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STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Pencik J, Schlederer M, Gruber W, Unger C, Walker SM, Chalaris A, Marié IJ, Hassler MR, Javaheri T, Aksoy O, Blayney JK, Prutsch N, Skucha A, Herac M, Krämer OH, Mazal P, Grebien F, Egger G, Poli V, Mikulits W, Eferl R, Esterbauer H, Kennedy R, Fend F, Scharpf M, Braun M, Perner S, Levy DE, Malcolm T, Turner SD, Haitel A, Susani M, Moazzami A, Rose-John S, Aberger F, Merkel O, Moriggl R, Culig Z, Dolznig H, Kenner L - Nat Commun (2015)

Bottom Line: However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit.STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases.Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Institute for Cancer Research, Waehringerstrasse 13A, 1090 Vienna, Austria.

ABSTRACT
Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

No MeSH data available.


Related in: MedlinePlus

Stat3 is a critical regulator of the ARF–Mdm2–p53 tumour suppressor pathway and senescence.(a) Haematoxilin/eosin (H&E) stains show higher grade PCa in Ptenpc−/−Stat3pc−/− mice compared with Ptenpc−/− mice. Scale bars, 100 μm. IHC analysis of p53, p19ARF and staining for senescence-associated-β-galactosidase activity in prostates from 19-week-old WT, Ptenpc−/− and Ptenpc−/−Stat3pc−/− mice. Scale bars, 100 μm; insets: × 600 magnification. (b) Western blot analysis showing pY-Stat3, Stat3, p53, p19ARF, Mdm2 and PML expression levels in Ptenpc−/−Stat3pc−/− compared with Ptenpc−/− mice. β-actin serves as a loading control. The remaining Stat3 bands in Ptenpc−/−Stat3pc−/− prostates are due to Stat3 stromal expression (Supplementary Fig. 2c). (c) Western blot analysis of Stat3 and p19ARF expression in prostates of 19-week-old WT or Stat3pc−/− mice. β-actin serves as a loading control. (d) qRT–PCR analysis of Stat3 and p19ARF mRNA expression in prostates of 19-week-old WT or Stat3pc−/− mice (n=5 each). Data were analysed by Student's t-test and are shown as mean±s.d. (e) Western blot analysis of Stat3 and p19ARF expression in WT, Stat3C/+ or Stat3C/C MEFs. (f) qRT–PCR analysis of Stat3 and p19ARF transcript levels in WT, Stat3C/+ and Stat3C/C MEFs (n=3 each). Data were analysed by Student's t-test and are shown as mean±s.d. (g) In vivo ChIP analysis of Stat3 binding to p19ARF and Socs3 promoters, respectively, in WT and Stat3pc−/− prostate tissue. Stat3 binding to the Socs3 (ref. 69) promoter, which is a direct Stat3 target served as a positive control. Data were normalized to Cis4200, which served as the negative control70. (h) In vivo ChIP analysis of Stat3 binding to the p19ARF and Socs3 promoters in PCa. Note the >15-fold enrichment of Stat3 bound to promoter fragments in Ptenpc−/− compared with Ptenpc−/−Stat3pc−/− tumours. Data in g and h were analysed by one-way analysis of variance with Tukey's multiple comparison test and shown as mean±s.d. (Primer pairs are listed in Supplementary Table 2).
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f4: Stat3 is a critical regulator of the ARF–Mdm2–p53 tumour suppressor pathway and senescence.(a) Haematoxilin/eosin (H&E) stains show higher grade PCa in Ptenpc−/−Stat3pc−/− mice compared with Ptenpc−/− mice. Scale bars, 100 μm. IHC analysis of p53, p19ARF and staining for senescence-associated-β-galactosidase activity in prostates from 19-week-old WT, Ptenpc−/− and Ptenpc−/−Stat3pc−/− mice. Scale bars, 100 μm; insets: × 600 magnification. (b) Western blot analysis showing pY-Stat3, Stat3, p53, p19ARF, Mdm2 and PML expression levels in Ptenpc−/−Stat3pc−/− compared with Ptenpc−/− mice. β-actin serves as a loading control. The remaining Stat3 bands in Ptenpc−/−Stat3pc−/− prostates are due to Stat3 stromal expression (Supplementary Fig. 2c). (c) Western blot analysis of Stat3 and p19ARF expression in prostates of 19-week-old WT or Stat3pc−/− mice. β-actin serves as a loading control. (d) qRT–PCR analysis of Stat3 and p19ARF mRNA expression in prostates of 19-week-old WT or Stat3pc−/− mice (n=5 each). Data were analysed by Student's t-test and are shown as mean±s.d. (e) Western blot analysis of Stat3 and p19ARF expression in WT, Stat3C/+ or Stat3C/C MEFs. (f) qRT–PCR analysis of Stat3 and p19ARF transcript levels in WT, Stat3C/+ and Stat3C/C MEFs (n=3 each). Data were analysed by Student's t-test and are shown as mean±s.d. (g) In vivo ChIP analysis of Stat3 binding to p19ARF and Socs3 promoters, respectively, in WT and Stat3pc−/− prostate tissue. Stat3 binding to the Socs3 (ref. 69) promoter, which is a direct Stat3 target served as a positive control. Data were normalized to Cis4200, which served as the negative control70. (h) In vivo ChIP analysis of Stat3 binding to the p19ARF and Socs3 promoters in PCa. Note the >15-fold enrichment of Stat3 bound to promoter fragments in Ptenpc−/− compared with Ptenpc−/−Stat3pc−/− tumours. Data in g and h were analysed by one-way analysis of variance with Tukey's multiple comparison test and shown as mean±s.d. (Primer pairs are listed in Supplementary Table 2).

