STAT5A/B Blockade Sensitizes Prostate Cancer to Radiation through Inhibition of RAD51 and DNA Repair

Organ-confined prostate cancer is typically treated with surgery or radiotherapy (1–4), while radiation alone or in combination with androgen deprivation therapy is a key treatment option for locally advanced prostate cancer (1–4). In addition, radiotherapy is elementary for postprostatectomy prostate cancer patients who have high-risk features, such as extracapsular extensions, positive surgical margins, or persistent PSA levels (1–4).

Irradiation, even with intensity-modulated conformal radiotherapy, is associated with significant toxicities to the surrounding tissues, which can cause debilitating side effects (5, 6). Identification of effective strategies to sensitize prostate cancer cells to radiation would allow the use of lower radiation doses by reducing radiation-associated side effects while enhancing prostate cancer cell death. Radiation induces double-strand breaks (DSB), which are the primary cause of cell death following radiation due to decreased DNA repair capacity of cancer cells (7–9). The major repair mechanisms of DSBs are homologous recombination (HR) repair and nonhomologous end joining (NHEJ) repair (7–9). HR repair occurs during late S- or G₂-phases of the cell cycle and uses sister chromatids as templates for DNA repair, which allows for nearly error-free repair of the damaged DNA (7–10). NHEJ, in contrast, occurs rapidly throughout the cell cycle (11, 12) and is error prone due to the nature of the repair by simple joining of the broken DNA ends (7–9, 13). Malignant tissues have an increased ratio of cells in S- and G₂-phase, leading them to favor HR repair over NHEJ repair (14).

HR DNA repair occurs through a highly regulated series of events, where the key protein controlling the DNA repair process is Rad51 recombinase (7–9, 15). The DNA breaks are initially recognized by the MRN (Mre11, Rad50, NbS1) protein complex, which together with phosphorylated CtIP (C-terminal binding protein interacting protein) and BRCA1 (Breast Cancer Susceptibility 1), generate the necessary 3′ single-stranded DNA (ssDNA) overhangs for HR repair (7–9, 15, 16). BRCA1 promotes this resection by dephosphorylating the inhibitory 53BP1 protein (7–9, 15–17). Following end resection, the MRN complex recruits and activates ATM (ataxia telangiectasia mutated) kinase, which phosphorylates histone H2AX on regions around DNA DSBs (18). Importantly, loading of Rad51 recombinase onto exposed ssDNA repair sites is carried out by BRCA1 and requires assistance of BRCA2 (16, 19). Rad51 is a requisite for HR DNA repair as it catalyzes DNA strand invasion and exchange (16, 20). Rad51 paralogs, including Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, have only a supporting role in this process by assisting Rad51 in the initiation and execution of HR DNA repair (21). BRCA1 and BRCA2 have a central role in assisting Rad51, and BRCA1/2-deficient cells have defective HR DNA repair (termed BRCAness), and consequently increased sensitivity to radiation, platinum chemotherapy, and PARP inhibitors (22). In contrast, Rad51 is indispensable for HR DNA repair (7–9, 15).

Stat5a/b is both a signaling protein and a transcription factor that mediates diverse cellular responses to cytokines and growth factors (23) and has been linked to DNA repair in chronic myeloid leukemia (24, 25). Stat5 comprises two highly homologous isoforms, Stat5a (94 kDa) and Stat5b (92 kDa), which are activated through phosphorylation of a conserved tyrosine residue, typically by Jak2, leading to formation of Stat5a/b dimers and subsequent translocation to the nucleus and binding to DNA (23). Stat5a/b is overexpressed in prostate cancer compared with normal prostate epithelium (26), and Stat5a/b levels positively correlate with Gleason grades of prostate cancer (26–29). Moreover, the Stat5a/b gene locus undergoes amplification during prostate cancer progression to castrate-resistant metastatic disease (28). Stat5a/b is critical for viability of prostate cancer cells in vitro and for prostate cancer xenograft tumor growth in vivo through both AR-dependent and AR-independent mechanisms (30–34). Stat5a/b inhibition suppresses growth of castrate-resistant prostate cancer following surgical castration (35), and Stat5a/b induces epithelial-to-mesenchymal transition, stem-like cancer cell properties, and distant metastases formation of prostate cancer in vivo (36). The significance of Stat5a/b in prostate cancer growth and progression, demonstrated in preclinical prostate cancer models, is corroborated by significant predictive value of active Stat5a/b for early disease recurrence of organ-confined prostate cancer in patients after intent-to-cure radical prostatectomy (29). Recently, we developed a family of novel small-molecule Stat5a/b inhibitors (IST5), with high efficacy of blocking Stat5a/b action in prostate cancer and Bcr-Abl–driven leukemias in vitro and in vivo (37).

