Enzalutamide-Induced Feed-Forward Signaling Loop Promotes Therapy-Resistant Prostate Cancer Growth Providing an Exploitable Molecular Target for Jak2 Inhibitors

Enzalutamide-liganded AR induces phosphorylation and activation of Stat5 in prostate cancer

IHC analyses of the activation status of Stat5 in prostate cancers of patients treated with enzalutamide show that the levels of nuclear active Stat5 were significantly (P < 0.0001) elevated in biopsies of enzalutamide-treated prostate cancers when compared with hormone-naïve prostate cancers of corresponding histologic grades (Fig. 1A; Supplementary Table S1A). In paired prostate cancer samples from patients before and after enzalutamide treatment (Supplementary Table S1B), active Stat5 levels were robustly elevated in the biopsies after enzalutamide treatment compared with the biopsies taken prior to enzalutamide treatment (Fig. 1B). In CWR22Pc and LAPC4 cell lines, enzalutamide induced a rapid increase in Stat5 phosphorylation at 6 hours that was sustained and further increased at day 7 (Fig. 1C). At the same time, phosphorylation of Stat3 was not induced by enzalutamide in the same cell lines (Fig. 1C). In a CWR22Pc cell subline expressing AR-F876L (CW22Pc-ENZ-R; ref. 17), which emerges in CWR22Pc cells surviving extended treatment with enzalutamide (≥6 months; ref. 18), active Stat5 levels were markedly higher compared with the parental CWR22Pc cells (Fig. 1C). Enzalutamide induction of Stat5 phosphorylation resulted in nuclear translocation of Stat5 to an extent comparable with cytokine-induced Stat5 nuclear translocation (Supplementary Fig. S1), indicating that enzalutamide induces formation of Stat5 dimers capable of nuclear localization in prostate cancer cells.

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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.

To investigate whether AR is required for enzalutamide induction of Stat5 signaling, we first analyzed enzalutamide activation of Stat5 in DU145 prostate cancer cell line which is AR-negative. In a parallel set of experiments, we inhibited AR expression in CWR22Pc and LAPC4 cells by genetic knockdown using lentiviral expression of AR shRNA. In the absence of AR, enzalutamide failed to induce Stat5 activation (Fig. 1D) demonstrating that enzalutamide induction of Stat5 signaling is not an AR-independent off-target effect of the enzalutamide compound. We next evaluated whether androgen deprivation, additional androgen treatment, or genetic knockdown of AR would lead to an increase in Stat5 phosphorylation in prostate cancer cells. CWR22Pc and LAPC4 cell were androgen-deprived, cultured with additional DHT supplementation (1.5 μmol/L), or AR was genetically depleted by lentiviral expression of AR shRNA (Fig. 1E). Androgen deprivation or AR knockdown did not stimulate Stat5 phosphorylation levels in prostate cancer cells. In addition, when the AR was liganded by DHT, Stat5 phosphorylation status remained unchanged. Taken together, these results indicate that enzalutamide-liganded AR induces sustained Stat5 activation in human prostate cancer.

Enzalutamide-induced Stat5 activation in prostate cancer cells occurs via Jak2

To assess whether enzalutamide-induced Stat5 activation in prostate cancer cells is dependent on Jak2 signaling, Jak2 activity was pharmacologically inhibited by Jak2 inhibitor AZD1480 (44) during enzalutamide treatment. AZD1480 blocked enzalutamide-induced Stat5 phosphorylation in CWR22Pc, LAPC4, and CWR22Pc-ENZ-R cells (Fig. 2A). To further assess the involvement of Jak2 in enzalutamide-induced Stat5 activation, Jak2 was genetically knocked down by lentiviral expression of Jak2 shRNA in CWR22Pc and LAPC4 cells for 3 days followed by enzalutamide treatment for 7 days (Fig. 2B). In the absence of Jak2, enzalutamide failed to induce Stat5 phosphorylation in both cell lines. Unexpectedly, the protein levels of Jak2 on day 7 were robustly increased in enzalutamide-treated CWR22Pc and LAPC4 cells compared with nontreated cells (Fig. 2B). Collectively, these results indicate that enzalutamide induction of Stat5 activation in prostate cancer cells requires Jak2.

