p27Kip1 in Proteasome Inhibitor-Induced Apoptosis

[Cell Cycle 2:5, 438-441; September/October 2003]; ©2003 Landes Bioscience

Perspective

The Role of p27Kip1 in Proteasome Inhibitor Induced Apoptosis

Previously published online as a Cell Cycle E-publication at: http://www.landesbioscience.com/journals/cc/tocnew25.php?volume=2&issue=5

KEY WORDS

The cyclin dependent kinase (CDK) inhibitor p27Kip1 is an important regulator of cell cycle progression controlling the transition from G1 to S-phase. A bipartite nuclear localization signal at the C-terminus enables p27Kip1 to translocate into the nucleus where it exerts its inhibitory activity. p27Kip1 can inhibit most CDK/Cyclin complexes but the main target of p27Kip1 appears to be CDK2 in the nucleus, where p27Kip1 integrates into a CDK2/cyclin containing complex and within this complex blocks CDK2 activity. As a consequence the retinoblastoma (Rb) protein cannot be phosphorylated to an extent that would allow dissociation of the transcription factor E2F from the Rb protein, a prerequisite for the synthesis of genes necessary for S-phase entry, and cells become arrestsed at G1. The G1 arrest observed in various tumor cell lines upon p27Kip1 overexpression has therefore been related to the inhibition of CDK2 activity.1-6 Functional p27Kip1 can also become stabilized by Transforming Growth Factor beta (TGF-β) or through contact inhibition by neighboring cells.7,8 p27Kip1 thus helps to restrict cell proliferation and contributes in a decisive way to the homeostasis within a given cell population. In contrast, when a cell is instructed to divide, the availability of functional p27Kip1 in the nucleus obviously has to be reduced to allow traversal of the cell into S-phase. p27Kip1 therefore has to be regulated in a cell cycle dependent fashion and the levels of p27Kip1 are oscillating during the cycle reaching a maximum at the G1/S transition. Two routes of modulating the nuclear levels of p27Kip1 have been identified in normal cells:

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apoptosis, proteasome, cdk inhibitor, p27Kip1, quiescence.

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Received 07/10/03; Accepted 07/12/03

Proteasome inhibitors are potent inducers of cell death. The cytotoxic effect of proteasome inhibitors in general appears to be selective for proliferating cells, while quiescent cells seem to be protected. Conflicting results have been reported on the role of the CKI p27Kip1 either in promoting or inhibiting apoptosis mediated by proteasome inhibitors and other drugs. Here I discuss the role of p27Kip1 in apoptosis and chemotherapy of cancer.

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Correspondence to: Hannes C.A. Drexler; Max Planck Institute for Physiological and Clinical Research; Dept. Molecular Cell Biology; D-61231 Bad Nauheim, Germany; Tel.: +49.6032.705296; Fax: +49.6032.72259; Email: [email protected]

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1. degradation mediated by the ubiquitin proteasome dependent pathway (UPP)9 and 2. blocking the import step of p27Kip1 into the nucleus which involves the Akt/PKB dependent phorphorylation of Serine 157 within the NLS of p27Kip1, leaving an inactivated p27Kip1 in the cytoplasm.

The p27Kip1 protein phosphorylated on Serine 157 apparently is not a target of the ubiquitination machinery.10-12 In order to be funneled into the UPP of degradation, p27Kip1 has to be phosphorylated first on Threonine 187 in a cyclin E dependent fashion, a prerequisite for being subsequently ubiquitinated with the help of SCFskp2 E3-ligase and then targeted to the proteasome, where it is finally degraded.13,14 In various tumor cells displaying a more aggressive phenotype levels of p27Kip1 are often dramatically reduced.15-17 The decrease in p27Kip1 levels has been related to an increased proteolytic activity of the proteasome itself.18 For these reasons overall levels of p27Kip1 have been inversely correlated with survival of patients suffering from various malignancies and have been regarded as a prognostic factor for patient survival.19-21 Provided that inactivation of p27Kip1 can also occur in the cytoplasm without proteolytic degradation, cytoplasmic localization of inactive p27Kip1 therefore has to be included as an additional negative factor determining patient survival. Investigations of the significance of UPP mediated protein degradation for cellular homeostasis have gained considerably during the last decade from the development of potent proteasome inhibitors (for reviews, see refs. 22,23,24). The most attractive feature of proteasome inhibitors regarding their potential use as anti-neoplastic drugs is certainly their capability to achieve rather selective growth inhibition and the induction of apoptosis in a wide variety of tumor cells, even in cells that are highly resistant to other commonly Cell Cycle

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THE ROLE OF p27Kip1 IN PROTEASOME INHIBITOR INDUCED APOPTOSIS

[p27Kip1]

Pros: Cons:

[p27Kip1] Pros: Cons:

Inhibition of cell cycle progression " retardation of tumor cell growth Reduced sensitivity to cytotoxic drugs " chemoresistance factor Increased sensitivity to cytotoxic agents " chemotherapy more successful Enhanced tumor cell proliferation " tumor growth accelerated

