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SIRNA-directed in vivo silencing of androgen receptor inhibits the growth of castration-resistant prostate carcinomas.

Compagno D, Merle C, Morin A, Gilbert C, Mathieu JR, Bozec A, Mauduit C, Benahmed M, Cabon F - PLoS ONE (2007)

Bottom Line: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth.Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo.The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

View Article: PubMed Central - PubMed

Affiliation: CNRS, University Paris-Sud, FRE2944, Epigenetics and Cancer, Institut André Lwoff, Villejuif, France.

ABSTRACT

Background: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated.

Methodology/principal findings: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis.

Conclusions/significance: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

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Silencing of AR in LNCaP cells and tumors.A: Control (cont)- panAR- or hAR-siRNA were transfected into human LNCaP or into mouse Sertoli TM4 cells. AR was immunodetected by western blot in cell lysates 2 days after removal of transfection medium. α-tubulin (tub) expression was used as a loading control. B: Relative PSA mRNA level in LNCaP cells transfected with control or hAR-siRNA and grown for 48 h in the absence of androgens or in the presence of R1881, 0.5 nM (mean±SE, n = 3 independent experiments). Similar results were obtained using the panAR-siRNA. **p<0.01 as compared to values in the absence of androgens. C: LNCaP cells were subcutaneously injected on day 0 to nude mice. Starting from day 51 (arrow), animals (5 per group) received a daily i.p. injection of 3 µg of cont- (black symbols) or panAR-siRNA (white symbols) diluted in 50 µl saline; tumor volume (cm3, mean±SE, n = 5). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors. D: Analysis of AR expression by immunohistochemistry (left panels) and apoptotic cells by TUNEL (right panels) in representative tumors collected at the end of the experiment shown in C. E: Mice bearing exponentially growing LNCaP tumors were randomized (12 mice per group) and received daily i.p. injections of cont-siRNA (black symbols), hAR-siRNA (white symbols) or an oral dose of 50 mg.kg−1 of bicalutamide (grey symbols); tumor volume (cm3, mean±SE). *p<0.05 and **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors. F: On the fourth day of treatment of the experiment shown in E, 6 mice in each group were sacrificed and AR and PSA mRNA levels were quantified in the tumors by qRT-PCR and normalized with cyclophilin A mRNA level. Results are expressed relative to the mean level in control tumors. **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors.
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pone-0001006-g001: Silencing of AR in LNCaP cells and tumors.A: Control (cont)- panAR- or hAR-siRNA were transfected into human LNCaP or into mouse Sertoli TM4 cells. AR was immunodetected by western blot in cell lysates 2 days after removal of transfection medium. α-tubulin (tub) expression was used as a loading control. B: Relative PSA mRNA level in LNCaP cells transfected with control or hAR-siRNA and grown for 48 h in the absence of androgens or in the presence of R1881, 0.5 nM (mean±SE, n = 3 independent experiments). Similar results were obtained using the panAR-siRNA. **p<0.01 as compared to values in the absence of androgens. C: LNCaP cells were subcutaneously injected on day 0 to nude mice. Starting from day 51 (arrow), animals (5 per group) received a daily i.p. injection of 3 µg of cont- (black symbols) or panAR-siRNA (white symbols) diluted in 50 µl saline; tumor volume (cm3, mean±SE, n = 5). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors. D: Analysis of AR expression by immunohistochemistry (left panels) and apoptotic cells by TUNEL (right panels) in representative tumors collected at the end of the experiment shown in C. E: Mice bearing exponentially growing LNCaP tumors were randomized (12 mice per group) and received daily i.p. injections of cont-siRNA (black symbols), hAR-siRNA (white symbols) or an oral dose of 50 mg.kg−1 of bicalutamide (grey symbols); tumor volume (cm3, mean±SE). *p<0.05 and **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors. F: On the fourth day of treatment of the experiment shown in E, 6 mice in each group were sacrificed and AR and PSA mRNA levels were quantified in the tumors by qRT-PCR and normalized with cyclophilin A mRNA level. Results are expressed relative to the mean level in control tumors. **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors.

Mentions: We used in this study RNA interference to investigate in vitro and in vivo the function of AR in prostate carcinomas. To establish the technical conditions and specificity of AR silencing, we first used the human androgen-dependent prostate tumor model LNCaP. Androgens stimulate LNCaP cells' proliferation whereas castration and the androgen antagonist bicalutamide inhibit the development of xenografted LNCaP tumors in mice [18]. We designed and synthesized two different siRNAs targeting the first exon of AR. The panAR-siRNA targets a sequence conserved between the human and mouse AR mRNAs. It silences AR expression in the mouse Sertoli TM4 as in the human LNCaP cell line (Figure 1A). In contrast, the hAR-siRNA, which targets the human sequence but presents 5 mismatches out of 19 with the mouse mRNA, inhibits AR expression in LNCaP but not in mouse TM4 cells (Figure 1A). Transfection of AR-siRNA in LNCaP cells strongly inhibits the androgen-induced transcription of Prostate Specific Antigen (PSA), a prototypic AR-target gene (Figure 1B).