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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.

We next investigated whether enzalutamide-liganded AR influences the phosphorylation status of Jak2 in prostate cancer cells. Enzalutamide induced a rapid phosphorylation of Jak2 already at 6 hours in both CWR22Pc and LAPC4 cells to almost the same extent as stimulation of the cells with the cytokine prolactin (Prl; Fig. 2C). Enzalutamide-induced Jak2 phosphorylation was sustained at 12 days and accompanied with a strong induction of Stat5 phosphorylation in both CWR22Pc and LAPC4 cells (Figs. 2C). To further examine whether AR is required for enzalutamide induction of Jak2 activation, AR was suppressed by lentiviral AR shRNA in CWR22Pc cells, which blocked the enzalutamide-induced Jak2 activation (Fig. 2D). In addition, AR agonists were not able to increase Jak2 phosphorylation in prostate cancer cells, because supplementation of culture medium with additional DHT (1.5 nmol/L) for 6 hours and 7 days cell did not elevate Jak2 phosphorylation in CWR22Pc prostate cancer cells (Fig. 2D, middle). To further understand whether enzalutamide-liganded AR induction of Jak2–Stat5 activation requires the presence of a cytokine receptor, we disrupted Prl receptor (PrlR) dimerization by a specific Prl antagonist LFA102 (43) for 6 hours and 7 days. As shown in Fig. 2D, enzalutamide failed to activate Jak2 and Stat5 in the presence of LFA102 suggesting that docking of Stat5 and Jak2 to the cytokine receptor complex is required for enzalutamide induction of Jak2–Stat5 signaling. In summary, the data shown here demonstrate that AR-liganded enzalutamide induces rapid and sustained Jak2 phosphorylation in prostate cancer cells that involves association of Jak2 with a cytokine receptor.

Enzalutamide induction of Jak2 phosphorylation in prostate cancer cells involves Jak2 phosphatases

To investigate the mechanisms underlying enzalutamide activation of Jak2 in prostate cancer cells, CWR22Pc cells were treated with cycloheximide (35 μmol/L) for 24 hours followed by enzalutamide (40 μmol/L) for 6 hours, which blocked enzalutamide-induced Jak2 phosphorylation (Fig. 2E). This suggests that enzalutamide induction of Jak2–Stat5 signaling is mediated by a protein that is regulated by enzalutamide and, therefore, requires ongoing protein synthesis. To evaluate whether enzalutamide induction of Jak2 phosphorylation is dependent on phosphatase activity in prostate cancer cells, we treated prostate cancer cells with a broad-spectrum phosphatase inhibitor vanadate (VAN). Pretreatment of CWR22Pc with VAN led to a general increase in Jak2 phosphorylation, as expected, but also attenuated the induction of Jak2 phosphorylation by enzalutamide (Fig. 2F) suggesting involvement of Jak2 phosphatases in enzalutamide induction of Jak2 phosphorylation. The key known Jak2 phosphatases expressed in solid tumors are SHP-2 (37), PTPϵ (38), and PTP1B (39, 40), while SHP-1 is expressed in hematopoietic cells. Using lentiviral expression of shRNA, we depleted each of the three phosphatases for 3 days prior to enzalutamide treatment (6 hours) of CWR22Pc and LAPC4 cells (Fig. 2G). In both prostate cancer cell lines, enzalutamide-induced Jak2 phosphorylation was significantly attenuated by depletion of either SHP-2 or PTPϵ. Genetic knockdown of PTPϵ resulted in generally elevated levels of Jak2 phosphorylation, which indicates that PTPϵ is a crucial negative regulator of Jak2 phosphorylation in prostate cancer cells. Importantly, when PTPϵ was genetically depleted in prostate cancer cells, there was no difference between enzalutamide treatment versus nontreated cells suggesting that the mechanism of action of enzalutamide on Jak2 phosphorylation involves suppression of PTPϵ activity (Fig. 2G, left). At the same time, genetic depletion of SHP-2 decreased enzalutamide induction of Jak2 phosphorylation while not increasing the general phosphorylation levels of Jak2 (Fig. 2G, middle). This, in turn, suggests that SHP-2 is a positive regulator of enzalutamide-induced Jak2–Stat5 signaling and is required for enzalutamide induction of Jak2 phosphorylation in prostate cancer cells. A positive regulatory role of SHP-2 for Jak2–Stat5 signaling has been previously reported for several different cell types (45, 46). Genetic knockdown of PTP1B did not affect Jak2 phosphorylation levels nor enzalutamide induction of Jak2 phosphorylation (Fig. 2G, right). In conclusion, these data indicate involvement of Jak2 phosphatases SHP-2 and PTPϵ in enzalutamide-induced Jak2 phosphorylation in prostate cancer cells.

