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IL6 sensitizes prostate cancer to the antiproliferative effect of IFNα2 through IRF9.

Erb HH, Langlechner RV, Moser PL, Handle F, Casneuf T, Verstraeten K, Schlick B, Schäfer G, Hall B, Sasser K, Culig Z, Santer FR - Endocr. Relat. Cancer (2013)

Bottom Line: Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines.Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2.We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Urology, Department of Urology, Innsbruck Medical University, Austria.

ABSTRACT
Development and progression of prostate cancer (PCa) are associated with chronic inflammation. The cytokine interleukin 6 (IL6) can influence progression, differentiation, survival, and angiogenesis of PCa. To identify novel pathways that are triggered by IL6, we performed a gene expression profiling of two PCa cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN) regulatory factor 9 (IRF9) was identified as one of the most prevalent IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN signaling and acts together with STAT1 and 2 to activate transcription of IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and protein levels by quantitative real-time PCR and western blot respectively in both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop displayed high expression of IRF9. A tissue microarray with 36 PCa tissues showed that IRF9 protein expression is moderately elevated in malignant areas and positively correlates with the tissue expression of IL6. Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines. Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2. We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

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Siltuximab counteracts the IL6-induced expression of IRF9 in LNCaP and MDA PCa 2b, but not in IL6-producing prostate cancer (PCa) cell lines or PCa tissue. (A) LNCaP and MDA PCa 2b cells were treated with 50 μg/ml control IgG, 5 ng/ml IL6, or 50 μg/ml Siltuximab for 48 h, as indicated. (B) IL6-producing LNCaP-IL6+, Du-145, and PC3 cells were treated with 50 μg/ml Siltuximab or control IgG for 48 h. (A and B) IRF9 expression was assessed by western blot (one representative blot is shown). Band intensity was quantified and mean±s.d., n=3 is shown. (C) Immunohistochemical staining for IRF9 of representative cores from PCa patients from a clinical phase 1 study with Siltuximab and evaluation of the staining intensity. Cohort 1: placebo group. Cohorts 2, 3, and 4: one, two, or three injections of Siltuximab (6 mg/kg, 29, 15, and 1 day prior to radical prostatectomy), respectively. (D) Representative western blot of IRF9 expression after 72-h downregulation of IL6. Band intensity was quantified and mean±s.d., n=3 is shown. *P<0.05.
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fig4: Siltuximab counteracts the IL6-induced expression of IRF9 in LNCaP and MDA PCa 2b, but not in IL6-producing prostate cancer (PCa) cell lines or PCa tissue. (A) LNCaP and MDA PCa 2b cells were treated with 50 μg/ml control IgG, 5 ng/ml IL6, or 50 μg/ml Siltuximab for 48 h, as indicated. (B) IL6-producing LNCaP-IL6+, Du-145, and PC3 cells were treated with 50 μg/ml Siltuximab or control IgG for 48 h. (A and B) IRF9 expression was assessed by western blot (one representative blot is shown). Band intensity was quantified and mean±s.d., n=3 is shown. (C) Immunohistochemical staining for IRF9 of representative cores from PCa patients from a clinical phase 1 study with Siltuximab and evaluation of the staining intensity. Cohort 1: placebo group. Cohorts 2, 3, and 4: one, two, or three injections of Siltuximab (6 mg/kg, 29, 15, and 1 day prior to radical prostatectomy), respectively. (D) Representative western blot of IRF9 expression after 72-h downregulation of IL6. Band intensity was quantified and mean±s.d., n=3 is shown. *P<0.05.

Mentions: It was previously reported that exogenous IL6 activity can be neutralized by the chimeric anti-IL6 antibody Siltuximab (Li et al. 2005). Therefore, we questioned whether the IL6 regulation of IRF9 can be inhibited by cotreatment of IL6 and Siltuximab. We found that in LNCaP and MDA PCa 2b cells cotreated with IL6 and Siltuximab, IRF9 expression returned almost to basal levels when compared with untreated cells (Fig. 4A). Addition of IL6 did not have any additional effect on IRF9 expression in the IL6-secreting LNCaP-IL6+, PC3, and Du-145 cell lines (Supplementary Figure S4A, see section on supplementary data given at the end of this article). Interestingly, treatment of those cells with Siltuximab did not reduce IRF9 protein expression, leading to the conclusion that extracellular inhibition of IL6 signaling is insufficient to reduce IRF9 expression (Fig. 4B).


