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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 suppresses colony formation and invasion.(a) shRNA-mediated knockdown of Stat3 in Pten−/− mouse PCa cells leads to robust decrease of Stat3 levels as demonstrated by western blotting. In all knockdown experiments, scrambled non-target shRNA served as a control (control shRNA/shcontrol). (b) Representative histology of increased matrigel invasion after shRNA-mediated knockdown of Stat3 in Pten-deficient (Pten−/−) mouse PCa cells. Quantification of relative invasion is shown (n=3). Scale bars, 100 μm. (c) Organotypic culture assays of Pten−/− mouse PCa cells in the absence of Stat3 showed capacity to invade into the fibroblast containing collagen gel (red arrows). Scale bars, 50 μm. The invasive area/total tumour cell area was quantified (control shRNA, n=4, shStat3 n=3). (d) Soft agar colony formation of primary Pten−/− mouse PCa cells with shStat3 and controls was quantified (n=3). Scale bars, 200 μm. Data were analysed by Student's t-test and are shown in c–e as mean±s.d. (e) Efficient STAT3 and PTEN siRNA-mediated knockdown of RWPE-1 cells was demonstrated by western blot. Scrambled non-target siRNA served as a control (control siRNA/siControl) (f) Organotypic culture of RWPE-1 cells in the presence and absence of STAT3 and/or PTEN cultivated in contact with human prostate stromal fibroblasts seeded in collagen I gels after 8 days of culture. Representative H&E stainings are shown, red arrows indicate invasion, scale bars, 100 μm. (n=3). (g) Quantification of invasion of RWPE-1 cells knocked down for PTEN and/or STAT3 using siRNA (number of invasive cells per mm, n=5 sections for each condition). Data were analysed by one-way analysis of variance with Tukey's multiple comparison test; error bars: s.d. (h) Crystal violet stains of focus formation of STAT3-V5 and empty vector transduced PC3 cells after 4 days of incubation (Supplementary Fig. 4c–e).
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f3: Stat3 suppresses colony formation and invasion.(a) shRNA-mediated knockdown of Stat3 in Pten−/− mouse PCa cells leads to robust decrease of Stat3 levels as demonstrated by western blotting. In all knockdown experiments, scrambled non-target shRNA served as a control (control shRNA/shcontrol). (b) Representative histology of increased matrigel invasion after shRNA-mediated knockdown of Stat3 in Pten-deficient (Pten−/−) mouse PCa cells. Quantification of relative invasion is shown (n=3). Scale bars, 100 μm. (c) Organotypic culture assays of Pten−/− mouse PCa cells in the absence of Stat3 showed capacity to invade into the fibroblast containing collagen gel (red arrows). Scale bars, 50 μm. The invasive area/total tumour cell area was quantified (control shRNA, n=4, shStat3 n=3). (d) Soft agar colony formation of primary Pten−/− mouse PCa cells with shStat3 and controls was quantified (n=3). Scale bars, 200 μm. Data were analysed by Student's t-test and are shown in c–e as mean±s.d. (e) Efficient STAT3 and PTEN siRNA-mediated knockdown of RWPE-1 cells was demonstrated by western blot. Scrambled non-target siRNA served as a control (control siRNA/siControl) (f) Organotypic culture of RWPE-1 cells in the presence and absence of STAT3 and/or PTEN cultivated in contact with human prostate stromal fibroblasts seeded in collagen I gels after 8 days of culture. Representative H&E stainings are shown, red arrows indicate invasion, scale bars, 100 μm. (n=3). (g) Quantification of invasion of RWPE-1 cells knocked down for PTEN and/or STAT3 using siRNA (number of invasive cells per mm, n=5 sections for each condition). Data were analysed by one-way analysis of variance with Tukey's multiple comparison test; error bars: s.d. (h) Crystal violet stains of focus formation of STAT3-V5 and empty vector transduced PC3 cells after 4 days of incubation (Supplementary Fig. 4c–e).

Mentions: To further dissect the tumour promoting effects of loss of Stat3, we established primary mouse Pten−/− PCa cells with stable, short hairpin RNA (shRNA) mediated knockdown of Stat3. Western blot and IHC analyses confirmed efficient Stat3 knockdown in these cells (Fig. 3a and Supplementary Fig. 4a). In line with our genetic data, Pten−/− mouse PCa cells with shStat3 knockdown were significantly more invasive in a transwell invasion assay compared with control cells (Fig. 3b). We verified the aggressive behaviour of PTEN–STAT3 double deficient tumour cells in an organotypic, physiologically relevant in vitro three-dimensional cancer model18. Pten−/−-shStat3 PCa cells displayed a more invasive phenotype compared with control cells in organotypic assays (Fig. 3c and Supplementary Fig. 4b). In addition, Pten−/−-shStat3 cells showed increased anchorage-independent cell growth in soft agar compared with Pten−/− PCa cells expressing non-targeting shRNA (control shRNA) (Fig. 3d). To corroborate our findings in human cells, combined knockdown of STAT3 and PTEN in human RWPE-1 prostate cells (Fig. 3e) increased invasiveness in organotypic assay compared with control and single knockdown cells (Fig. 3f,g). Notably, re-expression of STAT3 in human PC3 prostate carcinoma cells, which lack STAT3 expression (Supplementary Fig. 4c), led to significantly decreased cell numbers and reduced foci formation (Fig. 3h and Supplementary Fig. 4d,e). These data are consistent with a cell-autonomous tumour suppressive role of Stat3 in PCa.