SIRNA-directed in vivo silencing of androgen receptor inhibits the growth of castration-resistant prostate carcinomas.

Compagno D, Merle C, Morin A, Gilbert C, Mathieu JR, Bozec A, Mauduit C, Benahmed M, Cabon F - PLoS ONE (2007)

Silencing of AR in LNCaP cells and tumors.A: Control (cont)- panAR- or hAR-siRNA were transfected into human LNCaP or into mouse Sertoli TM4 cells. AR was immunodetected by western blot in cell lysates 2 days after removal of transfection medium. α-tubulin (tub) expression was used as a loading control. B: Relative PSA mRNA level in LNCaP cells transfected with control or hAR-siRNA and grown for 48 h in the absence of androgens or in the presence of R1881, 0.5 nM (mean±SE, n = 3 independent experiments). Similar results were obtained using the panAR-siRNA. **p<0.01 as compared to values in the absence of androgens. C: LNCaP cells were subcutaneously injected on day 0 to nude mice. Starting from day 51 (arrow), animals (5 per group) received a daily i.p. injection of 3 µg of cont- (black symbols) or panAR-siRNA (white symbols) diluted in 50 µl saline; tumor volume (cm3, mean±SE, n = 5). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors. D: Analysis of AR expression by immunohistochemistry (left panels) and apoptotic cells by TUNEL (right panels) in representative tumors collected at the end of the experiment shown in C. E: Mice bearing exponentially growing LNCaP tumors were randomized (12 mice per group) and received daily i.p. injections of cont-siRNA (black symbols), hAR-siRNA (white symbols) or an oral dose of 50 mg.kg−1 of bicalutamide (grey symbols); tumor volume (cm3, mean±SE). *p<0.05 and **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors. F: On the fourth day of treatment of the experiment shown in E, 6 mice in each group were sacrificed and AR and PSA mRNA levels were quantified in the tumors by qRT-PCR and normalized with cyclophilin A mRNA level. Results are expressed relative to the mean level in control tumors. **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1994591&req=5

pone-0001006-g001: Silencing of AR in LNCaP cells and tumors.A: Control (cont)- panAR- or hAR-siRNA were transfected into human LNCaP or into mouse Sertoli TM4 cells. AR was immunodetected by western blot in cell lysates 2 days after removal of transfection medium. α-tubulin (tub) expression was used as a loading control. B: Relative PSA mRNA level in LNCaP cells transfected with control or hAR-siRNA and grown for 48 h in the absence of androgens or in the presence of R1881, 0.5 nM (mean±SE, n = 3 independent experiments). Similar results were obtained using the panAR-siRNA. **p<0.01 as compared to values in the absence of androgens. C: LNCaP cells were subcutaneously injected on day 0 to nude mice. Starting from day 51 (arrow), animals (5 per group) received a daily i.p. injection of 3 µg of cont- (black symbols) or panAR-siRNA (white symbols) diluted in 50 µl saline; tumor volume (cm3, mean±SE, n = 5). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors. D: Analysis of AR expression by immunohistochemistry (left panels) and apoptotic cells by TUNEL (right panels) in representative tumors collected at the end of the experiment shown in C. E: Mice bearing exponentially growing LNCaP tumors were randomized (12 mice per group) and received daily i.p. injections of cont-siRNA (black symbols), hAR-siRNA (white symbols) or an oral dose of 50 mg.kg−1 of bicalutamide (grey symbols); tumor volume (cm3, mean±SE). *p<0.05 and **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors. F: On the fourth day of treatment of the experiment shown in E, 6 mice in each group were sacrificed and AR and PSA mRNA levels were quantified in the tumors by qRT-PCR and normalized with cyclophilin A mRNA level. Results are expressed relative to the mean level in control tumors. **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors.
Mentions: We used in this study RNA interference to investigate in vitro and in vivo the function of AR in prostate carcinomas. To establish the technical conditions and specificity of AR silencing, we first used the human androgen-dependent prostate tumor model LNCaP. Androgens stimulate LNCaP cells' proliferation whereas castration and the androgen antagonist bicalutamide inhibit the development of xenografted LNCaP tumors in mice [18]. We designed and synthesized two different siRNAs targeting the first exon of AR. The panAR-siRNA targets a sequence conserved between the human and mouse AR mRNAs. It silences AR expression in the mouse Sertoli TM4 as in the human LNCaP cell line (Figure 1A). In contrast, the hAR-siRNA, which targets the human sequence but presents 5 mismatches out of 19 with the mouse mRNA, inhibits AR expression in LNCaP but not in mouse TM4 cells (Figure 1A). Transfection of AR-siRNA in LNCaP cells strongly inhibits the androgen-induced transcription of Prostate Specific Antigen (PSA), a prototypic AR-target gene (Figure 1B).

Bottom Line: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth.Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo.The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

View Article: PubMed Central - PubMed

Affiliation: CNRS, University Paris-Sud, FRE2944, Epigenetics and Cancer, Institut André Lwoff, Villejuif, France.

ABSTRACT

Background: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated.

Methodology/principal findings: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis.

Conclusions/significance: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

Show MeSH
Related in: MedlinePlus