Enzalutamide-induced Stat5 activation increases Jak2 mRNA and protein levels and a formation of a positive feed-forward loop in prostate cancer cells

The finding demonstrating that enzalutamide may increase Jak2 protein levels in CWR22Pc and LAPC4 cells (Fig. 2B), led us to evaluate the effects of enzalutamide on phosphorylation, protein levels, and mRNA levels of Jak2 and Stat5 during a time-course ranging from 2 hours to 6 days in CWR22Pc and LAPC4 cells (Fig. 3A and B). Enzalutamide increased phosphorylation of Jak2 in both CWR22Pc and LAPC4 cells at 6 hours, which was accompanied by an increase in Stat5 phosphorylation. Activation of Stat5 reached maximum at 12–16 hours and was sustained at 6 days of exposure to enzalutamide (Fig. 3A). The levels of Jak2 protein, but not Stat5, increased starting at approximately 16 hours (CWR22Pc) and 6 hours (LAPC4) after initiation of the enzalutamide treatment. Concomitant with enzalutamide-induced activation of Jak2 and Stat5, Jak2 mRNA levels were increased by 70% at the 6-hour time-point of enzalutamide treatment, which was sustained at the day 6 (Fig. 3B). In parallel experiments, prostate cancer cells resistant to enzalutamide (CWR22Pc-ENZ-R) displayed markedly higher levels of Jak2 protein (Fig. 3A) and mRNA (Fig. 3B) in comparison with the parental CWR22Pc cells. While inducing Jak2 levels, enzalutamide did not induce Jak1 levels in CWR22Pc or LAPC-4 cells indicating specificity of enzalutamide regulation of the phosphorylation state of Jak2 (Fig. 3C).

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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.

To further understand the mechanisms underlying enzalutamide induction of Jak2 mRNA levels, we analyzed the significance of Stat5 in this process by genetic knockdown of Stat5 by lentiviral shRNA in CWR22Pc and LAPC4 cells. In the absence of Stat5, enzalutamide failed to induce Jak2 mRNA and protein expression and Jak2 mRNA levels were generally decreased by more than 90% (Fig. 3D). This finding suggests that Stat5 is critical for enzalutamide-induced upregulation of Jak2 mRNA expression in prostate cancer cells. Because Stat5 has not been shown to upregulate Jak2 mRNA expression in other cell types, we investigated this in prostate cancer cells in more detail. First, we suppressed Stat5 by lentiviral Stat5 shRNA in CWR22Pc and LAPC4 cells for 3 days and assessed Jak2 mRNA levels by qRT-PCR. Depletion of Stat5 led to a 60%–80% decrease in Jak2 mRNA levels in both CWR22PC and LAPC4 cells (Fig. 3E, left). In parallel experiments, lentiviral expression of constitutively active (CA) Stat5 (32) in CWR22Pc and LAPC4 cells increased Jak2 mRNA expression by 120%–160%, which was also reflected at Jak2 protein levels (Fig. 3E, middle). In addition, Prl-induced Stat5 activation increased Jak2 mRNA levels by 80%–120% in prostate cancer cells, which was blocked by genetic knockdown of Stat5 by shRNA (Fig. 3E, right). In summary, these results demonstrate that enzalutamide induces Jak2 mRNA and protein levels by Stat5-driven mechanisms in prostate cancer. Collectively, these findings indicate that enzalutamide induces a positive Jak2–Stat5 feed-forward loop in prostate cancer, depicted in Fig. 3F. In this proposed scheme, enzalutamide-liganded AR induces Jak2 phosphorylation in prostate cancer cells through regulation of the activity and function of Jak2 phosphatases. Activated Jak2 leads to phosphorylation of Stat5 which, in turn, increases Jak2 mRNA and protein levels. It is known that elevated Jak2 protein levels result in Jak2 autophosphorylation (41). In addition, increased levels of Jak2 are likely to result in upregulation of Jak2–Stat5 activation in prostate cancer cells induced by enzalutamide.

