A chimeric fusion of the human Achaete Scute Homolog 1 (hASH1) and the Enhancer of Zeste Homolog 2 (EZH2) promoters is a promising regulator of suicide gene therapy in small cell lung cancer (SCLC)

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Abstract:

SCLC is an aggressive disease with no satisfactory treatment presently available. Due to its disseminated nature SCLC must be treated systemically in order to target both the primary tumor and metastases. Transcriptionally targeted suicide gene therapy constitutes such a systemic strategy with a therapeutic potential for SCLC.

The hASH1 gene is almost exclusively inactive in adult normal human tissues but highly reactivated in certain neuroendocrine malignancies such as SCLC. The neuroendocrine specificity of hASH1 is mediated by the hASH1 promoter, which comprises a generally active enhancer and specific repressor regions confining gene expression exclusively to SCLC cells and certain cell types of neuroendocrine origin. The EZH2 gene is generally upregulated in many cancers by a transcriptional mechanism involving binding of E2F transcription factors to the promoter region.

The purpose of the current study has been to investigate the use of hASH1- and EZH2-promoter regions alone and in combination for regulating suicide gene therapy of SCLC.

Two promoter regions comprising 0,3kb and 0,7kb immediately upstream of – and including the hASH1 transcription start site were cloned. Both regions induced high gene activity in all tested hASH1-positive and two hASH1 negative SCLC cell lines, whereas gene activity was low or absent in one hASH1-negative SCLC and four non-neuroendocrine control cell lines tested.

To evaluate its therapeutic potential, the 0,7kb hASH1 proximal promoter region was fused to the Herpes Simplex Virus Thymidine Kinase (HSVTK) gene and SCLC and control cell lines were transfected, followed by administration of Ganciclovir (GCV). The hASH1-HSVTK construct conferred SCLC-cell death equivalent to that observed with an unspecific control construct. In contrast, transfected control cell lines were unaffected by GCV-treatment.

A 1,1kb EZH2 promoter was tested analogously. The EZH2-regulated reporter gene construct was highly active in the SCLC cell lines (up to 25 fold SV40-promoter activity), while moderate reporter gene activity was detected in the control cells (<12% of SV40 activity). However, in the cytotoxicity assay, the EZH2-promoter was fully capable of inducing cell death in a control cell line with moderate EZH2 promoter activity, suggesting lack of specificity.

Finally, the 0,7kb hASH1- and 1,1kb EZH2-promoters were fused in a chimeric construct and evaluated for SCLC specificity and activity. The chimeric construct demonstrated increased activity and cytotoxicity in SCLC cells when compared to the hASH1 construct alone, while fully retaining SCLC specificity as demonstrated by low reporter gene activity and lack of cytotoxicity in control cells.

In conclusion, the highly active and cancer specific hASH1EZH2 promoter construct constitutes a promising transcriptional regulator for gene therapy of SCLC and possibly other neuroendocrine malignancies.

Document Type: Abstract

DOI: http://dx.doi.org/10.1111/j.1600-0463.2008.001165_23.x

Publication date: May 1, 2008

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