Figure 1. p27Kip1 revisited: As high levels of p27Kip1 leading to cell cycle arrest can reduce the susceptibility of tumor cells to the cytotoxicity of proteasome ihibitors and other chemotherapeutic agents, the concept of nuclear p27Kip1 levels as prognostic factor for patient survival should be supplemented with the notion that p27Kip1 at the same time can have a decisive impact on the outcome of chemotherapy treatment.

used chemotherapeutic drugs25 and that lack a functional p53 tumor suppressor gene. Surprisingly tumor cells seem to be much more sensitive to this kind of blockage compared to normal cells, although protein degradation via the UPP is abrogated in normal cells too. Conflicting observations have been reported regarding the role of p27Kip1 in proteaseome inhibitor mediated apoptosis. Proteasome inhibitor induced apoptosis generally is accompanied by the accumulation of p27Kip1.26 As adenovirus-mediated overexpression of p27Kip1 in various tumor cell lines also increased the number of cells undergoing apoptosis,27,28 it has therefore been deduced that cell death by proteasome inhibitors could be due to the uncoordinated upregulation of p27Kip1. In contrast, tumor cells grown as spheroids also upregulate p27Kip1, but under these conditions show an increased resistance towards apoptosis induction.29,30 Similar observations have been made in different cancer cell lines engineered to overexpress p27Kip1,31-33 and more recently with K562 CML cells that were engineered to express p27Kip1 in an inducible fashion.34 In the latter study it was demonstrated that protection against proteasome inhibitor action was specifically linked to the expression levels of p27Kip1 and to arrest at the G1/S-transition and that an aphidicholin-mediated arrest in the S-Phase of the cell cycle could not substitute for the protection provided by p27Kip1. Likewise, primary endothelial cells which become contact inhibited upon reaching confluency display a remarkable degree of resistance against apoptosis induction by proteasome inhibitors in the presence of increased steady state levels of p27Kip1, when compared with their proliferating counterparts and a similar differential effect of proteasome inhibitors has been observed with proliferating vs. PMA-differentiated quiescent HL60 cells.35,36 Similarily, activated T lymphoyctes become arrested at G1 when deprived of IL-2 in a p27Kip1 dependent manner and survive, while lymphocytes deficient in p27Kip1 show an impaired ability to arrest and at the same time an enhanced susceptibility to initiate apoptosis.37 This study is also in line with the observation that p27Kip1 deficient fibroblasts displayed an increased tendency to undergo apoptosis upon growth factor removal, and that restoration of p27Kip1 expression was able to reduce the number of apoptotic cells to normal levels.38 How can p27Kip1 protect against apoptosis at least some cell types? As proteasome inhibitors not only block p27Kip1 degradation but also of a variety of other proteins regulating cell cycle progression, it is likely that the survival of a p27Kip1 expressing and arrested cell is far less sensitive to the disturbance of the delicate balance of www.landesbioscience.com

signals which is deciding over whether the cell should enter the next phase of the cycle or not. Synthesis of cyclin E, cyclin A, B-type cyclins and enzymes required for DNA replication can only occur when the restriction point has been traversed. Once a cell has passed this point, cell cycle progression will continue even if growth factors are withdrawn. Orderly progression through S and G2/M phase then involves tight regulation of especially CDK2 and CDK1 activity by the UPP-mediated degradation of their regulatory cyclin partners. It is easy to imagine that such a committed cell which is forced to proceed through mitosis is much more prone to the effects of aberrant stabilization of cyclins and consequently to uncoordinated CDK activation than a cell still arrested at the restriction point. In this scenario p27Kip1 might protect the cells simply by enforcing cellular hibernation, thus reducing the high competence of cycling cells to undergo apoptosis, particularly in response to proteasome inhibitors. Aberrant activation of CDKs, in particular of CDK1, CDK2 and has been shown to markedly sensitze cells to staurosprine and TNF-α induced apoptosis39,40 and to be increased in resting thymocytes undergoing apoptosis, in which p27Kip1 at the same time is being degraded.41 Cyclin E is upregulated following irradiation in multiple myeloma and lymphoma cells and and when overexpressed sensitized the lymphoma cells to apoptosis induction. In this case, apoptosis could be completely abolished by expression of a dominant negative CDK2.42 It therefore appears that deregulated CDK2 activity may represent the primary cause of apoptosis induction and that a p27Kip1 mediated sequestration and inactivation of CDK2/cyclin E and or CDK2/cyclin A complexes may be sufficient to ensure cell survival.38 Interestingly, primary neurons which are terminally differentiated and postmitotic are even protected by proteasome inhibitors against cell death induction when deprived of the survival factor NGF.43 Are there other possibilities how p27Kip1 expression can inhibit or delay cell death? p27Kip1 expression in U937 has been associated with a block in pro-caspase 2 processing, which in turn prevents cytochrome c release from the mitochondrium and caspase 3 activation.31 Analogous to p21Cip1/Waf1, which has been shown to interact with procaspase-3 by direct binding to the proform and thus blocking the formation of active caspase 3,44 it may be speculated that p27Kip1 could interact in a similar fashion with procaspase-2, thereby inhibiting the initiation of the apoptotic program. However, it is not known whether such a direct p27Kip1-mediated inhibition of procaspase 2 processing occurs in vivo and is of any physiological relevance. Once the apoptotic program has been initiated both p21Cip1/Waf1 and p27Kip1 themselves are cleaved in a caspase dependent fashion. While p21 fragmentation at least further enhances apoptosis in human endothelial cells,45 probably by reinforcing cdk2 activity, the p23 and p15 cleavage products of p27Kip1 processing have the opposite effect and significantly delay cell death.46 In myeloma cells the p23 fragment can be even generated in a caspase-dependent fashion without obvious apoptosis and induce a G1 growth arrest by inhibition of cyclin/CDK2 activity.47 Is the p27Kip1 associated growth arrest of the cell then the sole determinant of sensitivity against apoptosis induction by proteasome inhibitors? Clearly other parameters which may be cell type specific contribute to the adjustment of the threshold which determines the sensitivity of a particular cell type to proteasome inhibition. For instance, cell death induction in HeLa cells by proteasome inhibitors has been shown to occur in a p53 dependent fashion, whether the cells were cell cycle arrested or not.48 Another example, which at the

