Article Figures & Data
Figures
- Figure 1.
Enzalutamide (ENZ) induces Jak2–Stat5a/b signaling in prostate cancer. A, Active Stat5 levels were robustly elevated in prostate cancers from patients treated by enzalutamide compared with hormone-naïve prostate cancers, demonstrated by immunostaining of paraffin-embedded tissue sections (image magnification 40×; scale bar, 50 μm). B, Active Stat5 levels were elevated in paired biopsies after enzalutamide treatment compared with biopsies taken prior to enzalutamide treatment from the same patient (image magnification 40×; scale bar, 50 μm). C, Enzalutamide induces Stat5 phosphorylation in prostate cancer cells. Prostate cancer cells were treated with enzalutamide or vehicle for the indicated periods of time. Expression levels of active Stat5 and Stat3 were determined by immunoprecipitation (IP) of Stat5 and Stat3 followed by immunoblotting (WB) for pStat5a/b, pStat3, total Stat5, and total Stat3. Whole cell lysates (WCL) were immunoblotted for actin. Stat5 phosphorylation levels are elevated in enzalutamide-resistant (R) (CWR22Pc AR-F876L) cells compared with parental CWR22Pc cells (right). D, AR is required for enzalutamide induction of Stat5 phosphorylation. AR-negative DU145 prostate cancer cells were treated with enzalutamide or vehicle, and CWR22Pc and LAPC4 cells were transduced with lentiviral AR shRNA (shAR) or lentiviral shCtrl for 3 days followed by treatment with enzalutamide or vehicle for 7 days at the indicated concentrations. E, DHT, androgen deprivation, or genetic AR suppression do not increase Stat5 phosphorylation levels in prostate cancer cells. Prostate cancer cells were androgen deprived (charcoal-stripped FBS) or cultured with additional DHT (1.5 μmol/L) for 7 days. Alternatively, AR was suppressed by lentiviral AR shRNA for 7 days. Expression levels of active Stat5, Stat5, and Jak2 were determined by immunoprecipitation (IP) followed by immunoblotting (WB) with actin blotting as loading control.
- Figure 2.
Enzalutamide (ENZ)-liganded androgen receptor induces Jak2 phosphorylation in prostate cancer cells. A, CWR22Pc, LAPC4, and ENZ-R cells were cultured with enzalutamide or Jak2 inhibitor AZD1480 alone or in combination for 12 days at indicated concentrations. Expression levels of active Stat5 were determined by immunoprecipitation (IP) of Stat5 followed by immunoblotting (WB) for pStat5a/b and total Stat5. Whole cell lysates (WCL) were immunoblotted for pStat3, Stat3 and actin. B, Genetic knockdown of Jak2 blocks enzalutamide-induced Stat5 phosphorylation in prostate cancer cells. Jak2 was suppressed by lentiviral Jak2 shRNA versus shCtrl in prostate cancer cells for 3 days followed by enzalutamide or vehicle for 7 days. Active Stat5, Stat5, and Jak2 levels were evaluated by IP and Western blot analysis, as depicted. C, CWR22Pc and LAPC-4 cells were treated with enzalutamide or vehicle for 6 hours at the indicated concentrations. Control cells were stimulated with prolactin (Prl; 10 nmol/L) for 20 minutes as control for cytokine-induced Jak2 phosphorylation. Alternatively, prostate cancer cells were treated with enzalutamide or vehicle for 12 days or stimulated with Prl for 20 minutes at the indicated concentrations. Stat5 and Jak2 were immunoprecipitated and immunoblotted (WB) for pStat5, pJak2, total Stat5, and total Jak2. WCLs were immunoblotted for actin. D, AR is required for enzalutamide induction of Jak2 activation. AR was suppressed in CWR22Pc cells by lentiviral AR shRNA (shAR) versus shCtrl for 3 days followed by enzalutamide for 6 hours. In parallel experiments, CWR22Pc cells were cultured with 1.5 μmol/L DHT for 6 hours or 7 days. Enzalutamide induction of Jak2–Stat5 activation requires a cytokine receptor shown by treatment of CWR22Pc cells with enzalutamide alone or in combination with a prolactin receptor antagonist LFA102 for 6 hours or 7 days at the indicated concentrations. E, Enzalutamide induction of Jak2–Stat5 activation requires ongoing protein synthesis in prostate cancer cells. CWR22Pc cells were treated with cycloheximide (CHX; 35 μmol/L) for 24 hours followed by enzalutamide treatment (40 μmol/L) for 6 hours. F, Phosphatase inhibitor vanadate (VAN) abolished enzalutamide induction of Jak2 phosphorylation. CWR22Pc cells were treated with enzalutamide (6 hours) or VAN (2 hours) or vehicle alone or pretreated with VAN (2 hours) followed by enzalutamide (6 hours). G, Enzalutamide-induced Jak2 phosphorylation is decreased by depletion of either PTPϵ or SHP-2 phosphatases. CWR22Pc and LAPC4 cells were infected with lentivirus expressing shRNA targeting PTPϵ, SHP-2, PTP1B, or shCtrl for 3 days followed by enzalutamide or vehicle treatment for 6 hours at the indicated concentrations. IPs and WBs in were conducted as described in A.
