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Originally published In Press as doi:10.1074/jbc.M313816200 on February 6, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17515-17523, April 23, 2004
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RKIP Sensitizes Prostate and Breast Cancer Cells to Drug-induced Apoptosis*

Devasis Chatterjee,ab Yin Bai,c Zhe Wang,c Sandy Beach,c Stephanie Mott,a Rajat Roy,c Corey Braastad,a Yaping Sun,c Asok Mukhopadhyay,d Bharat B. Aggarwal,d James Darnowski,ab Panayotis Pantazis,ef James Wyche,e Zheng Fu,g Yasuhide Kitagwa,g Evan T. Keller,gh John M. Sedivy,i and Kam C. Yeungcj

From the aDepartment of Medicine, Brown University and Rhode Island Hospital, Providence, Rhode Island 02903, cDepartment of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804, dCytokine Research Section, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, eDepartment of Biology, University of Miami, Coral Gables, Florida 33146, fLaboratory of Pharmacology-Pharmacotechnology, 11BEAA Soranou Efessiou 4, Athens 11527, Greece, iDepartment of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, and gDepartment of Pathology, University of Michigan, Ann Arbor, Michigan 48109-0940

Received for publication, December 17, 2003 , and in revised form, January 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342–348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19–30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The very process of deregulated oncogene expression during cancer development also sensitizes cancer cells to apoptotic signals (1–3). Deregulated oncoproteins such as E1a and c-Myc promote apoptosis by activating multiple downstream pro-apoptotic effector pathways (4, 5). Additional mechanisms of sensitizing cancer cells to apoptosis by an activated oncoprotein have been described (6, 7). For example, E2F sensitized cells to apoptosis through down-regulation of anti-apoptotic signals (7). Here we show that cancer cells can also be sensitized to apoptosis by up-regulating the expression levels of RKIP (Raf kinase inhibitor protein).

RKIP was originally identified as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1 (8). RKIP also inhibits nuclear factor {kappa}B (NF-{kappa}B)1 signaling by negatively modulating the activating phosphorylation of IKK{alpha} and IKK{beta} via upstream kinases (9). Although the molecular mechanism by which RKIP inhibits the Raf and NF-{kappa}B signaling pathways has been partially delineated, little is known about the biological relevance of the inhibition of these pathways by RKIP. In addition to these functions, we presently demonstrate the rapid up-regulation of RKIP during induction of chemotherapy-triggered apoptosis in human prostate and breast cancer cells. However, in DNA-damaging agent-resistant cancer cells, treatment with the drugs does not up-regulate RKIP expression. Ectopic expression of RKIP sensitizes DNA damage agent-resistant cells to undergo apoptosis. Down-regulation of RKIP expression confers resistance to 9-nitrocamptothecin (9NC) by releasing its inhibitory constraint on two major survival pathways in cancer cells. Our studies suggest that RKIP represents a novel apoptotic marker in human cancer cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Plasmid Constructs, and Chemicals—The human breast cell lines 578T and 578Bst were purchased from American Type Culture Collection (Manassas, VA). A human breast cancer MCF7 cell subline resistant to 9NC treatment was a gift from Dr. Ray Frackelton (Brown University). The human prostate cell lines LNCaP, DU145, and PC3 were purchased from American Type Culture Collection. Early (<30)- or late (>100)-passage DU145 cells were not used for this study. The 9NC-resistant DU145 cell subline, RC1, was established by continuous exposure of DU145 cells to 9NC (10). All cell lines were grown in conditions suggested by American Type Culture Collection. MCF7 and prostate cells were grown in Iscove's medium and RPMI 1640 medium supplemented with 10% fetal bovine serum, respectively. 9-Nitrocamptothecin was suspended in polyethylene glycol-200 to form a stock that was stored at -20 °C until used; etoposide, Taxol, and cisplatin were purchased from Sigma. Ac-Leu-Leu-norleucinal was purchased from Calbiochem and dissolved in Me2SO. The mammalian expression plasmid CMV-HA-RKIP has been described previously (9). AS-hRKIP encompasses human RKIP nucleotides 1–429 cloned into pCMV5 in the antisense orientation (8). Expression plasmids pCMV-p65 and pCMV-I{kappa}B-SR are gifts from Dr. Albert Baldwin (University of North Carolina, Chapel Hill, NC). pCMV-FLAG-tBid and pBabeCrmA were kindly provided by Dr. Junying Yuan (Harvard Medical School). pBabemycRaf-CAAX was a gift from Dr. Mark Marshall (Lilly Research Laboratories, Indianapolis, IN). RKIP small interfering RNA was constructed by cloning the RKIP target sequence (186–205 bp, relative to the start codon) into pSUPER.retro (OligoEngine, Seattle, WA) according to the manufacturer's recommendations

