Inflammatory Cytokines and Survival Factors from Serum Modulate Tweak-Induced Apoptosis in PC-3 Prostate Cancer Cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the tumor necrosis factor superfamily. TWEAK activates the Fn14 receptor, and may regulate cell death, survival and proliferation in tumor cells. However, there is little information on the function and regulation of this system in prostate cancer. Fn14 expression and TWEAK actions were studied in two human prostate cancer cell lines, the androgen-independent PC-3 cell line and androgen-sensitive LNCaP cells. Additionally, the expression of Fn14 was analyzed in human biopsies of prostate cancer. Fn14 expression is increased in histological sections of human prostate adenocarcinoma. Both prostate cancer cell lines express constitutively Fn14, but, the androgen-independent cell line PC-3 showed higher levels of Fn14 that the LNCaP cells. Fn14 expression was up-regulated in PC-3 human prostate cancer cells in presence of inflammatory cytokines (TNFα/IFNγ) as well as in presence of bovine fetal serum. TWEAK induced apoptotic cell death in PC-3 cells, but not in LNCaP cells. Moreover, in PC-3 cells, co-stimulation with TNFα/IFNγ/TWEAK induced a higher rate of apoptosis. However, TWEAK or TWEAK/TNFα/IFNγ did not induce apoptosis in presence of bovine fetal serum. TWEAK induced cell death through activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-3, release of mitochondrial cytochrome C and an increased Bax/BclxL ratio. TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells under certain culture conditions. Further characterization of the therapeutic target potential of TWEAK/Fn14 for human prostate cancer is warranted.


Introduction
Prostate cancer is the second leading cause of cancer-related death in males [1]. Most cases of prostate cancer present as localized disease and may be cured with surgery and radiation. However, as is true with most solid malignancies, the development of metastatic disease is ultimately lethal. Despite active systemic therapies, the metastatic phenotype will drive in the development of resistance and disease progression. Moreover, systemic treatments in prostate cancer are limited. Until recently, there were only three FDA-approved chemotherapeutic agents for use in castrate-resistant prostate cancer (estramustine, mitoxantrone, and docetaxel) and two additional agents were approved in 2010 (sipuleucel-T and cabazitaxel [2]. However, there is still a clear need to develop additional systemic therapies. The growth of normal prostate epithelial cells is under the tight control of various growth factors, most notably androgens, castration leads to apoptosis of this cell population. Androgen-depletion has a similar effect on prostate cancers. However, following initial regression tumors often return in an androgen-depletion independent form that is frequently lethal. Thus, it is of particular interest to search for agents able to kill androgen-independent prostate cancer cells. Tumor necrosis factor (TNF) was originally described as a factor toxic for tumors [3,4]. It was later shown to belong to the TNF superfamily (TNFSF) of cytokines [5,6]. Many TNFSF cytokines regulate cell death and proliferation, as well as inflammation and may have a role in tumor biology, including prostate cancer biology [7][8][9]. As an example, recent attention has focused on the anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) [10,11]. However, in vivo prostate cancers express osteoprotegerin, a decoy receptor for both TRAIL and activator of nuclear factor-kB ligand (RANKL) [12]. TNFSF cytokines activate a family of receptors (TNFRSF) many of which carry a death domain (DD) and function as death receptors. Activation of death receptors in tumor cells by cytotoxic immune cells is the main mechanism by which the immune system eliminates malignant cells [13].
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, Apo3L, TNFSF12) is one of the most recent members of TNFSF to be identified [14,15]. TWEAK was originally described as an inducer of apoptosis in tumor cells. In addition, TWEAK can regulate cell proliferation, cell death, cell migration, cell differentiation, tissue regeneration, neoangiogenesis and inflammation [16][17][18][19][20][21][22][23][24]. TWEAK activates a single receptor, fibroblast growth factor-inducible-14 (Fn14, TWEAK receptor, TNFRSF12A, CD266). TWEAK activation of the Fn14 receptor results in apoptotic cell death of multiple tumor cell lines [22,[25][26][27][28][29]. Indeed, a phase I clinical trial of a humanized anti-TWEAK receptor monoclonal antibody in subjects with advanced solid tumors was recently completed [30]. However, TWEAK-Fn14 upregulates VEGF expression to foster ovarian cancer cell metastasis [31] and promotes breast cancer cell invasive capacity [32]. There is evidence that the different, even opposed, actions of TWEAK could be determined by the microenvironment. In this regard, TWEAK induces apoptosis in renal tubular cells in a proinflammatory environment, while, it promotes proliferation in presence of bovine fetal serum [33,34]. Prostate cancer cells have been shown to express Fn14 and high expression of Fn14 was significantly associated with higher prostate-specific antigen recurrence rate in patients who underwent radical prostatectomy [35]. Fn14 was highly expressed in androgen-independent prostate cancer cell lines, DU145 and PC-3, whereas expression was weak in androgen-sensitive LNCaP cells. A role for Fn14 in migration, invasion and proliferation was described in PC-3 cells [35].
We now explore the manipulation of the cell culture conditions as a potential tool to turn the TWEAK/Fn14 system against the tumor. We report that the inflammatory cytokines and survival factor from serum modulate the response of PC-3 cells to TWEAK. In the absence of serum TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells, but not in androgen-sensitive LNCaP cells. A better understanding of this regulation may turn a potential advantage of tumor cells into a therapeutic opportunity.