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 suppresses colony formation and invasion.(a) shRNA-mediated knockdown of Stat3 in Pten−/− mouse PCa cells leads to robust decrease of Stat3 levels as demonstrated by western blotting. In all knockdown experiments, scrambled non-target shRNA served as a control (control shRNA/shcontrol). (b) Representative histology of increased matrigel invasion after shRNA-mediated knockdown of Stat3 in Pten-deficient (Pten−/−) mouse PCa cells. Quantification of relative invasion is shown (n=3). Scale bars, 100 μm. (c) Organotypic culture assays of Pten−/− mouse PCa cells in the absence of Stat3 showed capacity to invade into the fibroblast containing collagen gel (red arrows). Scale bars, 50 μm. The invasive area/total tumour cell area was quantified (control shRNA, n=4, shStat3 n=3). (d) Soft agar colony formation of primary Pten−/− mouse PCa cells with shStat3 and controls was quantified (n=3). Scale bars, 200 μm. Data were analysed by Student's t-test and are shown in c–e as mean±s.d. (e) Efficient STAT3 and PTEN siRNA-mediated knockdown of RWPE-1 cells was demonstrated by western blot. Scrambled non-target siRNA served as a control (control siRNA/siControl) (f) Organotypic culture of RWPE-1 cells in the presence and absence of STAT3 and/or PTEN cultivated in contact with human prostate stromal fibroblasts seeded in collagen I gels after 8 days of culture. Representative H&E stainings are shown, red arrows indicate invasion, scale bars, 100 μm. (n=3). (g) Quantification of invasion of RWPE-1 cells knocked down for PTEN and/or STAT3 using siRNA (number of invasive cells per mm, n=5 sections for each condition). Data were analysed by one-way analysis of variance with Tukey's multiple comparison test; error bars: s.d. (h) Crystal violet stains of focus formation of STAT3-V5 and empty vector transduced PC3 cells after 4 days of incubation (Supplementary Fig. 4c–e).
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f3: Stat3 suppresses colony formation and invasion.(a) shRNA-mediated knockdown of Stat3 in Pten−/− mouse PCa cells leads to robust decrease of Stat3 levels as demonstrated by western blotting. In all knockdown experiments, scrambled non-target shRNA served as a control (control shRNA/shcontrol). (b) Representative histology of increased matrigel invasion after shRNA-mediated knockdown of Stat3 in Pten-deficient (Pten−/−) mouse PCa cells. Quantification of relative invasion is shown (n=3). Scale bars, 100 μm. (c) Organotypic culture assays of Pten−/− mouse PCa cells in the absence of Stat3 showed capacity to invade into the fibroblast containing collagen gel (red arrows). Scale bars, 50 μm. The invasive area/total tumour cell area was quantified (control shRNA, n=4, shStat3 n=3). (d) Soft agar colony formation of primary Pten−/− mouse PCa cells with shStat3 and controls was quantified (n=3). Scale bars, 200 μm. Data were analysed by Student's t-test and are shown in c–e as mean±s.d. (e) Efficient STAT3 and PTEN siRNA-mediated knockdown of RWPE-1 cells was demonstrated by western blot. Scrambled non-target siRNA served as a control (control siRNA/siControl) (f) Organotypic culture of RWPE-1 cells in the presence and absence of STAT3 and/or PTEN cultivated in contact with human prostate stromal fibroblasts seeded in collagen I gels after 8 days of culture. Representative H&E stainings are shown, red arrows indicate invasion, scale bars, 100 μm. (n=3). (g) Quantification of invasion of RWPE-1 cells knocked down for PTEN and/or STAT3 using siRNA (number of invasive cells per mm, n=5 sections for each condition). Data were analysed by one-way analysis of variance with Tukey's multiple comparison test; error bars: s.d. (h) Crystal violet stains of focus formation of STAT3-V5 and empty vector transduced PC3 cells after 4 days of incubation (Supplementary Fig. 4c–e).
Mentions: To further dissect the tumour promoting effects of loss of Stat3, we established primary mouse Pten−/− PCa cells with stable, short hairpin RNA (shRNA) mediated knockdown of Stat3. Western blot and IHC analyses confirmed efficient Stat3 knockdown in these cells (Fig. 3a and Supplementary Fig. 4a). In line with our genetic data, Pten−/− mouse PCa cells with shStat3 knockdown were significantly more invasive in a transwell invasion assay compared with control cells (Fig. 3b). We verified the aggressive behaviour of PTEN–STAT3 double deficient tumour cells in an organotypic, physiologically relevant in vitro three-dimensional cancer model18. Pten−/−-shStat3 PCa cells displayed a more invasive phenotype compared with control cells in organotypic assays (Fig. 3c and Supplementary Fig. 4b). In addition, Pten−/−-shStat3 cells showed increased anchorage-independent cell growth in soft agar compared with Pten−/− PCa cells expressing non-targeting shRNA (control shRNA) (Fig. 3d). To corroborate our findings in human cells, combined knockdown of STAT3 and PTEN in human RWPE-1 prostate cells (Fig. 3e) increased invasiveness in organotypic assay compared with control and single knockdown cells (Fig. 3f,g). Notably, re-expression of STAT3 in human PC3 prostate carcinoma cells, which lack STAT3 expression (Supplementary Fig. 4c), led to significantly decreased cell numbers and reduced foci formation (Fig. 3h and Supplementary Fig. 4d,e). These data are consistent with a cell-autonomous tumour suppressive role of Stat3 in PCa.

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