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. 1998 Jan 1;101(1):153–159. doi: 10.1172/JCI831

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

R K Ravi 1, E Weber 1, M McMahon 1, J R Williams 1, S Baylin 1, A Mal 1, M L Harter 1, L E Dillehay 1, P P Claudio 1, A Giordano 1, B D Nelkin 1, M Mabry 1
PMCID: PMC508551  PMID: 9421477

Abstract

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.

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Selected References

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  1. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995 Nov 17;270(46):27489–27494. doi: 10.1074/jbc.270.46.27489. [DOI] [PubMed] [Google Scholar]
  2. Avruch J., Zhang X. F., Kyriakis J. M. Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci. 1994 Jul;19(7):279–283. doi: 10.1016/0968-0004(94)90005-1. [DOI] [PubMed] [Google Scholar]
  3. Carney D. N., Gazdar A. F., Bepler G., Guccion J. G., Marangos P. J., Moody T. W., Zweig M. H., Minna J. D. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 1985 Jun;45(6):2913–2923. [PubMed] [Google Scholar]
  4. Carson E. B., McMahon M., Baylin S. B., Nelkin B. D. Ret gene silencing is associated with Raf-1-induced medullary thyroid carcinoma cell differentiation. Cancer Res. 1995 May 15;55(10):2048–2052. [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Dulić V., Lees E., Reed S. I. Association of human cyclin E with a periodic G1-S phase protein kinase. Science. 1992 Sep 25;257(5078):1958–1961. doi: 10.1126/science.1329201. [DOI] [PubMed] [Google Scholar]
  7. Harbour J. W., Lai S. L., Whang-Peng J., Gazdar A. F., Minna J. D., Kaye F. J. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science. 1988 Jul 15;241(4863):353–357. doi: 10.1126/science.2838909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hengst L., Reed S. I. Translational control of p27Kip1 accumulation during the cell cycle. Science. 1996 Mar 29;271(5257):1861–1864. doi: 10.1126/science.271.5257.1861. [DOI] [PubMed] [Google Scholar]
  9. Hensel C. H., Hsieh C. L., Gazdar A. F., Johnson B. E., Sakaguchi A. Y., Naylor S. L., Lee W. H., Lee E. Y. Altered structure and expression of the human retinoblastoma susceptibility gene in small cell lung cancer. Cancer Res. 1990 May 15;50(10):3067–3072. [PubMed] [Google Scholar]
  10. Hirakawa T., Ruley H. E. Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1519–1523. doi: 10.1073/pnas.85.5.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. King R. W., Jackson P. K., Kirschner M. W. Mitosis in transition. Cell. 1994 Nov 18;79(4):563–571. doi: 10.1016/0092-8674(94)90542-8. [DOI] [PubMed] [Google Scholar]
  12. Koff A., Giordano A., Desai D., Yamashita K., Harper J. W., Elledge S., Nishimoto T., Morgan D. O., Franza B. R., Roberts J. M. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science. 1992 Sep 18;257(5077):1689–1694. doi: 10.1126/science.1388288. [DOI] [PubMed] [Google Scholar]
  13. Kuo W. L., Abe M., Rhee J., Eves E. M., McCarthy S. A., Yan M., Templeton D. J., McMahon M., Rosner M. R. Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells. Mol Cell Biol. 1996 Apr;16(4):1458–1470. doi: 10.1128/mcb.16.4.1458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Li H., Kawasaki H., Nishida E., Hattori S., Nakamura S. Ras-regulated hypophosphorylation of the retinoblastoma protein mediates neuronal differentiation in PC12 cells. J Neurochem. 1996 Jun;66(6):2287–2294. doi: 10.1046/j.1471-4159.1996.66062287.x. [DOI] [PubMed] [Google Scholar]
  15. Mabry M., Nakagawa T., Baylin S., Pettengill O., Sorenson G., Nelkin B. Insertion of the v-Ha-ras oncogene induces differentiation of calcitonin-producing human small cell lung cancer. J Clin Invest. 1989 Jul;84(1):194–199. doi: 10.1172/JCI114140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mal A., Poon R. Y., Howe P. H., Toyoshima H., Hunter T., Harter M. L. Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFbeta-treated cells. Nature. 1996 Mar 21;380(6571):262–265. doi: 10.1038/380262a0. [DOI] [PubMed] [Google Scholar]
  17. Marshall M. Interactions between Ras and Raf: key regulatory proteins in cellular transformation. Mol Reprod Dev. 1995 Dec;42(4):493–499. doi: 10.1002/mrd.1080420418. [DOI] [PubMed] [Google Scholar]
  18. Mitsudomi T., Viallet J., Mulshine J. L., Linnoila R. I., Minna J. D., Gazdar A. F. Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene. 1991 Aug;6(8):1353–1362. [PubMed] [Google Scholar]
