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The AP-1 transcription factor complex has been implicated in a variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. We previously established that activation of the AP-1 family member JunD contributes to deregulated expression of the anti-apoptotic IL-6 gene in prostate cancer cells. We now show that inhibition of JunD in prostate cancer cells results in GADD45α and γ dependent induction of cell death and inhibition of tumor growth that is mediated at least partially via c-Jun N-terminal kinase (JNK) and p38 kinase activation. Apoptosis induction by dominant negative JunD and JNK and p38 kinase activation are impeded upon knock down of GADD45α and γ expression by small interfering RNA, most vividly demonstrating the central role of GADD45α and γ in JunD-mediated escape of prostate cancer cells from programmed cell death.
Prostate cancer is the most prevalent malignancy in older men and a frequent cause of death. As a result of an aging population, improvements in early detection and advances in cardiovascular disease management rates of prostate cancer are increasing. Despite recent breakthroughs in identifying specific prostate cancer genes such as the Ets fusion proteins and PTEN mutations and the proven relevance of androgen receptor-dependent gene regulation in prostate cancer development and progression,1–6 very few therapeutic successes have been achieved in treating advanced hormone-refractory prostate cancer.
A variety of signaling pathways have been implicated in prostate cancer progression including the interleukin-6 (IL-6) pathway.7,8 Plasma IL-6 and soluble IL-6 receptor (IL-6sR) levels are significantly elevated in patients with metastatic, hormone-refractory prostate cancer and IL-6 and IL-6sR levels in blood independently predict malignant prostate cancer progression and poor outcome in patients with localized tumors.9–11 Hormone-refractory prostate cancer and bone metastases express increased levels of IL-6.12–14 IL-6 enhances proliferation, escape from programmed cell death and angiogenesis as well as chemoresistance of prostate cancer cells and thus, combined with its effects on bone metabolism, inflammation and other effects on the micro-environment elicits multifaceted tumor and metastasis promoting effects on prostate cancer.15,16 In addition, IL-6 induces androgen synthesis in prostate cancer cells through induction of steroidogenic enzymes and androgen receptor-dependent gene expression due to STAT3-mediated androgen receptor (AR) activation.17–19
We previously reported that increased expression of the IL-6 gene in prostate cancer is primarily due to activation of NFκB p50 and p65 and the activating protein-1 (AP-1) transcription factor heterodimer of JunD and Fra-1.20 The AP-1 transcription factor family forms heterodimeric complexes of members of the JUN family (c-Jun, JunB, v-Jun and JunD) with members of the FOS (c-Fos, Fra1, Fra2 and FosB), ATF/CREB (ATF1-4, ATF-6, β-ATF and ATFx) and JDP family (JDP2).21–23 Each dimeric complex may be functionally distinct, playing a role in either transcriptional activation or repression, and regulating distinct sets of genes in response to various stimuli.24,25 AP-1 activity can be modulated by interactions with NFκB,26 different members of the mitogen-activated protein kinase (MAPK) family, and phosphoinositide-3-kinase (PI3K) signaling proteins (reviewed in ref. 27). AP-1 complexes play critical roles in cell proliferation, differentiation, transformation and apoptosis and several members of the AP-1 family have been identified as oncogenes. Enhanced expression of c-Jun has been associated with recurrence of the disease, and suggested to be a marker of high-risk prostate cancer.28 JunD has been shown to be an essential mediator for the androgen-induced increase in reactive oxygen species levels in androgen-sensitive LNCaP prostate cancer cells.29 Besides, JunD has been demonstrated to create complexes in situ with the androgen receptor.30 Recently, Kajanne et al.31 demonstrated that PI3K-dependent activation of Fra-1 and Fra-2 in complexes with JunD plays an essential role in prostate cancer proliferation and conferring protection against cell death by gamma-radiation exposure. Our previously report that aberrant activation of JunD and Fra-1 in androgen-insensitive prostate cancer cells results in deregulated IL-6 expression provides further support for the notion that JunD and Fra-1 are critical for prostate cancer cell proliferation and escape from programmed cell death.20
In this study, we sought to further clarify the relevance of JunD in escape from programmed cell death of androgen-insensitive prostate cancer cells and to determine the molecular mechanisms underlying the anti-apoptotic effects mediated by JunD. We present here data clearly demonstrating that JunD inhibition induces apoptosis in prostate cancer cells and inhibits tumor growth and IL-6 expression in prostate cancer xenografts. Our results establish that apoptosis induction by dominant negative JunD is due to induction of growth arrest- and DNA-damage-inducible proteins (GADD) 45α and γ proteins. Furthermore, we demonstrate that GADD45α and γ-dependent JNK and p38 activation contributes to apoptosis induction in prostate cancer cells. Our data offer a strong rational for targeting hormone-refractory prostate cancer by therapeutic inactivation of JunD-dependent pathways.