Cell Surface Fn14 Expression
Cells were plated at a density of 8610 4 cells in twelve-well plates. Following stimulation they were detached with 2 mM EDTA/1%BSA in PBS, washed, and resuspended in PBS/1% BSA and blocked for 4 min. Then cells were incubated with 1 mg/ ml anti-Fn14 ITEM4 antibody (eBioscience, San Diego, CA) or an isotype-matched control antibody for 30 min on ice. Cells were washed twice, blocked for 4 min and incubated with Alexa488labeled goat anti-mouse IgG antibody (1/300, Invitrogen, Carlsbad, CA) for 45 min on ice in the dark. Following two additional washes with PBS/1%BSA, cells were resuspended in 1% filtered paraformaldehyde in PBS and analyzed by flow cytometry using BD CellQuest Software (BD Biosciences, San Jose, CA) [39].

RNA Extraction and Real-Time Polymerase Chain Reaction
Total RNA was extracted from cells by the TRI Reagent method (Sigma) and 1 mg of RNA was reverse transcribed with High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Pre-developed primer and probe assays for Fn14, and GAPDH (human) were from Applied (Applied Biosystems). Quantitative PCR was performed by 7500 Real Time PCR System with the Prism 7000 System SDS Software (Applied Biosystems) and RNA expression of different genes was corrected for GAPDH [40].

Apoptosis and Cell Death
10,000 cells were seeded in 12-well plates (Costar, Cambridge, MA) in 10% FCS RPMI overnight. In some cases they were rested in serum-free medium for 24 hour. Thereafter, stimuli were added to subconfluent cells. Apoptosis was characterized by morphologic and functional criteria. Nuclei of formalin-fixed cells were stained with DAPI (Sigma) to observe the typical morphological changes, as previously described [33,42]. For assessment of apoptosis by flow cytometry adherent cells were pooled with spontaneously detached cells, and incubated in 100 mg/mL propidium iodide (PI), 0.05% NP-40, 10 mg/mL RNAse A in PBS at 4uC for .1 h. This assay permeabilizes the cells, thus PI stains both live and dead cells. The percentage of apoptotic cells with decreased DNA staining (hypodiploid cells) was counted by flow cytometry using BD CellQuest Software (BD Biosciences) [33,34,42].
To quantify cell death, cells were resuspended in 100 ml of PBS and stained with 100 mg/mL PI just before to flow cytometry. This assay is based on the known ability of PI to enter in dead cells. The percentage of dead cells (stained with PI) and live cells (not stained cells) was counted by flow cytometry using BD FACS Diva Software (BD Biosciences).

