[Cell Cycle 5:12, 1262-1264, 15 June 2006]; ©2006 Landes Bioscience
Epigenetic Inactivation Implies a Tumor Suppressor Function in Hematologic Malignancies for Polo-Like Kinase 2 But Not Polo-Like Kinase 3 Extra View
ABSTRACT
The Polo-Like kinases (Plk) are a family of highly conserved cell cycle kinases, of which there are four members in humans. Whilst many studies support an oncogenic role for Plk1 in neoplasia, there is little definitive evidence at present to support involvement of the other family members in human cancer. Both Plk2 and Plk3 function in pathways of DNA damage response. Plk2 is a target gene for p53 and imposes a G2 checkpoint. More recent evidence reveals a novel function for Plk2 in mediating apoptosis in highgrade B lymphomas. Epigenetic inactivation of Plk2 via aberrant CpG methylation in the transcriptional regulatory elements of the gene is a common event in B cell neoplasia, whereas epigenetic inactivation of Plk3 is exceedingly rare in lymphomas. Further, in every case lacking Plk2 expression, there is concomitant overexpression of Plk3, consistent with functional degeneracy between the two proteins. These results imply that Plk2 may function as a tumor suppressor in hematologic neoplasia and have pharmaco-epigenomic implications.
Original manuscript submitted: 04/11/006 Manuscript accepted: 04/12/06
ON
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2813
polo-like kinases, Methylation, transcriptional silencing, tumor suppressor gene
THE POLO-LIKE KINASES (Plk): A FAMILY OF MULTI-FUNCTIONAL CELL CYCLE KINASES
.D
KEY WORDS
The Polo-like kinases (Plks) are a family of multi-functional, serine-threonine kinases that participate in cell cycle regulation and cellular response to stresses such as DNA damage. For a comprehensive review of the involvement of the Plks in cell division, see the recent review in Cell Cycle by van de Weerdt and Medema.1 The founder member of the Plk family is Polo, a protein originally identified in Drosophila melanogaster. Polo mutants display defects in mitosis.2 The Plk family is highly conserved in evolution, with homologues described in organisms from yeast to mammals. There are four Plk family members in mammalian cells: Plk1, Plk2 (Snk), Plk3 (Fnk/Prk) and Plk4 (Sak). Each of the four Plk family members has an N-terminal catalytic domain and a C-terminal Polo box, which mediates localization of the kinase to key structures in mitosis.3 There are 2 Polo-box motifs in Plk1, Plk2 and Plk3, but only a single such motif in Plk4. Plk1 is the best-characterized human Plk and has multiple roles in cell cycle regulation. Both Plk2 and Plk3 were originally identified as immediate early-transcripts following serum stimulation of quiescent mouse fibroblasts.4,5 More recent evidence shows that Plk2 mRNA is also induced in lymphocytes by B cell mitogens such as Epstein Barr virus infection, CD40 ligand and PMA.6 Together, these observations suggest that Plk2 is likely to be an immediate-early transcript in multiple cell types. Consistent with this hypothesis, Plk2 is required for centriole duplication near the G1-S transition7,8 suggesting a critical role for the protein in cellular proliferation, but Plk2-/- mice are viable.9 The viability of Plk2-/cells implies complementation either by a Plk family member or another cell cycle kinase of the functions contributed to replication by Plk2.7,8 Plk3 has functions in regulation of mitosis and DNA damage-induced checkpoint activation.1,10 More recently, Plk4 has been implicated in regulation of centriole duplication.11 Abundance, sub-cellular localization and hence activity of the Plks are meticulously controlled during the cell cycle. At least one of the ubiquitin ligases which target Plk2 has been identified. This is the ring E2 ubiquitin ligase hVPS18.12
IEN
CE
ACKNOWLEDGEMENTS
RIB
*Correspondence to: Tim Crook; Cancer Genetics and Epigenetics Laboratory; The Toby Robins Breakthrough Breast Cancer Centre; Institute for Cancer Research; London SW3 6JB England; Tel.: +44.20.7352.8133; Fax +44.20.7153.5340; Email:
[email protected]
IST
†These authors contributted equally to this work.
