Identification of <em>CDK4 </em>as a target of c-MYC

H Hermeking

C Rago

M Schuhmacher

Q Li

W Tam+6 authors

^{Howard Hughes Medical Institute,}^{The Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine, 424 North Bond Street, Baltimore, MD 21231;}^{Institut für Klinische Molekularbiologie und Tumorgenetik, GSF-Forschungsinstitut, Marchioninistrasse 25, D-81377 Munich, Germany;}^{Program of Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and}^{Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912}

^{To whom reprint requests should be sent at present address: Molecular Oncology, Max Planck Institute for Biochemistry, Am Klopferspitz I8A, D-82152 Martinsried/Munich, Germany. E-mail:}moc.loa@gnikemrehH.

Contributed by Bert Vogelstein

Accepted 1999 Dec 30.

Abstract

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

Abstract

The protooncogene c-MYC has been implicated in a variety of human and experimental tumors (for review see refs. 1–4). In some cases, the overexpression of c-MYC can be traced to genetic alterations of the oncogene itself, whereas in others, this dysregulation is caused by genetic defects in upstream regulators of c-MYC expression. In either case, the ability of c-MYC to promote proliferation through cell-cycle reentry seems critical to its oncogenic function. Accordingly, expression of c-MYC is induced by a variety of mitogens and repressed under conditions of growth arrest. Furthermore, ectopic c-MYC expression, in some cases, can promote reentry of resting cells into the cell cycle and facilitate proliferation in the absence of external growth factors (5).

The c-MYC gene encodes a transcription factor of the helix–loop–helix leucine zipper class (for review see refs. 1 and 2). c-MYC binds to E-boxes (CACGTG) in the vicinity of target genes, which are then activated. The DNA binding activity requires dimerization with another helix–loop–helix leucine zipper protein called MAX. MAX also can interact with transcriptional repressors such as MAD and Mxi1, which presumably down-regulate expression of c-MYC target genes. Despite many advances and identification of a number of c-MYC target genes, the direct mediators of c-MYC's effects on cell-cycle reentry have not yet been identified.

Acknowledgments

We thank Tong-Chuan He for experimental advice, Lin Zhang for RNA samples, Christoph Lengauer for retroviral constructs, Jim Flook for assistance with fluorescence-activated cell sorter analysis, Katrin Berns and Rene Bernards for MADMYC plasmids, and Kornelia Polyak and members of our laboratories for critical reading of this manuscript. This work was supported by National Institutes of Health Grants CA-57345 (to K.W.K.), CA57341 (to C.V.D.), GM-41690 (to J.M.S.), and GM-07601 (to M.K.M.). A.J.O. was supported by a postdoctoral fellowship from the Ministerio Educación y Cultura de España. K.W.K. received research funding from Genzyme Molecular Oncology (Genzyme). Under a licensing agreement between The Johns Hopkins University and Genzyme, the SAGE technology was licensed to Genzyme for commercial purposes, and K.W.K and B.V. are entitled to a share of royalties received by the University from sales of the licensed technology. The SAGE technology is freely available to academia for research purposes. K.W.K. and B.V. are consultants to Genzyme. The Johns Hopkins University, K.W.K., and B.V. own Genzyme stock, which is subject to certain restrictions under University policy. The terms of this arrangement are being managed by the University in accordance with its conflict-of-interest policies.

Acknowledgments

Abbreviations

SAGE	serial analysis of gene expression
MBS	c-MYC binding sites
HUVEC cells	human umbilical vein cord cells
HA	hemagglutinin
GFP	green fluorescent protein
Ad	adenovirus

Abbreviations

Footnotes

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AF224272","term_id":"7141289"}}AF224272 and {"type":"entrez-nucleotide","attrs":{"text":"AF223390","term_id":"7340079"}}AF223390).

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.050586197.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.050586197