Transfection and Reporter Gene Assays—For transient transfection studies, 3 x 105 cells were transfected with a total of 2 µg of plasmid DNA using LipofectAMINE Plus Reagent (Invitrogen), except for 578T, which was transfected with Geneporter (Gene Therapy Systems, San Diego, CA). Forty to 48 h after transfection, both adherent and floating cells were collected for analyses. A Renilla luciferase gene (Promega) driven by the constitutive thymidine kinase promoter was included in all transfection experiments as an internal control to correct for transfection efficiency. Dual luciferase assays were performed using a kit obtained from Promega.

Immunoblot Analyses—Total cell extracts were prepared as described previously (11), and protein concentrations of lysates were determined using the Bradford assay kit (Bio-Rad). Proteins (10–50 µg) were separated by SDS-PAGE and electrophoretically transferred from the gel to nitrocellulose membranes (Amersham Biosciences). Proteins recognized by the antibodies were detected by enhanced chemiluminescence reagents (Amersham Biosciences). The poly(ADP-ribose) polymerase (PARP) monoclonal antibody was from Zymed Laboratories Inc., the I{kappa}B{alpha} antibody was from Upstate Biotechnology (Lake Placid, NY), MEK was from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-MEK and phospho-I{kappa}B{alpha} antibodies were from Cell Signaling (Beverly, MA), and the antibody to actin was purchased from Sigma. The antibody to RKIP has been described previously (8). The antibody to caspase-8 (C15) was generously provided by Dr. Peter H. Krammer (German Cancer Research Center, Heidelberg, Germany).

Immunohistochemistry of Human Tissue—Radical prostatectomy specimens were obtained at the time of surgery from prostate cancer patients at the University of Michigan and frozen in liquid nitrogen within 30 min after surgical excision as described previously (12). Histological confirmation of both tumor and normal regions of each prostate gland and tumor grading were performed as described previously (13). Written informed consent was obtained from all patients, and all tissue procurement was approved by the University of Michigan Institutional Review Board. Frozen tissue blocks were sectioned on a cryostat, and frozen sections were placed on slides, which were immersed in acetone at 4 °C for 15 min and air dried for 30 s and then stored in phosphate-buffered saline until immunostaining was performed. RKIP protein expression was detected using a rabbit polyclonal anti-RKIP antibody (1:400 dilution; Upstate Biotechnology) followed by the streptavidin-biotin method, which included horseradish peroxidase and diaminobenzidine chromogen (Histostain Kit; Zymed Laboratories Inc.). Immunostaining intensity was scored by a urologist (Y. K.) blinded to Gleason score, tumor size, and clinical outcome as described previously (13). Briefly, the spectrum of staining intensity in all the samples was evaluated, and then each sample scored as negative (score 1), weak (score 2), moderate (score 3), or strong (score 4), based on the amount of stain detected.

Cell Death/Survival Analyses—At various times after treatment with the indicated drugs, adherent and floating cells were collected for analysis. Apoptosis was assayed by staining the cells with annexin-V and 7-amino-actinomycin D (7-AAD) and quantified by a Guava personal cytometer (Guava, Burlingame, CA). Alternatively, total cell extracts were prepared and analyzed for both PARP and caspase-8 cleavage by immunoblotting. To determine fractions of apoptotic cells in transient transfection experiments, cells were co-transfected with a membrane-targeted EYFP expression plasmid (Clontech). Forty to 48 h after transfection, adherent and floating cells were stained with cy5-annexin V (Pharmingen). YFP- and annexin-V-positive cells were quantified by a Guava personal cytometer (Guava). For viable cell counting, 3 x 105 cells were transfected with a total of 2 µg of plasmid DNA, 0.2 µg of pCMV-LacZ, and 1.8 µg of effector plasmids using LipofectAMINE Plus Reagent (Invitrogen). Forty to 48 h after transfection, the cells were fixed and stained with X-gal. Viable blue-stained cells, determined by their morphology, were counted from 20 randomly selected fields. Data are displayed as mean ± S.D. of at least three independent experiments.