  19. Morgan D. O. Principles of CDK regulation. Nature. 1995 Mar 9;374(6518):131–134. doi: 10.1038/374131a0. [DOI] [PubMed] [Google Scholar]
  20. Nakagawa T., Mabry M., de Bustros A., Ihle J. N., Nelkin B. D., Baylin S. B. Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5923–5927. doi: 10.1073/pnas.84.16.5923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ohtsubo M., Theodoras A. M., Schumacher J., Roberts J. M., Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995 May;15(5):2612–2624. doi: 10.1128/mcb.15.5.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pang L., Sawada T., Decker S. J., Saltiel A. R. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995 Jun 9;270(23):13585–13588. doi: 10.1074/jbc.270.23.13585. [DOI] [PubMed] [Google Scholar]
  23. Polyak K., Kato J. Y., Solomon M. J., Sherr C. J., Massague J., Roberts J. M., Koff A. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 1994 Jan;8(1):9–22. doi: 10.1101/gad.8.1.9. [DOI] [PubMed] [Google Scholar]
  24. Polyak K., Lee M. H., Erdjument-Bromage H., Koff A., Roberts J. M., Tempst P., Massagué J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994 Jul 15;78(1):59–66. doi: 10.1016/0092-8674(94)90572-x. [DOI] [PubMed] [Google Scholar]
  25. Pumiglia K. M., Decker S. J. Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):448–452. doi: 10.1073/pnas.94.2.448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Resnitzky D., Reed S. I. Different roles for cyclins D1 and E in regulation of the G1-to-S transition. Mol Cell Biol. 1995 Jul;15(7):3463–3469. doi: 10.1128/mcb.15.7.3463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reynisdóttir I., Polyak K., Iavarone A., Massagué J. Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta. Genes Dev. 1995 Aug 1;9(15):1831–1845. doi: 10.1101/gad.9.15.1831. [DOI] [PubMed] [Google Scholar]
  28. Ridley A. J., Paterson H. F., Noble M., Land H. Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation. EMBO J. 1988 Jun;7(6):1635–1645. doi: 10.1002/j.1460-2075.1988.tb02990.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Samuels M. L., Weber M. J., Bishop J. M., McMahon M. Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase. Mol Cell Biol. 1993 Oct;13(10):6241–6252. doi: 10.1128/mcb.13.10.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schauer I. E., Siriwardana S., Langan T. A., Sclafani R. A. Cyclin D1 overexpression vs. retinoblastoma inactivation: implications for growth control evasion in non-small cell and small cell lung cancer. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7827–7831. doi: 10.1073/pnas.91.16.7827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Seger R., Krebs E. G. The MAPK signaling cascade. FASEB J. 1995 Jun;9(9):726–735. [PubMed] [Google Scholar]
  32. Serrano M., Lin A. W., McCurrach M. E., Beach D., Lowe S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 1997 Mar 7;88(5):593–602. doi: 10.1016/s0092-8674(00)81902-9. [DOI] [PubMed] [Google Scholar]
  33. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995 May 15;9(10):1149–1163. doi: 10.1101/gad.9.10.1149. [DOI] [PubMed] [Google Scholar]
  34. Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
  35. Takahashi T., Nau M. M., Chiba I., Birrer M. J., Rosenberg R. K., Vinocour M., Levitt M., Pass H., Gazdar A. F., Minna J. D. p53: a frequent target for genetic abnormalities in lung cancer. Science. 1989 Oct 27;246(4929):491–494. doi: 10.1126/science.2554494. [DOI] [PubMed] [Google Scholar]
  36. Toyoshima H., Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994 Jul 15;78(1):67–74. doi: 10.1016/0092-8674(94)90573-8. [DOI] [PubMed] [Google Scholar]
  37. Tsai L. H., Lees E., Faha B., Harlow E., Riabowol K. The cdk2 kinase is required for the G1-to-S transition in mammalian cells. Oncogene. 1993 Jun;8(6):1593–1602. [PubMed] [Google Scholar]
  38. Vindeløv L. L., Christensen I. J., Nissen N. I. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry. 1983 Mar;3(5):323–327. doi: 10.1002/cyto.990030503. [DOI] [PubMed] [Google Scholar]
  39. Williams N. G., Roberts T. M. Signal transduction pathways involving the Raf proto-oncogene. Cancer Metastasis Rev. 1994 Mar;13(1):105–116. doi: 10.1007/BF00690421. [DOI] [PubMed] [Google Scholar]
  40. Wood K. W., Qi H., D'Arcangelo G., Armstrong R. C., Roberts T. M., Halegoua S. The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5016–5020. doi: 10.1073/pnas.90.11.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]

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