase inhibitors block MYC induction by ST. Experiments were carried out as in Figure 2 except that 50 mM AKT inhibitor (PI3 kinase inhibitor LY294002, Cell signalling #9901), or Acknowledgements 10 mM MEK inhibitor (UO126, Cell signalling #999) were added 1 h before cell harvesting. (b) Kinase inhibitors and MYC inhibitor block ST-induced transactivation of TERT and cyclin D1. Kinase We are grateful to Peter Angel (DKFZ) and to Roland Foisner inhibitors were used as in (a), MYC inhibitor (10 mM H1-S6A, F8A and Ernst Mu¨ llner from our department for kindly providing from Biomol Res. Lab. Plymouth Meeting, PA, USA) was added reagents. This work was supported by FWF and the together with Dexamethasone to serum-starved 3T3ST cells for Herzfelder Foundation. 28 h. Semiquantitative RT–PCR was performed as described (Klucky et al., 2004). HPRT served as loading control. (c)ST activates b-catenin-dependent TCF transcription factor. TCF References reporter construct (wt or mutant from Upstate, Charlottesville, VA, USA #17–285) was cotransfected with pCIneoSTwt or Barker N, Morin PJ, Clevers H. (2000). The Yin-Yang of mutants into REF 52 cells. Assays were carried out as in Figure 1b. TCF/beta-catenin signalling. Adv Cancer Res 77:1–24.

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