Mentions: Since loss of Pten triggers senescence thereby restricting cancer progression and metastasis11, we next tested whether Stat3 exerts a tumour suppressive function by activating senescence-inducing programmes in Ptenpc−/−PCa cells19 at an early stage of PCa development (19 weeks). Senescence is generally characterized by upregulation of p53, cyclin-dependent kinase inhibitor 1 (Cdkn1, p21), promyelocytic leukaemia protein (PML) and elevated senescence-associated-β-galactosidase activity20. Of note, Ptenpc−/−Stat3−/− tumours lacked p21 expression, displayed reduced numbers of PML nuclear bodies and decreased SA-β-Gal activity compared with Ptenpc−/− tumours (Fig. 4a,b and Supplementary Fig. 5a,b), suggesting Stat3 as a novel mediator of senescence in response to loss of Pten. Senescence associated with loss of Pten was shown to be bypassed by deletion of p53 leading to early lethality11. We show here that loss of Stat3 and Pten revealed a phenotype strikingly similar to that of p53 and Pten loss11. Intriguingly, Stat3 and Pten deletion resulted in downregulation of p53 expression in the prostate epithelium, which was accompanied by the loss of p19ARF (Fig. 4a,b). The p53 expression in the tumour stromal cells remained unchanged (Supplementary Fig. 5c). Since p19ARF is a critical regulator of Mdm2 degradation21, our results suggest that the tumour suppressive capacity of Stat3 in senescent tumour cells22 may rely on the p19ARF–Mdm2–p53 tumour suppressor axis.


STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Pencik J, Schlederer M, Gruber W, Unger C, Walker SM, Chalaris A, Marié IJ, Hassler MR, Javaheri T, Aksoy O, Blayney JK, Prutsch N, Skucha A, Herac M, Krämer OH, Mazal P, Grebien F, Egger G, Poli V, Mikulits W, Eferl R, Esterbauer H, Kennedy R, Fend F, Scharpf M, Braun M, Perner S, Levy DE, Malcolm T, Turner SD, Haitel A, Susani M, Moazzami A, Rose-John S, Aberger F, Merkel O, Moriggl R, Culig Z, Dolznig H, Kenner L - Nat Commun (2015)