The promoter region of Rad51 contains Stat5a/b response elements (24, 25), which led us to explore the possibility that Stat5a/b may regulate HR DNA repair and radiation response in prostate cancer. Here, we demonstrate, for the first time, that Stat5a/b is required for Rad51 expression in prostate cancer, and Stat5a/b inhibition sensitizes prostate cancer to radiation in vitro and in vivo through suppression of HR DNA repair. Stat5a/b activation increased, whereas genetic or pharmacologic Stat5a/b inhibition decreased Rad51 expression in prostate cancer cells and in clinical prostate cancers. Moreover, Stat5a/b inhibition suppressed HR DNA repair in prostate cancer as indicated by decreased Rad51 foci formation, increased DNA damage, and decreased repair of I-SceI–generated DSBs. Importantly, genetic or pharmacologic Stat5a/b inhibition sensitized both prostate cancer cells and prostate cancer xenograft tumors to radiation, while not affecting the radiation sensitivity of the surrounding tissues. In summary, this work introduces the novel concept of a critical role of Jak2–Stat5a/b signaling for HR DNA repair in prostate cancer through induction of Rad51 expression. Conceptually, inhibition of Jak2–Stat5a/b signaling represents a novel strategy to introduce transient BRCA-ness in prostate cancer to sensitize prostate cancer to radiation by impairing HR DNA repair and, therefore, may provide an adjuvant therapy for radiation treatment of prostate cancer.

CWR22Rv1, PC-3, DU145, LNCaP (all from ATCC, between 2003 and 2006), LAPC-4 (provided by Dr. Charles Sawyers, Memorial Sloan Kettering Cancer Center, New York, NY) and CWR22Pc (38). Prostate cancer cells were cultured in RPMI1640 medium (Mediatech) containing 10% FBS (Gemini), 2 mmol/L l-glutamine (Mediatech), and penicillin/streptomycin (50 IU/mL and 50 μg/mL, respectively; Mediatech). LNCaP and CWR22Pc cells were cultured in the presence of 0.5 and 0.8 nmol/L DHT, respectively. All cell lines included in this study have been authenticated on a regular basis in the users' laboratory. The testing has been conducted by DNA fingerprinting, isozyme collection, observation of characteristic cell morphology, hormone/growth factor responsiveness, and the expression of cell line–specific markers, such as PSA, hormone receptors, Stat3/Stat5, Erk1/2 protein, and Akt. All cell lines were tested for mycoplasma contamination (PCR Mycoplasma Detection Set; Takara Bio Inc.,) every 3 months. IST5-002 (Fox Chase Chemical Diversity; ref. 37), AZD1480 (35) and ruxolitinib (MedChem Express), and MG132 and cycloheximide (Calbiochem) were dissolved according to the manufacturers' instructions.

Detailed information is provided in Supplementary Materials and Methods.

CWR22Rv1 cells expressing wild-type (WT) Stat5b, DNStat5a/b, or LacZ [adenoviral gene transfer at multiplicity of infection (MOI) = 5] were irradiated with a single dose of 10 Gy, mixed with 0.7% low melting point agarose (Sigma-Aldrich), and spread on CometSlide microscope slides (Trevigen), followed by cell lysis. After electrophoresis, slides were stained with ethidium bromide, and comets were scored (100 cells per treatment) under a fluorescence microscope (HistoRx Inc.), followed by analysis using CometScore 15 software (TriTek Corp.). The comet parameter (olive tail moment) reflects the amount of unrepaired DNA released from the cells (39).

Detailed information is provided in Supplementary Materials and Methods.

Stat5a/b was depleted by lentiviral expression of Stat5a/b shRNA (shStat5a/b) versus nontarget shRNA in CWR22Rv1 cells for 72 hours, followed by irradiation with a single dose of 10 Gy. Cells were harvested 10, 20, 30, or 60 minutes after irradiation, mixed with CleanCut agarose (0.75%), and transferred to agarose embedded DNA plug molds (CHEF genomic DNA Plug Kit; Bio-Rad). For 0-minute incubation, cells were irradiated on ice. DNA was lysed and digested with Proteinase K, incubated overnight (50°C), and run in 0.5% Pulsed Field Certified Agarose using CHEF Mapper XA Pulsed Field Electrophoresis System (Bio-Rad) in 0.5% TBE at the following conditions: 0.9 V/cm of voltage, 60 degrees angle time for 75 minutes, 120 degrees angle time for 65 hours, and 25 mA at 14°C with linear ramping factors. Quantitative analysis of the intensity of stained and released broken DNA in the gel was performed by Scion 2 software (Meyer Instruments, Inc.).