Stat5 promotes viability of prostate cancer cells surviving enzalutamide treatment

Having established that enzalutamide induces Jak2–Stat5 signaling in prostate cancer, we investigated whether active Stat5 promotes CRPC growth during enzalutamide treatment. First, constitutively active (CA) Stat5 (32) was expressed in CWR22Pc and LAPC4 cells using lentivirus during treatment of the cells with increasing doses of enzalutamide ranging from 5 to 40 μmol/L for 6 days. Expression of CAStat5 increased the fraction of viable CWR22Pc and LAPC4 cells at all concentrations of enzalutamide with the maximum increase of 60% (P < 0.0001) and 45% (P < 0.0001) in CWR22Pc and LAPC4 cells (Fig. 4A, B, D, and E). The efficacy of lentiviral CAStat5 expression in prostate cancer cells was confirmed by immunoblotting (Fig. 4C and F).

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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).

To investigate the efficacy of inhibition of Jak2–Stat5 signaling in eliminating prostate cancer cells that survive enzalutamide treatment, we used a specific Stat5 inhibitor IST5-002 (47) or Jak2 inhibitor AZD1480 (44). Prostate cancer cells were exposed to enzalutamide for 5 days followed by treatment with IST5-002 or AZD1480 for 5 or 10 days (Fig. 5A). Control cells were grown in culture medium (MOCK), in the presence of vehicle (VEH), enzalutamide, IST5-002 (IST5), AZD148, or enzalutamide combined with IST5-002 (ENZ + IST5) or AZD1480 (ENZ + AZD1480) for 5, 10, or 15 days, as depicted in Fig. 5A. In comparison with the control group (enzalutamide followed by vehicle: ENZ > vehicle), CWR22Pc cells treated at the start of the experiment with enzalutamide and later switched to IST5-002 after 5 days (ENZ > IST5-002) incurred additional loss of viability at both time points with the maximal reduction of 50% at 10 days of enzalutamide treatment (15-day timepoint; P < 0.001; Fig. 5B). Notably, IST5-002 alone or in combination with enzalutamide was more efficacious (P < 0.001) in reducing viability of CWR22Pc cells than enzalutamide applied as a single treatment (Fig. 5B). This may be due to enzalutamide induction of hyperactive Jak2–Stat5 signaling loop, which leads to Stat5 promotion of prostate cancer cell viability and growth. Moreover, when the cells were pretreated with enzalutamide, inhibition of Stat5 was less efficient in reducing prostate cancer cell viability compared with Stat5 inhibition monotherapy or combination treatment of the cells with both IST5-002 and enzalutamide (Fig. 5B). Similarly, this may be explained by enzalutamide activation of positive Jak2–Stat5 feed-forward loop, which sets a barrier for a second-line therapy to overcome. Viability of CWR22Pc in each group was visualized and quantified by cell-cycle analysis and crystal violet staining (Fig. 5C and D).

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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.

