Multiple pathways regulated by the tumor suppressor PP2A in transformation

Review

Multiple pathways regulated by the tumor suppressor PP2A in transformation Jukka Westermarck1,2 and William C. Hahn3,4 1

Institute of Medical Technology, University of Tampere and Tampere University Hospital, 33520 Tampere, Finland ˚ bo Akademi University, 20520 Turku, Finland Centre for Biotechnology, University of Turku and A 3 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA 4 Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02139, USA 2

Reversible protein phosphorylation plays a central role in regulating intracellular signaling. Dysregulation of the mechanisms that regulate phosphorylation plays a direct role in cancer initiation and maintenance. Although abundant evidence supports the role of kinase oncogenes in cancer development, recent work has illuminated the role of specific protein phosphatases in malignant transformation. Protein phosphatase 2A (PP2A) is the major serine-threonine phosphatase in mammalian cells. Inactivation of PP2A by viral oncoproteins, mutation of specific subunits or overexpression of endogenous inhibitors contributes to cell transformation by regulating specific phosphorylation events. Here, we review recent progress in our understanding of how PP2A regulates mitogenic signaling pathways in cancer pathogenesis and how PP2A activity is modulated in human cancers. Introduction Phosphorylation plays a critical role in the regulation of cell physiology, and dysregulation of the mechanisms that control specific phosphorylation events contributes to many disease states. Although much is known regarding alterations in kinase function in diseases such as cancer, the role of specific phosphatases in these same processes remains poorly characterized. Indeed, initial in vitro biochemical studies using purified enzymes suggested that serine-threonine phosphatases show relatively promiscuous activity on a range of phosphorylated substrates [1]; however, recent work indicates that phosphatases might exhibit a more narrow range of substrate specificity in vivo. Protein phosphatase 2A (PP2A) refers to a large family of heterotrimeric serine-threonine phosphatases that account for the majority of serine-threonine phosphatase activity in eukaryotic cells [1]. The PP2A core enzyme consists of a catalytic C subunit, also called PP2AC or PPP2C, and a structural A subunit, also known as PR65 or PPP2R1 (Figure 1). In mammals, two distinct genes encode closely related versions of the PP2A A (Aa and Ab) [2,3] and C (Ca and Cb) subunits [4,5]. A third subunit (B) binds the A-C heterodimer, and these B subunits regulate Corresponding author: Hahn, W.C. ([email protected]).

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both the substrate specificity and localization of PP2A complexes. Four families of B subunits have been identified to date: B/B55/PR55/PPP2R2 [6–9], B0 /B56/PR61/PPP2R5 [10–12], B00 /PR72/PPP2R3 [13–16] and B000 /PR93/SG2NA/ PR110/Striatin [17]. The crescent-shaped A subunit consists of 15 nonidenticalrepeated domains, which are found in the huntingtin, elongation factor 3, PP2A A subunit and the TOR kinase proteins (HEAT) (Figure 1). Mutagenesis and recent structural studies have shown that B subunits bind to the Nterminal HEAT repeats 2–8 of the A subunit, whereas the C subunit interacts with the carboxyl-terminal HEAT repeats 11–15 of the A subunit [18,19]. The A subunit primarily serves a structural role, and specific single amino acid alterations in either of the Aa or Ab subunits disrupt the binding of specific B subunits [20–23], suggesting that the A subunits regulate PP2A holoenzyme composition [24]. The C subunit shares sequence homology with other serinethreonine phosphatases, such as protein phosphatase 1 (PP1) and PP2B (calcineurin) [25]. Recent structural work suggests that post-translational modifications of the C subunit regulate holoenzyme assembly [26]. In addition to these canonical PP2A complexes, a heterodimer related to PP2A is also formed through the interactions of the C subunit with the Tap42/a4 protein instead of the structural A subunit [27]. The Tap42/a4PP2A C phosphatase serves to repress apoptosis in mammalian cells through negative regulation of c-Jun and p53 [28]. The combinatorial assembly of these various A, B and C subunits permits the formation of many distinct PP2A complexes, and various PP2A complexes have been implicated in the control of a diverse array of cellular processes, including cell proliferation, survival, adhesion and cytoskeletal dynamics. In particular, recent work has elucidated roles for PP2A in various aspects of malignant transformation. Here, we review recent progress in our understanding of how PP2A acts as a tumor suppressor (Box 1). Viral oncoproteins, PP2A and cell transformation DNA tumor viruses, such as adenovirus, polyomavirus and simian virus 40 (SV40), transform mammalian cells