Assessment of Apoptosis by Annexin V-FITC
Briefly, 5610 5 cells were washed with ice-cold PBS, resuspended in 100 ml binding buffer, and, stained with 2.5 ml of FITC-Annexin V (Myltenyi Biotec, Bergisch Gladbach, Germany). The cells were incubated for 15 min at 37uC in the dark. Then, 200 ml of binding buffer containing PI (20 mg/mL) was added just before flow cytometry. The cells were analyzed using FACS Canto  Immunostaining Cells plated onto Labtek TM slides were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100/ PBS, washed in PBS and incubated with rabbit polyclonal antiactive caspase 3 (1:100, Cell Signaling) or anti-cytochrome C (1:500, BDPharmigen) followed by Alexa-488 or Alexa-633 conjugated secondary antibody respectively (1:300, Invitrogen). Nuclei were counterstained with propidium iodide or DAPI respectively.

Immunohistochemistry
Fn14 immunohistochemistry was performed in 5 biopsies from patients diagnosed of prostate adenocarcinoma, aged 65 to 77 years. The Ethics Committee of Fundacion Jimenez Diaz approved the study, and informed consent was obtained from each subject. The antigenic epitope retrieval was performed in 3 mm thick sections of paraffin-embedded tissue using a PTlink device (with a high pH solution, 95uC, 20 min). The tissue slides were incubated for 30 min at room temperature with the primary antibody, rabbit polyclonal anti-Fn14 (1:100, Cell Signaling). For immunohistochemical staining EnvisionFLEX + visualization system was used, in a DAKO Autostainerplus platform. The tissue sections were subsequently counterstained with hematoxylin. Same sections were incubated without the primary antibody as negative controls.

Statistics
Statistical analysis was performed using SPSS 11.0 statistical software. Results are expressed as mean 6SEM for protein and mRNA expression experiments and as mean6 SD for flow cytometry experiments. Significance at the p,0.05 level was assessed by Students t test for two groups of data and ANOVA for three of more groups.

Expression of Fn14 in Prostate Cancer
Prostate adenocarcinoma biopsies displayed a similar pattern of Fn14 expression (Figure 1). Fn14 expression was negative in normal prostate epithelium, mildly positive in high-grade prostatic intraepithelial neoplasia (PIN) foci and very positive in prostate adenocarcinoma. This suggests that the cell culture observation that prostate carcinoma cell lines express Fn14 is clinically relevant and is concordant with prior reports [35].

Constitutive Fn14 and TWEAK Expression in Human Prostate Cancer Cells
First, we studied the expression of TWEAK and Fn14, the TWEAK receptor, in two different human prostate cancer cell lines, PC-3 and LNCaP. PC-3 is an androgen-independent cell line, whereas LNCaP is an androgen-sensitive cell line. Both cell lines constitutively expressed Fn14 at the mRNA (Figure 2.A) and protein levels (Figure 2.B). Although, basal Fn14 expression, either mRNA levels or protein levels, was significantly higher in PC-3 cells compared to LNCaP cells. Moreover, basal levels of TWEAK also were higher in PC-3 cells compared to LNCaP cells, as assessed by RT-PCR (Figure 2.A).
Fn14 expression was clearly increased by fetal bovine serum (FBS) in PC3 cells, and, weakly in LNCaP cells (Figure 2.B). These results are similar to those reported by Huang et al [35], and, suggest that Fn14 may have a role in prostate cancer because is highly expressed in the more aggressive malignant cells. Finally, we demonstrated by flow cytometry that PC-3 cells expressed Fn14 in cell surface (Figure 2.C).

TWEAK Increased Fn14 and MCP-1 Expression in Prostate Cancer Cells
TWEAK is a multifunctional cytokine that can induce inflammatory molecule production in numerous cell types [40]. We stimulated prostate cancer cells with TWEAK and measured Fn14 and MCP-1 mRNA levels by RT-PCR. We observed that TWEAK increased Fn14 and MCP-1 expression in both cell lines, demonstrating that, the two cell types have functional TWEAK receptor ( Figure 3). However, the time course differed between the two cell lines, being more transient in LNCaP cells that in PC-3 cells.