We previously demonstrated that concomitant deregulated activation of NFκB p50 and p65 and AP-1 JunD and Fra-1 in androgen-insensitive prostate cancer cells results in deregulated IL-6 expression via transactivation of the IL-6 promoter.20 Since IL-6 is a pro-survival factor for prostate cancer cells and recent data indicate that JunD confers radio-resistance in prostate cancer cells, we further explored the relevance of JunD for cell proliferation and apoptosis in prostate cancer cells. DU145 prostate cancer cells, which constitutively express active AP-1 Fra-1 and JunD,20 were infected with an adenovirus expressing dominant negative JunD (Ad-DNJunD) and an adenovirus encoding the β-galactosidase gene (Adβ-gal) as infection control. Blockage of JunD in DU145 cells resulted in inhibition of cell proliferation (Fig. 1A) and apoptosis induction (Fig. 1B) as early as 48 h after infection that was significantly more pronounced at 72 h postinfection. These data provide clear evidence that JunD activation in prostate cancer cells participates in escape from programmed cell death of prostate cancer cells.
The three GADD45α, β and γ proteins respond to various environmental stresses mediating the activation of both p38 and JNK pathways via MTK1/MEKK4 kinase. GADD45 proteins have been correlated with the regulation of cell cycle arrest, tumorigenesis (upon response to oncogenic stress), maintenance of genomic stability, and other fundamental cellular processes such as survival, apoptosis and senescence.32 Furthermore, the GADD45α and γ family members have been implicated in NFκB induction of cell survival in cancer cells.33 To gain further insights into the relevance of JunD for prostate cancer cell survival, DU145 prostate cancer cells were infected with AdDNJunD or an adenovirus carrying the β-galactosidase gene (Adβ-gal) as a control. Real time PCR shows that blockage of JunD results in upregulation of both GADD45α and γ and slight downregulation of GADD45β starting 24 h after infection when compared with Adβ-gal control (Fig. 2A). To confirm that protein expression correlates with mRNA expression we determined by western blot analysis employing GADD45-specific antibodies whether the blockage of JunD modulates protein expression. Our analysis of GADD45 family protein levels in protein extracts obtained from DU145 cells infected with the same virus combination as above revealed that overexpression of dominant negative JunD leads to drastic activation of GADD45α and γ proteins with no significant differences detected in GADD45β protein levels (Fig. 2B). Since inhibition of JunD increases GADD45α and γ, we expected that activation of JunD will induce opposite effects. To explore this notion we performed real time PCR analysis on total RNA isolated from DU145 cells transfected with expression vectors for AP-1 JunD and Fra1. Concomitant expression of both AP-1 members repressed GADD45α and γ gene expression without affecting GADD45β expression (Fig. 2C).
To determine whether blockage of JunD has an effect on tumor formation in vivo, DU145 cells infected with AdDNJunD and Adβ-gal, as well as uninfected cells, were orthotopically implanted into the prostate of SCID mice. Two months later, the mice were examined for tumor formation, tumor weight, metastasis and IL-6 expression in the serum. In contrast to the β-galactosidase adenovirus infected cells, JunD blockage led to drastic reduction in tumor weight, lymph node metastasis and IL-6 expression (Fig. 3). These results most clearly demonstrate that JunD plays an important role in tumor growth and IL-6 expression in prostate cancer and that targeting JunD may provide a novel therapeutic entry point for prostate cancer.