Reverse Transcription-PCR Assays—DU145 cells were treated with 9NC for 24 h. Total RNA was isolated, and 1 µg was used for first-strand synthesis, which then served as a template for PCR reactions using a 1st Strand cDNA Synthesis kit purchased from Roche Applied Science. The RKIP PCR primers were as follows: hrkip1 (sense in exon 1), 5'-CCTCGCCATGCCGGTGGACC-3'; hrkip2 (antisense in exon 1), 5'-CCGCCGCCCCGGCGTAGGTG-3'; hrkip3 (sense in exon 2), 5'-GGATGCTCCCAGCAGGAAGG-3'; and hrkip4 (antisense in exon 3), 5'-CCTTGGGAGGCCCCGAGCCC-3'. The {beta}-actin PCR primers were 5'-ATCTGGCACCAGACCTTCTACAATGAGCTGCG-3' and 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'. The PCR products were analyzed on a 2% agarose gel containing ethidium bromide.

Retrovirus-mediated Gene Transfer—pBabe-puro-based retroviral constructs encoding p65, Raf-CAAX, and CrmA were used to generate retroviruses using the 293GPG packaging cell line as described previously (14). Infected cells were cultured in puromycin selection medium for 3 days, and drug-resistant cells were pooled. The percentage of retrovirus-infected cells ranged from 60% to 80%, as estimated in parallel infections using retroviruses expressing green fluorescent protein. Expression of relevant genes was confirmed by immunoblotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The DU145 human prostate carcinoma cell line undergoes extensive apoptosis when treated with the clinically relevant anti-cancer drug 9NC (10, 11). 9NC inhibits topoisomerase I, thereby introducing single-strand breaks into the DNA molecule (15). Apoptosis was measured by binding to 7-AAD and annexin-V (16, 17), as well as by the cleavage of caspase-8 and PARP, a downstream physiological target of the caspase-8 cleavage cascade (Fig. 1a, left panel and b, top panels). In contrast, apoptosis was not induced in a 9NC-resistant DU145 subline, RC1, treated with 9NC concentrations up to 1 µM for 48 h (Fig. 1a, right panel and b, bottom panels).



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  		FIG. 1.
DNA-damaging drugs up-regulate RKIP in prostate cancer cells. DU145 and RC1 cells were treated with 50 nM 9NC (+) or carrier (-) for 24 h. Extracts were prepared after 24 h for (a) immunoblot analysis to examine PARP and caspase-8, (b) cytometric analysis to examine binding to 7-AAD and annexin-V, (insets in c) immunoblot analysis to examine the expression of RKIP and I{kappa}B proteins, and (d) immunoblot analysis with antibodies specific for actin and phosphorylated H2A.X. c, NF-{kappa}B activities of DU145 and RC1 cells were measured with an NF-{kappa}B luciferase reporter. Cells were transfected with an NF-{kappa}B reporter (NF-{kappa}BX3Luc) (9). Twenty-four h after transfection, cells were treated with 9NC (50 nM) for an additional 24 h before being harvested for luciferase assays. e and f, RKIP sensitizes RC1 cells to apoptosis. RC1 cells were transfected with the indicated expression plasmids or empty control vector. Twenty-four h after transfection, cells were treated with 9NC (100 nM) for an additional 24 h before being harvested. Extracts were prepared for (e) cytometric analysis to examine binding to 7-ADD and annexin-V and (f) immunoblot blot analysis using PARP-specific antibodies. g, the pan-caspase inhibitor Z-VAD-FMK does not block 9NC-induced RKIP expression in DU145 cells. DU145 cells were treated with a combination of 9NC (50 nM) and Z-VAD-FMK (20 µM) or carrier (-), as indicated, for 24 h. Extracts were prepared after 24 h for cytometric analysis to examine binding to 7-AAD and annexin-V (% Apoptosis) and immunoblot analysis to examine caspase-8, RKIP, and actin expression.