Stat3 is a critical regulator of the ARF–Mdm2–p53 tumour suppressor pathway and senescence.(a) Haematoxilin/eosin (H&E) stains show higher grade PCa in Ptenpc−/−Stat3pc−/− mice compared with Ptenpc−/− mice. Scale bars, 100 μm. IHC analysis of p53, p19ARF and staining for senescence-associated-β-galactosidase activity in prostates from 19-week-old WT, Ptenpc−/− and Ptenpc−/−Stat3pc−/− mice. Scale bars, 100 μm; insets: × 600 magnification. (b) Western blot analysis showing pY-Stat3, Stat3, p53, p19ARF, Mdm2 and PML expression levels in Ptenpc−/−Stat3pc−/− compared with Ptenpc−/− mice. β-actin serves as a loading control. The remaining Stat3 bands in Ptenpc−/−Stat3pc−/− prostates are due to Stat3 stromal expression (Supplementary Fig. 2c). (c) Western blot analysis of Stat3 and p19ARF expression in prostates of 19-week-old WT or Stat3pc−/− mice. β-actin serves as a loading control. (d) qRT–PCR analysis of Stat3 and p19ARF mRNA expression in prostates of 19-week-old WT or Stat3pc−/− mice (n=5 each). Data were analysed by Student's t-test and are shown as mean±s.d. (e) Western blot analysis of Stat3 and p19ARF expression in WT, Stat3C/+ or Stat3C/C MEFs. (f) qRT–PCR analysis of Stat3 and p19ARF transcript levels in WT, Stat3C/+ and Stat3C/C MEFs (n=3 each). Data were analysed by Student's t-test and are shown as mean±s.d. (g) In vivo ChIP analysis of Stat3 binding to p19ARF and Socs3 promoters, respectively, in WT and Stat3pc−/− prostate tissue. Stat3 binding to the Socs3 (ref. 69) promoter, which is a direct Stat3 target served as a positive control. Data were normalized to Cis4200, which served as the negative control70. (h) In vivo ChIP analysis of Stat3 binding to the p19ARF and Socs3 promoters in PCa. Note the >15-fold enrichment of Stat3 bound to promoter fragments in Ptenpc−/− compared with Ptenpc−/−Stat3pc−/− tumours. Data in g and h were analysed by one-way analysis of variance with Tukey's multiple comparison test and shown as mean±s.d. (Primer pairs are listed in Supplementary Table 2).
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f4: Stat3 is a critical regulator of the ARF–Mdm2–p53 tumour suppressor pathway and senescence.(a) Haematoxilin/eosin (H&E) stains show higher grade PCa in Ptenpc−/−Stat3pc−/− mice compared with Ptenpc−/− mice. Scale bars, 100 μm. IHC analysis of p53, p19ARF and staining for senescence-associated-β-galactosidase activity in prostates from 19-week-old WT, Ptenpc−/− and Ptenpc−/−Stat3pc−/− mice. Scale bars, 100 μm; insets: × 600 magnification. (b) Western blot analysis showing pY-Stat3, Stat3, p53, p19ARF, Mdm2 and PML expression levels in Ptenpc−/−Stat3pc−/− compared with Ptenpc−/− mice. β-actin serves as a loading control. The remaining Stat3 bands in Ptenpc−/−Stat3pc−/− prostates are due to Stat3 stromal expression (Supplementary Fig. 2c). (c) Western blot analysis of Stat3 and p19ARF expression in prostates of 19-week-old WT or Stat3pc−/− mice. β-actin serves as a loading control. (d) qRT–PCR analysis of Stat3 and p19ARF mRNA expression in prostates of 19-week-old WT or Stat3pc−/− mice (n=5 each). Data were analysed by Student's t-test and are shown as mean±s.d. (e) Western blot analysis of Stat3 and p19ARF expression in WT, Stat3C/+ or Stat3C/C MEFs. (f) qRT–PCR analysis of Stat3 and p19ARF transcript levels in WT, Stat3C/+ and Stat3C/C MEFs (n=3 each). Data were analysed by Student's t-test and are shown as mean±s.d. (g) In vivo ChIP analysis of Stat3 binding to p19ARF and Socs3 promoters, respectively, in WT and Stat3pc−/− prostate tissue. Stat3 binding to the Socs3 (ref. 69) promoter, which is a direct Stat3 target served as a positive control. Data were normalized to Cis4200, which served as the negative control70. (h) In vivo ChIP analysis of Stat3 binding to the p19ARF and Socs3 promoters in PCa. Note the >15-fold enrichment of Stat3 bound to promoter fragments in Ptenpc−/− compared with Ptenpc−/−Stat3pc−/− tumours. Data in g and h were analysed by one-way analysis of variance with Tukey's multiple comparison test and shown as mean±s.d. (Primer pairs are listed in Supplementary Table 2).
Mentions: Since loss of Pten triggers senescence thereby restricting cancer progression and metastasis11, we next tested whether Stat3 exerts a tumour suppressive function by activating senescence-inducing programmes in Ptenpc−/−PCa cells19 at an early stage of PCa development (19 weeks). Senescence is generally characterized by upregulation of p53, cyclin-dependent kinase inhibitor 1 (Cdkn1, p21), promyelocytic leukaemia protein (PML) and elevated senescence-associated-β-galactosidase activity20. Of note, Ptenpc−/−Stat3−/− tumours lacked p21 expression, displayed reduced numbers of PML nuclear bodies and decreased SA-β-Gal activity compared with Ptenpc−/− tumours (Fig. 4a,b and Supplementary Fig. 5a,b), suggesting Stat3 as a novel mediator of senescence in response to loss of Pten. Senescence associated with loss of Pten was shown to be bypassed by deletion of p53 leading to early lethality11. We show here that loss of Stat3 and Pten revealed a phenotype strikingly similar to that of p53 and Pten loss11. Intriguingly, Stat3 and Pten deletion resulted in downregulation of p53 expression in the prostate epithelium, which was accompanied by the loss of p19ARF (Fig. 4a,b). The p53 expression in the tumour stromal cells remained unchanged (Supplementary Fig. 5c). Since p19ARF is a critical regulator of Mdm2 degradation21, our results suggest that the tumour suppressive capacity of Stat3 in senescent tumour cells22 may rely on the p19ARF–Mdm2–p53 tumour suppressor axis.

Bottom Line: However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit.STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases.Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Institute for Cancer Research, Waehringerstrasse 13A, 1090 Vienna, Austria.

ABSTRACT
Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

No MeSH data available.


Related in: MedlinePlus