1471-4914/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2008.02.001 Available online 10 March 2008

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Figure 1. The PP2A family of serine-threonine phosphatases. PP2A is a heterotrimeric complex composed of a structural A subunit (green), a catalytic C subunit (pink) and one of several B regulatory subunits (yellow, orange, red and blue). B subunits regulate the activity and localization of PP2A complexes. Several forms of each of these subunits exist in humans, and thus many different enzymatic complexes can be formed. Recent structural studies confirm that the B subunits and the SV40 ST (purple) bind the A subunit at overlapping binding sites, while the C subunit binds HEAT repeats 11–15 in the A subunit. The PP2A subunits and ST are not drawn to scale.

through the actions of dominant acting oncoproteins that deregulate key host cell pathways that control proliferation and replication. Each of these viruses produces proteins that target the same host cell proteins, suggesting that these viruses induce cell transformation by perturbing a shared set of signaling pathways. The SV40 genome is divided into an Early Region (SV40 ER) and a Late Region based on the timing when each region is expressed after SV40 infection. In cells that are transformed by SV40 infection, only the SV40 ER is expressed, and expression of the ER suffices to induce cell transformation. The SV40 ER encodes three proteins through alternative splicing: SV40 large T (LT), small t (ST) and 17 kT antigens (Figure 2) [29]. These three proteins share their N-terminal ends, but both the LT and 17 kT antigens harbor a LXCXE motif that mediates binding to the retinoblastoma (pRB) family members pRB, p107 and p130 (reviewed in [29,30]). In addition, LT binds to the tumor suppressor protein p53, and several lines of evidence indicate that the binding of LT to pRB and p53 is Box 1. What is a tumor suppressor? Tumor suppressor genes encode proteins or functional RNA molecules whose normal function is to repress cancer initiation or maintenance. Although first identified as gene products that regulate cell-cycle progression or promote apoptosis, tumor suppressors also regulate responses to DNA damage or inhibit tumor progression. Germline mutations that result in the production of inactive tumor suppressor gene products can result in an increased susceptibility to cancer development. Alternatively, somatic mutations of tumor suppressor genes cooperate with other genetic alterations, such as the activation of oncogenes, to contribute to tumor formation. Although inactivation of tumor suppressor genes usually requires mutation or loss of both alleles, recent work suggests that haploinsufficiency can also result impair tumor suppressor function.

the primary mechanism by which LT contributes to cell transformation [15,31,32]. By contrast, the carboxyterminal end of ST is unique from LT and 17 kT (Figure 2). This ST domain mediates binding to PP2A by displacing B subunits from the PP2A A-C heterodimer [33,34] (Figure 1). The polyomavirus middle and small t antigens [35,36] and the adenoviral E4orf4 proteins [37] also bind PP2A. The co-expression of LT with an oncogenic allele of H-Ras transforms several types of rodent cells [38,39]. By contrast, human cells are more resistant to transformation [40,41]. Although expression of LT permits human cells to bypass senescence, immortalization requires the additional expression of the human telomerase catalytic subunit (hTERT) (reviewed in [42]). In addition to LT and hTERT, the coexpression of ST and an oncogenic allele of Ras are necessary to generate transformed human fibroblasts capable of tumor formation [43–45]. Examination of ST mutants has provided important clues to the functions of ST during cell transformation. ST mutants that cannot bind PP2A also fail to transform human cells expressing LT, hTERT and H-Ras, demonstrating that this interaction is required for ST-mediated transformation [43,45–47]. By contrast, an ST mutant that contains only the PP2A-binding domain (amino acids 88– 174) retains the ability to induce transformation [43]. These observations suggest that the binding of ST to PP2A is necessary for its transforming activity. PP2A subunits are tumor suppressor genes ST, PP2A Aa and B56g Several structural and biochemical studies demonstrate that ST contacts the Aa-C heterodimer at a site overlapping the HEAT repeats where the PP2A B subunits bind, thereby displacing B subunits (Figure 1). To determine 153

