Background The cyclin-D/CDK4,6/p16INK4a/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. Methodology/Principal Findings We have shown here that the tumor suppressor p16INK4a protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16INK4a stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16INK4a, using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3′UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16INK4a -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. Conclusion/Significance These findings show that the cyclin-dependent kinase inhibitor p16 INK4a is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.
References
[1]
Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10: 789–799.
[2]
Nevins JR (2001) The Rb/E2F pathway and cancer. Hum Mol Genet 10: 699–703.
[3]
Kaye FJ (2002) RB and cyclin dependent kinase pathways: defining a distinction between RB and p16 loss in lung cancer. Oncogene 21: 6908–6914.
[4]
Giacinti C, Giordano A (2006) RB and cell cycle progression. Oncogene 25: 5220–5227.
[5]
Crosby ME, Almasan A (2004) Opposing roles of E2Fs in cell proliferation and death. Cancer Biol Ther 3: 1208–1211.
[6]
Nahle Z, Polakoff J, Davuluri RV, McCurrach ME, Jacobson MD, et al. (2002) Direct coupling of the cell cycle and cell death machinery by E2F. Nat Cell Biol 4: 859–864.
[7]
Serrano M, Hannon GJ, Beach D (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366: 704–707.
[8]
Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, et al. (1994) A cell cycle regulator potentially involved in genesis of many tumor types. Science 264: 436–440.
[9]
Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, et al. (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368: 753–756.
[10]
Pei XH, Xiong Y (2005) Biochemical and cellular mechanisms of mammalian CDK inhibitors: a few unresolved issues. Oncogene 24: 2787–2795.
[11]
Ruas M, Peters G (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378: F115–177.
[12]
Ortega S, Malumbres M, Barbacid M (2002) Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602: 73–87.
[13]
Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, et al. (2001) Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413: 86–91.
[14]
Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A (2001) Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 413: 83–86.
[15]
Holst CR, Nuovo GJ, Esteller M, Chew K, Baylin SB, et al. (2003) Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia. Cancer Res 63: 1596–1601.
[16]
Vernell R, Helin K, Muller H (2003) Identification of target genes of the p16INK4A-pRB-E2F pathway. J Biol Chem 278: 46124–46137.
[17]
Johnson DG (1995) Regulation of E2F-1 gene expression by p130 (Rb2) and D-type cyclin kinase activity. Oncogene 11: 1685–1692.
[18]
Hiyama H, Iavarone A, LaBaer J, Reeves SA (1997) Regulated ectopic expression of cyclin D1 induces transcriptional activation of the cdk inhibitor p21 gene without altering cell cycle progression. Oncogene 14: 2533–2542.
[19]
Tashiro E, Maruki H, Minato Y, Doki Y, Weinstein IB, et al. (2003) Overexpression of cyclin D1 contributes to malignancy by up-regulation of fibroblast growth factor receptor 1 via the pRB/E2F pathway. Cancer Res 63: 424–431.
[20]
Al-Mohanna MA, Al-Khalaf HH, Al-Yousef N, Aboussekhra A (2007) The p16INK4a tumor suppressor controls p21WAF1 induction in response to ultraviolet light. Nucleic Acids Res 35: 223–233.
[21]
Wang W, Furneaux H, Cheng H, Caldwell MC, Hutter D, et al. (2000) HuR regulates p21 mRNA stabilization by UV light. Mol Cell Biol 20: 760–769.
[22]
Al-Zoghaibi F, Ashour T, Al-Ahmadi W, Abulleef H, Demirkaya O, et al. (2007) Bioinformatics and experimental derivation of an efficient hybrid 3′ untranslated region and use in expression active linear DNA with minimum poly(A) region. Gene 391: 130–139.
[23]
Al-Mohanna MA, Manogaran PS, Al-Mukhalafi Z, Al-Hussein AK, Aboussekhra A (2004) The tumor suppressor p16(INK4a) gene is a regulator of apoptosis induced by ultraviolet light and cisplatin. Oncogene 23: 201–212.
[24]
Li C, Hung Wong W (2001) Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol 2: RESEARCH0032.
[25]
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80.