Detailed information is provided in Supplementary Materials and Methods.

Detailed information is provided in Supplementary Materials and Methods.

Stat5a/b shRNA or control shRNA were expressed using lentivirus in CWR22Rv1 cells, irradiated with a single dose of 10 Gy after 48 hours, and immunostained with primary anti-Rad51 pAb (Santa Cruz Biotechnology), fluorescein-conjugated goat anti-rabbit secondary antibody (Vector Labs), followed by analysis using a Zeiss LSM 510 laser-scanning microscope (Zeiss). For quantification, 200 nuclei were analyzed per each treatment group and cells with more than five Rad51-positive foci were counted as Rad51 foci–positive cells.

Detailed information is provided in Supplementary Materials and Methods.

CWR22Rv1 cells stably expressing pDR-GFP reporter were established (CWR22Rv1/pDR-GFP). LacZ and CAStat5a/b were introduced to CWR22Rv1/pDR-GFP cells using adenoviral vector at MOI = 5, and genetic knockdown was achieved by lentiviral expression of Stat5a/b shRNA (shStat5a/b) versus control shRNA (shCtrl). After 48 hours, cells were transiently transfected using X-fect (Clontech) with 10 μg pCbA-Sce, which contains I-SceI cDNA to generate DSBs in Sce-GFP cDNA (40, 41). After 72 hours, the amount of functional GFP reflecting HR DNA repair was analyzed by FACS. Ethanol-fixed cells were stained with propidium iodide and treated with RNase (Sigma Aldrich), followed by analysis by BD LSR II flow cytometry using the FL-2 channel.

Detailed information is provided in Supplementary Materials and Methods.

CWR22Rv1, CWR22Pc, and DU145 cells were irradiated with 0 to 8 Gy and seeded at various densities. After 21 days, colonies were stained for 30 minutes with 0.25% crystal violet (Sigma-Aldrich) and counted (minimum 50 cells/colony). Plating efficiency (colonies counted/cells seeded × 100) and survival fraction [colonies counted/cells seeded × (plating efficiency/100)] were calculated for each group.

Detailed information is provided in Supplementary Materials and Methods.

Castrated athymic nude male mice (Taconic), cared for according to institutional guidelines, were implanted with sustained-release DHT pellets (90-day release, 1 pellet/mouse; Innovative Research of America) 7 days before prostate cancer cell inoculation. CWR22Rv1 cells (20 × 10⁶) in 0.2 mL Matrigel (BD Biosciences) were injected subcutaneously into the right flank of each mouse (n = 10/group, one tumor/mouse), and the treatments were initiated when tumors reached 100 mm³. Daily intraperitoneal injections of IST5-002 (10 or 20 mg/kg; ref. 37) were initiated 3 days prior to irradiation, which was administered at a dose of 2 Gy daily for 3 consecutive days to more closely mimic fractionated radiation. Radiation was performed on anesthetized mice (75 mg/kg ketamine/0.35 mg/kg acepromazine) using an X-ray machine (Gulmay Medical) operating at 250 kV, 10 mA, with 2-mm aluminum filtration, and effective photon energy of approximately 90 keV. Tumor volumes were measured 3 times weekly and calculated using the formula V = (π/6) × d₁ × (d₂)², with d₁ and d₂ being two perpendicular tumor diameters.

The lower abdomens of athymic nude male mice were irradiated at 2 Gy/day for 3 consecutive days (250 kVp, approximate dose rate 1.4 Gy/minute, 50 cm distance, 2 mm copper filter; Pantak). Half of the mice were injected intraperitoneally with vehicle or IST5-002 (20 mg/kg) for 3 days before irradiation and for 3 additional days during the irradiation, while control mice did not receive irradiation. On day 7, the animals were euthanized. Cryosections (7 μm) of small intestines were immunostained for Ki-67 (mAb; Abcam), CD45 (pAb; R&D Systems), and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL; Apo-Green Detection Kit; Biotool). For quantitation of apoptotic and proliferating cells in the crypts, the total number of crypt-associated cells was assessed on three independent microscopic (20×) fields for each assay based on DAPI staining, followed by counting of either TUNEL⁻, CD45, or Ki-67⁺ crypt-associated cells on each image.

Detailed information is provided in Supplementary Materials and Methods.