 
Because NF-{kappa}B is a regulator of apoptosis (18), we analyzed both cytoplasmic and nuclear extracts of 9NC-treated DU145 and RC1 cells for NF-{kappa}B activation. NF-{kappa}B activity was consistently up-regulated, as indicated by increased expression of an NF-{kappa}B reporter and I{kappa}B degradation in 9NC-treated cells (Fig. 1c, right panel). No difference was detected in extracts from the DU145 cells before and after 9NC treatment (Fig. 1c, left panel). In acknowledgment of the anti-apoptotic effects of NF-{kappa}B, we reasoned that increased NF-{kappa}B activity might account for the resistant phenotype of RC1 cells to 9NC treatment. In agreement with this reasoning, inhibition of NF-{kappa}B activity by ectopic expression of I{kappa}B-SR, a super-repressor of NF-{kappa}B (19, 20) in RC1 cells, re-sensitized the cells to 9NC (Fig. 1e, top panels and f).

Considering the inhibitory effects of RKIP on NF-{kappa}B signaling (9), we examined the expression of RKIP in these two cell lines before and after 9NC treatment. Comparable RKIP levels were detected in RC1 extracts prepared from cells before and after 9NC treatment, but RKIP expression was robustly increased in 9NC-treated DU145 cells (insets in Fig. 1c). To investigate whether the induction of RKIP expression resulted from a DNA damage response or was a consequence of apoptosis, we blocked 9NC-mediated apoptosis in DU145 cells with the pan-caspase inhibitor Z-VAD-FMK (21). As expected, inhibiting the activities of caspases, the major executors of apoptosis in mammalian cells, protected DU145 cells from apoptosis (Fig. 1g). Importantly, Z-VAD-FMK treatment did not block 9NC-induced RKIP expression, indicating that induction of RKIP was not the consequence of apoptosis (Fig. 1g).

We have shown recently that unlike DU145 cells, RC1 cells contain a mutated topoisomerase I, and this has been associated with development of resistance to 9NC (22). To determine whether 9NC introduces DNA strand breaks in RC1 cells, we monitored the extent of H2A.X phosphorylation with H2A.X phospho-specific antibodies. H2A.X is a histone variant, which is rapidly phosphorylated at serine 139 in response to DNA damage and is indicative of a cell committed to the apoptotic pathway (23). After treatment with 9NC, the histone variant H2A.X was robustly phosphorylated in DU145 cells but remained unphosphorylated in RC1 cells (Fig. 1d). The data described above therefore suggest that RKIP is the downstream target of a DNA damage-mediated signaling pathway initiated by 9NC and that this pathway is defective in RC1 cells.

The known protective effects of NF-{kappa}B, the strong induction of RKIP in the 9NC-treated DU145 cell line, and the loss of this effect in the RC1 cell line imply that one biological role of RKIP may be to exert a repressive effect on the NF-{kappa}B pathway in certain situations, allowing apoptosis to proceed. To assess whether RKIP was functionally linked to the apoptosis-resistant phenotype, we transfected RC1 cells with an RKIP expression vector and subsequently treated them with 9NC. Expression of RKIP re-sensitized the cells to 9NC-mediated apoptosis (Fig. 1, e and f). It is important to note that overexpression of neither RKIP nor I{kappa}B-SR alone fully induces apoptosis in RC1 cells (Fig. 1e, bottom panels).

To determine whether induction of RKIP expression is chemotherapeutic agent-specific, we examined the effects of other DNA-damaging agents on DU145 cells. Both cisplatin and etoposide up-regulated RKIP expression and caused apoptosis in DU145 cells (Fig 2a and b). Remarkably, the onset of apoptosis, as indicated by percentage of apoptotic cells, coincided with a robust induction of RKIP expression (Fig. 2, a and b). The human prostate carcinoma cell lines PC3 and LNCaP were also investigated for RKIP expression after 9NC treatment. No significant changes were observed in LNCaP cells, but an increase in RKIP expression was readily observed in PC3 cells after 9NC treatment (Fig. 2c). We next investigated at which level the expression of RKIP was regulated. Semiquantitative reverse transcription-PCR demonstrated a significant increase of RKIP mRNA after challenge with 9NC (Fig. 2d). The observed difference in the amounts of RKIP mRNA in untreated versus treated cells was not the result of global alteration of gene expression because the level of the {beta}-actin control was unchanged. Because RKIP expression was low in some prostate cancer cells in vitro, we decided to examine expression of RKIP in primary prostate tumors. The clinical relevance of RKIP expression to prostate cancer was evaluated by comparing RKIP protein expression in clinical samples of normal prostate with prostate cancer tissue. RKIP staining intensities were 4, 4, 4, 4, and 3 for five normal prostate samples and 2, 2, 2, 3, and 4 for five primary prostate tumors of Gleason score 5 or higher. A representative result is shown in Fig. 2e. These results demonstrate that RKIP staining intensity is decreased in primary prostate tumors compared with normal prostate.