IL6 sensitizes prostate cancer to the antiproliferative effect of IFNα2 through IRF9.

Erb HH, Langlechner RV, Moser PL, Handle F, Casneuf T, Verstraeten K, Schlick B, Schäfer G, Hall B, Sasser K, Culig Z, Santer FR - Endocr. Relat. Cancer (2013)

Siltuximab counteracts the IL6-induced expression of IRF9 in LNCaP and MDA PCa 2b, but not in IL6-producing prostate cancer (PCa) cell lines or PCa tissue. (A) LNCaP and MDA PCa 2b cells were treated with 50 μg/ml control IgG, 5 ng/ml IL6, or 50 μg/ml Siltuximab for 48 h, as indicated. (B) IL6-producing LNCaP-IL6+, Du-145, and PC3 cells were treated with 50 μg/ml Siltuximab or control IgG for 48 h. (A and B) IRF9 expression was assessed by western blot (one representative blot is shown). Band intensity was quantified and mean±s.d., n=3 is shown. (C) Immunohistochemical staining for IRF9 of representative cores from PCa patients from a clinical phase 1 study with Siltuximab and evaluation of the staining intensity. Cohort 1: placebo group. Cohorts 2, 3, and 4: one, two, or three injections of Siltuximab (6 mg/kg, 29, 15, and 1 day prior to radical prostatectomy), respectively. (D) Representative western blot of IRF9 expression after 72-h downregulation of IL6. Band intensity was quantified and mean±s.d., n=3 is shown. *P<0.05.
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Related In: Results  -  Collection

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fig4: Siltuximab counteracts the IL6-induced expression of IRF9 in LNCaP and MDA PCa 2b, but not in IL6-producing prostate cancer (PCa) cell lines or PCa tissue. (A) LNCaP and MDA PCa 2b cells were treated with 50 μg/ml control IgG, 5 ng/ml IL6, or 50 μg/ml Siltuximab for 48 h, as indicated. (B) IL6-producing LNCaP-IL6+, Du-145, and PC3 cells were treated with 50 μg/ml Siltuximab or control IgG for 48 h. (A and B) IRF9 expression was assessed by western blot (one representative blot is shown). Band intensity was quantified and mean±s.d., n=3 is shown. (C) Immunohistochemical staining for IRF9 of representative cores from PCa patients from a clinical phase 1 study with Siltuximab and evaluation of the staining intensity. Cohort 1: placebo group. Cohorts 2, 3, and 4: one, two, or three injections of Siltuximab (6 mg/kg, 29, 15, and 1 day prior to radical prostatectomy), respectively. (D) Representative western blot of IRF9 expression after 72-h downregulation of IL6. Band intensity was quantified and mean±s.d., n=3 is shown. *P<0.05.
Mentions: It was previously reported that exogenous IL6 activity can be neutralized by the chimeric anti-IL6 antibody Siltuximab (Li et al. 2005). Therefore, we questioned whether the IL6 regulation of IRF9 can be inhibited by cotreatment of IL6 and Siltuximab. We found that in LNCaP and MDA PCa 2b cells cotreated with IL6 and Siltuximab, IRF9 expression returned almost to basal levels when compared with untreated cells (Fig. 4A). Addition of IL6 did not have any additional effect on IRF9 expression in the IL6-secreting LNCaP-IL6+, PC3, and Du-145 cell lines (Supplementary Figure S4A, see section on supplementary data given at the end of this article). Interestingly, treatment of those cells with Siltuximab did not reduce IRF9 protein expression, leading to the conclusion that extracellular inhibition of IL6 signaling is insufficient to reduce IRF9 expression (Fig. 4B).

Bottom Line: Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines.Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2.We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Urology, Department of Urology, Innsbruck Medical University, Austria.

ABSTRACT
Development and progression of prostate cancer (PCa) are associated with chronic inflammation. The cytokine interleukin 6 (IL6) can influence progression, differentiation, survival, and angiogenesis of PCa. To identify novel pathways that are triggered by IL6, we performed a gene expression profiling of two PCa cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN) regulatory factor 9 (IRF9) was identified as one of the most prevalent IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN signaling and acts together with STAT1 and 2 to activate transcription of IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and protein levels by quantitative real-time PCR and western blot respectively in both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop displayed high expression of IRF9. A tissue microarray with 36 PCa tissues showed that IRF9 protein expression is moderately elevated in malignant areas and positively correlates with the tissue expression of IL6. Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines. Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2. We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

Show MeSH
Related in: MedlinePlus