To investigate whether Rad51 expression is regulated by Jak2–Stat5a/b signaling in prostate cancer, we first evaluated basal expression levels of Rad51, BRCA1, and BRCA2 in a panel of prostate cancer cell lines, as shown in Fig. 1A. Stat5a/b was inhibited by genetic knockdown of Stat5a/b by lentiviral expression of Stat5a/b shRNA or transfection with Stat5a/b siRNA, which downregulated Rad51 levels in DU145 and CWR22Rv1 cells (Fig. 1B). Similarly, Stat5a/b inhibition by antisense oligodeoxynucleotides (ODN) for 72 hours decreased Rad51 protein levels markedly in CWR22Rv1 and LNCaP cells compared with mismatch ODN (Fig. 1B). Conversely, adenoviral expression of constitutively active Stat5a/b (CAStat5a/b) in CWR22Pc and CWR22Rv1 cells resulted in a robust increase in Rad51 expression (Fig. 1B). Importantly, direct inhibition of Stat5a/b by a specific small-molecule Stat5a/b inhibitor, IST5-002 (37), caused a dose-dependent reduction in Rad51 levels in CWR22Rv1, CWR22Pc, LAPC-4, DU145, and LNCaP cells at 72 hours (Fig. 1C). Collectively, these results indicate that Stat5a/b is critical for Rad51 protein expression in prostate cancer cells.

To evaluate whether Stat5a/b induction of Rad51 occurs via Jak2 signaling, Jak2 was inhibited in CWR22Pc and CWR22Rv1 cells by genetic or pharmacologic knockdown. Lentiviral expression of Jak2 shRNA suppressed Rad51 in both cell lines (Fig. 1D). Similarly, inhibition of Jak2 by pharmacologic Jak2 inhibitors AZD1480 (0.8 μmol/L) or ruxolitinib (0.4 μmol/L) resulted in a robust decrease in Rad51 protein expression in both cell lines at 72 hours (Fig. 1D), indicating that Stat5a/b upregulation of Rad51 occurs through Jak2-dependent processes. To determine whether Stat5a/b is critical for Rad51 expression in patient-derived prostate cancers, we utilized 3D tumor explant culture system of patient-derived prostate cancers ex vivo, which we have rigorously described previously (26, 35–37, 42). All tissue components of prostate cancer, including epithelium and stroma, are retained in this culture system, thus offering a more physiologic model of prostate cancer growth than prostate cancer cell lines. Prostate cancers from four patients (Table 1) were cultured ex vivo in 3D explant cultures and treated with IST5-002 (25 μmol/L) or AZD1480 (25 μmol/L) for 7 days (37). As shown in Fig. 1E, inhibition of Jak2–Stat5a/b signaling by IST5-002 or AZD1480 potently reduced Rad51 expression in the clinical prostate cancers tested. Collectively, these data indicate that Jak2–Stat5a/b signaling is an inducer of Rad51 expression not only in human prostate cancer cell lines but also in clinical prostate cancers. Moreover, these data show that Rad51 levels in prostate cancer can be effectively reduced by pharmacologic inhibitors of Jak2–Stat5a/b signaling pathway.

We next sought to determine whether Stat5a/b regulates Rad51 at the mRNA or protein level in prostate cancer. First, to assess whether loss of Rad51 protein after Stat5a/b knockdown was due to increased flux of Rad51 through the proteasome, we genetically inhibited Stat5a/b for 72 hours, followed by treatment with proteasome inhibitor MG132 (10 μmol/L) for 6 hours (Fig. 2A). Suppression of proteasomal degradation did not reverse downregulation of Rad51 protein induced by genetic depletion of Stat5a/b by shRNA (Fig. 2A). To further evaluate whether Rad51 is downregulated by Stat5a/b knockdown at the protein level, new protein synthesis was restricted by cycloheximide (35 μmol/L) for 6 hours, which lowered the Rad51 levels as expected. However, Rad51 levels were not rescued by inhibition of the proteasome with MG132, indicating that Stat5a/b does not protect Rad51 from proteasomal degradation (Fig. 2A, right). At the same time, inhibition of Stat5a/b by shRNA resulted in a marked decrease in Rad51 mRNA expression in both CWR22Rv1 (P < 0.001) and DU145 (P < 0.01) cells compared with cells expressing control shRNA at 72 hours after genetic Stat5a/b depletion, as indicated by quantitative RT-PCR using cells with genetic Rad51 knockdown as a positive control for the assay (Fig. 2B). Effective Stat5 knockdown was verified by immunoblotting (Fig. 2B). In summary, these data indicate that Stat5a/b regulates Rad51 expression in prostate cancer cells at the mRNA level.

