Aberrant Regulation of Survivin by the RB/E2F Family of Proteins

Survivin is a putative oncogene that is aberrantly expressed in cancer cells. It has been hypothesized to play a central role in cancer progression and resistance to therapy in diverse tumor types. Although some of the transcriptional processes regulating its expression have been established, the diversity of genes that may be controlling the levels of its expression in both normal cells as well as in cancer cells has not been fully explored. The most common genetically mutated pathways in human malignancies are the p53 tumor suppressor pathway and the RB/E2F pathway. Both of these pathways, when intact, provide essential checkpoints in the maintenance of normal cell growth and protect the cell from DNA damage. Using non-transformed embryonic fibroblasts, we provide evidence of a molecular link between the regulation of survivin transcription and the RB/E2F family of proteins. We demonstrate that both pRB and p130 can interact with the survivin promoter and can repress survivin transcription. We also show that the E2F activators (E2F1, E2F2, and E2F3) can bind to the survivin promoter and induce survivin transcription. Genetically modified cells that harbor deletions in various members of the RB/E2F family confirm our data from the wild-type cells. Our findings implicate several members of the RB/E2F pathway in an intricate mechanism of survivin gene regulation that, when genetically altered during the process of tumorigenesis, may function within cancer cells to aberrantly alter survivin levels and enhance tumor progression. Survivin is a putative oncogene that is aberrantly expressed in cancer cells. It has been hypothesized to play a central role in cancer progression and resistance to therapy in diverse tumor types. Although some of the transcriptional processes regulating its expression have been established, the diversity of genes that may be controlling the levels of its expression in both normal cells as well as in cancer cells has not been fully explored. The most common genetically mutated pathways in human malignancies are the p53 tumor suppressor pathway and the RB/E2F pathway. Both of these pathways, when intact, provide essential checkpoints in the maintenance of normal cell growth and protect the cell from DNA damage. Using non-transformed embryonic fibroblasts, we provide evidence of a molecular link between the regulation of survivin transcription and the RB/E2F family of proteins. We demonstrate that both pRB and p130 can interact with the survivin promoter and can repress survivin transcription. We also show that the E2F activators (E2F1, E2F2, and E2F3) can bind to the survivin promoter and induce survivin transcription. Genetically modified cells that harbor deletions in various members of the RB/E2F family confirm our data from the wild-type cells. Our findings implicate several members of the RB/E2F pathway in an intricate mechanism of survivin gene regulation that, when genetically altered during the process of tumorigenesis, may function within cancer cells to aberrantly alter survivin levels and enhance tumor progression. Survivin is a gene with structural and functional similarities to both the inhibitor of apoptosis gene family, which specifically blocks the downstream effectors of cell death, and the chromosomal passenger proteins, which play an essential role in cytokinesis (1Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1756) Google Scholar, 2Li F. Ackermann E.J. Bennett C.F. Rothermel A.L. Plescia J. Tognin S. Villa A. Marchisio P.C. Altieri D.C. Nat. Cell Biol. 1999; 1: 461-466Crossref PubMed Scopus (559) Google Scholar, 3Uren A.G. Wong L. Pakusch M. Fowler K.J. Burrows F.J. Vaux D.L. Choo K.H. Curr. Biol. 2000; 10: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 4Skoufias D.A. Mollinari C. Lacroix F.B. Margolis R.L. J. Cell Biol. 2000; 151: 1575-1582Crossref PubMed Scopus (237) Google Scholar). Survivin expression is cell cycle-regulated with the highest levels reported in G2/M and lower levels in G1 (1Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1756) Google Scholar, 5Kobayashi K. Hatano M. Otaki M. Ogasawara T. Tokuhisa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1457-1462Crossref PubMed Scopus (233) Google Scholar). This cell cycle specificity has been attributed to two cell cycle-dependent elements (CDEs) 1The abbreviations used are: CDE, cell cycle-dependent element; MEF, mouse embryonic fibroblast; FBS, fetal bovine serum; REF, rat embryonic