- Figure 3.
Enzalutamide (ENZ) induces a formation of a positive Jak2–Stat5 feed-forward loop in prostate cancer. A, Enzalutamide-induced Jak2 phosphorylation is accompanied by an increase in Stat5 phosphorylation and Jak2 protein levels in prostate cancer cells. CWR22Pc, LAPC-4, and ENZ-R cells were treated with enzalutamide or vehicle during a time-course at the indicated concentrations. Prostate cancer cells were stimulated with Prl (10 nmol/L) as control for Jak2 and Stat5 phosphorylation. The phosphorylation status of Stat5 and Jak2 was determined by IP of Stat5 and Jak2 followed by immunoblotting for pStat5, total Stat5, pJak2, and total Jak2. WCLs were immunoblotted for actin. B, Enzalutamide induces Jak2 mRNA levels in prostate cancer cells. CWR22Pc and LAPC-4 cells were treated with enzalutamide or vehicle for indicated periods of time or infected with lentiviral Jak2 shRNA (shJak2) versus shCtrl (Jak2 mRNA control; 3 days) followed by qRT-PCR. C, Jak2, but not Jak1, levels are induced by enzalutamide. Prostate cancer cells were treated with enzalutamide or vehicle for 12 days. Jak1 and Jak2 were immunoprecipitated and blotted for Jak1 and Jak2. WCLs were immunoblotted for actin. D, Enzalutamide increases Jak2 protein levels by Stat5-driven upregulation of Jak2 mRNA expression in prostate cancer cells. When Stat5 was depleted in prostate cancer cells by lentiviral Stat5 shRNA (shStat5) or shCtrl for 3 days followed by enzalutamide or vehicle for 7 days, enzalutamide failed to increase Jak2 mRNA levels. For Jak2 mRNA control, Jak2 was suppressed by lentiviral Jak2 shRNA (shJak2) or shCtrl (left) for 3 days. Jak2 mRNA levels were determined by qPCR and protein levels by IP and Western blot analysis (WB) as described in A. E, CWR22Pc and LAPC-4 cells were infected with lentiviral Stat5 shRNA (shStat5), shCtrl (top and bottom panels), constitutively active (CA) Stat5 or GFP for 3 days (middle). After 3 days, prostate cancer cells were treated with Prl for 3 days (bottom). IP, immunoblotting (WB), and qPCR were conducted as described in A and B. F, Schematic representation of the proposed enzalutamide induction of hyperactivated Jak2–Stat5 feed-forward loop in prostate cancer.
- Figure 4.
Stat5 promotes viability of prostate cancer cells during enzalutamide (ENZ) treatment. Constitutively active (CA) Stat5 or GFP was lentivirally expressed in CWR22Pc and LAPC-4 cells for 2 days followed by enzalutamide treatment (5, 10, 20, 30, and 40 μmol/L) or vehicle for 6 days (A and D). The fractions of viable cells were determined by counting three separate fields from each of the three parallel wells, and the averages are indicated by a line graph with SDs. Crystal violet staining (B and E) and immunoblotting of whole cell lysates (WCL) for Stat5 and actin on day 6 (C and F).
- Figure 5.
Pharmacologic inhibition of Jak2–Stat5 signaling increases death of prostate cancer cells when used in combination with enzalutamide (ENZ) or alone after enzalutamide. A, The experimental design for a sequential treatment of prostate cancer cells with AR inhibition followed by genetic or pharmacologic knockdown of Stat5. B, Stat5 inhibitors alone or in combination with enzalutamide are more effective than enzalutamide alone in suppressing prostate cancer cell viability. Sequential therapy with enzalutamide followed by IST5-002 decreases viability of prostate cancer cells surviving enzalutamide treatment. CWR22Pc cells were treated at the start of the experiment (day 0) with vehicle, enzalutamide (40 μmol/L) for 5 days, IST5-002 (12.5 μmol/L) alone or in combination, as indicated or left untreated (MOCK). For sequential treatment, cells were treated at the start of the experiment (day 0) with enzalutamide (40 μmol/L) followed by treatment with vehicle (ENZ > vehicle) or IST5-002 (12.5 μmol/L; ENZ > IST5-002) for 5 or 10 days. The fractions of viable cells are indicated by columns with SDs. Activation of Stat5 was determined by IP of followed by immunoblotting (WB) for pStat5 and total Stat5. WCLs were immunoblotted for actin. C, FACS analysis of cell-cycle distribution at the indicated timepoints. D, Pictures of prostate cancer cells stained with crystal violet after the treatments, as described in B. E, Combination of enzalutamide with AZD1480 is more effective than enzalutamide alone in decreasing viability of prostate cancer cells surviving enzalutamide treatment. Sequential treatment of CWR22Pc cells with enzalutamide (40 μmol/L) for 5 days followed by AZD1480 (0.8 μmol/L) for 5 or 10 days suppresses viability of prostate cancer cells surviving enzalutamide treatment. CWR22Pc cells were treated and analyzed for viable cells as described in A. F, FACS analysis of cell-cycle distribution at the indicated timepoints. G, Crystal violet staining of the parallel wells. H, IST5-002 and AZD1480 decreased viability of both parental CWR22Pc cells and ENZ-R cells (AR-F876L) after 5 and 10 days of treatment. Enzalutamide decreased viability of parental cells but did not affect viability of ENZ-R cells after 5 or 10 days of treatment. The fractions of viable cells are indicated by columns with SDs.