Because Stat5a/b regulates Rad51 expression in prostate cancer, we investigated whether Stat5a/b is involved in radiation-induced DNA repair in prostate cancer. We first analyzed radiation-induced DNA damage repair using the neutral comet assay. In this assay, damaged DNA is released from the cell, generating a “tail” (termed the olive tail moment), the length and density of which reflects unrepaired DNA (43). Stat5a/b signaling was inhibited in CWR22Rv1 cells by adenoviral expression of DNStat5a/b, with AdLacZ, AdWTStat5b, or mock-infected cells as controls for 48 hours. As shown in Fig. 3A, CWR22Rv1 cells displayed similar amounts of DNA damage regardless of the status of Stat5a/b signaling activity 1 hour after irradiation at a dose of 10 Gy. Most DNA damage was repaired at 24 hours when Stat5a/b signaling was intact (Fig. 3A). In contrast, Stat5a/b inhibition by AdDNStat5a/b resulted in a 13-fold increase in the olive tail moment 24 hours after irradiation when compared with the AdLacZ control group (P < 0.001; Fig. 3A). In conclusion, Stat5a/b inhibition resulted in decreased repair of radiation-induced DNA damage, indicating that Stat5a/b regulates radiation response of prostate cancer cells.

Given that Stat5a/b upregulates Rad51 expression in prostate cancer cells (Fig. 1), we next investigated whether Stat5a/b regulates loading of Rad51 onto dsDNA breaks and formation of Rad51 repair foci (7–9, 15) in prostate cancer cells following radiation. Stat5a/b signaling was inhibited by RNAi in CWR22Rv1 cells for 48 hours. Following irradiation with 10 Gy, inhibition of Stat5a/b suppressed Rad51 foci formation by 75% compared with nontarget control shRNA (shCtrl; P < 0.001; Fig. 3B), indicating that Stat5a/b induces DNA repair through a Rad51-dependent mechanism.

To directly assess whether Stat5a/b upregulates radiation-induced DNA repair through promotion of the HR DNA repair pathway, we utilized the Sce-GFP assay, which is based on a recombination reporter pDR-GFP plasmid containing an inactive GFP gene (Sce-GFP) and a fragment of the GFP gene as a donor for homologous repair (40, 41). The Sce-GFP cassette has an inactivating insertion, which consists of two STOP codons and a restriction site for the rare cutting endonuclease, I-SceI. When I-SceI is expressed in pDR-GFP expressing cells, it inflicts DSBs within the Sce-GFP fragment and provides a signal for HR DNA repair. This is followed by reconstruction of functional GFP, which is readily detectable by fluorescence microscopy or FACS (40, 41). Stat5a/b signaling was increased by adenoviral expression of CAStat5a/b (AdLacZ as control) in CWR22Rv1 cells stably expressing pDR-GFP. In parallel experiments, Stat5a/b was inhibited in CWR22Rv1/pDR-GFP cells by lentiviral expression of Stat5a/b shRNA. After 48 hours, DNA DSBs were introduced by transfection of the cells with pCbA-SceI for 72 hours. Compared with LacZ control, Stat5a/b activation resulted in an approximately 40% increase in HR DNA repair (P < 0.01; Fig. 3C; left). At the same time, Stat5a/b inhibition reduced HR DNA repair by approximately 30% compared with cells expressing nontarget shRNA (P < 0.01; shCtrl; Fig. 3C; right). To mechanistically test the linkage of Stat5a/b with Rad51 expression and HR DNA repair in prostate cancer cells, introduction of CAStat5a/b using adenovirus (AdLacZ as control) led to 52% increase (P < 0.001) in HR DNA repair in CWR22Rv1 cells (Fig. 3D). Genetic knockdown of Rad51 by lentiviral expression of Rad51 shRNA reversed (P < 0.001) CAStat5a/b induction of HR DNA repair. As control, depletion of Rad51 by lentiviral Rad51 shRNA decreased HR DNA repair below the basal levels in control groups (AdLacZ + shCtrl, shCtrl; P < 0.001; Fig. 3D). Together, these data support the concept that Stat5a/b upregulates HR repair through induction of Rad51 expression in prostate cancer cells.