OT D
Cancer Genetics and Epigenetics Laboratory; The Toby Robins Breakthrough Breast Cancer Centre; Institute for Cancer Research; London, England
UT E
.
Paul Smith† Nelofer Syed† Tim Crook*
©
20
06
LA
ND
ES
BIO
SC
Tim Crook is Clinical Research Fellow of Cancer Research UK.
1262
Cell Cycle
2006; Vol. 5 Issue 12
Tumor Suppressor Function of Polo Like Kinases
Plk1: A CANDIDATE ONCOGENE Plk1 has several properties consistent with a putative oncogenic function. Overexpression of Plk1 has been reported in several cancers, including carcinomas of the head and neck, breast and lung13 and, as we showed recently, in many B lymphomas.6 Overexpression of Plk1 is reported to correlate with metastasis and poor prognosis in some cancers. The mechanism(s) underlying overexpression of Plk1 have not been unequivocally established in every case. In cycling cells, Plk1 is ubiquitinated by the Anaphase Promoting Complex (APC) and there is also some evidence that Plk1 may be a substrate for the E3 ubiquitin ligase activity of the mitotic checkpoint encoded by the Chfr gene (Checkpoint with Forkhead and Ring finger).14 Expression of Chfr is frequently lost in human tumors, affording an attractive possible explanation that degradation of Plk1 is inhibited or reduced in cancer. In B lymphomas, however, Chfr is invariably unmethylated and expressed, implying that increased Chfr-dependent degradation does not underlie overexpression of Plk1. Consistent with this observation, overexpression of Plk1 in B lymphomas is associated with upregulation of Plk1 mRNA.6
Plk2 AND Plk3: POTENTIAL TUMOR SUPPRESSOR GENES
The proposed role for Plk2 in cellular replication perhaps suggests at first glance that the protein might possess oncogenic activity, like Plk1. Other evidence, however, more strongly supports a tumor suppressor function, at least in some tissues. Plk2 expression is directly upregulated by wild-type p53 after DNA damage and in these circumstances activates a G2 checkpoint.15 In this respect, Plk2 resembles 14-3-3-σ, also a transcriptional target for p53 that mediates a G2 checkpoint.16 We and others have shown that 14-3-3σ is subject to methylation-dependent transcriptional silencing in a variety of human cancers.17,18 Further support for a potential tumor suppressor function for Plk2 is the ability of the protein, when ectopically expressed, to induce apoptosis in B lymphoma cell lines lacking endogenous expression of the gene.6 This compares with the G2 arrest seen when endogenous Plk2 is upregulated following genotoxic stress in carcinoma cell lines expressing wild-type p53.15 In any case, induction by p53/p73 and activation of cell cycle arrest and/or apoptosis are clearly useful attributes for a potential tumor suppressor. Plk3 also has some biological properties that would be consistent with a tumor suppressor role. For example, like Plk2, expression of Plk3 can induce cell cycle arrest and apoptosis.19 Activation of Plk3 occurs in response to DNA damage and the protein then mediates phosphorylation of substrate proteins which themselves participate in the DNA damage response. Establishment of a tumor suppressor function for Plk2 and/or Plk3 requires evidence of somatic inactivation by genetic and/or epigenetic mechanisms in human cancer. Using the differential cloning technique of subtraction suppression polymerase chain reaction, we identified Plk2 as a gene whose mRNA is dramatically downregulated in Burkitt lymphoma cell lines relative to immortalized B lymphoblastoid cell lines and primary B lymphocytes.6 Analysis of other B cell-derived lymphomas including mantle cell lymphomas, diffuse large B cell lymphomas and B lymphoproliferative disease occurring in chronically immunosuppressed renal allograft recipients, revealed that Plk2 was also downregulated in a subset of such cancers, yet the gene was unmethylated and expressed (albeit at low levels) in normal B cells isolated from peripheral blood. Interestingly, abundant expression was detected in rapidly proliferating, immortalized B lymphoblastoid www.landesbioscience.com