Footnotes

References

1. Amati B, Alevizopoulos K, Vlach J. Front Biosci. 1998;3:250–268.[PubMed]
2. Dang C V. Mol Cell Biol. 1999;19:1–11.
3. Eick D, Hermeking H. Trends Genet. 1996;12:4–6.[PubMed]
4. Garte S. Crit Rev Oncog. 1993;4:435–449.[PubMed]
5. Eilers M, Schirm S, Bishop J M. EMBO J. 1991;10:133–141.
6. Mateyak K M, Obaya A J, Adachi S, Sedivy J M. Cell Growth Differ. 1997;8:1039–1048.[PubMed]
7. Pear W S, Nolan G P, Scott M L, Baltimore D. Proc Natl Acad Sci USA. 1993;90:8392–8396.
8. He T C, Zhou S, da Costa L T, Yu J, Kinzler K W, Vogelstein B. Proc Natl Acad Sci USA. 1998;95:2509–2514.
9. Hermeking H, Wolf D A, Kohlhuber F, Dickmanns A, Billaud M, Fanning E, Eick D. Proc Natl Acad Sci USA. 1994;91:10412–10416.
10. Berns K, Hijmans E M, Bernards R. Oncogene. 1997;15:1347–1356.[PubMed]
11. Hermeking H, Lengauer C, Polyak K, He T C, Zhang L, Thiagalingam S, Kinzler K W, Vogelstein B. Mol Cell. 1997;1:3–11.[PubMed]
12. Velculescu V E, Zhang L, Vogelstein B, Kinzler K W. Science. 1995;270:484–487.[PubMed]
13. Elkahloun A G, Krizman D B, Wang Z, Hofmann T A, Roe B, Meltzer P S. Genomics. 1997;42:295–301.[PubMed]
14. Kohlhuber F, Hermeking H, Graessmann A, Eick D. J Biol Chem. 1995;270:28797–28805.[PubMed]
15. He T C, Sparks A B, Rago C, Hermeking H, Zawel L, da Costa L T, Morin P J, Vogelstein B, Kinzler K W. Science. 1998;281:1509–1512.[PubMed]
16. Shim H, Dolde C, Lewis B C, Wu C S, Dang G, Jungmann R A, Dalla-Favera R, Dang C V. Proc Natl Acad Sci USA. 1997;94:6658–6663.
17. Matsushime H, Ewen M E, Strom D K, Kato J Y, Hanks S K, Roussel M F, Sherr C J. Cell. 1992;71:323–334.[PubMed]
18. Ewen M E, Sluss H K, Whitehouse L L, Livingston D M. Cell. 1993;74:1009–1020.[PubMed]
19. Alexandrow M G, Kawabata M, Aakre M, Moses H L. Proc Natl Acad Sci USA. 1995;92:3239–3243.
20. Latham K M, Eastman S W, Wong A, Hinds P W. Mol Cell Biol. 1996;16:4445–4455.
21. Hermeking H, Funk J O, Reichert M, Ellwart J W, Eick D. Oncogene. 1995;11:1409–1415.[PubMed]
22. Wang J, Xie L Y, Allan S, Beach D, Hannon G J. Genes Dev. 1988;12:1769–1774.
23. Holland E C, Hively W P, Gallo V, Varmus H E. Genes Dev. 1998;12:3644–3649.
24. Schuhmacher M, Staege M S, Pajic A, Polack A, Weidle U H, Bornkamm G W, Eick D, Kohlhuber F. Curr Biol. 1999;9:1255–1258.[PubMed]
25. Mateyak M K, Obaya A J, Sedivy J M. Mol Cell Biol. 1999;19:4672–4683.
26. Erisman M D, Rothberg P G, Diehl R E, Morse C C, Spandorfer J M, Astrin S M. Mol Cell Biol. 1985;5:1969–1976.
27. Augenlicht L H, Wadler S, Corner G, Richards C, Ryan L, Multani A S, Pathak S, Benson A, Haller D, Heerdt B G. Cancer Res. 1997;57:1769–1775.[PubMed]
28. Zhang T, Nanney L B, Luongo C, Lamps L, Heppner K J, DuBois R N, Beauchamp R D. Cancer Res. 1997;57:169–175.[PubMed]
29. Zhang T, Nanney L B, Peeler M O, Williams C S, Lamps L, Heppner K J, DuBois R N, Beauchamp R D. Cancer Res. 1997;57:1638–1643.[PubMed]
30. Rane S G, Dubus P, Mettus R V, Galbreath E J, Boden G, Reddy E P, Barbacid M S, Rane G. Nat Genet. 1999;22:44–52.[PubMed]
31. Tsutsui T, Hesabi B, Moons D S, Pandolfi P P, Hansel K S, Koff A, Kiyokawa H. Mol Cell Biol. 1999;19:7011–7019.
32. Steiner P, Philipp A, Lukas J, Godden-Kent D, Pagano M, Mittnacht S, Bartek J, Eilers M. EMBO J. 1995;14:4814–4826.
33. Vlach J, Hennecke S, Alevizopoulos K, Conti D, Amati B. EMBO J. 1996;15:6595–6604.
34. Reynisdottir I, Polyak K, Iavarone A, Massague J. Genes Dev. 1995;9:1831–1845.[PubMed]
35. Serrano M, Hannon G J, Beach D. Nature (London) 1993;366:704–707.[PubMed]
36. Serrano M, Lin A W, McCurrach M E, Beach D, Lowe S W. Cell. 1997;88:593–602.[PubMed]
37. Koh J, Enders G H, Dynlacht B D, Harlow E. Nature (London) 1995;375:506–510.[PubMed]
38. Lukas J, Parry D, Aagaard L, Mann D J, Bartkova J, Strauss M, Peters G, Bartek J. Nature (London) 1995;375:503–506.[PubMed]
39. Haas K, Staller P, Geisen C, Bartek J, Eilers M, Moroy T. Oncogene. 1997;15:179–192.[PubMed]
40. Rao R N, Stamm N B, Otto K, Kovacevic S, Watkins S A, Rutherford P, Lemke S, Cocke K, Beckmann R P, Houck K, et al Oncogene. 1999;18:6343–6356.[PubMed][Google Scholar]