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  		FIG. 2.
Expression of RKIP in prostate cancer cells and primary prostate tumors. a and b, DU145 cells were treated with cisplatin (1 µg/ml) and etoposide (2 µM) for 24 and 48 h. Extracts were prepared for (a) immunoblot analysis to examine PARP, caspase-8, tubulin, and RKIP expression (*, marks a protein that is cross-reactive with the PARP antibody) and (b) cytometric analysis to examine binding to 7-AAD and annexin-V (% Apoptosis). c, immunoblot analysis of extracts from LNCaP and PC3 cells treated with 9NC (50 nM) for 24 or 48 h to examine PARP and RKIP expression. d, 9NC increases the total steady-state RKIP mRNA in DU145. Reverse transcription-PCR was performed on total RNA isolated from DU145 cells treated with 9NC (50 nM) for 48 h and examined for the expression of RKIP and actin (control) mRNA. e, immunohistochemical staining for RKIP in human clinical tissue samples. Frozen samples of normal prostate or primary prostate tumors were stained with a goat polyclonal antibody against RKIP and counterstained with hematoxylin. Scale bar, 25 µm.

 
To examine whether up-regulation of RKIP is the cause or consequence of DNA damage-induced apoptosis in DU145 cells, we simultaneously monitored RKIP expression and extent of apoptosis at various times after 9NC treatment, as measured by the cleavage of PARP and binding to 7-AAD/annexin-V. Although substantial cleavage of PARP was detected as early as 12 h after 9NC treatment, it required 24 h of treatment for a significant portion of the cell population to stain positive for both 7-AAD and annexin-V, entering the final stage of apoptosis (Fig. 3, a and b). Consistent with a causal role of RKIP in 9NC-induced apoptosis, the peak RKIP expression preceded the onset of extensive apoptosis (Fig. 3, a and b). Furthermore, overexpression of RKIP in DU145 cells could induce apoptosis almost as efficiently as t-Bid, a constitutively activated pro-apoptotic molecule (24). Apoptosis was measured using various approaches, including counting the number of viable cells, monitoring the extent of cleavage of caspase-8 and PARP, and evaluating binding to annexin-V (Fig. 3, c-e). Taken together, these data strongly suggest that RKIP is the cause of 9NC-mediated apoptosis in DU145 cells. We reasoned that if RKIP is the cause of apoptosis in DU145 cells, then we should be able to protect these cells from apoptosis by blocking the 9NC-triggered increase in RKIP expression. Indeed, down-regulation of RKIP expression by antisense RKIP expression vector and RKIP small interfering RNA blocked 9NC-induced apoptosis in DU145 as measured by PARP cleavage and by the number of viable cells (Fig. 3, f-h).



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  		FIG. 3.
RKIP induces apoptosis in DU145 cells. a, DU145 cells were treated for the indicated times with 9NC (50 nM). Extracts were prepared for (a) flow cytometric analysis to examine binding to 7-AAD and annexin-V (percentage of apoptosis) and (b) immunoblot analysis to examine PARP, actin, and RKIP expression. c, immunoblot analysis of cells extracts from DU145 cells transfected with HA-RKIP or empty control vector to examine the cleavage of PARP and caspase-8 with specific antibodies. d, DU145 cells were transfected with pCMV-LacZ and the indicated expression vectors or empty vector control. Forty to 48 h after transfection, the cells were fixed and stained with X-gal. Viable blue-stained cells, determined by their morphology, were counted from 20 randomly selected fields. Data are displayed as mean ± S.D. of at least three independent experiments. e, flow cytometric analysis of annexin-V staining of DU145 cells transfected with (0.1 µg) pCMV-mEYFP and different expression vectors (0.9 µg) as indicated. The percentage of annexin-V+/EYPF+ (top right quadrant) and annexin-V-/EYFP+ (bottom right quadrant) cells is shown in the top corner of each quadrant. f and g, ectopic expression of antisense RKIP RNA blocks 9NC-induced apoptosis in DU145 cells. DU145 cells were transfected with an RKIP antisense construct or empty vector control, and 24 h after transfection, cells were treated with 9NC (50 nM) for an additional 24 h before they were harvested for (f) immunoblot analysis with specific antibodies and (g) analysis of cell death by trypan blue staining. h, reduction of RKIP by small interfering RNA blocks 9NC-induced apoptosis in DU145 cells. DU145 cells were stably transduced with RKIP-small interfering RNA expression vector by retroviral infection. Stable clones were treated with 9NC (50 nM) for 24 h, and extracts were prepared (inset) for immunoblot analysis to examine RKIP and tubulin expression levels and flow cytometric analysis to examine binding to 7-AAD and annexin-V (percentage of apoptosis).

