Gemcitabine-mediated tumour regression and p53-dependent gene expression: implications for colon and pancreatic cancer therapy
R Hill, M Rabb, P A Madureira, D Clements, S A Gujar, D M Waisman, C A Giacomantonio, P W K Lee, R Hill, M Rabb, P A Madureira, D Clements, S A Gujar, D M Waisman, C A Giacomantonio, P W K Lee
Abstract
Gemcitabine is a chemotherapeutic that is widely used for the treatment of a variety of haematological malignancies and has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Combinational gemcitabine regimes (e.g.with doxorubicin) are being tested in clinical trials to treat a variety of cancers, including colon cancer. The limited success of these trials has prompted us to pursue a better understanding of gemcitabine's mechanism of cell killing, which could dramatically improve the therapeutic potential of this agent. For comparison, we included gamma irradiation that triggers robust cell cycle arrest and Cr(VI), which is a highly toxic chemical that induces a robust p53-dependent apoptotic response. Gemcitabine induced a potent p53-dependent apoptosis that correlated with the accumulation of pro-apoptotic proteins such as PUMA and Bax. This is accompanied by a drastic reduction in p2l and 14-3-3σ protein levels, thereby significantly sensitizing the cells to apoptosis. In vitro and in vivo studies demonstrated that gemcitabine required PUMA transcription to instigate an apoptotic programme. This was in contrast to Cr(VI)-induced apoptosis that required Bax and was independent of transcription. An examination of clinical colon and pancreatic cancer tissues shows higher p53, p21, 14-3-3σ and Bax expression compared with matched normal tissues, yet there is a near absence of PUMA protein. This may explain why gemcitabine shows only limited efficacy in the treatment of these cancers. Our results raise the possibility that targeting the Bax-dependent cell death pathway, rather than the PUMA pathway, could result in significantly improved patient outcome and prognosis for these cancers.
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References
- Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell. 1992;70:523–526.
- Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 1995;55:5187–5190.
- Wahl GM, Carr AM. The evolution of diverse biological responses to DNA damage: insights from yeast and p53. Nat Cell Biol. 2001;3:E277–E286.
- Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature. 2007;445:656–660.
- Pietenpol JA, Tokino T, Thiagalingam S, el-Deiry WS, Kinzler KW, Vogelstein B. Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc Natl Acad Sci USA. 1994;91:1998–2002.
- Zupnick A, Prives C. Mutational analysis of the p53 core domain L1 loop. J Biol Chem. 2006;281:20464–20473.
- Qian H, Wang T, Naumovski L, Lopez CD, Brachmann RK. Groups of p53 target genes involved in specific p53 downstream effects cluster into different classes of DNA binding sites. Oncogene. 2002;21:7901–7911.
- Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell. 2001;7:683–694.
- Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang L. PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci USA. 2003;100:1931–1936.
- Chipuk JE, Green DR. Cytoplasmic p53: bax and forward. Cell Cycle. 2004;3:429–431.
- Chipuk JE, Green DR. p53's believe it or not: lessons on transcription-independent death. J Clin Immunol. 2003;23:355–361.
- Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, et al. p53 has a direct apoptogenic role at the mitochondria. Mol Cell. 2003;11:577–590.
- Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science. 2004;303:1010–1014.
- Erster S, Mihara M, Kim RH, Petrenko O, Moll UM. In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation. Mol Cell Biol. 2004;24:6728–6741.
- Leu JI, Dumont P, Hafey M, Murphy ME, George DL. Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol. 2004;6:443–450.
- Abbruzzese JL. New applications of gemcitabine and future directions in the management of pancreatic cancer. Cancer. 2002;95 (4 Suppl:941–945.
- Macdonald JS. Clinical overview: adjuvant therapy of gastrointestinal cancer. Cancer Chemother Pharmacol. 2004;54 (Suppl 1:S4–11.