Given that enzalutamide induces a marked increase in Jak2 phosphorylation in prostate cancer cells, we next assessed the efficacy of a sequential application of enzalutamide followed by AZD1480 (44) as a representative of Jak2 inhibitors. Compared with control (ENZ > vehicle), CWR22Pc cells initially receiving enzalutamide at the start of the experiment for 5 days and switched to AZD1480 for 5 or 10 days (ENZ > AZD1480) exhibited additional loss of 15%–20% of cell viability at both time points (P < 0.05; Fig. 5E–G). Stat5 inhibition was verified by immunoblotting in cells receiving AZD1480, combined enzalutamide and AZD1480, or enzalutamide followed by AZD1480 (Fig. 5E). In summary, Jak2 inhibition by AZD1480 induced extensive death of CWR22Pc cells that survived enzalutamide treatment.

To investigate whether prostate cancer cells encompassing AR with the mutation conferring enzalutamide resistance are sensitive to inhibition of Jak2–Stat5 signaling with cell viability as the endpoint, Jak2–Stat5a/b signaling was inhibited in both parental CWR22Pc and ENZ-R (AR-F876L) cells by AZD1480 or IST5-002. While parental CWR22Pc cells remained sensitive to enzalutamide, viability of enzalutamide-resistant (AR-F876L) CWR22Pc cells was unaffected by enzalutamide, as expected (Fig. 5H). Viability of both parental and enzalutamide-resistant CWR22Pc cells was decreased by both IST5-002 (12.5 μmol/L; P < 0.001) or AZD1480 (P < 0.001; 0.8 μmol/L) at 10 days, indicating that enzalutamide-resistant CWR22Pc cells remained sensitive to Stat5a/b inhibition (Fig. 5H). Enzalutamide-resistant cells displayed uniformly high levels of active Stat5 (Fig. 5H). Collectively, these findings support the concept of an emergence of sustained upregulation of Jak2–Stat5 activation during long-term enzalutamide treatment. To summarize, these data demonstrate that active Stat5 increases viability and growth of prostate cancer cells during enzalutamide treatment, and pharmacologic inhibition of Jak2–Stat5 signaling induces death of prostate cancer cells surviving enzalutamide treatment.

Stat5 inhibition decreases growth of enzalutamide-resistant prostate cancer xenograft tumors and patient-derived prostate cancers in tumor explant cultures ex vivo

Given that Stat5a inhibition potently induced apoptotic death of prostate cancer cells depleted of AR signaling, we sought to extend our in vitro findings to prostate cancer xenograft tumor growth in vivo. Castrated athymic nude mice, implanted with sustained-release DHT pellets to normalize circulating androgen levels, were inoculated subcutaneously with CWR22Pc cells. CWR22Pc tumors are known to initially display androgen-sensitive growth and regress upon castration, but later recur as castrate-resistant tumors (42). The mice were treated with bicalutamide, enzalutamide, or IST5-002 daily for 32 days with vehicle or surgical castration as control groups (Fig. 6A). Tumors in the castration group rapidly resumed the same growth rate as tumors in the vehicle-treated mice, suggesting the presence of adrenal and/or intracrine androgen synthesis. As expected, enzalutamide was more effective than bicalutamide in blocking androgen-sensitive CWR22Pc tumor growth (P < 0.001; Fig. 6A). In line with the higher efficacy of IST5-002 than enzalutamide in inducing death of prostate cancer cells in vitro shown in Fig. 5, IST5-002 was superior to enzalutamide (P < 0.05) at suppressing in vivo androgen-sensitive growth of CWR22Pc xenograft tumors in the presence of circulating androgens (Fig. 6A). IHC analyses showed decreased nuclear Stat5 content within tumor cells treated with IST5-002, while nuclear Stat5 levels were elevated in tumors treated with enzalutamide (Supplementary Fig. S2A). Collectively, these results indicate that Stat5 inhibition by IST5-002 blocked CWR22Pc xenograft tumor growth equally or more effectively than enzalutamide.

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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).