fibroblast; EMEM, Eagle's minimal essential medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; CHR, cell cycle gene homology region. that are upstream of the transcription start site (6Otaki M. Hatano M. Kobayashi K. Ogasawara T. Kuriyama T. Tokuhisa T. Biochim. Biophys. Acta. 2000; 1493: 188-194Crossref PubMed Scopus (57) Google Scholar, 7Li F. Altieri D.C. Biochem. J. 1999; 344: 305-311Crossref PubMed Scopus (290) Google Scholar). Mutation within the CDEs abolishes the cell cycle specificity of survivin expression. Survivin is highly expressed in a variety of transformed cell lines, including human cancers of the lung, colon, pancreas, breast, brain, and bone marrow (for review, see Ref. 8Altieri D.C. Oncogene. 2003; 22: 8581-8589Crossref PubMed Scopus (832) Google Scholar). High levels of expression within cancer cells correlate with an adverse clinical outcome (8Altieri D.C. Oncogene. 2003; 22: 8581-8589Crossref PubMed Scopus (832) Google Scholar). Data from an in vivo tumor model support a mechanism whereby its targeted disruption specifically eliminates tumor cells by a proapoptotic mechanism (9Mesri M. Wall N.R. Li J. Kim R.W. Altieri D.C. J. Clin. Investig. 2001; 108: 981-990Crossref PubMed Scopus (361) Google Scholar). Survivin also plays a critical role in normal development, as genetic disruption is lethal early in the embryo (65). Postnatally, survivin is expressed in a few select tissues, including bone marrow stem cells, vascular endothelial cells, colonic epithelium, and the ependyma and choroid plexus of the brain (10Altura R.A. Olshefski R.S. Jiang Y. Boue D.R. Br. J. Cancer. 2003; 89: 1743-1749Crossref PubMed Scopus (47) Google Scholar, 11Gianani R. Jarboe E. Orlicky D. Frost M. Bobak J. Lehner R. Shroyer K.R. Hum. Pathol. 2001; 32: 119-125Crossref PubMed Scopus (167) Google Scholar, 12Fukuda S. Foster R.G. Porter S.B. Pelus L.M. Blood. 2002; 100: 2463-2471Crossref PubMed Scopus (127) Google Scholar, 13Conway E.M. Zwerts F. Van Eygen V. DeVriese A. Nagai N. Luo W. Collen D. Am. J. Pathol. 2003; 163: 935-946Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The pattern of survivin expression suggests that it plays a critical role in the maintenance of actively dividing cells and tissues. The RB/E2F pathway plays an essential role in cell cycle regulation and maintenance of cellular homeostasis (14Sherr C.J. McCormick F. Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar, 15Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (617) Google Scholar). Mutations in genes within this pathway are found at high rates in almost all types of cancer, attesting to their importance in cell growth control (15Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (617) Google Scholar, 16Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (757) Google Scholar). RB and its structurally and functionally related family members, p107 and p130, have similar properties of binding to the E2F family members and of inhibiting cell cycle progression when ectopically expressed (17Sage J. Mulligan G.J. Attardi L.D. Miller A. Chen S. Williams B. Theodorou E. Jacks T. Genes Dev. 2000; 14: 3037-3050Crossref PubMed Scopus (514) Google Scholar). The E2F family of proteins includes both the transcriptional activators (E2F1, E2F2, and E2F3) and the repressors (E2F4 and E2F5) (18Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 11-20Crossref PubMed Scopus (975) Google Scholar). These proteins bind to specific promoter regions within diverse gene targets. The interaction of RB with the E2F activators normally results in an inhibition of E2F-mediated transactivation and a cell growth arrest at the G1 phase of the cell cycle (19Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1181) Google Scholar, 20Kaelin Jr., W.G. Pallas D.C. DeCaprio J.A. Kaye F.J. Livingston D.M. Cell. 1991; 64: 521-532Abstract Full Text PDF PubMed Scopus (469) Google Scholar, 21Bandara L.R. La Thangue N.B. Nature. 1991; 351: 494-497Crossref PubMed Scopus (352) Google Scholar). RB family mutants that lack the ability to cause growth arrest also lack the ability to bind E2F and inhibit E2F-dependent transcription. Such mutants have been isolated from many different human tumors (22Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4360) Google Scholar, 23Paggi M.G. Baldi A. Bonetto F. Giordano A. J. Cell. Biochem. 1996; 62: 418-430Crossref PubMed Scopus (193) Google Scholar). The viral oncoprotein E1A also binds and sequesters the RB family members, resulting in the release of E2F transcriptional activity, induction of DNA synthesis, and transformation of mammalian cells (24Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1369) Google Scholar, 25Samuelson A.V. Lowe S.