To elucidate the relevance of GADD45 genes in dominant negative JunD mediated-apoptosis in prostate cancer cells, we used siRNAs to inhibit the endogenous expression of GADD45α, β and γ genes in DU145 cells infected with AdDNJunD and Adβ-gal. siRNA oligonucleotides specific for the different members of the GADD45 family, as well as a siRNA oligonucleotide against GFP as control, were stably expressed by lentivirus vectors. siRNA oligonucleotides were previously validated in control experiments by our group.33 Inhibition of DNJunD-mediated GADD45α and γ upregulation almost completely abolished apoptosis induction, whereas blocking of GADD45β had only a marginal effect on apoptosis induction (Fig. 4). These data provide the strongest evidence that the GADD45α and γ genes are critical mediators of apoptosis induction upon inhibition of JunD, and the escape from programmed cell death in prostate cancer cells is at least partially dependent on JunD-mediated downregulation of GADD45α and γ expression.
GADD45α and γ have been shown to activate both JNK and p38 signaling pathways,34 and we have demonstrated that JNK activation by GADD45α and γ is essential for apoptosis induction upon inhibition of NFκB in various types of cancer.33 Cell survival mediated by NFκB relies on the inhibition of GADD45α and γ proteins in cancer cells,33 and on induction of GADD45β in inflammatory conditions, which suggests that depending on the particular cell context and signaling pathways involved different GADD45 proteins mediate cell survival mechanisms.33,35 To investigate the potential involvement of JNK and/or p38 kinase activation in apoptosis induction mediated by dominant negative JunD, we infected DU145 cells with AdDNJunD and Adβ-gal, and evaluated JNK and p38 kinase activity using in vitro kinase assays. Compared with the control, both JNK and p38 pathways were induced by dominant negative JunD as enhanced phosphorylation of c-jun due to activated JNK was observed only in cells expressing dominant negative mutant JunD (Fig. 5A). Similarly enhanced ATF2 phosphorylation due to p38 activation was present only in DU145 cells expressing dominant negative mutant JunD (Fig. 5A).
To establish the relevance of JNK and p38 activation in mediating apoptosis, we incubated DU145 cells infected with AdDNJunD and Adβ-gal in the absence or presence of a specific JNK (JNKII SP600125) or p38 (SB202190) inhibitor. Compared with the control, apoptosis of DU145 cells expressing dominant negative JunD was strongly reduced, but not completely abolished in both JNK and p38 inhibitor-treated cells (Fig. 5B), demonstrating that both JNK and p38 contribute to dominant negative JunD mediated apoptosis. To investigate whether JNK activation in DNJunD mediated apoptosis is dependent on GADD45α and γ upregulation, DU145 cells were analyzed for JNK and p38 activation after infection with AdDNJunD and Adβ-gal, and lentivirus vectors encoding siRNAs against the GADD45 family members or lentiviruses encoding GFP as control. Our data demonstrate that siRNA mediated inhibition of upregulation of GADD45α and γ expression in response to dominant negative JunD drastically reduced JNK activation. However, p38 activation remained at the same levels suggesting that other activators of the p38 pathway may be involved.
In this study, we have demonstrated that AP-1 family member JunD mediated cell survival effects in prostate cancer cells are required on GADD45α and γ family members. We demonstrate that downregulation of GADD45α and γ protein expression by JunD is an essential step in AP-1 dependent escape from programmed cell death of prostate cancer cells. JNK activation has been shown to contribute to apoptosis induction upon inhibition of NFκB in cancer cells.33 Here, we show that JNK activity contributes to apoptosis of prostate cancer cells in response to JunD inhibition as well and, similar to NFκB signaling, is dependent on GADD45α and γ, but not β, corroborating the important role of the JNK pathway in cell survival of prostate cancer.