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Figure 2. The SV40 Early Region. The SV40 Early Region encodes three proteins through alternative splicing: the Large T antigen (LT), the 17 kT antigen and the small t antigen (ST). These three proteins share an 82 residue amino acid N-terminal end (gray). LT and 17 kT each harbor the LXCXE motif, which mediates pRB binding. ST has a unique carboxyterminal end (light green) that mediates binding to PP2A. LT also contains a bipartite p53-binding domain. The SV40 genomic DNA is depicted above the blue line. Proteins are shown below the blue line. Proteins that bind to the SV40 Early Region are shown above the genomic DNA, above their respective binding domains.

whether ST induces cell transformation by displacing specific B subunits, Chen et al. showed that suppression of the B56g PP2A subunit, but not the B55a subunit, sufficed to induce cell transformation in human cells expressing LT, hTERT and H-Ras [48]. Because ST displaces a fraction of each of the B subunits bound to the Aa-C heterodimer, these observations suggest that displacement of B subunits is not specific for B56g-containing PP2A complexes. Instead, PP2A complexes that contain B56g regulate the activity of substrates that suppress transformation. These observations demonstrated that ST induces cell transformation by disrupting PP2A complexes containing B56g. More generally, these findings confirm that PP2A B subunits regulate the activity and localization of the PP2A A-C dimer and that displacement of B subunits alters the activity of the A-C dimer. Despite these findings, it remains unclear whether loss of B56g occurs in human cancers. While one group reported higher expression levels of the PP2A B56g mRNA in human melanoma cell lines compared to normal melanocytes [49], a second group observed decreased expression in primary human melanoma samples compared to melanocytic nevi [50]. In addition, many lung cancer cell lines appear to lack B56g protein expression, and overexpression of PP2A B56g in such lung cancer cell lines partially reverses the tumorigenic phenotype of these cells [48]. Thus, while some data suggests that loss of B56g contributes to cancer development, further work is necessary to determine whether B56g is a tumor suppressor gene. By contrast, somatic mutations of the PP2A Aa and Ab subunits have been reported in human lung, breast and colon cancers as well as melanomas, albeit at low frequency [22,51–54]. Indeed, complete loss of Aa in fly, murine and human cells induces cell death by apoptosis [20,55,56], showing that Aa is an essential gene product. Biochemical studies confirm that PP2A Aa mutations disrupt the ability of such mutants to form PP2A complexes [57], and overexpression of these mutants fails to induce cell transformation [20,22]. However, reduction of wild-type PP2A Aa expression by 50% substituted for ST in the transformation of human cells expressing LT, hTERT and HRas. Overexpression of wild-type PP2A Aa inhibited cell transformation induced by its partial suppression, whereas cancer-associated PP2A Aa mutants failed to reverse the transforming phenotype, confirming that the level of functional PP2A Aa mediates cell transformation 154

[20]. These observations suggest that cancer-associated PP2A Aa mutations contribute to human cell transformation by creating a state of haploinsufficiency. Cancer-associated PP2A Aa mutants are defective in binding to all B56 family members [20,22]. Thus, a mutation in one Aa allele effectively decreases by half the amount of Aa available for PP2A B56 subunit binding. In human cells, suppression of wild-type Aa expression by 50% leads to complete loss of Aa–C-B56g heterotrimeric complexes [20]. Thus, suppression of B56g or PP2A Aa haploinsufficiency both result in the loss of PP2A complexes that contain B56g. Identifying the specific substrates affected by these changes is an active area of investigation. These observations suggest that PP2A Aa–C-B56g complexes regulate the phosphorylation state of specific substrates that suppress cell transformation. At present, the specific substrates of PP2A Aa–C-B56g complexes are unknown. ST expression [58,59], depletion of B56g-con-