To evaluate whether Stat5a/b regulates NHEJ DNA repair in prostate cancer cells, we next measured DNA fragmentation following irradiation by pulsed field gel electrophoresis. This assay quantifies the amount of dsDNA damage based on the pattern of migratory DNA. The repair kinetics within a window of 0 to 60 minutes after irradiation reflects NHEJ repair of dsDNA breaks (11, 12). Inhibition of Stat5a/b expression by lentiviral expression of Stat5a/b shRNA (48 hours) had no effect (P = 0.88) on dsDNA break repair in CWR22Rv1 cells irradiated with 20 Gy for the indicated times (Fig. 3E). Specifically, the DNA fragmentation pattern of cells depleted of Stat5a/b by shRNA was similar to that of nonirradiated controls 60 minutes after the irradiation (Fig. 3E). These results demonstrate that Stat5a/b does not influence dsDNA break repair through the NHEJ DNA repair pathway.

Having established that Stat5a/b upregulates HR DNA repair in prostate cancer cells, we investigated whether Stat5a/b inhibition sensitizes prostate cancer cells to radiation-induced cell death. Stat5a/b signaling was inhibited by IST5-002 (37) in CWR22Rv1, CWR22Pc, and DU145 cells for 48 hours, followed by irradiation with 0 or 3 Gy, and immunostaining for the dsDNA break marker γH2AX 4 hours after irradiation. Treatment of CWR22Rv1, CWR22Pc, and DU145 cells with IST5-002 resulted in a marked increase in γH2AX levels in irradiated cells in a dose-dependent manner (Fig. 4A), indicating that accumulation of dsDNA breaks is associated with Stat5a/b inhibition. To further evaluate Stat5a/b inhibition as a strategy to sensitize prostate cancer cells to radiation, Stat5a/b was disrupted by the expression of AdDNStat5a/b in CWR22Rv1 cells for 48 hours, followed by irradiation (0, 1, 2, 3, 4, 5, 6, and 8 Gy) with assessment of clonogenic survival by crystal violet staining after 21 days. Stat5a/b inhibition by genetic depletion potently reduced clonogenic survival of prostate cancer cells compared with controls (P < 0.001; ref. Fig. 4B). To evaluate whether pharmacologic inhibition of Stat5a/b also sensitized prostate cancer cells to radiation, Stat5a/b was inhibited in CWR22Rv1 cells by IST5-002 (48 hours), followed by irradiation with 0, 1, 2, 3, 3.5, or 4 Gy. Inhibition of Stat5a/b by IST5-002 robustly reduced the fraction of surviving cell clones from approximately 0.25 to 0.1, 0.08 to 0.02, and 0.02 to <0.01 at irradiation doses of 2, 3, and 4 Gy, respectively (P < 0.01; Fig. 4B). Similarly, CWR22Pc and DU145 cells demonstrated significantly decreased clonogenic survival after treatment with IST5-002 and irradiation with 0, 0.5, 1.5, 2.5, or 3 Gy (P < 0.001; Fig. 4C). Collectively, these data show that Stat5a/b inhibition effectively sensitizes prostate cancer cells to radiation induced.

To evaluate whether Stat5a/b inhibition sensitizes prostate cancer to radiation in vivo, CWR22Rv1 prostate cancer cells were inoculated subcutaneously into castrated male athymic nude mice supplemented with sustained-release DHT pellets to normalize circulating androgen levels. Once the tumors reached 5 to 6 mm in diameter, Stat5a/b was inhibited by IST5-002 at two different doses (10 and 20 mg/kg), which by themselves are insufficient to significantly suppress tumor growth if administered without radiation (Fig. 5A). Three days after Stat5a/b inhibitor treatment was started, the tumors were irradiated for three consecutive days at 0 or 2 Gy. Tumor growth was suppressed by approximately 40% to 45% on day 29 in mice treated with 10 mg/kg IST5-002 combined with radiation compared with mice receiving radiation alone (P = 0.07; Fig. 5A). At the same time, treatment with 20 mg/kg IST5-002 combined with radiation suppressed growth of the tumors by 60% compared with mice treated with radiation only (P < 0.01) or IST5-002 (20 mg/kg) without radiation (P < 0.01; Fig. 5A). A significant loss (80%; P < 0.001) in cell viability was associated with irradiation combined with 20 mg/kg IST5-002 compared with vehicle control, and 50% decrease (P < 0.01) compared with radiation alone (Fig. 5B, left). IHC analysis of tumor sections showed significant decrease in nuclear active Stat5a/b levels in all tumors treated with IST5-002 compared with vehicle or irradiation (P < 0.001), with the greatest decrease evident in tumors of mice treated with IST5-002 (20 mg/kg; Fig. 5B, right). As expected, radiation alone did not affect nuclear active Stat5a/b levels. At the same time, radiation increased both γH2AX (P < 0.01) and Rad51 (P < 0.001) levels in CWR22Rv1 tumors. The γH2AX levels in irradiated tumors, indicating DNA breaks, were further increased by IST5-002 compared with either vehicle or irradiation-only groups (P < 0.001), whereas Rad51 levels were decreased in the tumors treated by IST5-002 compared with vehicle group (P < 0.01; Fig. 5B). In summary, these results demonstrate that inhibition of Stat5a/b by IST5-002 sensitizes prostate cancer xenograft tumors to radiation, while preventing nuclear localization of Stat5a/b, leading to accumulation of dsDNA breaks and suppression of Rad51 expression.