AP-1 proteins play a role during normal cellular development and transformation, and AP-1 complexes bind to palindromic DNA sequences, resulting in the transcriptional regulation of various target genes.24 AP-1 proteins that lack potent transactivation domains, such as Fra-1, Fra-2, JunB and JunD express weak transforming activities.36 For example, mice that lack JunD are viable and do not form tumors spontaneously.37 Some AP-1 proteins that lack transforming activity can actually suppress tumorigenesis.38 The decision as to whether a particular AP-1 complex is oncogenic or anti-oncogenic may depend on the opposing activities of different AP-1 proteins, and is probably influenced by tumor type, tumor stage and the genetic background. Indeed, the role that JunD plays in the cellular environment is dependent on its partner's identity in the AP-1 heterodimer-complex such as Fra-1, on post-transcriptional and post-translational regulation and additional transcription factors that synergize with JunD protein. For example, AP-1 activity is not only regulated as a result of formation of a particular AP-1/DNA complex but also by interactions with NFκB and MAPK or PI3K signaling pathways.27 Shin et al. recently implicated AP-1 proteins and certain AP-1/DNA complexes, including JunD, in the malignant transformation of pancreatic cancer cells.
Cooperative interactions between transcription factors due to direct protein-protein interactions or via indirect effects on the DNA structure are common mechanisms of transcriptional regulation. Many transcription factors have their DNA binding sites adjacent to DNA binding sites for other transcription factors or overlapping with other binding sites. Depending on the sequence context and the particular cellular environment, binding of two distinct transcription factor complexes result in higher affinity interaction, synergistic repression or activation of specific target genes.40 Combinatorial control of transcriptional regulation has been demonstrated to affect transcription of a large number of enhancers or promoters. Combinations of binding sites for AP-1 and NFκB occur in a large number of promoter/enhancer elements, and functional cooperation between both is decisive for controlled expression of many genes, including several cytokines.
In this regard, we previously showed that combined aberrant activation of NFκB p50 and p65 and AP-1 JunD and Fra-1 in androgen-insensitive prostate cancer cells is the primary culprit for deregulated expression of IL-6, which plays a role in prostate cancer survival and progression.20 IL-6 is one of several proteins that activates the androgen receptor in hormone refractory prostate cancer, and it was recently suggested that IL-6 production promotes cell growth and escape from programmed cell death in hormone refractory prostate cancer via an autocrine and paracrine mechanism.41 Similarly to deregulated IL-6 expression in prostate cancer, here we demonstrate that JunD plays an essential role in cell survival and proliferation of prostate cancer cells that constitutively express active AP-1 and NFκB.20 We previously reported that NFκB-mediated cell-survival mechanisms in various types of cancer were dependent on GADD45α and γ family members,33 suggesting that both transcription factors, NFκB and JunD, are key players in repressing GADD45α and γ gene expression in prostate cancer cells. Interestingly, while in inflammatory cells NFκB-dependent survival mechanisms are primarily due to upregulation of GADD45β, in cancer cells downregulation of GADD45α and γ gene expression by JunD and NFκB is the major mechanisms for escape from programmed cell death. We did not observe a major contribution of GADD45β to apoptosis induction upon inhibition of JunD, but instead an essential role of GADD45α and γ proteins in apoptosis induction-mediated by JunD inhibition. Blockage of JunD activity by its dominant negative mutant inhibits prostate cancer tumor growth in an orthotopic xenograft model, demonstrating that JunD is an essential mediator of prostate cancer progression.
GADD45 proteins respond to environmental stresses mediating the activation of both p38 and JNK pathways via MTK1/MEKK4 kinase.34 The induction of JNK and p38 pathways modulates cell cycle decisions and promotes cell survival or apoptosis depending on the cell type and stimulus. JunD is phosphorylated by JNK at serines 90 and 100 and at threonine 117, which positively regulates trans-activation activity.42 JunD phosphorylation is decreased by the downregulation of JNK mediated by estrogen, which also decreases the expression of JunD transcripts, indicating an autocrine loop in the regulation of JunD mRNAs.43 We previously also demonstrated that JNK physically interacts with p53 and mediates p53 serine-15 phosphorylation in prostate cancer cells leading to p53 stabilization, increased p53 binding to its target genes, and induction of apoptosis in the context of the NFκB/IκB signaling pathway.44 Our findings demonstrate that blockage of JunD activity leads to induction of JNK and p38 activity and apoptosis. Cells expressing the dominant negative JunD and treated with JNK and p38 inhibitors show strongly reduced, but not abolished, apoptosis induction, which suggests that JNK and p38 contribute in a major form to apoptosis induction, although additional pathways cannot be excluded. These observations prompted us to investigate whether JNK and p38 activation and apoptosis induction are dependent of GADD45α and γ activity. Blockage of JunD leads to GADD45α and γ dependant induction of apoptosis that, interestingly, relies only on JNK activity as blockage of GADD45 gene expression by siRNA approaches only abrogates JNK activity while p38 activation is not altered. These data indicate that inhibition of JunD induces a pro-apoptotic pathway that is GADD45α and γ and JNK dependent and an additional pathway that involves p38 activation via a GADD45-independent mechanism.