Figure 3. Tumor suppressor activities of the PP2A A subunits. The two PP2A A subunits (Aa and Ab) are structurally similar. (a) However, PP2A Aa complexes containing B56g regulate the phosphorylation of Akt by regulating a yet unknown protein (polygon containing ‘?’). Cancer-associated Aa mutations (denoted by *) lead to haploinsufficiency, loss of Aa complexes containing B56g and eventually increased phosphorylation of Akt. Expression of ST also inhibits Aa complexes containing B56g (not shown). (b) By contrast, PP2A Ab binds and directly dephosphorylates the small GTPase RalA. Cancer-associated PP2A Ab mutations (denoted by *) usually involve both alleles and lead to complete loss of function of PP2A and increased RalA phosphorylation. Dotted lines represent dephosphorylation events. The B subunit depicted in (b) has not yet been identified.

Review taining PP2A complexes [20] and Aa haploinsufficiency [20] all lead to activation of the Akt pathway, implicating activation of Akt as an essential step in human cell transformation induced by loss of Aa–C-B56g complexes (Figure 3a). Indeed, either activated phosphatidylinositol 3-kinase (PI3K) or a combination of the activated PI3K effectors Akt and Rac1 can substitute for ST to induce human cell transformation [59]. However, B56g could also contribute to tumor development through regulation of two other well-known tumor suppressors, APC and p53, which have been shown to bind the B56g regulatory subunit. For example, PP2A B56g inhibits formation of APC–axin complexes, leading to destabilization of the b-catenin protein [60]. As a result, overexpression of B56g reduces the abundance of b-catenin and inhibits transcription of b-catenin target genes. In addition to B56g, the PR72 and PR130 B subunits have recently been shown to regulate WNT signaling through direct interactions with Naked cuticle [61,62]. Because the WNT/b-catenin pathway is implicated in the initiation of many epithelial cancers [63], these interactions might contribute to cell transformation; however, the relative contributions of these various PP2A complexes in regulating WNT-b-catenin in specific human cancers remain unknown. PP2A B56g complexes might regulate p53 by at least two distinct mechanisms. PP2A B56g-containing complexes dephosphorylate p53 at Thr55, which prevents its proteasome-mediated degradation and inhibits cell proliferation in part by inducing the expression of p21 [64]. Moreover, a p53 target gene, cyclin G, recruits PP2A B56g to a complex containing p53 and Mdm2, which leads to the modulation of Mdm2 phosphorylation. Inhibition of Mdm2 by PP2A B56g–cyclin G complexes results in stabilization of p53 [65]. Although these observations suggest that inactivation of PP2A complexes perturbs p53 activation, this effect fails to transform human cells in the absence of direct inhibition of p53 by LT or other means [15,44,66]. Further work is necessary to understand how these and perhaps other PP2A Aa–C-B56g substrates induce malignant transformation. PP2A Ab, RalA and cell transformation In addition to mutations in PP2A Aa, somatic alterations of the PP2A structural subunit Ab (PPP2R1B) have been found in colon, lung and breast cancers [51–54]. Similar to what was observed in tumors harboring PP2A Aa mutations, cancer-associated Ab mutations result in alleles that are unable to form PP2A complexes [21,23]. However, point mutations in one Ab allele are commonly accompanied by loss of the second Ab allele [51–54]. These studies indicate that Ab is genetically inactivated in a subset of human cancers. Functionally, suppression of Ab expression cooperated with LT, hTERT and H-Ras to induce transformation of human cells, and the introduction of wild-type Ab into lung carcinoma cells expressing biallelic Ab mutations partially reverses this tumorigenic phenotype [23]. Together, these observations implicate PP2A Ab as a tumor suppressor gene. Moreover, although PP2A Aa and Ab, are 86% identical at the amino acid level [13], these two structural isoforms form distinct PP2A