Regulation of Fn14 Expression in PC-3 Cells
In various cell types Fn14 expression is dependent on the microenvironment [33,34,43,44]. FBS, which is rich in growth and survival factors, increased Fn14 expression in PC-3 cells at the mRNA, as assessed by RT-PCR (Figure 4.A) and protein levels, as measured by western blot (Figure 4.B, E).
The inflammatory cytokines TNFa and IFNc also upregulated Fn14 mRNA (Figure 4.C) and protein (Figure 4.D, E) expression in PC-3 cells. The time course of Fn14 upregulation in response to TNFa/IFNc was delayed with respect to observations in other cell types [33]. Lethal Effect of TWEAK in Human Prostate Cancer Cells TWEAK can induce apoptosis, survival or even proliferation in different tumor cell lines and other cells [27,33,34]. We studied the lethal effect of TWEAK over prostate cancer cell lines. TWEAK (100 ng/mL) induced apoptosis in PC-3 cultured in the absence of FBS. The lethal effect was prevented by FBS ( Figure 5.A). In some cases, TWEAK-induced cell death requires co-incubation with sensitizers, such as the inflammatory cytokines TNFa and IFNc [19,33]. In this regard, co-incubation of TWEAK with TNFa/IFNc strongly increased apoptosis in PC-3 cells (Figure 5.A). Neither TNFa nor IFNc, alone or together, induced apoptosis in PC-3 cells (data not shown). However, TWEAK did not induce apoptosis in serum depleted LNCaP cells, neither alone, nor in combination with TNFa/ IFNc ( Figure 5.B). These results are concordant with the effect of TWEAK observed in renal tubular cells, where, TWEAK induces proliferation in non-stressed cells, but, induces apoptosis in the presence of inflammatory cytokines [33,34]. TWEAK-

Fn14 Mediates the Lethal Effect of TWEAK Over PC-3 Cells
While Fn14 is the only characterized TWEAK receptor, there is evidence for a second TWEAK receptor, and TWEAK can bind to CD163 [45,46]. Therefore, we investigated whether TWEAK induces apoptosis in PC-3 cells through Fn14 activation. Pre-treatment with neutralizing anti-Fn14 (ITEM-2) or anti-TWEAK antibodies prevented TWEAK- (Figure 6.A) or TWEAK/TNFa/IFNc-induced apoptosis (Figure 6.B-C). These results suggest that TWEAK induces apoptosis in PC-3 cells through Fn14 activation. However, TWEAK may induce apoptosis through recruitment of endogenous TNF/TNF receptor 1 (TNFR1) [47]. To rule out this mechanism, we pre-stimulated PC-3 cells with an anti-TNF neutralizing antibody. Anti-TNF treatment did not prevent TWEAKinduced apoptosis in PC-3 cells (Figure 6.A).

Characterization of TWEAK-induced Apoptosis in PC-3 Cells
TWEAK-induced apoptosis in PC-3 cells was assessed both by the presence of hypodiploid cells measured by flow cytometry and by the typical morphology (nuclear shrinkage, condensation and fragmentation as well as decreased cell size) (Figure 7.A-B). In Annexin V/PI assays, TWEAK increased the percentage of apoptotic cells in PC-3 cells cultured the in absence of serum, and this was more evident in cells treated with TWEAK/TNFa/IFNc. However, neither TWEAK nor TWEAK/TNFa/IFNc increased the number of apoptotic cells in the presence of serum (Figure 7.C). In addition, TWEAK did not increase Annexin V/PI positive cells in the LNCaP cell line (data not shown). We then studied the levels of proteins of the Bcl2 family. TWEAK and TWEAK/TNFa/IFNc increased proapoptotic Bax levels ( Figure 8.A), downregulated antiapoptotic BclxL levels ( Figure 8.B), and increased the final Bax/BclxL ratio (Figure 8.C). These results indicate that TWEAK modulates proteins of the Bcl2 family to favor the apoptosis cell death. Moreover, a Bax inhibitor peptide (P5) dose-dependently decreased TWEAK-induced apoptosis in PC3 cells, further suggesting recruitment of Bax and the mitochondrial pathway (Figure 8.D). To confirm the involvement of the mitochondrial apoptosis pathway we performed immunofluorescence of cytochrome C (Cyt C). Unstimulated cells showed mitochondrial Cyt C staining, whereas, in presence of TWEAK/TNFa/IFNc some cells showed Cyt C release, indicating that this pathway is activated (Figure 9).