While JunD and most likely Fra-1 elicit anti-apoptotic activities, at least in prostate cancer cells, not all members of the AP-1 family share the same activity. For example, c-Fos at least in the context of Trail-induced apoptosis of prostate cancer cells is essential, but not enough, to regulate TRAIL-induced apoptosis.45,46 Recently, Zhang et al.47 reported that NFκB inhibition and c-Fos activation significantly enhance prostate cancer cells sensitivity to TNFα or TRAIL-induced apoptosis. Thus, different members of the AP-1 family can trigger diverse cellular responses under specific stimuli or particular treatments.
In conclusion, our results establish the role of JunD in the maintenance of prostate cancer cell survival, controlling GADD45α or γ gene expression, JNK activity and apoptosis, supporting the notion of JunD as a novel therapeutic target in prostate cancer.
The prostate cancer cell lines DU145 and PC-3 were obtained from American Type Culture Collection (Rockville, MD). DU145 cells were grown in MEM (Life Technologies, Inc.) and PC-3 were grown in HAMS F-12 medium (BioWhitaker, Walkersville, MD). The media were supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Inc.,), 50 units/ml penicillin, and 50 µg/ml streptomycin (all from Life Technologies, Inc.). The cells were maintained in a 5% CO2-humidified incubator at 37°C.
The adenovirus encoding dominant negative JunD and the β-gal gene were generated using the ADENO-X system from Clontech Laboratories, Inc. The dominant negative JunD mutant was obtained from the pCMV5-JundΔ plasmid, kindly provided by Dr. Lester F. Lau, University of Illinois.48 The fragment encoding the β-gal gene was excised from the pCMVβ plasmid (Clontech Laboratories, Inc.). Adenovirus construction has been previously described by Zerbini et al.20 Adenovirus infection was performed at a MOI of 100.20
DU145 cells infected with an adenovirus expressing dominant negative JunD (Ad5CMV-DNJunD) or an adenovirus encoding β-galactosidase gene (Adβ-gal) or uninfected cells (2 × 106 in 50 µL of MEM) were used for implantation (eight mice per group) and were carefully injected under the prostatic capsule of 8-week-old male severe combined immunodeficient (SCID) beige mice as described previously in references 33–49. Six weeks after cancer cell implantation, phlebotomy was done by accessing the retroorbital venous plexus to obtain 150 µL of blood from each mouse. Serum IL-6 level was measured by an IL-6 specific ELISA to estimate the tumor-take rate and tumor size. At the end of the experiment (8 weeks), animals were sacrificed and tumors were carefully dissected and weighed. Lymph nodes and lungs were collected to determine metastases.