 
The NF-{kappa}B and Raf signal pathways are both important in determining cell survival in many different cell types (18, 25–27). The significance of these signaling pathways in DU145 cell survival was investigated by a loss-of-function approach. We interfered with the NF-{kappa}B and Raf signaling pathways in DU145 cells by ectopically expressing either I{kappa}B-SR or a dominant-negative mutant of Raf (RafDN) (28). As expected, we observed inhibition of an NF-{kappa}B reporter and down-regulation of the Raf signaling pathway as measured by MEK dephosphorylation in I{kappa}B-SR- or RafDN mutant-transfected cells (data not shown). Importantly, down-regulation of either pathway was accompanied by an increase in apoptosis as measured by a reduction in the number of viable cells (Fig. 4a). It is also noteworthy that the pro-apoptotic effects of either I{kappa}B-SR or RafDN are comparable with that of the well-characterized pro-apoptotic protein t-Bid in DU145 cells (Fig. 4a). Conversely, ectopic expression of the activated Raf (Raf-CAAX) by retroviral transduction partially protected the DU145 cells from 9NC-induced apoptosis (Fig. 4b, row 2). The survival-promoting effects of Raf-CAAX are comparable with that of ectopic expression of CrmA, a known inhibitor of caspase-8 (Fig. 4b, row 6) (29). Unexpectedly, expression of p65 or p52 either failed to protect or minimally protected the DU145 cells from apoptosis (Fig. 4b, rows 3 and 4). We speculate that the failure is probably because neither p65 nor p52 is constitutively active. In addition, we observed that the expression levels obtained with retroviral transduction are not as high as transient expression driven by cytomegalovirus promoter (data not shown). None-theless, the expression of p65 augmented the survival effects of activated Raf, resulting in the total protection of cells from apoptosis (Fig. 4b, row 5), and thus supports our argument that NF-{kappa}B is an important survival factor in DU145 cells. We next investigated the possibility that RKIP induces apoptosis by antagonizing either one of the two survival pathways. As shown in Fig. 4, c and d, RKIP-induced apoptosis was relieved by overexpression of the p65 subunit of NF-{kappa}B or the constitutively active form of Raf (BXB). The effects of p65 and BXB on RKIP-induced apoptosis are not due to a lower RKIP expression in combined transfection because RKIP levels are similar as detected by immunoblotting (Fig. 4d, bottom panel). This indicates that NF-{kappa}B as well as Raf up-regulation has an apoptosis-sparing effect. In agreement with our hypothesis that RKIP induces apoptosis by antagonizing Raf and NF-{kappa}B signaling, overexpression of RKIP in DU145 cells diminished the phosphorylation of both MEK and I{kappa}B-{alpha} (Fig. 4e).