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complexes and appear to regulate different signaling pathways (Figure 3). Recent work has identified the small GTPase RalA as a protein that interacts with PP2A Ab but not Aa [23]. RalA has been implicated in the regulation of a wide range of cell processes, including exocyst function, transcription and cell motility [67]. PP2A Ab-containing complexes dephosphorylate RalA at serines 183 and 194, and this dephosphorylation event downregulates RalA activity (Figure 3b). RalA expression and activity is crucial for transformation mediated by loss of Ab function. Indeed, wild-type RalA but not RalA phosphorylation site mutants (S183A and S194A) mediate cell transformation induced by suppression of PP2A Ab expression, and lung cancer cell lines that harbor inactivating mutations of PP2A Ab exhibit constitutive phosphorylation of RalA. These observations suggest that accumulation of phospho-RalA in PP2A Ab-deficient cells promotes cell transformation. These findings are consistent with prior work that has implicated RalA in the transformed phenotype of many cancer cell lines [68–70]. In addition, several groups have demonstrated that Ral-guanine nucleotide dissociation stimulator (Ral-GDS) signaling contributes directly to the transformation induced by Ras signaling [44,71,72]. The RalA GTPase has been implicated in regulation of several signaling pathways relevant to transformation [67,73,74], including the activation of phospholipase D1 and Src kinase [75,76], vesicle transport [77], increased cell motility [78–81] and anchorage-independent growth [68,70,82]. However, the downstream events of RalAmediated transformation remain undefined. Moreover, it is clear that the signaling pathways perturbed by PP2A and Ras in transformation are intertwined, and further work is necessary to define how these two pathways contribute to cancer development. PP2A inhibitor proteins in cellular transformation Recent reports have indicated that, in addition to genetic alterations of PP2A subunits, overexpression of endogenous cellular proteins might also inhibit PP2A tumor suppressor activity. Here, we summarize recent work that implicates newly identified PP2A inhibitor proteins in promoting malignant growth (Figures 4 and 5). I2PP2A/SET In addition to viral oncoproteins and chemical inhibitors that have been used experimentally to inhibit PP2A [83,84], two endogenous protein inhibitors, tentatively designated I1PP2A and I2PP2A, were identified as noncompetitive inhibitors of PP2A activity toward several phosphorylated substrates in vitro [85]. Although this original report indicated that I1PP2A and I2PP2A do not inhibit other serine-threonine phosphatases, subsequent work has shown that I1PP2A and I2PP2A also associate with and modify the substrate specificity of PP1 [86]. In the presence of Mn+2, I1PP2A and I2PP2A selectively stimulate PP1 activity toward phosphorylated myelin basic protein and histone H1 [86]. These findings suggest that I1PP2A and I2PP2A reciprocally regulate the two major mammalian serine-threonine phosphatases, PP1 and PP2A. 155

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Figure 4. Signaling pathways targeted by PP2A inhibitor protein I2PP2A/SET. (a) PP2A inhibition by I2PP2A/SET stimulates BCR-ABL-mediated signaling. (i) BCR-ABL protein stability is negatively regulated by SHP-1-mediated tyrosine dephosphorylation. (ii) In BCR-ABL positive cells, BCR-ABL kinase activity stimulates I2PP2A/SET expression. (iii) I2PP2A/SET inhibits PP2A activity, and (iv) thereby attenuates SHP-1 activity and BCR-ABL tyrosine dephosphorylation. (v) The consequence of I2PP2A/SET expression is stabilization of BCR-ABL and increased activity of indicated downstream signaling pathways. (b) PP2A inhibition by I2PP2A/SET also stimulates activity of the MEK-ERK-MAPK pathway and c-Jun phosphorylation. Question marks denote that the direct target(s) of the I2PP2A/SET inhibited PP2A activity in MEK-ERK and JNK-c-Jun pathways have not been identified.