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12094-12099Crossref PubMed Scopus (89) Google Scholar, 26Flint J. Shenk T. Annu. Rev. Genet. 1997; 31: 177-212Crossref PubMed Scopus (185) Google Scholar). Overexpression of E2F1, E2F2, and E2F3 can induce entry into the cell cycle and can lead to transformation of primary cells (27Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (505) Google Scholar). Deregulated E2F transcriptional activity can also trigger apoptosis through p53-dependent and -independent pathways, although the individual mechanisms and E2F species that play the major role in these processes have not been identified (18Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 11-20Crossref PubMed Scopus (975) Google Scholar). Our data, in wild-type cells and in cells that have been genetically modified to delete pRb, the E2Fs, and p53, suggests that survivin and the RB/E2F proteins interact via a mechanism that involves multiple RB/E2F members acting as repressors and activators of survivin transcription. We hypothesize that disruption of upstream elements within the RB pathway such as the deletion of RB, the amplification of CDK4, or the deletion of p16INK4A, perhaps in the early stages of tumorigenesis, may result in the deregulation of survivin, leading to chromosomal instability and an accumulation of mutations that confer resistance to therapy. Cells and Culture Conditions—Primary mouse embryonic fibroblasts (MEFs) harboring genetic deletions of the E2F activators (E2F1, E2F2, and E2F3), as well as pRB and p53, from harboring these A. L. Saavedra H.I. P. Y. M. Robinson M.L. Leone G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). These as and with an by of of the gene of delete the genes in cells with a of an that within the and the cell of these genes from early primary in with and embryonic and cell in modified Eagle's with and human embryo fibroblasts in Eagle's minimal essential with and cells at and cell growth arrest cells at in with and in cell cycle cells by and with of a gene and wild-type or survivin promoter by the of Tokuhisa of (6Otaki M. Hatano M. Kobayashi K. Ogasawara T. Kuriyama T. Tokuhisa T. Biochim. Biophys. Acta. 2000; 1493: 188-194Crossref PubMed Scopus (57) Google Scholar). of wild-type E1A and the of E1A by the of K. B. B. Oncogene. 2000; PubMed Scopus Google Scholar). 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Biol. 1997; PubMed Scopus Google Scholar). Cells to by and by from the and and activity to isolated from to and to a with a of survivin or by G. J. L. S. Williams R.S. Nevins J.R. Genes Dev. 1998; PubMed Scopus Google Scholar). of by a and to a a The in with at The with primary at a or at and with or at a the in and to a as an control and with mouse modified from a from the of T. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). cells in with and by induction into the cell cycle with at of or of at and by of Cells with by and in and at Cells by in and with a to release by in in the of and through a and with a high the chromatin in and with and by with of E2F3 p130 p107 or pRB at with and to and the at The by and DNA with and with A. the and the at The chromatin used as a control the and The DNA and by The and of human used to DNA the binding site to and to The binding site and the survivin transcription start site identified a S. Chen J. M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). in and results with DNA as the the Foster Scholar). at and at the of and results used as the p130, and results at used as the to mouse survivin to the the used by the to expression levels and used to mouse survivin used as the control to the by an in and the of the Foster with the as that the of and or in the of and Cell of Survivin the of non-transformed human embryonic fibroblasts as a model our we the cell cycle of survivin expression in these cells. human fibroblasts to in EMEM, and by the with EMEM, Cells with and at various Although or survivin expression be in cells expression as cells the cell cycle with levels at this the of cells in G2/M as by of cells in These the cell cycle-dependent expression pattern of survivin in cells, reported in cell types. the cell cycle-dependent expression of survivin via transcriptional non-transformed fibroblasts with by a of the survivin promoter of to through the (6Otaki M. Hatano