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first glance argues against a general role of p27Kip1 in protecting against the effect of proteasome inhibitors are B lymphoma cells (B-CLL), which are largely quiescent due to high amounts of p27Kip1 and yet have proven to be exquisitely sensitive to apoptosis induction by proteasome inhibitors.49,50 Resting B-CLL cells, however, stimulated with CpG-oligonucleotides and Il-2 are able to resume proliferation which is accompanied by a reduction of p27Kip1 levels and these cycling B-CLL cells now are again more sensitive to proteasome inhibitors compared to their non-proliferating counterparts.51 A major consequence of the anti-apoptotic properties of p27Kip1 is that it appears that high levels of p27Kip1 in tumor cells may not be always good news for cancer patients: high levels of active p27Kip1 within tumor cells may indicate that this tumor—although less aggressive and more slowly growing—may be more difficult to attack by proteasome inhibitor treatment or other chemotherapeutic drugs. Conversely, the opposite would be true for tumors displaying low levels of p27Kip1: These tumors come along with a bad prognosis for the patients a priori, as their growth rate is higher and also because they seem to be more prone to formation of metastasis, but on the other hand these tumors may be much more approachable by treatment with proteasome inhibitors and potentially with other chemotherapeutic drugs as well (Fig. 1). p27Kip1 therefore could represent a double-edged sword under conditions, where increased amounts of p27Kip1 in tumor cells translates into suppression of cdk activity and growth arrest and it will be important to determine what impact this condition will have on the efficacy of tumor therapy. Clinical trails of the proteasome inhibitor Velcade™ in multiple myeloma patients have so far provided a stunningly high success rate with complete remission of disease in some of the patients52 and have led to recent FDA approval of the drug. Yet there were also patients who were weakly responding to the treatment and it is important whether these weak responses can be attributed to increased levels of p27Kip1 expression and the presence of a subfraction of cells in a non-cycling refractory state. R eferences 1. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff. A p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev 1994; 8:9-22. 2. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 Cyclin-Cdk protein kinase activity, is related to p21. Cell 1994; 78:67-74. 3. Kwon TK, Nagel JE, Buchholz MA, Nordin AA. Characterization of the murine cyclin-dependent kinase inhibitor gene P27(Kip1). Gene 1996; 180:113-20. 4. Dirks PB, Patel K, Hubbard SL, Ackerley C, Hamel PA and Rutka JT. Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells. Oncogene 1997; 15:2037-48. 5. Blain SW, Montalvo E, Massague J. Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) with Cyclin A-Cdk2 and Cyclin D2-Cdk4. J Biol Chem 1997; 272:25863-72. 6. Yang HY, Shao RP, Hung MC, Lee MH. p27 Kip1 inhibits HER2/neu-mediated cell growth and tumorigenesis. Oncogene 2001; 20:3695-702. 7. Reynisdottir I, Polyak K, Iavarone A, Massague J. Kip-Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta. Genes Develop 1995; 9:1831-45. 8. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff. A P27-Kip1, a cyclin-Cdk inhibitors, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Develop 1994; 8:9-22. 9. Pagano M, Tam SW, Theodoras AM, Beerromero P, Delsal G, Chau V, Yew PR, Draetta GF, Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 1995; 269:682-5. 10. Viglietto G, Motti ML, Bruni P, Melillo RM, D'Alessio A, Califano D, Vinci F, Chiappetta G, Tsichlis P, Bellacosa A, Fusco A, Santoro M. Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer. Nat Med 2002; 8:1136-44. 11. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 2002; 8:1153-60.

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