Although the physiological function of I1PP2A and I2PP2A remains incompletely understood, abnormal expression of I2PP2A occurs in some human cancers. Based on the analysis, using cDNA microarrays, of differential gene expression between normal and cancer tissues, the I2PP2A/SET mRNA is overexpressed in several types of malignant tumors (http://www.oncomine.org). In particular, high levels of I2PP2A/SET mRNA expression have been observed in malignant brain tumors, tumors of the head and neck region, testicular cancers and in different types of hematological malignancies [87–90]. Consistent with a proposed role for I2PP2A/SET in regulating cell proliferation, I2PP2A/SET mRNA expression was markedly reduced in cells rendered quiescent by serum starvation, contact inhibition or differentiation [91]. The mitogen-activated protein kinase (MAPK) signaling pathways have been established as important targets for PP2A tumor suppressor activity. Inhibition of PP2A by ST or by chemical inhibitors such as okadaic acid has been shown to stimulate activator protein-1 (AP-1) transcription-factor-complex-mediated gene expression through enhanced MAPK pathway activity [84,92,93]. In consonance with these observations, overexpression of I2PP2A/SET was shown to induce phosphorylation of cJun at serine 63 and threonine 73, as well as to increase 156

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Figure 5. PP2A inhibitor CIP2A stabilizes Myc protein. (a) Myc is phosphorylated on threonine 58 (T58) and serine 62 (S62) by GSK-3 and ERK kinases, respectively. (b) PP2A binds to the N-terminus of phosphorylated Myc via B56a. (c) CIP2A directly interacts with Myc and inhibits PP2A-mediated S62 dephosphorylation. Inhibition of S62 dephosphorylation results in Myc protein stabilization. (d) In the absence of CIP2A, PP2A dephosphorylates S62, which targets Myc phosphorylated at T58 for ubiquitination and for proteolytic degradation.

AP-1 activity [94] (Figure 4b). However, whether I2PP2A/ SET directly inhibits PP2A-mediated c-Jun dephosphorylation or whether the observed increase in c-Jun phosphorylation was due to stimulation of JNK (c-Jun N-terminal kinase) MAPK activity, was not addressed in this study [94]. More recently, I2PP2A/SET overexpression was also demonstrated to activate MEK (mitogen-activated/ extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase)-MAPK signaling and thereby inhibit death-receptor-elicited apoptosis in HeLa cells [95]. I2PP2A is a truncated form of the myeloid-leukemiaassociated protein SET, which is expressed in a subset of acute non-lymphocytic myeloid leukemias as the fusion protein SET-Nup214 [96]. These results implicate the inhibition of PP2A activity by I2PP2A/SET in acute myeloid leukemogenesis [96]. Recent work has identified I2PP2A/SET as overexpressed in chronic myelogenous leukemia (CML) and shown that I2PP2A/SET expression correlated with expression and oncogenic activity of the BCR-ABL kinase [97]. Moreover, forced expression of BCRABL in lymphoid precursors stimulated I2PP2A/SET expression, and inhibition of BCR-ABL activity by the small-molecule BCR-ABL inhibitor imatinib markedly inhibited I2PP2A/SET expression. Interestingly, BCRABL-induced overexpression of I2PP2A/SET results in increased BCR-ABL stability, as depletion of I2PP2A/ SET by RNA interference resulted in proteolytic degradation of BCR-ABL and subsequent inhibition of BCRABL-mediated activation of several signaling pathways involved in CML pathogenesis (Figure 4a). Specifically, activation of PP2A by depletion of I2PP2A/ SET, by overexpression of the PP2A C subunit or by

