Influence of small interfering RNA corresponding to ets homologous factor on senescence-associated modulation of prostate carcinogenesis

Chaehwa Park,Won Ki Kang,Inkyoung Lee

doi:10.1158/1535-7163.mct-06-0570

Abstract

Senescence is thought to be an inherent tumor-suppressive mechanism. In the process of identifying senescence-associated genes, we found significant suppression of the ets homologous factor (EHF) in cancer cells in a state of DNA damage-induced senescence. In this study, we show that EHF provides substantial drug resistance in PC-3 prostate cancer cells by inhibiting senescence and cell cycle arrest. Knockdown of EHF by small interfering RNA inhibited cell proliferation and induced a premature cellular senescence characterized by hypophosphorylation of Rb and increased level of p27, with concomitant decreases of cyclin A, cdc2, and E2F1. Telomeric repeat amplification protocol analysis showed that transient EHF knockdown significantly decreased telomerase activity, whereas this activity was increased by overexpression of EHF. In vivo tumorigenesis analyses revealed that tumors derived from EHF knockdown cells were significantly smaller than those derived from control cells (P < 0.0001). Further, the preestablished tumors were reduced after the injection of small interfering RNA corresponding to EHF (P = 0.0122). Collectively, these observations indicate that aberrant expression of EHF and the subsequent disruption of p27-mediated senescence and telomerase activity is likely to contribute significantly to tumor progression, and furthermore that EHF might be a promising target for future cancer therapeutics.

Highlights

Senescence, which is considered a major determinant of treatment outcome in cancer therapy (1 – 3), plays a pivotal role in safeguarding higher organisms against tumorigenesis by suppressing the emergence of immortal cells [4, 5]
To determine whether inhibition of ets homologous factor (EHF) is a cause or consequence of cell growth arrest and senescence, we investigated the effects of EHF knockdown on the expression levels of cell cycle proteins in primary mouse embryonic fibroblasts (MEF)
We found that treatment of MEFs with the EHF-targeting small interfering RNA (siRNA) resulted in decreased EHF, enlargement and flattening of the cells, and positive SA-h-Gal activity

Summary

Introduction

Senescence, which is considered a major determinant of treatment outcome in cancer therapy (1 – 3), plays a pivotal role in safeguarding higher organisms against tumorigenesis by suppressing the emergence of immortal cells [4, 5]. Senescence in human cells is associated with specific physiologic and morphologic changes, including reduced proliferation, shortened telomeres, a flat and enlarged cell shape, and the appearance of senescenceassociated h-galactosidase (SA-h-Gal) activity [4]. The growth arrest seen during senescence has been associated with up-regulation of various negative mediators of the cell cycle, including p53; pRb; and the cyclin-dependent kinase inhibitors p21CIP1, p27Kip, and p16INK4a (5 – 9). In a cDNA microarray hybridization analysis, we found that DNA damage – induced senescence selectively inhibits a set of genes, including the ets homologous factor (EHF), which is encoded by a divergent ets gene located at human chromosome 11p12. Most ets proteins are oncoproteins; they are upregulated in proliferating cells and may be activated by chromosomal translocations in human malignancies (12 – 14). A single study has shown that EHF is aberrantly expressed in cancers [15], but the precise functions of EHF in modulating senescence and/or carcinogenesis remain unknown

Methods

Results

Conclusion