M. Kobayashi K. Ogasawara T. Kuriyama T. Tokuhisa T. Biochim. Biophys. Acta. 2000; 1493: 188-194Crossref PubMed Scopus (57) Google Scholar). These cells by as and at various Although levels of activity be in cells into the cell cycle by to a in activity similar a promoter a within the in in a in activity in cells. in activity the of These with reported in cell (6Otaki M. Hatano M. Kobayashi K. Ogasawara T. Kuriyama T. Tokuhisa T. Biochim. Biophys. Acta. 2000; 1493: 188-194Crossref PubMed Scopus (57) Google that in non-transformed cells transcriptional regulation of survivin through that an region. of these promoter elements is to induce its expression during entry into the cell that E2F is a major the expression of many cell cycle-regulated we an to to its role in the control of survivin expression. Overexpression of in cells in an induction of survivin expression the of a transcriptional of the E2F proteins the survivin the ability of various E2F family members to a wild-type or survivin in these cells. of of E2F1, E2F2, and expression to a in wild-type survivin activity that levels This an DNA survivin at this site to E2F mutants of and of survivin activity that E2F-mediated regulation of survivin expression of the E2F transactivation with the the survivin cells apoptosis expression of survivin Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). proapoptotic by the reported RB by E1A to the of Survivin the that survivin is by the RB family of proteins, a oncogene of binding to and RB and its family members, into both and cells, and survivin expression by expression of E1A in cells to a in survivin levels to an control the specific of E1A that to this we a of E1A that E1A of binding to the RB family of proteins. We the transcriptional of wild-type E1A with of the E1A in survivin cells with these by and in E1A expression to a in activity, the RB of E1A of survivin The ability of E1A to induce survivin activity when the promoter not these results that wild-type E1A survivin expression through the of RB its family members and that this regulation an These data that the RB family of proteins can repress survivin transcription. of the E2F Survivin the molecular between the RB/E2F pathway and survivin we used genetically modified that harbor deletions of of the RB/E2F pathway as well as of the tumor suppressor cells from mouse harboring or targeted genetic deletions in the p53, pRb, and E2F the and with an by of of the gene of two cell from primary used to confirm our findings in the primary cells. of a expression into primary cells in the deletion of the in as by of into the primary cells as cell Cells and of survivin expression cell is in and a also used to survivin transcription levels with that used expression to p53 or cancer cell p53 J. Shenk T. Annu. Rev. Genet. 1997; 31: 177-212Crossref PubMed Scopus (185) Google Scholar, A. M. S. L. P. S. Oncogene. 2002; PubMed Scopus Google genetic deletion of p53 in non-transformed fibroblasts in a induction of survivin and by deletion of RB also to an induction of survivin in these cells. deletion of p53 or pRB in with a deletion of the E2F E2F1, E2F2, and to a by of survivin transcription. This that or of the E2F activators play a role in survivin We the role of E2F3 in survivin transcription primary cells harboring a targeted deletion this of E2F3 has been in amplification and a also in cells with survivin expression H.I. Maiti B. Timmers C. R. Y. K. Leone G. Cancer Cell. 2003; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the E2F3 gene in a of survivin a role of E2F3 in the of survivin transcription. The E2F to the Survivin in provide evidence a interaction between the survivin promoter and members of the RB/E2F family, we in vivo DNA binding of the E2F and the E2F and as well as of the RB family members RB and p130 with the survivin promoter by in human embryonic fibroblasts cells by and at two and our cells are in G1 at and are in phase of the cell cycle survivin the binding site used in to the binding in binding levels as cells from a to a of the that and E2F3 bind to the survivin promoter with a in binding entry into the cell cycle p130 with the survivin promoter when the cells are in a with the survivin promoter also with an in binding when cells are This binding activity is with an RB of survivin that through binding of the RB proteins to the E2F binding of the E2F proteins, and also used in Both and bind to the survivin when cells are these findings that survivin transcription is by the E2F proteins and by the E2F the in with the RB proteins. The functional of survivin within both normal cells and transformed to be deletion of the survivin results in early embryonic with a disruption of and in the cells A.G. Wong L. Pakusch M. Fowler K.J. Burrows F.J. Vaux D.L. Choo K.H. Curr. Biol. 2000; 10: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). This that reported chromosomal passenger proteins and the family of the control of and cytokinesis R. C. J. Cell Sci. 1999; PubMed Google Scholar). It is also similar to that of a survivin in disruption results in of and and in cytokinesis A. Vaux D. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in cells support a role survivin in regulating processes D.A. Mollinari C. Lacroix F.B. Margolis R.L. J. Cell Biol. 2000; 151: 1575-1582Crossref PubMed Scopus (237) Google Scholar). in cancer cells an function survivin as a cell by mouse of survivin inhibiting apoptosis through with D. Kim Tognin S. Marchisio P.C. Altieri D.C. J. Clin. Investig. 2001; 108: PubMed Scopus Google Scholar). The RB/E2F pathway is in all human malignancies (14Sherr C.J. McCormick F. Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar, 15Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (617) Google Scholar). Although survivin is aberrantly expressed in the of human the mechanism of its in these tumors has not been It is that high levels of expression as a result of gene as of the survivin have not been regulation of survivin expression at the transcription as a of the deregulation of upstream transcriptional these upstream members of mutated transcription of proteins in cancer, including p53 and The survivin gene has cell within its promoter that control in the G2/M phase of the cell Mutation within these results in the of cell cycle and a transcription induction (6Otaki M. Hatano M. Kobayashi K. Ogasawara T. Kuriyama T. Tokuhisa T. Biochim. Biophys. Acta. 2000; 1493: 188-194Crossref PubMed Scopus (57) Google Scholar). This with genes that also elements J. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google suggests that survivin transcription is actively the G1 phase of the cell of this a of within the region. It has been that the p53 and tumor suppressor genes can repress survivin transcription S. Chen J. M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. S. L. P. S. Oncogene. 2002; PubMed Scopus Google Scholar, K. C. A. M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This mechanism is an transactivation within these proteins and a functional has been to function in survivin regulation by K. C. A. M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This result in the of RB, the release of E2F activators an interaction with the E2F as and to bind to E2F p53 or a in human cancer, not be and RB be leading to the release of the E2F activators and the induction of survivin transcription. these support the findings in this RB to the regulation of survivin transcription. Our in non-transformed embryonic fibroblasts that several RB/E2F proteins survivin expression. We show that RB proteins can repress survivin transcription as by a in survivin activity of an We also show that both pRB and p130 an upstream survivin DNA promoter that an binding we show that primary cells from genetically have levels of survivin control cells with an to the of survivin we demonstrate that several E2F proteins can induce survivin and E2F3 induce survivin through binding to its promoter as by both the and by the This E2F-mediated transcriptional induction is the E2F is the importance of this in transcriptional The E2F3 the expression of two genes G. J. L. S. Williams R.S. Nevins J.R. Genes Dev. 1998; PubMed Scopus Google and or both be in survivin regulation during different of the cell Using an to E2F3 that the not the we show that binds to survivin in deletion of E2F3 in cells from with genetically targeted deletions of both the and the genes results in a of survivin transcription. E2F3 has been to control its disruption may through survivin to with a resulting in and genetic instability in cancer cells H.I. Maiti B. Timmers C. R. Y. K. Leone G. Cancer Cell. 2003; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). our data support the that of survivin expression can as a of genetic within the RB/E2F pathway during the process of these are common in all human it is to hypothesize that multiple within these pathways, such as deletions or mutations or or of CDK4, may be some of the regulating survivin expression. These findings have in the of in in highly We We