Intestinal epithelial cells undergo apoptosis if exposed to critical amounts of radiation during pelvic radiation, which leads to radiation-induced acute side effects, including diarrhea and potentially serious late toxicities, such as bowel obstruction and/or perforation of the intestinal wall (5, 6). To evaluate whether IST5-002 combined with radiation is associated with increased damage to the intestinal epithelium, mice were treated with vehicle or IST5-002 while receiving lower abdominal irradiation with 2 Gy (no irradiation as control). Overall, the structure and morphology of the intestinal crypts harboring intestinal stem cells were similar in mice receiving radiation alone or IST-002 with radiation (Fig. 5C). Inhibition of Stat5a/b by IST5-002 did not lead to sensitization of the cells in the intestinal crypts to radiation, as shown by lack of increase in TUNEL positivity indicative of apoptotic cells (Fig. 5C). Moreover, proliferating cells within the crypts were not decreased by IST5-002 combined with radiation compared with radiation only (Fig. 5C). Collectively, these data demonstrate that Stat5a/b inhibition sensitizes prostate cancer to radiation in vitro and in vivo without increased damage to the gastrointestinal mucosa.

Radiotherapy is the mainstay of the treatment for locally advanced prostate cancer (1, 3) and one of the key treatment options for organ-confined prostate cancer (1). Radiotherapy can be associated with debilitating side effects due to unintentional radiation delivered to neighboring organs, especially the bowel and rectum (5, 6). Therefore, identification of new strategies to selectively sensitize prostate cancer to radiation would allow the use of lower radiation doses resulting in less damage to the normal tissues surrounding prostate and thus fewer side effects. In this study, we show, for the first time, that Jak2–Stat5a/b signaling in prostate cancer induces HR DNA repair through upregulation of Rad51. Inhibition of Stat5a/b depletes Rad51 in prostate cancer cells, disrupts HR DNA repair, and sensitizes prostate cancer to radiation and, therefore, may provide an adjuvant therapy for radiation, improve outcomes, and reduce radiation-induced side effects.

One of the key findings of the work presented here is the concept that Stat5a/b is a critical inducer of Rad51 and HR DNA repair in prostate cancer. We demonstrate that Stat5a/b upregulates Rad51 expression in a panel of prostate cancer cell lines, in prostate cancer xenograft tumors in vivo and in clinical patient-derived prostate cancers ex vivo in 3D explant cultures. Stat5a/b regulation of Rad51 was independent of BRCA1/2 status of prostate cancer, demonstrating that Stat5a/b has a primary role in controlling Rad51 expression in prostate cancer. We further show that Rad51 induction by Stat5a/b occurs at the mRNA rather than the protein level, consistent with the previous findings of the presence of Stat5a/b response element in the regulatory regions of the Rad51 gene (24). Genetic or pharmacologic knockdown of Jak2 by small-molecule Jak2 inhibitors significantly suppressed Rad51 levels in prostate cancer cells, indicating that Stat5a/b promotion of Rad51 expression occurs through Jak2-dependent mechanisms. In addition, a specific small-molecule inhibitor of Stat5a/b, IST5-002 (37), effectively inhibited Rad51 expression both in vitro and in vivo in prostate cancer xenograft tumors. Most importantly, IST5-002 and AZD1480, both, robustly decreased Rad51 levels in patient-derived prostate cancers ex vivo in 3D explant cultures, indicating that Stat5 regulation of Rad51 extends to clinical prostate cancers. Collectively, this work introduces a novel concept of Jak2–Stat5a/b signaling pathway as a critical regulator of Rad51 expression in prostate cancer.

