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Mechanisms underlying lack of insulin-like growth factor-binding protein-3 expression in non-small-cell lung cancer

Oncogene volume 23, pages 6569–6580 (2004)Cite this article

Abstract

Expression of insulin-like growth factor-binding protein-3, which (IGFBP-3) inhibits the proliferation of non-small-cell lung cancer (NSCLC) cells by inducing apoptosis, is lost in about half of stage I NSCLC cases. Since promoter methylation can silence gene expression, we investigated whether hypermethylation of the IGFBP-3 promoter is involved in loss of IGFBP-3 expression in NSCLC. We found the IGFBP-3 promoter to be methylated in seven of 13 NSCLC cell lines and in 16 of 23, seven of 9, eight of 11, and six of six tumor specimens from patients with stage I, II, III, and IV NSCLC, respectively. Methylation status correlated with IGFBP-3 mRNA and protein levels in a subset of NSCLC cell lines tested in our study. However, treatment with 5′-aza-2′-deoxycytidine (5′-aza-dC) restored IGFBP-3 expression in four of seven NSCLC cell lines with the methylated promoter, suggesting that multiple mechanisms regulate IGFBP-3 expression in NSCLC. Gel shift and chromatin immunoprecipitation assays showed that methylation of the Sp-1/Sp-3-binding element in the IGFBP-3 promoter influenced the binding of Sp-1, methyl-CpG-binding protein-2 (MeCP2), and histone deacetylase (HDAC). A luciferase construct expressing IGFBP-3 promoter in which the Sp-1/Sp-3 binding element was methylated showed significantly reduced transcriptional activity. The reduction in promoter activity was further suppressed by overexpression of MeCP2, which was rescued by 5′-aza-dC. Thus interference with Sp-1 transactivation by MeCP2 may contribute to the transcriptional defect of IGFBP-3 expression in NSCLC cells with methylated promoter.

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Abbreviations

IGFBP-3:

insulin-like growth factor-binding protein-3

NSCLC:

non-small-cell lung cancer

HDAC:

histone deacetylase

MeCP2:

methyl-CpG-binding protein-2

TRD:

transcription repression domain

FCS:

fetal calf serum

SDS:

sodium dodecyl sulfate

ECL:

enhanced chemiluminescence

GAPDH:

glyceraldehyde-3 phosphate dehydrogenase

5′-aza-dC:

5′-aza-2′-deoxycytidine

PCR:

polymerase chain reaction

ChIP assay:

chromatin immunoprecipitation assay

MSP:

methylation-specific polymerase chain reaction

MBD:

methyl CpG-binding domain

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Acknowledgements

This work was supported by National American Cancer Society RSG-04-082-01-TBE 01 (to H-Y Lee), National Institutes of Health Grants1RO1 CA100816-01A1 (to H-Y Lee), and Department of Defense grant DAMD 17-01-1-0689 (to WK Hong) from the Department of Defense.

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Authors and Affiliations

  1. Department of Thoracic Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Box 432, 1515 Holcombe Boulevard, Houston, 77030, TX, USA

    Yoon Soo Chang, Luo Wang, Young-Ah Suh, Li Mao, Waun Ki Hong & Ho-Young Lee

  2. Department of Pediatrics, The Texas Children's Liver Center, Baylor College of Medicine, Houston, 77030, TX, USA

    Saul J Karpen

  3. Hematology and Medical Oncology, Translational Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA

    Fadlo R Khuri

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  1. Yoon Soo Chang
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Correspondence to Ho-Young Lee.

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Chang, Y., Wang, L., Suh, YA. et al. Mechanisms underlying lack of insulin-like growth factor-binding protein-3 expression in non-small-cell lung cancer. Oncogene 23, 6569–6580 (2004). https://doi.org/10.1038/sj.onc.1207882

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