[26]
Wu Z, Irizarry RA (2004) Preprocessing of oligonucleotide array data. Nat Biotechnol 22: 656–658; author reply 658.
[27]
Wu Z, Irizarry RA (2005) Stochastic models inspired by hybridization theory for short oligonucleotide arrays. J Comput Biol 12: 882–893.
[28]
McConnell BB, Gregory FJ, Stott FJ, Hara E, Peters G (1999) Induced expression of p16(INK4a) inhibits both CDK4- and CDK2- associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes. Mol Cell Biol 19: 1981–1989.
[29]
Barreau C, Paillard L, Osborne HB (2006) AU-rich elements and associated factors: are there unifying principles? Nucleic Acids Res 33: 7138–7150.
[30]
Lal A, Mazan-Mamczarz K, Kawai T, Yang X, Martindale JL, et al. (2004) Concurrent versus individual binding of HuR and AUF1 to common labile target mRNAs. EMBO J 23: 3092–3102.
[31]
Wang W, Caldwell MC, Lin S, Furneaux H, Gorospe M (2000) HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation. EMBO J 19: 2340–2350.
[32]
Al-Ahmadi W, Al-Haj L, Al-Mohanna FA, Silverman RH, Khabar KS (2009) RNase L downmodulation of the RNA-binding protein, HuR, and cellular growth. Oncogene 28: 1782–1791.
[33]
Zhang W, Wagner BJ, Ehrenman K, Schaefer AW, DeMaria CT, et al. (1993) Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol Cell Biol 13: 7652–7665.
[34]
Al-Ahmadi W, Al-Ghamdi M, Al-Haj L, Al-Saif M, Khabar KS (2009) Alternative polyadenylation variants of the RNA binding protein, HuR: abundance, role of AU-rich elements and auto-Regulation. Nucleic Acids Res 37: 3612–3624.
[35]
Wagoner J, Austin M, Green J, Imaizumi T, Casola A, et al. (2007) Regulation of CXCL-8 (interleukin-8) induction by double-stranded RNA signaling pathways during hepatitis C virus infection. J Virol 81: 309–318.
[36]
Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ (1996) Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase CDK2 and CDK4 in human breast carcinoma cells. Cancer Res 56: 2973–2978.
[37]
Sedlacek HH (2001) Mechanisms of action of flavopiridol. Crit Rev Oncol Hematol 38: 139–170.
[38]
Mazan-Mamczarz K, Kuwano Y, Zhan M, White EJ, Martindale JL, et al. (2009) Identification of a signature motif in target mRNAs of RNA-binding protein AUF1. Nucleic Acids Res 37: 204–214.
[39]
Stanelle J, Stiewe T, Theseling CC, Peter M, Putzer BM (2002) Gene expression changes in response to E2F1 activation. Nucleic Acids Res 30: 1859–1867.
[40]
Matsumura I, Tanaka H, Kanakura Y (2003) E2F1 and c-Myc in cell growth and death. Cell Cycle 2: 333–338.
[41]
Wang W, Martindale JL, Yang X, Chrest FJ, Gorospe M (2005) Increased stability of the p16 mRNA with replicative senescence. EMBO Rep 6: 158–164.
[42]
Chang N, Yi J, Guo G, Liu X, Shang Y, et al. (2010) HuR uses AUF1 as a cofactor to promote p16INK4 mRNA decay. Mol Cell Biol 30: 3875–3886.
[43]
Wang W, Yang X, Cristofalo VJ, Holbrook NJ, Gorospe M (2001) Loss of HuR is linked to reduced expression of proliferative genes during replicative senescence. Mol Cell Biol 21: 5889–5898.
[44]
Ghosh S, Karin M (2002) Missing pieces in the NF-kappaB puzzle. Cell 109: S81–96.
[45]
Singh P (2007) Role of Annexin-II in GI cancers: interaction with gastrins/progastrins. Cancer Lett 252: 19–35.
[46]
Sharpless E, Chin L (2003) The INK4a/ARF locus and melanoma. Oncogene 22: 3092–3098.
[47]
Putzer BM (2007) E2F1 death pathways as targets for cancer therapy. J Cell Mol Med 11: 239–251.
