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Mol Cancer Ther. 2006;5:3191-3196
© 2006 American Association for Cancer Research

Research Articles: Therapeutics, Targets, and Development

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

Chaehwa Park1, Inkyoung Lee2 and Won Ki Kang3

1 Cancer Center, Samsung Medical Center, Samsung Biomedical Research Institute; 2 Samsung Biomedical Research Institute; and 3 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Requests for reprints: Chaehwa Park or Won Ki Kang, Cancer Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Seoul 135-710, Korea. Phone: 82-2-3410-3458; Fax: 82-2-3410-6808. E-mail: cpark{at}smc.samsung.co.kr or wkkang{at}smc.samsung.co.kr

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. [Mol Cancer Ther 2006;5(12):3191–6]

Introduction

Senescence, which is considered a major determinant of treatment outcome in cancer therapy (1–3), also 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 senescence-associated ß-galactosidase (SA-ß-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, p27Kip1, and p16INK4a (5–9).

To improve our understanding of how malignant cells escape senescence, it may be useful to define the genes that control cellular senescence. 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. The ets family transcription factors are characterized by a conserved DNA-binding domain known as the ETS domain (10, 11). Most ets proteins are oncoproteins; they are up-regulated in proliferating cells and may be activated by chromosomal translocations in human malignancies (12–14). To date, 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.

In the present study, we explored the role of EHF in regulating cell proliferation and senescence and sought to identify some of the involved mechanisms. Specifically, we investigated the effects of EHF expression on the cellular response to DNA-damaging agents in PC-3 human cancer cells. Our results revealed that even in the presence of an intact pRb pathway, EHF expression led to decreased senescence and increased doxorubicin resistance. Furthermore, EHF expression down-regulated multiple genes involved in growth arrest or senescence, leading to decrease of p27 in doxorubicin-treated cells. Knockdown of p27 effectively blocked senescence induced by small interfering RNA (siRNA)–mediated knockdown of EHF, supporting the notion that p27 is critical for the effects of EHF on senescence. Finally, we showed that telomerase activity was up-regulated by EHF expression and that differential expression of EHF affected in vivo tumorigenesis in athymic nude mice. Collectively, these findings strongly suggest that the effect of EHF on cell proliferation and senescence contributes to tumor progression in vivo.

Materials and Methods

Cell Culture and Transfection
Human prostate carcinoma (DU145 and PC-3) and breast cancer cells (MDA-MB468) were grown in RPMI 1640 (Life Technologies Life Science, Grand Island, NY). The 21-nucleotide-long siRNAs corresponding to EHF (siEHF) and a control scrambled siRNA (siScr) were purchased from Dharmacon (Lafayette, CO). Cells were transfected with siRNA or pCMV/EHF using the Amaxa electroporation system, according to the supplier's protocol (Amaxa, Gaithersburg, MD).

Reagents and Vectors
Doxorubicin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside were purchased from Sigma (St. Louis, MO). Antibodies against Rb, p16, p21, p27, E2F-1, cyclin A, cdc2, Flag, and ß-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The full-length EHF open reading frame was obtained from normal fibroblast mRNA using a reverse transcription-PCR–based cloning technique, and inserted into pCMVTaq4C (Invitrogen, Carlsbad, CA).

SA-ß-Gal Staining
Cells were seeded into six-well plates in RPMI 1640 and transfected with siRNA (100 nmol/L), and SA-ß-Gal staining was done as previously described (9). Senescence was scored based on the percentage of the population that exhibited a SA-ß-Gal activity, and the results were photographed under phase-contrast microscopy.

Telomerase Assay
The telomere repeat amplification protocol was done using a Telo TAGGG telomerase ELISA kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Briefly, the telomerase extension reaction was done at 25°C for 30 min, and PCR was done using the provided primers and 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s.

In vivo Tumorigenesis Analyses
We examined in vivo tumorigenesis of PC-3 cells expressing EHF-targeting or control siRNAs. Briefly, 1.5 x 106 cells were implanted s.c. into the flanks of 4-week old female athymic nu/nu mice (Charles River Laboratories, Atsugi, Japan). For a siEHF treatment model, the mice received intratumoral injections of EHF siRNA at 3 weeks after PC-3 injection. Mice were maintained and sacrificed according to institutional guidelines, and the procedures were approved by the Institutional Committee on the Use and Care of Animals and Recombinant DNA research.

