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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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Identification of IRF9 as an IL6-regulated gene in LNCaP and MDA PCa 2b cells. (A) LNCaP and MDA PCa 2b cells were treated for 18 h with 5 ng/ml IL6 and profiled on Affymetrix microarrays. These volcano plots show the results of a test for differential expression between IL6-treated and untreated cells, with the significance (P value) in the Y-axis and the log fold change in the X-axis. IRF9 is among the top genes with the smallest P values regulated by IL6 in both cell lines. (B) To validate results from the microarray experiment, LNCaP and MDA PCa 2b cells were treated with 5 ng/ml IL6 for 18 h and QRT-PCR was performed. Values indicated are mean±s.e.m., n=3. (C) Protein regulation of IRF9 through IL6 treatment (5 ng/ml for 48 h) was controlled by western blotting. A representative western blot of three independent experiments is shown. *P<0.05; **P<0.01.
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fig1: Identification of IRF9 as an IL6-regulated gene in LNCaP and MDA PCa 2b cells. (A) LNCaP and MDA PCa 2b cells were treated for 18 h with 5 ng/ml IL6 and profiled on Affymetrix microarrays. These volcano plots show the results of a test for differential expression between IL6-treated and untreated cells, with the significance (P value) in the Y-axis and the log fold change in the X-axis. IRF9 is among the top genes with the smallest P values regulated by IL6 in both cell lines. (B) To validate results from the microarray experiment, LNCaP and MDA PCa 2b cells were treated with 5 ng/ml IL6 for 18 h and QRT-PCR was performed. Values indicated are mean±s.e.m., n=3. (C) Protein regulation of IRF9 through IL6 treatment (5 ng/ml for 48 h) was controlled by western blotting. A representative western blot of three independent experiments is shown. *P<0.05; **P<0.01.

Mentions: To identify novel genes regulated by IL6 in PCa cell lines, we cultured LNCaP and MDA PCa 2b cells for 18 h in the presence or absence of 5 ng/ml IL6 and performed gene expression profiling on Affymetrix microarrays. The data have been submitted to GEO database (accession number GSE47973). We used a two-group limma comparison in both cell lines to identify genes that are regulated by IL6 treatment. We detected substantial gene expression changes induced in the LNCaP cell line, where 672 genes are significant at adjusted P value ≤0.05. No genes met this criterion for MDA PCa 2b cell line; however, 931 genes were found to be significantly differentially expressed at P value ≤0.05. The volcano plot in Fig. 1A shows the fold changes and P values of all genes. The genes with the most significant P values and/or the largest fold changes are depicted with their names. The top genes regulated by IL6 according to the P values are listed in Table 1. For further investigation, we selected IRF9 as the gene regulated by IL6 in both LNCaP and MDA PCa 2b. To confirm the IL6 regulation of IRF9 in LNCaP and MDA PCa 2b cells, we performed QRT-PCR analysis. As shown in Fig. 1B, IRF9 was found to be significantly increased in IL6-treated LNCaP and MDA PCa 2b cells. Additionally, we performed western blot analysis to confirm that IL6 also increases the protein levels of IRF9. When exposed to IL6 for 48 h, both cell lines showed an increase in IRF9 protein expression (Fig. 1C). Altogether, we concluded that under our experimental conditions, IL6 upregulates IRF9 in LNCaP and MDA PCa 2b cells at the mRNA and protein levels.


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)

Identification of IRF9 as an IL6-regulated gene in LNCaP and MDA PCa 2b cells. (A) LNCaP and MDA PCa 2b cells were treated for 18 h with 5 ng/ml IL6 and profiled on Affymetrix microarrays. These volcano plots show the results of a test for differential expression between IL6-treated and untreated cells, with the significance (P value) in the Y-axis and the log fold change in the X-axis. IRF9 is among the top genes with the smallest P values regulated by IL6 in both cell lines. (B) To validate results from the microarray experiment, LNCaP and MDA PCa 2b cells were treated with 5 ng/ml IL6 for 18 h and QRT-PCR was performed. Values indicated are mean±s.e.m., n=3. (C) Protein regulation of IRF9 through IL6 treatment (5 ng/ml for 48 h) was controlled by western blotting. A representative western blot of three independent experiments is shown. *P<0.05; **P<0.01.
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Related In: Results  -  Collection

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Show All Figures
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fig1: Identification of IRF9 as an IL6-regulated gene in LNCaP and MDA PCa 2b cells. (A) LNCaP and MDA PCa 2b cells were treated for 18 h with 5 ng/ml IL6 and profiled on Affymetrix microarrays. These volcano plots show the results of a test for differential expression between IL6-treated and untreated cells, with the significance (P value) in the Y-axis and the log fold change in the X-axis. IRF9 is among the top genes with the smallest P values regulated by IL6 in both cell lines. (B) To validate results from the microarray experiment, LNCaP and MDA PCa 2b cells were treated with 5 ng/ml IL6 for 18 h and QRT-PCR was performed. Values indicated are mean±s.e.m., n=3. (C) Protein regulation of IRF9 through IL6 treatment (5 ng/ml for 48 h) was controlled by western blotting. A representative western blot of three independent experiments is shown. *P<0.05; **P<0.01.
Mentions: To identify novel genes regulated by IL6 in PCa cell lines, we cultured LNCaP and MDA PCa 2b cells for 18 h in the presence or absence of 5 ng/ml IL6 and performed gene expression profiling on Affymetrix microarrays. The data have been submitted to GEO database (accession number GSE47973). We used a two-group limma comparison in both cell lines to identify genes that are regulated by IL6 treatment. We detected substantial gene expression changes induced in the LNCaP cell line, where 672 genes are significant at adjusted P value ≤0.05. No genes met this criterion for MDA PCa 2b cell line; however, 931 genes were found to be significantly differentially expressed at P value ≤0.05. The volcano plot in Fig. 1A shows the fold changes and P values of all genes. The genes with the most significant P values and/or the largest fold changes are depicted with their names. The top genes regulated by IL6 according to the P values are listed in Table 1. For further investigation, we selected IRF9 as the gene regulated by IL6 in both LNCaP and MDA PCa 2b. To confirm the IL6 regulation of IRF9 in LNCaP and MDA PCa 2b cells, we performed QRT-PCR analysis. As shown in Fig. 1B, IRF9 was found to be significantly increased in IL6-treated LNCaP and MDA PCa 2b cells. Additionally, we performed western blot analysis to confirm that IL6 also increases the protein levels of IRF9. When exposed to IL6 for 48 h, both cell lines showed an increase in IRF9 protein expression (Fig. 1C). Altogether, we concluded that under our experimental conditions, IL6 upregulates IRF9 in LNCaP and MDA PCa 2b cells at the mRNA and protein levels.

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