Review inhibition of BCR-ABL with imatinib resulted in inhibition of MYC expression and dephosphorylation of STAT5 (signal transducers and activators of transcription 5), ERK, Akt, BAD (Bcl-2-associated death promoter), Jak2 and pRB. Surprisingly, proteolytic degradation of BCR-ABL in response to I2PP2A/SET depletion was dependent on the activity of the tyrosine phosphatase SHP-1 (SH2domain-containing protein tyrosine phosphatase-1). Indeed, I2PP2A/SET depletion resulted in BCR-ABL tyrosine dephosphorylation, and PP2A-induced BCR-ABL degradation was blocked by treatment with an SHP-1 inhibitor. Although the mechanism by which PP2A activation induces SHP-1-mediated BCR-ABL tyrosine dephosphorylation remains unknown, it remains possible that PP2A directly activates SHP-1 tyrosine phosphatase activity [97]. Nevertheless, the consequence of I2PP2A/ SET-mediated PP2A inhibition in BCR-ABL-expressing cells is increased colony-forming ability and tumor growth that is dependent upon I2PP2A/SET expression [97]. These observations establish a role for I2PP2A/SET in promoting BCR-ABL stability and CML progression through inhibition of PP2A tumor suppressor activity. Cancerous inhibitor of PP2A (CIP2A) Myc is overexpressed in a wide variety of human cancers, ranging from 50% of hepatocellular cancers to up to 90% of gynecological cancers [98]. Although gene amplifications or translocations often explain this increase in Myc expression, in some studies there is a clear discrepancy between cMyc mRNA and Myc protein expression levels [99,100], suggesting that Myc levels are also regulated at the posttranscriptional level. In vitro, Myc exhibits a very short protein half-life in non-transformed cells but has been shown to have an extended half-life in some cancer cell lines [101–103]. Myc stability is regulated in part through phosphorylation of two residues, threonine 58 and serine 62. Phosphorylation of threonine 58 by GSK-3 is required for MYC degradation [104], whereas Ras-mediated activation of the ERK/MAPK pathway induces MYC phosphorylation at serine 62 (S62) and correlates with increased stability of Myc [104,105]. PP2A complexes containing B56a regulate the phosphorylation status of S62 [106–108]. Consistent with these observations, expression of a Myc mutant (MycT58A), which is resistant to proteolytic degradation, substituted for ST in transformation in cells expressing LT, hTERT and H-Ras [107]. These observations link inhibition of PP2A tumor suppressor activity, Myc stabilization and human cell transformation. More recently, a novel PP2A-interacting protein displaying Myc stabilization activity in human transformed cells was recently identified through an affinity purification approach [109]. This protein, previously called p90 [110] and now designated cancerous inhibitor of PP2A (CIP2A), was shown to immunoprecipitate with endogenous PP2A complexes and endogenous Myc, and depletion of CIP2A resulted in a significant increase in PP2A activity measured from Myc immune complexes. In addition, CIP2A depletion induced Myc S62 dephosphorylation and subsequent Myc protein destabilization (Figure 5). Moreover, overexpression of CIP2A resulted in increased steady-state expression of Myc. Because sup-