IL-6 in the serum of SCID mice was assayed by an IL-6 ELISA (BioSource International, Camarillo, CA) as previously described by Zerbini et al.20
Total RNA was harvested using QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen). cDNA was generated from 2 µg of total RNA using Ready-to-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech). SYBR Green I-based real-time PCR was performed on a MJ Research DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research Inc. Walthan, MA). All PCR mixtures contained PCR buffer [final concentration, 10 mM TRIS-HCl (pH 9.0), 50 mM KCl, 2 mM MgCl2 and 0.1% Triton X-100], 250 µM deoxynucleoside triphosphate (Roche Molecular Biochemicals), 0.5 µM of each PCR primer, 0.5× SYBR Green I (Molecular Probes), 5% DMSO and 1 unit of Taq DNA polymerase (Promega, Madison, WI) with 2 µl of cDNA in a 25-µl final volume reaction mix. The samples were loaded into wells of Low Profile 96-well micro-plates. After an initial denaturation step for 1 min at 94°C, conditions for cycling were 35 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. The fluorescence signal was measured right after incubation for 5 sec at 75°C following the extension step, which eliminates possible primer dimer formation. At the end of the PCR cycles, a melting curve was generated to identify specificity of the PCR product. For each run, serial dilutions of hGAPDH plasmids were used as standards for quantitative measurement of the amount of amplified DNA. For normalization of each sample, hGAPDH primers were used to measure the amount of hGAPDH cDNA. All samples were run in triplicates, and the data were presented as ratio of Gene:GAPDH and then as a percentage of β-gal control sample or DU145 cells control sample. The sequences of the primers were as follows: for GADD45α, sense 5′-GCC TGT GAG TGA GTG CAG AA-3′; antisense 5′-ATC TCT GTC GTC GTC CTC GT-3′; for GADD45β, sense 5′-TCG GAT TTT GCA ATT TCT CC-3′; antisense 5′-GGA TGA GCG TGA AGT GGA TT-3′; for GADD45γ, sense 5′-CTG CAT GAG TTG CTG CTG TC-3′; antisense 5′-TTC GAA ATG AGG ATG CAG TG-3′; and for hGAPDH, sense 5′-CAA AGT TGT CAT GGA TGA CC-3′; antisense 5′-CCA TGG AGA AGG CTG GGG-3′.
Whole cell lysates were prepared in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na PPi, 1 mM 3-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride]. Thirty µg of protein were electrophoresed in a 10% acrylamide-SDS gel. Proteins were electroblotted onto a polyvinylidene difluoride membrane in a 50 mM Tris-base, 20% methanol and 40 mM glycine electrophoresis buffer. Membranes were incubated in 5% nonfat dry milk in TBST (60 mM Trisbase, 120 mM NaCl and 0.2% Tween 20) for 1 h. Blots were probed with anti-GADD45α, -β or -γ antibodies (Santa Cruz Biotechnology) overnight at 4°C in 2% BSA in TBST and then incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) in 5% dry milk in TBST for 1 h at room temperature. Bound antibodies were detected by chemiluminescence with ECL detection reagents (Amersham Pharmacia Biotech) and visualized by autoradiography.
JNK and p38 kinase activities were measured by using the stress-activated protein kinase SAPK/JNK assay kit and p38 MAP Kinase assay kit (Cell Signaling Technology), respectively, according to the manufacturer's protocol.
Proliferation assays were performed by using the Rapid Cell Viability assay (Oncogene Research Products, San Diego) according to the manufacturer's protocol. Apoptosis was assayed in tissue culture supernatants by using the Apoptotic Cell Death Detection ELISA (Roche) and/or the Cell Death Detection (Nuclear Matrix Protein, San Diego, CA) ELISA (EMD Biosciences) according to the manufacturer's protocol.
Apoptosis assay of DU145 cells was measured as described above according to the manufacturer's protocol. Briefly, DU145 cells were infected with an adenovirus expressing dominant negative JunD and an adenovirus encoding β-galactosidase gene (control), and treated with the JNK inhibitor JNKII SP600125 (100 nM) or the p38 inhibitor SB202190 (100 nM). Apoptosis was measured 24, 48 and 72 h after infection.
The oligonucleotides for the three GADD45 family members have been described in reference 33. RNA duplexes (50 µmol/L) were transfected into cells using TKO transfection reagent (Mirus, Madison, Wl) and tested for specificity and efficiency.50
The lentiviruses encoding siRNA against the three GADD45 family members have been described in reference 33. The LV-siRNA GFP construct (control) was kindly donated by Dr. Oded Singer (Salk Institute for Biological Studies).
We acknowledge fruitful discussions with Drs. Franck Grall, Jon Jones and Mehmet Inan. J.F.V. was recipient of a CAPES international fellowship (1102-08-7). This work was supported by the Dana Farber/Harvard Cancer Center Prostate Cancer and Breast Cancer SPORE grants from the National Cancer Institute (NIH P50 CA090381 (T.A.L.) and NIH P50 CA89393 (T.A.L.) and the Prostate Cancer Foundation (T.A.L.).