- Figure 6.
Second-line treatment by pharmacologic inhibitors of Jak2–Stat5 signaling suppresses growth of both androgen-dependent and enzalutamide (ENZ)-resistant xenograft tumors in nude mice and in patient0-derived prostate cancers ex vivo in tumor explant cultures. A, Inhibition of Stat5 by IST5-002 suppressed androgen-sensitive CWR22Pc xenograft tumor growth more effectively than bicalutamide (BIC) or enzalutamide, as shown by tumor growth curves (top) and representative tumor images (bottom). CWR22Pc cells were inoculated subcutaneously (s.c.) into flanks of castrated athymic nude mice supplied with sustained release DHT pellets. Mice were surgically castrated or treated daily with vehicle, bicalutamide (30 mg/kg), enzalutamide (30 mg/kg), or IST5-002 (IST5; 50 mg/kg), and tumor growth rates were calculated for each treatment group and are presented as fold changes in tumor volume (volume at timepoint/volume at treatment start). B, Experimental design for the sequential therapy of the xenograft tumors in vivo. A two-phase in vivo experiment using vehicle or enzalutamide as first-line therapy (phase I, 13 days) and vehicle, enzalutamide, AZD1480, IST5-002, or ENZ + IST5-002 as second-line therapy (phase II, 18 days). On day 31, mice were sacrificed and CWR22Pc xenograft tumors collected for analyses. Sequential, second-line therapy with IST5-002 (IST5) or ENZ + IST5-002 (IST5) effectively suppressed the growth of androgen-sensitive (left; C) and enzalutamide-resistant (right; D). CWR22Pc xenograft tumors, as shown by tumor growth curves (top) and representative tumor images (bottom). CWR22Pc cells were inoculated subcutaneously into flanks of castrated athymic nude mice supplied with sustained release DHT pellets. Mice were treated with vehicle or enzalutamide (30 mg/kg) for 13 days. On day 13, mice were randomly distributed into subgroups and treated daily for an additional 18 days with vehicle, enzalutamide (30 mg/kg), AZD1480 (30 mg/kg), IST5-002 (IST5; 50 mg/kg), or enzalutamide (30 mg/kg) + IST5-002 (IST5; 50 mg/kg). Tumor growth rates are calculated and presented as described in A. E, To test the efficacy of a second-line therapy with combined enzalutamide and IST5-002 (IST5) versus enzalutamide monotherapy in clinical prostate cancers, seven localized prostate cancers obtained from radical prostatectomies were cultured ex vivo in tumor explant cultures in the presence of vehicle (8 days), enzalutamide (40 μmol/L) alone (8 days), or enzalutamide (40 μmol/L) as first-line therapy (4 days) followed by enzalutamide (40 μmol/L) and IST5-002 (IST5; 25 μmol/L) as a second-line therapy (4 days). Second-line therapy with combined enzalutamide and IST5-002 (IST5) induced cell death in clinical prostate cancers more effectively than enzalutamide alone, as demonstrated by extensive loss of viable acinar epithelium. The number of viable epithelial cells in the explants at the end of the cultures was counted and presented as percentage per epithelial cells prior to culture. Representative histologies are shown (image magnification 40×; scale bar, 50 μm). F, Levels of nuclear Stat5 in the prostate cancer explants were determined by immunostaining using biotin–streptavidin–amplified peroxidase-antiperoxidase immunodetection at the end of the cultures and expressed as percentages of Stat5-positive cells per 100 epithelial cells in explants for each treatment group (12–20 explants; image magnification 40×; scale bar, 50 μm).
Additional Files
Supplementary Data
- Supplementary Data - Supplementary Material and Methods
- Supplementary Figure 1 - ENZ-induced activated Stat5 is capable of nuclear translocation in PC cells.
- Supplementary Figure 2 - Levels of nuclear Stat5 in xenograft tumors.
- Supplementary Table 1 - Supplementary Table 1 a and b: Characteristics of PCs from patients.
- Supplementary Table 2 - Characteristics of patient-derived PCs tested ex vivo in 3D tumor explant cultures for responsiveness to Stat5-inhibitor IST5 in combination with ENZ as a second-line treatment after ENZ.
- Supplementary Table 3 - Antibodies used in the study.
Volume 19, Issue 1