cell lines (LCL), consistent with a role for Plk2 in mitosis. These observations raise the possibility that epigenetic mechanisms might underlie loss of expression of expression of Plk2. Whilst these experiments were in progress, we had independently identified Plk2 as a candidate epigenetically regulated gene in Burkitt lymphoma using 5' deazacytidine and micro-array analysis to “unmask” genes subject to methylation-dependent transcriptional silencing. These findings imply that methylation-dependent silencing, occurring during malignant transformation of B lymphocytes, underlies loss of expression of the gene. Bisulphite sequencing and methylation specific PCR analyses of the CpG rich sequences in the 5' end of the Plk2 gene did indeed reveal the presence of dense aberrant CpG methylation in B lymphoma cell lines lacking expression of Plk2. Indeed, a perfect correlation was observed between methylation of the Plk2 CpG island and silencing of gene transcription, whereas the CpG island is not detectably methylated in normal B lymphocytes and EBV-immortalized LCL. In contrast to Plk2, only a single lymphoma was identified in which Plk3 was transcriptionally down regulated. Like Plk2, the 5' end of the Plk3 gene also contains a CpG island. However, there was no evidence in our studies of methylation-dependent silencing of Plk3 in any B lymphomas or other hematologic malignancies analysed, including the single lymphoma cell line lacking Plk3 expression. We have found no mutations in either Plk2 or Plk3 in any lymphoma cell lines or primary cell lines. The absence of genetic and epigenetic change in Plk3 is in contrast to Plk2 and implies that loss of Plk2 rather than Plk3 confers a strong selective advantage during neoplastic development in B lymphocytes. Loss of Plk2 expression by transcriptional silencing in a high-grade lymphoma such as BL, which is highly proliferative, emphasizes that Plk2 null cells are fully replication competent and raises the question of how cells compensate for the lack of Plk2. It has been proposed that Plk3, which is known to be expressed in early phases of the cell cycle, may functionally compensate for the absence of Plk2 in nonexpressing cells.7 The invariable loss of Plk2 in BL cell lines allows testing of this hypothesis. Only a single B lymphoma cell line (follicular lymphoma, DOHHC1) was identified from 22 analysed in which Plk3 was not expressed. Remarkably, this was one of only two B lymphoma cell lines from 22 analysed that expressed Plk2.6 This perfect reciprocal correlation between loss of Plk2 expression and overexpression of Plk3 in this panel of lymphoma cell lines is striking. A second possible candidate for complementing Plk2 loss is Plk1. Although Plk1 was detectable in the majority of lymphoma cell lines lacking Plk2, overexpression was less uniform than Plk3 and in some Plk2 non-expressing cells Plk1 was also undetectable.6 Together, these data strongly support the model that Plk3 can functionally compensate for Plk2. Although methylation-dependent silencing of Plk2 is a frequent occurrence in B cell lymphomas, methylation was not detected in breast or head and neck cancers and the mRNA of Plk2 is frequently expressed in these cancers at levels comparable to, or greater than, matched normal tissues.6 Together, these observations suggest that Plk2 has a tumor suppressor function only in hematologic malignancies, and clearly question the candidacy of Plk2 as a tumor suppressor gene in solid tumors. It is unlikely that loss of function mutations are the cause of inactivation of Plk2 because we have failed to detect any sequence changes in analysis of a panel of breast and squamous carcinoma cell lines. Additional studies will be required to definitively exclude loss of Plk2 function by other mechanisms in solid tumors.