TWEAK-induced Apoptosis in PC-3 Cells is Caspase Dependent
Next, we studied the mechanisms of TWEAK-induced apoptosis in PC-3 cells. Pretreatment with a pan-caspase inhibitor, zVAD, prevented TWEAK/TNFa/IFNc-induced apoptosis in PC-3 cells, indicating that the apoptosis is caspases-dependent ( Figure 10.A). Western blot showed processing of pro-caspase 3 to yield active caspasa-3 in presence of TWEAK at 6 hours. This effect was stronger and earlier in presence of TWEAK/TNFa/ IFNc (Figure 10.B). In presence of 10%FBS the TWEAK/ TNFa/IFNc combination barely activated caspase 3 (Figure 10.C). Immunofluorescence using an anti-active caspase-3 antibody confirmed caspase-3 activation (Figure 10.D). In some cells stimulated with TNFSF cytokines, caspases inhibition prevents apoptotic cell death, but, induces necrotic cell death [33]. In cell death assays staining with PI, zVAD protected from the cell death induced by TWEAK/TNFa/IFNc at the same level that anti-TWEAK antibody. This result indicates that caspase inhibition does not induce necrosis in PC-3 cells stimulated with TWEAK ( Figure 11).

Discussion
The main finding is that cell culture conditions and, possibly the in vivo microenvironment can be manipulated to sensitize androgen-independent prostate cancer cells to TWEAK-induced apoptosis. Inflammatory cytokines and serum regulate both the expression of Fn14 in human cancer cell line PC-3, as well as the cell sensitivity to the lethal effect of TWEAK. Interestingly, the PC-3 androgen-independent cell line was sensitive to the lethal effect of TWEAK when cultured in the absence of the survival and mitogenic factors contained in serum. Moreover, an inflammatory milieu composed of the combination of TNFa/IFNc increased the lethal effect of TWEAK over these cells. This offers new potential therapeutic opportunities for androgen-independent prostate cancer.