Results

Identification of EHF as a Senescence-Associated Molecular Marker
DNA damage–induced senescence can be triggered by treatment of human cancer cells with a low dose of doxorubicin (16). We found that EHF expression was decreased in senescence as shown in Fig. 1A . To determine whether inhibition of 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 siRNA resulted in decreased EHF, enlargement and flattening of the cells, and positive SA-ß-Gal activity (ref. 3; Supplementary Fig. S1A).4


Figure 1
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  		Figure 1. EHF as a senescence-associated molecular marker. A, time course expression of EHF RNA in DNA damage–induced PC-3 cell senescence (top). Doxorubicin (10 nmol/L) treatment was done for 5 d and the cells were harvested at the indicated intervals and evaluated for EHF expression by reverse transcription-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Expression of EHF in PC-3 after transfection of siRNAs (100 nmol/L) was examined by reverse transcription-PCR (bottom). Reverse transcription-PCR was done using primers designed from the coding region of the human EHF cDNA (sense, 5'-TGCTGGTGAATGCCCTCTACT-3' and antisense, 5'-CGGTCATTCCCAGGTTCTCTA-3'). The PCR conditions consisted of 25 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, followed by a final incubation at 72°C for 10 min. B, effects of siEHF on the proliferation of PC-3. PC-3 cells were transfected with PBS, siScr, or siEHF. Columns, percentage survival of cells counted 3 d after the transfection; bars, SD. SA-ß-Gal staining of PC-3 was done 5 d after siRNA (100 nmol/L) transfection. C, siRNA-mediated knockdown of EHF-induced senescence in PC-3 (p53 and p16 deficient), DU145 (p53 and pRb mutant), and MDA-MB468 (p53 and pRb mutant). D, EHF overexpression rescued siEHF-induced senescence and the corresponding SA-ß-Gal activity.

 
To further characterize the nature of the senescence caused by siEHF, we examined the expression of several cell cycle regulatory proteins in EHF-depleted primary cells. Our results revealed that EHF-knockdown cells had increased levels of p16 and p27 expression but decreased levels of Rb phosphorylation, cyclin A, cdc2, and E2F1 (Supplementary Fig. S1B)4. Interestingly, EHF knockdown did not trigger increased p21 expression.

EHF Knockdown Inhibits Growth and Causes Premature Senescence in PC-3 Cells
We next examined the effect of siEHF on PC-3 human prostate cancer cells. Reverse transcription-PCR analysis revealed that siEHF-transfected PC-3 cells showed decreased EHF mRNA expression within 1 day of transfection (Fig. 1A), indicating successful knockdown. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that siEHF specifically inhibited the proliferation of PC-3 cells, whereas the negative control siRNA (siScr) did not (Fig. 1B). Notably, the accumulation of SA-ß-Gal activity was siEHF dose-dependent (Fig. 1B). Previously, we observed DNA damage–induced cellular senescence in PC-3 (p53- and p16-deficient) and MDA-MB468 (p53- and pRb-mutated), but not in DU145 (Rb-, p53-, and p16-mutated) cells (9). In contrast, PC-3, DU145, and MDA-MB468 (p53- and pRb-mutated) cells transfected with siEHF all displayed senescent morphologies and positive SA-ß-Gal staining (Fig. 1C). As shown in Fig. 1D, EHF overexpression effectively rescued siEHF-induced senescence and the corresponding SA-ß-Gal positivity, indicating that these biological changes were EHF mediated. Consistent with our findings in primary MEFs, we observed that EHF-knockdown PC-3 cells showed increased p27 protein expression and decreased Rb phosphorylation (Fig. 2A ). The expression level of p27 protein increased gradually after siEHF transfection and remained high for the 6-day experimental period (Fig. 2A).


Figure 2
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  		Figure 2. Effects of p27 on siEHF-induced cellular senescence of PC-3 cells. A, the siRNA-mediated EHF knockdown modulates cell cycle gene expression. PC-3 cells were seeded at a concentration of 2 x 105/mL before siEHF transfection and harvested at the indicated intervals. The cells were lysed and 20 µg of protein were loaded into each lane. Western blots were hybridized with the antibodies shown on the right. ß-Actin was used as a loading control. B, Western blot analysis of lysates from PC-3 cells transfected with siRNAs. 100 nmol/L siScr or siEHF, with or without sip27, were cotransfected into PC-3 cells. After 1 d, cells were harvested and analyzed for p27 expression. C and D, percentages of cells that were positively stained for SA-ß-Gal. PC-3 cells were stained for SA-ß-Gal 5 d after transfection with EHF siRNA (100 nmol/L).

 
SiEHF-Induced Senescence Requires p16 or p27
Because EHF knockdown resulted in increased levels of cell cycle regulatory proteins, we next examined whether these proteins were required for siEHF-induced senescence. More specifically, because p27 expression is enhanced by siEHF in low-passage MEF (P2) and PC-3 cells (Supplementary Fig. S1B;4 Fig. 2A), we hypothesized that EHF modulates senescence-associated cell cycle arrest by regulating p27. Our results revealed that the presence of sip27 prevented accumulation of p27 protein and reduced the SA-ß-Gal–positive population in MEFs and PC-3 cells, compared with controls transfected with siEHF alone (Supplementary Fig. S2;4 Fig. 2). These findings suggest that the EHF protein might enable cells to evade premature senescence at least in part via suppression of p27. Cotransfection of MEFs with siEHF and sip16 resulted in a partial rescue of siEHF-induced premature senescence (Supplementary Fig. S2),4 indicating that p16 could potentially play a role in EHF-modulated senescence.