To assess therapeutic efficacy of sequential targeting of AR and Stat5 in prostate cancer tumors in vivo, we designed a two-phase in vivo experiment, depicted in Fig. 6B. Mice received first-line therapy of vehicle or enzalutamide daily until emergence of resistance to enzalutamide occurred (day 13), followed by randomization and switch to a second-line therapy (vehicle, enzalutamide, AZD1480, IST5-002, or ENZ + IST5-002) daily for additional 18 days. Tumors in mice receiving vehicle during the first phase represent androgen-sensitive prostate cancer growth (Fig. 6C), and final fold changes in tumor volume (volume at day 31/day 0 ± SEM) at the end of the second phase (day 31) were as follows: vehicle > vehicle, 41.6 ± 1.9; vehicle > ENZ, 29.8 ± 2.5; vehicle > AZD1480, 23.7 ± 0.9; vehicle > IST5-002, 18.9 ± 2.0; vehicle > ENZ + IST5-002, 14.6 ± 2.0 (Fig. 6C). As expected, enzalutamide reduced androgen-sensitive CWR22Pc xenograft tumor growth significantly compared with vehicle-treated tumors (P = 0.05). At the same time, Stat5 inhibition by IST5-002 alone suppressed androgen-sensitive prostate cancer xenograft tumor growth equally effectively as enzalutamide when compared with the control group (vehicle; P < 0.01). However, the growth of androgen-sensitive prostate cancer tumors was suppressed more effectively by combination of IST5-002 with enzalutamide when compared with enzalutamide alone (P = 0.02; Fig. 6C). In conclusion, while enzalutamide alone suppressed growth in the presence of androgens as expected, combination of enzalutamide with IST5-002 was remarkably more effective than enzalutamide alone.

Prostate cancer tumors in mice receiving enzalutamide during the first phase developed resistance as evidenced by regrowth of tumors in enzalutamide-treated mice starting on day 13. At the end of the second-line treatment on day 31, final fold changes in enzalutamide-resistant tumor volumes were as follows: ENZ > vehicle, 22.8 ± 2.3; ENZ > ENZ, 13.5 ± 1.5; ENZ > AZD1480, 8.9 ± 1.3; ENZ > IST5-002, 7.3 ± 0.9; ENZ > ENZ + IST5-002, 4.9 ± 0.6 (Fig. 6D). Importantly, IST5-002 alone suppressed enzalutamide-resistant prostate cancer tumor growth. At the same time, combined enzalutamide with IST5-002 was most effective in suppressing enzalutamide-resistant CRPC tumor growth when compared with continued enzalutamide treatment (P < 0.01). IHC analysis of Stat5 demonstrated that enzalutamide increased nuclear Stat5 in both androgen-induced and castrate-resistant tumors (Supplementary Fig. S2). In conclusion, growth of enzalutamide-resistant CRPC xenograft tumors was most effectively suppressed by a second-line therapy of combined enzalutamide and IST5-002.

To evaluate the efficacy of combined enzalutamide and IST5-002 as a second-line therapy in clinical patient-derived prostate cancers, we utilized an ex vivo 3D tumor explant culture system of patient-derived prostate cancers, which we have established and characterized previously (25, 32, 47–49). Crucial interactions between prostate epithelium and stroma are retained in tumor explant cultures and, therefore, it is considered a more physiologic model of prostate cancer growth than cell lines or primary cell cultures. Prostate cancers from seven individual patients (Supplementary Table S2) were cultured ex vivo in tumor explant cultures with enzalutamide alone for 4 days followed by combined enzalutamide and IST5-002 for 4 days with explants cultured with enzalutamide or vehicle alone for 8 days as control groups. Prostate cancers receiving combined enzalutamide and IST5-002 as the second-line therapy demonstrated extensive loss of viable epithelium at the end of the culture period, with only 15% viable cells at the end of the treatment period compared with enzalutamide monotherapy (30%; P < 0.05) or vehicle (80%; P < 0.0001; Fig. 6E). Nuclear levels of active Stat5 evaluated by IHC, were increased in the explants treated with enzalutamide compared with vehicle-treated explants (P = 0.05). At the same time, treatment of the explants with the combination of enzalutamide and IST5-002 as a second-line therapy reduced the nuclear active Stat5 levels compared with enzalutamide (P = 0.05; Fig. 6F). Overall, these data suggest that second-line therapy with combined enzalutamide and IST5-002 was more effective than enzalutamide alone in inducing epithelial cell death in patient-derived clinical prostate cancers ex vivo.