PC-3 cells than in LNCaP cells. LNCaP cells expressed noninducible, low levels of Fn14. Both LNCaP and PC-3 cells respond
to the pro-inflammatory effect of TWEAK. However, the apoptotic response of both prostate cancer cell lines to TWEAK differed. LNCaP cells were resistant to TWEAK-induced apoptosis, both in the presence or absence of TNFa/IFNc. More interesting were the results obtained in androgen-independent PC-3 cells, since androgen independent prostate cancer poses a more significant therapeutic challenge. Serum-deprived PC-3 cells were spontaneously sensitive to TWEAK-induced apoptosis and this effect was increased in an inflammatory milieu. Huang et al [35] reported that Fn14 increased proliferation and prevented basal apoptosis in PC-3 cells. However, these results were obtained in PC-3 cells cultured in the presence of FBS, and we observed the lethal effect of TWEAK over PC-3 cells cultured in the absence of serum. This phenomenon has already been observed for nonprostate cells. Thus, TWEAK alone induced proliferation, not cell death, in cultured, non-tumor, non-stressed renal tubular epithelial cells [34]. Upregulation of the Fn14 receptor by the growth factors present in serum increased the sensitivity of tubular cells to TWEAK-induced proliferation [34]. By contrast, upregulation of Fn14 expression in serum-deprived tubular cells by inflammatory cytokines (TNFa/IFNc) changed the response to TWEAK from proliferation to cell death [33,34].
An inflammatory milieu resulted in a sustained increase in the expression of Fn14 in PC-3 cells. However, similar to observation in tubular cells [34], the level of Fn14 expression may not be the single mechanism implicated in TWEAK sensitization, as serum also increased Fn14 expression but did not sensitize to cell death by TWEAK in PC-3 cells. A similar pattern of sensitivity to another TNFSF cytokine has been described: PC-3 were reported to be highly sensitive to TRAIL-induced apoptosis, and LNCaP to be resistant [48]. Although not confirmed by all authors [49] this observation may have a biological basis: androgens positively regulate the expression of the antiapoptotic FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP) [50], which blocks transmission of the lethal signal from TNFRSF receptors that possess a death domain (DD) [51]. This information is pertinent despite Fn14 lacking a DD because one of the potential pathways for TWEAK-induced apoptosis is sensitization to minor amounts of other TNFSF cytokines present in the microenvironment [52].
We also characterized the molecular and cellular mechanism of the cell death induced by TWEAK in PC-3 cells. PC-3 treated with TWEAK showed characteristics of apoptosis, such as presence of hypodiploid cells, the typical morphology (nuclear shrinkage, condensation and fragmentation as well as decreased cell size) and Annexin V/PI staining. Furthermore, we observed that TWEAK modulated proteins of Bcl2 family, increasing the Bax/BclxL ratio, and a Bax inhibitor peptide dose-dependently prevented cell death. TWEAK-induced apoptosis in PC-3 cells was caspase-dependent. Different to other cell systems caspase inhibition did not sensitize PC-3 cells to necrotic death [33].
Natural sources of TWEAK in the prostate may include leukocytes. Thus, T cells express an array of lethal cytokines, such as TRAIL and TWEAK that are functional in target cell killing [53,54]. Furthermore, TWEAK mediates the anti-tumor effect of tumor-infiltrating macrophages [55]. Since these are inflammatory cells, this information, together with our observation, suggests that TWEAK may have a role in natural defenses against prostate cancer. In addition, TWEAK circulates in serum [56]. However, circulating TWEAK levels may decrease under diverse pathological circumstances, including chronic kidney disease, vascular injury and diabetes [57][58][59]. Interestingly, diabetes appears to worsen the outcomes of prostate cancer [60,61]. It would be worth measuring circulating TWEAK levels in metastatic prostate  carcinoma, since low TWEAK availability might be one of the factors facilitating tumor expansion once it reaches an androgenresistant stage. In addition, CD163, a scavenger TWEAK receptor expressed by certain macrophages [45] and may compromise TWEAK availability in the context of an inflamed prostate. In this regard, a biological therapy approach destined to activate the Fn14 receptor in androgen resistant prostate cancer may complement natural anti-tumor defenses. The use of an activating antibody may bypass regulatory or maladaptive mechanisms that lower TWEAK levels.
The recent availability of Fn14 targeting antibodies may enhance the spectrum of prostate tumors sensitive to TWEAK/ Fn14 modulating therapies. Two different anti-Fn14 antibodies have shown anti-tumor activity in cultured cells and experimental models [26] [62,63]. A phase 1 dose escalation trial of PDL192 humanized anti-TWEAK receptor monoclonal antibody in subjects with advanced solid tumors was recently completed [30]. An agonistic anti-Fn14 antibody undergoing clinical trials appears to have dual mechanisms of action, where binding to the target activates Fn14 and also recruits the immune system to mediate antibody-dependent cellular cytotoxicity (ADCC) activity to help destroy the tumor [26]. Recruitment of ADCC may sensitize to killing by this antibody even Fn14-expressing, TWEAK-resistant cells.
In conclusion, TWEAK activation of the Fn14 receptor induced apoptosis in androgen-independent prostate cancer cells, when stimulated with inflammatory cytokines and deprived of survival factors. Since that TWEAK/Fn14 system was previously reported to promote prostate cancer cell proliferation in the presence of serum, this information may have therapeutic consequences for treatment of androgen-independent prostate cancer by designing maneuvers that sensitize tumor cells to TWEAK-induced apoptosis or by the use of agonistic anti-Fn14 antibodies [26].