- Okusaka T, Ishii H, Funakoshi A, Ueno H, Furuse J, Sumii T. A phase I/II study of combination chemotherapy with gemcitabine and 5-fluorouracil for advanced pancreatic cancer. Jpn J Clin Oncol. 2006;36:557–563.
- Marsman DS, Barrett JC. Apoptosis and chemical carcinogenesis. Risk Anal. 1994;14:321–326.
- O'Brien TJ, Fornsaglio JL, Ceryak S, Patierno SR. Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae. DNA Repair (Amst) 2002;1:617–627.
- Salnikow K, Zhitkovich A, Costa M. Analysis of the binding sites of chromium to DNA and protein in vitro and in intact cells. Carcinogenesis. 1992;13:2341–2346.
- O'Brien TJ, Ceryak S, Patierno SR. Complexities of chromium carcinogenesis: role of cellular response, repair and recovery mechanisms. Mutat Res. 2003;533:3–36.
- Laronga C, Yang HY, Neal C, Lee MH. Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression. J Biol Chem. 2000;275:23106–23112.
- Hill R, Leidal AM, Madureira PA, Gillis LD, Cochrane HK, Waisman DM, et al. Hypersensitivity to chromium-induced DNA damage correlates with constitutive deregulation of upstream p53 kinases in p21-/- HCT116 colon cancer cells. DNA Repair (Amst) 2008;7:239–252.
- Javelaud D, Besancon F. Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio. J Biol Chem. 2002;277:37949–37954.
- Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene. 2001;20:1803–1815.
- Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, et al. 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell. 1997;1:3–11.
- Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B. 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature. 1999;401:616–620.
- Hill R, Leidal AM, Madureira PA, Gillis LD, Waisman DM, Chiu A, et al. Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNA-PK) and differential induction of p53 target genes. DNA Repair (Amst) 2008;7:1484–1499.
- Hill R, Madureira PA, Waisman DM, Lee PW. DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death. Oncotarget. 2011;2:1094–1108.
- Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991;51 (23 Pt 1:6304–6311.
- Price BD, Youmell MB. The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage. Cancer Res. 1996;56:246–250.
- Suzuki A, Tsutomi Y, Akahane K, Araki T, Miura M. Resistance to Fas-mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene. 1998;17:931–939.
- Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science. 1991;253:49–53.
- Erster S, Slade N, Moll UM. Mutational analysis of p53 in human tumors: direct DNA sequencing and SSCP. Methods Mol Biol. 2003;234:219–230.
- Singh J, Carlisle DL, Pritchard DE, Patierno SR. Chromium-induced genotoxicity and apoptosis: relationship to chromium carcinogenesis (review) Oncol Rep. 1998;5:1307–1318.
- Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol. 2012;52:1213–1225.
- Ewer MS, Ewer SM. Cardiotoxicity of anticancer treatments: what the cardiologist needs to know. Nat Rev Cardiol. 2010;7:564–575.
- Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gallinger S, et al. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 2007;25:1960–1966.
- Van CE, Verslype C, Grusenmeyer PA. Lessons learned in the management of advanced pancreatic cancer. J Clin Oncol. 2007;25:1949–1952.
- Okusaka T, Furuse J, Funakoshi A, Ioka T, Yamao K, Ohkawa S, et al. Phase II study of erlotinib plus gemcitabine in Japanese patients with unresectable pancreatic cancer. Cancer Sci. 2011;102:425–431.
- Kindler HL, Ioka T, Richel DJ, Bennouna J, Letourneau R, Okusaka T, et al. Axitinib plus gemcitabine versus placebo plus gemcitabine in patients with advanced pancreatic adenocarcinoma: a double-blind randomised phase 3 study. Lancet Oncol. 2011;12:256–262.
- Grayson S, Elias PM. Human epidermal proteolipids: isolation, partial characterization, and subcellular localization. J Invest Dermatol. 1988;90:185–192.
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408.
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