Inhibition of Stat5a/b suppressed HR DNA repair and sensitized prostate cancer cells and xenograft tumors to radiation through downregulation of Rad51 expression. Mechanistic association of Rad51 to Stat5a/b regulation of HR DNA repair was demonstrated in a functional assay of HR DNA repair, where Stat5a/b-induced increase in HR-DNA repair was abolished by genetic knockdown of Rad51. At the same time, Stat5a/b inhibition sensitized prostate cancer cells and prostate xenograft tumors to radiation. In these experiments, IST5-002 was administered at low doses, which were sufficient to reduce Stat5a/b action but were insufficient to significantly block growth of prostate cancer cells and xenograft tumors alone without radiation. Concomitant with Stat5a/b suppression, Rad51 expression was decreased, whereas γ-H2AX immunostaining marking dsDNA breaks was increased, indicating involvement of Rad51 suppression in sensitization of prostate cancer to radiation in vivo. Importantly, IST5-002 did not sensitize epithelial cells in the crypts of small intestines of mice to radiation, implying specificity of Stat5a/b regulation of Rad51 to prostate cancer tissues, which may be due to both elevated Rad51 and Stat5a/b levels in prostate cancer (26–29, 44). A recent study reported reduction of radiation-induced intestinal injury induced by Stat5a/b (45). However, both the experimental systems as well as the designs were different between our study and the work by Gilbert and colleagues (45), where Gilbert and colleagues used single doses ranging from 8.5 to 20 Gy, while we used a cumulative dose of 6 Gy (3 × 2 Gy). Of note, only moderate effects on crypt numbers and morphology were observed at the lowest dose (8.5 Gy), consistent with the results presented here. Furthermore, transient pharmacologic inhibition of Stat5a/b activity is distinct from genetic knockout of Stat5 in gastrointestinal stem cells in that, absent radiation, the knockout mice exhibited altered expression of differentiation makers potentially predisposing these cells to more pronounced radiation damage. In conclusion, our results support the concept that Stat5a/b inhibition may provide a strategy to sensitize prostate cancer to radiation while sparing the surrounding normal tissues.

Establishment of a pharmacologic strategy to suppress Rad51 expression and HR DNA repair by targeting Jak2–Stat5a/b signaling in prostate cancer has high translational significance. This is because active Stat5a/b and Rad51, both, are highly expressed in malignant, but not in normal prostate epithelium (26–29). Moreover, potent inhibitors targeting Jak2–Stat5a/b signaling are in clinical development and can be readily used to suppress HR DNA repair in prostate cancer. Conceptually, depletion of Rad51 by Stat5a/b inhibition induces transient BRCA-ness in prostate cancer cells to impair HR DNA repair of radiation-induced dsDNA breaks (14, 19, 46). The term BRCA-ness refers to dysfunctional BRCA1/2, which leads to defects in HR DNA repair through compromising Rad51 function, and it is known to sensitize cancer cells to therapies inducing dsDNA breaks such as radiation and platinum chemotherapy (46). Moreover, BRCA1/2-defective cancer cells have increased sensitivity to PARP inhibitors (22, 47), which suppress nucleotide excision repair leading to accumulation of ssDNA breaks that convert to dsDNA breaks during DNA replication (48), making the cancer cells increasingly dependent on HR DNA repair (49). Future studies need to address whether Stat5a/b inhibitor–induced BRCA-ness will also sensitize prostate cancer cells to PARP inhibitors and platinum-based chemotherapy.

U. Rodeck reports receiving commercial research grants from REATA Pharmaceuticals. K.A. Iczkowski is a consultant/advisory board member for 3D Biopsy, LLC and MicroGen DX. No potential conflicts of interest were disclosed by the other authors.

Conception and design: U. Rodeck, C. Bergom, M.T. Nevalainen

Development of methodology: M.T. Nevalainen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Maranto, V. Udhane, D.T. Hoang, L. Gu, V. Alexeev, K. Malas, K. Cardenas, J.R. Brody, K.A. Iczkowski, K. Jacobsohn, S.M. Schmitt, M.T. Nevalainen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Maranto, V. Udhane, D.T. Hoang, L. Gu, V. Alexeev, W. See, M.T. Nevalainen

Writing, review, and/or revision of the manuscript: V. Alexeev, J.R. Brody, U. Rodeck, C. Bergom, K.A. Iczkowski, K. Jacobsohn, W. See, S.M. Schmitt, M.T. Nevalainen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Maranto, V. Udhane, D.T. Hoang, L. Gu, K. Cardenas, S.M. Schmitt, M.T. Nevalainen

Study supervision: M.T. Nevalainen

This work is supported in part by grants from the NIH/NCI to M.T. Nevalainen (7R01CA113580-10, 5R21CA178755-02), Advancing a Healthier Wisconsin (#5520368), and grants to C. Bergom (86-004-26, 8KL2TR000056), U. Rodeck (W81XWH-12-1-0477), J.R. Brody (1R01CA212600-01), and D.T. Hoang (5F31CA180626-03).

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