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pression of CIP2A failed to affect PP2A activity toward MDM2, CIP2A appears to selectively target Myc-associated PP2A activity. Based on a combination of immunoprecipitation analysis and in vitro PP2A activity measurements, CIP2A inhibits the catalytic activity of the PP2A C subunit in the Myc–PP2A complex [109]. The ability of CIP2A to inhibit PP2A C appears to be dependent on the interaction of CIP2A with Myc. Indeed, CIP2A and Myc interact, and CIP2A binding to MYC was abrogated by mutation of PP2A target S62. Together, these observations identify CIP2A as an endogenous PP2A inhibitor that inhibits MYC S62 dephosphorylation [109]. Consistent with these biochemical experiments, overexpression of CIP2A in human cells expressing LT, hTERT and H-Ras [109] led to cell transformation, demonstrating that CIP2A can replace either ST or MycT58A. Moreover, CIP2A depletion was shown to significantly inhibit tumor growth of both HeLa cells and low passage squamous cell carcinoma cells isolated from human head and neck tumors (HNSCCs). In HNSCC tumors, CIP2A is expressed in epithelial cancer cells but is absent in tumor stroma, as well as normal skin. In addition to HNSCC, overexpression of CIP2A has been demonstrated in gastric and colon cancer samples [109,110]. By contrast, in most of the normal tissues studied, very low expression levels of CIP2A were detected [109]. These observations implicate the dephosphorylation of Myc S62 as one mechanism by which PP2A functions as a tumor suppressor. Further studies are necessary to understand how PP2A complexes containing B56a are recruited to MYC and how CIP2A is regulated in both normal and malignant cells. Other endogenous PP2A inhibitor proteins In addition to I2PP2A/SET and CIP2A, other endogenous proteins inhibit PP2A activity in cancer cells. For example, the PP2A C methylesterase protein phosphatase methylesterase-1 (PME-1) preferentially associates with the inactive form of the PP2A C subunit and inhibits PP2A by demethylation of the PP2A C C-terminal leucine 307 [111,112]. Another endogenous PP2A inhibitor with a potential link to human carcinogenesis is type 2A-interacting protein (TIP), which was shown to be expressed in human cancer cells and to interact with the PP2A holoenzyme in HEK-293 cells [113]. Importantly, in an in vitro assay, TIP directly inhibited PP2A activity. Functionally, TIP depletion resulted in a significant decrease in the phosphorylation of newly identified 32 kD target protein substrate of ataxia-telangiectasia mutated (ATM)/ATMand Rad3-related (ATR) kinases [113]. Conclusions These observations firmly establish PP2A as an important regulator of signaling pathways involved in oncogenesis. Multiple members of the PP2A phosphatase family act as tumor suppressors, and aberrant expression of endogenous PP2A inhibitors appears to contribute to transformation in some human cancers. As a large family of abundantly expressed serine-threonine phosphatases, PP2A regulates myriad signaling pathways, and recent work suggests that specific PP2A complexes regulate 157

Review the activity of specific substrates, many of which are involved in cell transformation. For this reason, it is no longer appropriate to refer to PP2A in generic terms. Instead, identifying the specific PP2A complexes, particularly the composition of B subunits, is necessary when studying the role of PP2A. Although phosphatases remain difficult targets for inhibition by small molecules, understanding the phosphorylation events regulated by specific PP2A complexes might provide insight into new targets for new cancer therapeutics. In particular, the identification of PP2A inhibitor proteins expressed in malignant cells might provide novel opportunities for targeted cancer therapies. For example, the identification of kinases that phosphorylate RalA should lead to the development small molecule inhibitors that achieve the same tumor suppressor activity of PP2A Ab-containing PP2A complexes. Thus, the identification of phosphatase substrates might identify new targets to repress tumor maintenance. Alternatively, it might be possible to identify small molecules that induce PP2A activity. For example, inhibition of I2PP2A/SET in BCR-ABL-positive CML [97] or CIP2A in HNSCC [109] might reactivate the tumor suppressive effects of PP2A in these situations [97,109]. Indeed, the myriocin analog FTY720, a synthetic nontoxic drug with high oral bioavailability, activates PP2A in cell lines derived from CML and Philadelphia chromosome positive acute lymphoblastic leukemia (ALL) patients [114]. FTY720-elicited PP2A activation resulted in inactivation of BCR-ABL-mediated survival signaling in CML and ALL cells and dramatically suppressed in vivo leukemogenesis in mice without any obvious toxicity [114]. These findings suggest that other compounds that reactivate PP2A in malignant cells will be found. It is clear that future studies are necessary to identify alterations in PP2A and/or its regulators, as these alterations might serve as or reveal biomarkers for patients who might benefit from targeted therapies. Acknowledgements We apologize to our colleagues whose work could not be cited due to space limitations. This work was supported in part by grants from the Academy of Finland (projects 878179, 8212695) (J.W.), Pirkanmaa Hospital District (competitive research funding) (J.W.), Sigrid Juse´lius Foundation (J.W.), Emil Aaltonen Foundation (J.W.), Cancer Research Foundation of Finland (J.W.) and the U.S. National Cancer Institute (P01 CA50661) (W.C.H.).

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