Cell Cycle
1263
Tumor Suppressor Function of Polo Like Kinases
LOSS OF Plk2 IN HUMAN CANCER MAY HAVE CLINICAL IMPLICATIONS The frequent loss of Plk2 expression may have important implications in mechanisms of anti-cancer drug activity and predictive pharmaco-epigenomics. RNAi-dependent downregulation of Plk2 in carcinoma cells causes them to undergo mitotic catastrophe when treated with the microtubule acting drug paclitaxel.15 B lymphoma lines that express Plk2 are significantly less sensitive to paclitaxel (taxol) and docetaxel (taxotere), than lines in which expression is silenced by methylation-dependent transcriptional silencing. If future studies of solid tumors do identify cases with inactivation of Plk2, it may be that such cancers will show increased sensitivity to taxanes, relative to those with intact Plk2. As such, analysis of Plk2 might in these circumstances have utility as a predictive marker of clinical response to taxanes. References 1. Van de Weerdt BCM, Medema RH. Polo-like kinases: A team in control of the division. Cell Cycle 2006; 5:1-12. 2. Fenton B, Glover DM. A conserved mitotic kinase active at late anaphase-telophase in syncitial Drosophila embryos. Nature 1993; 363:637-640. 3. Nigg EA. Polo-like kinases: Positive regulators of cell division from start to finish. Curr Opin Cell Biol 2004; 10:776-83. 4. Simmons DL, Neel BG, Stevens R, Evett G, Erikson L. Identification of an early growth-response gene encoding a novel putative serine/threonine kinase. Mol Cell Biol 1992; 12:4164-9. 5. Donohue PJ, Alberts GF, Guo Y, Winkles JA. Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase. J Biol Chem 270:10351-7. 6. Syed N, Smith P, Sullivan A, Spender LC, Dyer M, Karran L, O’Nions J, Allday M, Hoffmann I, Crawford D, Griffin B, Farrell PJ, Crook T. Transcriptional silencing of Polo-like kinase 2 (Snk/Plk2) is a frequent event in B-cell malignancies. Blood 2006; 107:250-6. 7. Warnke S, Kemmler S, Hames R, Tsai HL, Hoffmann-Rohrer U, Fry Am, Hoffman I. Polo-like kinase-2 is required for centriole duplication in mammalian cells. Current Biol 2004; 14:1200-7. 8. Hoffman I. Playing polo in G1: A novel function of polo like kinase 2 in centriole duplication. Cell Cycle 2004; 3:76-7. 9. Ma S, Charron J, Erikson R. Role of Plk2 (Snk) in mouse development and cell proliferation. Mol Cell Biol 2003; 23:6936-43. 10. Jiang N, Wang X, Jhanwar-Uniyal M, Darzynkiewicz Z, Dai W. Polo box domain of Plk3 functions as a centrosome localization signal, overexpression of which causes mitotic arrest, cytokinesis defects and apoptosis. J Biol Chem 2006; 281:10577-82. 11. Habedanck R, Stierhof YD, Wilkinson CJ, Nigg EA. The Polo kinase Plk4 functions in centriole duplication. Nature Cell Biology 2005; 7:1140-6. 12. Yogosawa S, Hatakeyama S, Nakayama KI, Miyoshi H, Kohsaka S, Akazawa C. Ubiquitylation and degradation of polo-like kinase SNK by hVPS18, a RING-E2 type ubiquitin ligase. J Biol Chem 2005; 280:41619-27. 13. Winkles JA, Alberts GF. Differential regulation of polo-like kinase 1, 2, 3 and 4 gene expression in mammalian cells and tissues. Oncogene 2005; 24:260-6. 14. Kang D, Chen J, Wong J, Fang G. The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition. J Cell Biol 2002; 156:249-59. 15. Burns TF, Fei P, Scata KA, Dicker DT, El-Deiry WS. Silencing of the novel p53 target gene Snk/Plk2 leads to mitotic catastrophe in paclitaxel (Taxol)-exposed cells. Mol Cell Biol 2003; 23:5556-71. 16. Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW, Vogelstein B. 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell 1997; 1:3-11. 17. Gasco M, Sullivan A, Repellin C, Brooks L, Farrell PJ, Tidy JA, Dunne B, Gusterson B, Evans DJ, Crook T. Coincident inactivation of 14-3-3 sigma and p16INK4a is an early event in vulval squamous neoplasia. Oncogene 2002; 21:1876-81. 18. Gasco M, Bell AK, Heath V, Sullivan A, Smith P, Hiller L, Yulug I, Numico G, Merlano M, Farrell PJ, Tavassoli M, Gusterson B, Crook T. Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: Association with p16INK4a silencing and human papillomavirus negativity. Cancer Res 2002; 62:2072-6. 19. Wang Q, Chen J, Fukasawa K, Naik U, Traganos F, Darzynkiewicz Z, Jhanwar-Uniyal M, Dai W. Cell cycle arrest and apoptosis induced by human polo-like kinase 3 is mediated through perturbation of microtubule integrity. Mol Cell Biol 2002; 22:3450-9.
1264
Cell Cycle
2006; Vol. 5 Issue 12
