Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3

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Abstract

Allelic loss at the short arm of chromosome 3 is one of the most common and earliest events in the pathogenesis of lung cancer, and is observed in more than 90% of small-cell lung cancers (SCLCs) and in 50–80% of non-small-cell lung cancers1,2 (NSCLCs). Frequent and early loss of heterozygosity and the presence of homozygous deletions suggested a critical role of the region 3p21.3 in tumorigenesis2,3,4 and a region of common homozygous deletion in 3p21.3 was narrowed to 120 kb (ref. 5). Several putative tumour-suppressor genes located at 3p21 have been characterized, but none of these genes appear to be altered in lung cancer. Here we describe the cloning and characterization of a human RAS effector homologue (RASSF1) located in the 120-kb region of minimal homozygous deletion. We identified three transcripts, A, B and C, derived from alternative splicing and promoter usage. The major transcripts A and C were expressed in all normal tissues. Transcript A was missing in all SCLC cell lines analysed and in several other cancer cell lines. Loss of expression was correlated with methylation of the CpG-island promoter sequence of RASSF1A. The promoter was highly methylated in 24 of 60 (40%) primary lung tumours, and 4 of 41 tumours analysed carried missense mutations. Re-expression of transcript A in lung carcinoma cells reduced colony formation, suppressed anchorage-independent growth and inhibited tumour formation in nude mice. These characteristics indicate a potential role for RASSF1A as a lung tumour suppressor gene.

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Figure 1: Location of genes and deletions in 3p21.3 and amino acid comparison.
Figure 2: Northern-blot analysis of RASSF1A, RASSF1B and RASSF1C transcripts in human tissues and cancer cell lines.
Figure 3: Expression of RASSF1 in normal and tumour cell lines.
Figure 4: Methylation analysis of RASSF1A.
Figure 5: Re-expression of transcript A by treatment with 5-aza-2′-deoxycytidine (5-Aza-CdR).
Figure 6: Effect of RASSF1A expression on cell growth characteristics.

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References

  1. 1

    Whang-Peng, J. et al. Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23). Science 215, 181–182 (1982).

  2. 2

    Kok, K., Naylor, S.L. & Buys, C.H.C.M. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv. Cancer Res. 71, 27–92 (1997).

  3. 3

    Hung, J. et al. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA 273, 558–563 (1995).

  4. 4

    Wistuba, I.I. et al. Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma. Oncogene 18, 643–650 (1999).

  5. 5

    Sekido, Y. et al. Cloning of a breast cancer homozygous deletion junction narrows the region of search for a 3p21.3 tumor suppressor gene. Oncogene 16, 3151–3157 (1998).

  6. 6

    Wei, M.-H. et al. Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG. Cancer Res. 56, 1487–1492 (1996).

  7. 7

    Vavvas, D., Li, X., Avruch, J. & Zhang, X.-F. Identification of Nore1 as a potential Ras effector. J. Biol. Chem. 273, 5439–5442 (1998).

  8. 8

    Ponting, C.P. & Benjamin, D.R. A novel family of Ras-binding domains. Trends Biochem. Sci. 21, 422–425 (1996).

  9. 9

    Newton, A.C. Seeing two domains. Curr. Biol. 5, 973–976 (1995).

  10. 10

    Baylin, S.B., Herman, J.G., Graff, J.R., Vertino, P.M. & Issa, J.P. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res. 72, 141–196 (1998).

  11. 11

    Jones, P.A. & Laird, P.W. Cancer epigenetics comes of age. Nature Genet. 21, 163–167 (1999).

  12. 12

    Costello, J.F. et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nature Genet. 24, 132–138 (2000).

  13. 13

    Eng, C., Herman, J.G. & Baylin, S.B. A bird's eye view of global methylation. Nature Genet. 24, 101–102 (2000).

  14. 14

    Clark, S.J., Harrison, J., Paul, C.L. & Frommer, M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997 (1994).

  15. 15

    Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

  16. 16

    Szabo, P.E. & Mann, J.R. Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting. Genes Dev. 9, 1857–1868 (1995).

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Acknowledgements

We thank C.A. Tessler for technical support. This work was supported by a grant from the University of California Tobacco Related Disease Research Program (6RT-0361 to G.P.P.).

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Correspondence to Gerd P. Pfeifer.

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