Stat5 promotes metastatic behavior of human prostate cancer cells in vitro and in vivo

Abstract

There are no effective therapies for disseminated prostate cancer. Constitutive activation of Stat5 in prostate cancer is associated with cancer lesions of high histological grade. We have shown that Stat5 is activated in 61% of distant metastases of clinical prostate cancer. Active Stat5 increased metastases formation of prostate cancer cells in nude mice by 11-fold in an experimental metastases assay. Active Stat5 promoted migration and invasion of prostate cancer cells, and induced rearrangement of the microtubule network. Active Stat5 expression was associated with decreased cell surface E-cadherin levels, while heterotypic adhesion of prostate cancer cells to endothelial cells was stimulated by active Stat5. Activation of Stat5 and Stat5-induced binding of prostate cancer cells to endothelial cells were decreased by inhibition of Src but not of Jak2. Gene expression profiling indicated that 21% of Stat5-regulated genes in prostate cancer cells were related to metastases, while 7.9% were related to proliferation and 3.9% to apoptosis. The work presented here provides the first evidence of Stat5 involvement in the induction of metastatic behavior of human prostate cancer cells in vitro and in vivo. Stat5 may provide a therapeutic target protein for disseminated prostate cancer.

Figure 1

Stat5 is active in distant human prostate cancer metastases, and Stat5 induces migration and microtubule formation of human prostate cancer cells. (A) (i) Activation of Stat5 in prostate cancer metastases to bone (left panel) and lymph nodes (right panel) was analyzed by immunohistochemistry. Bar, 50 mm. (ii) Stat5a or Stat5b was immunoprecipitated (IP) from LNCaP, DU145, PC-3, and CWR22Rv1 cells, and was blotted with anti-phospho-Stat5a/b. Filters re-blotted with anti-Stat5ab mAb and whole cell lysates (WCLs) were immunoblotted with anti-actin pAb. (iii) Pc-3 cells were infected with Ad-activeStat5 (Stat5aS710F) at different MOIs as indicated. Whole cell lysates of PC-3 cells or DU145 cells were immunoblotted with anti-phospho-Stat5ab mAb, anti-Stat5ab pAb, or anti-actin pAb. DU145 (B) and PC-3 cells (C) were infected with Ad-wild-type Stat5a (AdWTStat5a), AdWTStat5b, Ad-activeStat5, or Ad-β-galactosidase (MOI 10). Identical scratches were made in parallel wells, and the cells were fixed and photographed, and wound sizes were measured at the indicated time points (i and ii). For Boyden chamber assays (iii and iv), DU145 (B) and Pc-3 (C) cells were infected with AdWTStat5a, AdWTStat5b, Ad-activeStat5, or Ad-β-galactosidase (MOI 10). DU145 (A) and PC-3 (B) cell motility through uncoated filters (iii) or Matrigel-coated filters (iv) in three individual experiments (mean±s.d). (D) Stat5 induces rearrangement of the microtubule network of human prostate cancer cells while not affecting the organization of the actin cytoskeleton. DU145 and PC-3 cells infected with Ad-activeStat5 or Ad-β-galactosidase were immunostained with α-tubulin mAb (i and ii) or rhodamine-conjugated phalloidin (iii and iv) using FITC-conjugated secondary antibodies.

Figure 2

Active Stat5 regulates cell surface E-cadherin expression and heterotypic adhesion of prostate cancer cells, and Stat5 induces experimental prostate cancer metastases to the lungs of nude mice. (A) (i) LNCaP, DU145, and PC-3 cells were immunostained for E-cadherin using FITC-conjugated secondary antibodies (green). DNA was stained with propidium iodide (red). Total E-cadherin levels in LNCaP, DU145, and PC-3 cells were determined by immunoblotting WCLs with anti-E-cadherin mAb (lower panel). (ii) LNCaP cells were infected with Ad-activeStat5 or Ad-β-galactosidase (MOI 10), and after 48 h, the cells were immunostained for E-cadherin as described above. (iii) LNCaP cells were biotinylated, and E-cadherin was immunoprecipitated (IP) and detected by western blotting (WB) as a 120 kDa protein using streptavidin–HRP (upper panel). WCLs of the same samples were immunoblotted with anti-Stat5a/b and anti-actin antibodies. (iv) Total cellular levels of E-cadherin were determined by immunoprecipitation of parallel samples with E-cadherin mAb and immunoblotting with anti-E-cadherin mAb. (B) DU145, PC-3, and LNCaP cells were infected with Ad-activeStat5 or Ad-β-galactosidase (MOI 10). After 72 h, the cells were stained with a fluorescent dye and allowed to adhere to HUVECs (i–iii) or HBMECs (iv–vi), and the adhered cells were quantitated. (C) Active Stat5 increases metastases formation of DU145 human prostate cancer cells in the lungs of athymic nude mice. Mice were injected with DU145 cells infected with Ad-activeStat5 or Ad-β-galactosidase (MOI 10) through the tail vein. The lungs were scored for surface metastases (i). Representative photographs of India Ink-stained lungs (ii).

Figure 3

Identification of Stat5-regulated genes in human prostate cancer cells. (A) DU145 cells were transfected with Stat5a/b siRNA or scrambled siRNA as control. After 48 h, cell lysates were immunoblotted with anti-Stat5a/b mAb and re-blotted with anti-actin antibody. (B) Transcriptional profiles of Stat5-regulated genes in DU145 prostate cancer cells related to metastases, proliferation, or apoptosis out of a total of 777 genes that were differentially expressed between control siRNA and Stat5 siRNA using false discovery rate <0.01 on the full dataset of 15 992 genes. (C) A heatmap showing the top 50 differentially expressed genes by Stat5 (determined by the smallest P values) of the metastases-related group of genes. In the heatmap, red represents higher expression, whereas green represents lower expression.

Figure 4

Stat5 activation and heterotypic adhesion of prostate cancer cells were related to Src kinase activation rather than to Jak2 activation. (A) CWR22Rv1 (i), DU145 (ii), and LNCaP (MOI 10) (iii) cells were infected with AdDNJak2, AdWTJak2, or AdLacZ for 48 h, after which, immunoprecipitated Stat5a and Stat5b were immunoblotted with anti-phospho-Stat5a/b mAb, and the filters were re-blotted with anti-Stat5a/b mAb. WCLs of the same samples were blotted for actin. (B) DU145 cells were treated with pharmacological inhibitors of RhoA (Y27632) and Rac1 (553502) for 48 h. Stat5a and Stat5b were immunoprecipitated and immunoblotted with anti-phospho-Stat5a/b mAb and anti-Stat5a/b mAb. (C) DU145 (i) and LNCaP (ii and iii) cells were treated with pharmacological Src inhibitor PP2 for 72 or 48 h (iii) using PP3 as a control compound. Levels of active Stat5a and Stat5b were analyzed by immunoprecipitation and western blotting as described above. (D) Pharmacological inhibition of Src kinases attenuates heterotypic adhesion of DU145 cells to endothelial cells. DU145 cells were treated with PP2 (20 μM) using PP3 as the control for 72 h, after which, the cells were stained with a fluorescent dye and allowed to bind to HUVECs (i) or HBMECs (ii) for 60 min before quantitation of the adhered cells.