EHF Suppresses Cell Cycle Arrest and Induction of Senescence in Cancer Cells
Next, we attempted to determine whether EHF overexpression could prevent DNA damage–induced senescence in cancer cells. As shown in Fig. 3 , doxorubicin-treated PC-3 cells overexpressing EHF had better growth and survival (Fig. 3A) and decreased DNA damage–induced accumulation of SA-ß-Gal (Fig. 3B) versus doxorubicin-treated control cancer cells. Fluorescence-activated cell sorting analysis of EHF-transfected cells confirmed that EHF overexpression relieved doxorubicin-induced, senescence-associated cell cycle arrest (Fig. 3C). Consistent with the cell cycle data, Western blot analysis revealed that EHF-overexpressing cells showed decrease in p27, as well as increased Rb phosphorylation versus control (Fig. 3D). These results strongly indicate that EHF overexpression suppresses p27 induction, cell cycle arrest, and senescence in doxorubicin-treated PC-3 cancer cells.


Figure 3
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  		Figure 3. Prevention of doxorubicin-induced senescence via EHF overexpression. A, EHF expression vector (2 µg) or control vector (2 µg) was transfected into 2 x 106 PC-3 cells, which were then plated onto cell culture dishes. The next day, the cells were treated doxorubicin (10 nmol/L) for 5 d and were assessed for SA-ß-Gal activity. B, cell growth detailing the effect of EHF expression on the proliferation of PC-3 cells. EHF- or vector-transfected cells were treated with 10 nmol/L doxorubicin (doxo) on the following day and were counted at days 4, 5, and 6. C, effect of EHF expression on the cell cycle of senescence-induced PC-3 cells. EHF- or vector-transfected cells were treated with doxorubicin (10 nmol/L) for 5 d. Cells were fixed with 70% ethanol and incubated with RNase A and the DNA-intercalating dye propidium iodide. Fluorescence-activated cell sorting analysis of EHF-transfected cells showed that doxorubicin-induced cell cycle arrest was relieved by EHF expression. Results are from a representative experiment from three independent experiments. Means and SDs are given in the table. D, EHF modulates cell cycle gene expression in DNA damage–induced PC-3 cell senescence. Doxorubicin (10 nmol/L) treatment was done, and the cells were harvested at the indicated days and evaluated for cell cycle gene expression by Western blot analysis with the antibodies shown on the left. Levels of ectopic EHF expression were examined by immunoblotting with an anti-Flag antibody. ß-Actin was used as a loading control. V, vector; VD, vector + doxurubicin; ED, EHF + doxorubicin.

 
EHF Modulates Telomerase Activity and In vivo Tumorigenicity
The function of telomerase has been extensively studied in relation to development, aging, and cancer (17). To examine whether ectopic expression of EHF affects telomerase activity, we established PC-3 clones stably overexpressing EHF and a negative control stably containing the empty vector. Interestingly, our results revealed that telomerase activity was significantly and dose-dependently higher in EHF-overexpressing cells relative to controls (Supplementary Fig. S3;4 Fig. 4A ), and this increased telomerase activity could be dose-dependently decreased by transfection of siEHF (Fig. 4B). These results indicate that telomerase activity can be modulated according to EHF expression in PC-3 cancer cells.


Figure 4
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  		Figure 4. EHF-mediated control of telomerase activity and in vivo tumorigenesis. A, overexpression of EHF increases telomerase activity. Stable PC-3 cell clones [EHF and vector (vec)] were harvested at 60 population doublings (60PD) and telomerase activities were measured using telomere repeat amplification protocol ELISA. Cell lysates from stable PC-3 cell lines were analyzed for ectopic EHF expression by Western blotting. B, EHF-directed siRNA decreases telomerase activity in a siEHF concentration-dependent manner. PC-3 cells transfected with siRNAs (EHF and control) were harvested 3 d after transfection and the telomerase activities were estimated using telomere repeat amplification protocol ELISA. PC, positive control; NC, negative control. C, siRNA of EHF suppresses tumor growth in nude mice. For each injection, 1.5 x 106 PC-3 cells were transfected with siRNA against control or EHF and implanted s.c. into the flanks of 4-week-old female athymic nu/nu mice. Ten mice were used for each group. Gross tumors shown are representative of groups immediately after resection. P < 0.0001, t test comparing siScr to siEHF xenografts. D, effects of siEHF injection on the growth of established tumor. siEHF or control siRNA was given twice after the injection of CT-26 cells (arrow). The siEHF was administered as a mixture of 100 nmol/L siEHF in 100 µL of Effectene per injection. Four mice were used for each group. Bars, SE. P = 0.0122, t test comparing siScr to siEHF xenografts. Tumor sizes in two dimensions were measured with calipers, and volumes were calculated with the formula (L x W2) x 0.5, where L is length and W is width.

 
Last, we investigated whether EHF could affect tumor formation and progression in vivo. When PC-3 cells transfected with siEHF or control siRNA were injected s.c. into athymic nude mice, the control cells formed tumors ~289 ± 39 mm3 in size over 6 weeks, whereas the siEHF-treated cells developed tumors that were only 118 ± 23 mm3 in size during the same period (P < 0.0001; Fig. 4C). Further, the preestablished tumors progressed much slowly after the injection of siEHF, suggesting that EHF can be a promising target for future cancer therapeutics (P = 0.0122; Fig. 4D).

Discussion

For cells to be transformed, they must bypass the program leading to premature senescence, most likely via mutation or aberrant expression of genes that affect the senescence-associated pathways. Therefore, identification of molecules responsible for controlling premature senescence may help us understand how oncogenes promote uncontrolled cell growth and may facilitate the development of new cancer treatment modalities.

In the present study, we examined the consequences of EHF knockdown in primary MEFs as well as human cancer cells. Using siRNA specific for EHF, we showed that EHF knockdown caused cell growth arrest, and that this seems to occur via senescence. Additional experiments revealed that EHF levels decreased in response to doxorubicin, whereas ectopic overexpression of EHF prevented doxorubicin-induced premature senescence, suggesting that EHF may negatively regulate senescence in human cancer cells. Although ets family proteins have previously been associated with cell proliferation and transformation (12–14), this is the first study to show that EHF is capable of modulating telomere activity, cell cycle arrest, and premature senescence. Furthermore, this work provides in vivo evidence of differential tumorigenesis according to EHF expression.

To understand how EHF regulates growth and transformation, it is important to determine which proteins mediate these processes. Although replicative senescence has previously been shown to be associated with increased expression of tumor suppressors, such as p16, p21, pRb, and p53 (5–7), we herein show that the premature senescence induced by siEHF was associated with up-regulation of p16 and p27 but not p21. Our results are consistent with the models of premature senescence described by te Poele et al. (2) and Alexander and Hinds (18), in which senescent cells leads to induction of genes such as p16 and p27, respectively.

In the present work, we showed that knockdown of p27 by sip27 prevented siEHF-induced senescence, supporting the notion that p27 plays a critical role in the induction or maintenance of cellular senescence. This is consistent with previous studies showing that the p27 protein, which acts as an inhibitor of cdk2, is linked with cell cycle arrest and premature senescence (5, 18). p53, pRb, and p16 have all been associated with fibroblast senescence; however, PC-3 cells do not have functional p53 or p16, and the DU145 cell line contains mutations in p53, pRb, and p16, but both of these cell lines showed EHF-associated senescence. Considering that the p16/Rb tumor-suppressor pathway is frequently dysregulated in human cancers, it seems logical to hypothesize that p27 may be a critical target of the EHF pathway in cancer cells. Our findings indicate that cells having increased EHF expression are resistant to growth arrest and are able to proliferate in the presence of DNA damage levels that would normally induce senescence. However, all these processes are strictly dependent on the presence of p27 or p16, as evidenced by the prevention of siEHF-induced senescence by siRNA knockdown of p27 or p16.

Because telomerase modulates the expression of growth-controlling genes (19) and plays important roles in regulating cell growth and senescence, we then examined the effect of EHF on telomerase activity. The results showed a correlation between EHF expression and telomerase, strengthening the idea that EHF plays a key role in premature senescence.

In sum, we herein show for the first time that the proliferation of cancer cells could be controlled by modulation of EHF. This finding is especially significant because our results suggest that EHF plays a role in the escape from premature senescence, and in tumor formation by cancer cells in vivo. Intriguingly, Rb, p53, and p16 were not strictly required for siEHF-induced senescence, and EHF knockdown led to decreased telomerase activity. As cancer cells are largely deficient in functional Rb, p53, and p16, it would seem that depletion of a protein such as EHF might be a more feasible strategy for recovery of tumor-suppressor function during cancer treatment. On the basis of our results, we conclude that EHF not only affects the progression of the cell cycle but also constitutes a pivotal factor for senescence suppression, thereby contributing to carcinogenesis.

Footnotes

Grant support: Samsung Biomedical Research Institute grant C-A6-401-1.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Supplementary material for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Back

Received 9/14/06; accepted 10/27/06.

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
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