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  		FIG. 4.
RKIP induces apoptosis by blocking two major survival pathways in DU145 cells. a, down-regulation of Raf and NF-{kappa}B signaling pathways induces apoptosis in DU145 cells. DU145 cells were transfected with pCMV-LacZ and the indicated expression vectors or empty vector control. Forty to 48 h after transfection, the cells were fixed and stained with X-gal. Viable blue-stained cells were determined as described in Fig. 2b. b, ectopic expression of Raf-CAAX and p65 protected DU145 cells from 9NC-induced apoptosis. Raf-CAAX, p65, p52, and CrmA were ectopically expressed in DU145 by retroviral infection. Infected cells were selected with puromycin for 3 days. For Raf-CAAX- and p65-infected cells, cells were selected with puromycin and G415 for 5 days. Selected cells were pooled and treated with 9NC (50 nM) for 24 h. Extracts were prepared for flow cytometric analysis to examine binding to 7-AAD and annexin-V (percentage of apoptosis). c and d, up-regulation of Raf and NF-{kappa}B signaling pathways abrogates RKIP-induced apoptosis in DU145 cells. c, DU145 cells were transfected with different combinations of expression vectors as indicated. Forty to 48 h after transfection, cell extracts were prepared for immunoblot analysis to examine the cleavage of PARP with specific antibodies. d, top panel, DU145 cells were transfected with pCMV-LacZ and different combinations of expression vectors as indicated. Forty to 48 h after transfection, the cells were fixed and stained with X-gal. Viable blue-stained cells were determined as described in Fig. 2b. To detect RKIP levels in the combination transfection, a duplicated set of transfected cells was harvested for immunoblot with anti-RKIP and anti-tubulin antibody (bottom panel). e, expression of RKIP down-regulates both MEK and I{kappa}B-{alpha} phosphorylation. To measure the expression levels of I{kappa}B, cells were treated with proteasome inhibitor ALLN (50 µM) for 2 h before being harvested. The phosphorylation of I{kappa}B-{alpha} was examined by immunoblot analysis with phospho-specific antibodies. The phosphorylation of MEK was examined by immunoblot analysis with phospho-specific antibodies.

 
An aberrant up-regulation of NF-{kappa}B activity is often observed in breast cancer cell lines (30, 31). Nuclear extracts prepared from normal, untransformed breast epithelial 578Bst cells displayed low levels of NF-{kappa}B DNA binding activity, whereas elevated levels of activity were detected in extracts from the breast tumor cell line 578T (26). Inhibition of this NF-{kappa}B activity by microinjection of I{kappa}B protein into breast cancer cell lines induced apoptosis (26). Consistent with these studies, we also observed elevated levels of NF-{kappa}B activity as measured by the phosphorylation of I{kappa}B (Fig. 5a). In agreement with the observed inhibitory role of RKIP on NF-{kappa}B signaling (9), higher levels of NF-{kappa}B activity in 578T correlated with decreased expression of RKIP (Fig. 5a). The converse was observed in the normal cell line 578Bst (Fig. 5a). Consistent with the decreased levels of RKIP, the Raf signaling pathway was comparatively elevated in 578T, as measured by the activities of transcriptional factor AP-1 and phosphorylation of MEK (Fig. 5a). Next we determined whether the expression levels of RKIP could be up-regulated by DNA-damaging agents. Unlike the prostate cancer DU145 cells, RKIP protein expression in 578T did not change after treatment with the DNA-damaging drugs 9NC, cisplatin, and etoposide (Fig. 5c). Furthermore, 578T cells did not undergo apoptosis after 9NC treatment as indicated by the lack of PARP cleavage and binding to annexin-V (Fig. 5, b and c). The nonresponsiveness to DNA damage-induced apoptosis was not due to mutations in the apoptotic pathways in 578T cancer cells because apoptosis was induced by another pro-apoptotic drug, Taxol (Fig. 5, b and c). Finally, restoring the expression levels of RKIP by ectopic expression triggered apoptosis (Fig. 5d), comparable with that triggered by ectopically expressed I{kappa}B super-repressor I{kappa}B-SR (Fig. 5d, compare lanes 2 and 3 with lanes 4 and 5). Similar results were observed with the MCF7 breast cancer cell line (Fig. 5, e-h).



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  		FIG. 5.
RKIP sensitizes breast cancer cells to apoptosis. a, NF-{kappa}B and Raf signaling pathways are up-regulated in 578T cells. Total cell lysates were prepared from serum-starved 578T or 578Bst cells. The phosphorylation of I{kappa}B-{alpha} was used as the readout of NF-{kappa}B signaling pathway. To measure the expression levels of I{kappa}B, cells were treated with proteasome inhibitor ALLN (50 µM) for 2 h before being harvested. The phosphorylation of I{kappa}B-{alpha} was examined by immunoblot analysis with phospho-specific antibodies. Raf kinase activities were measured by an AP-1 luciferase reporter and the phosphorylation of MEK. The phosphorylation of MEK was examined by immunoblot analysis with phospho-specific antibodies. To measure the AP-1 activities, 578T or 578Bst cells were transfected ith AP-1 or control reporter constructs as indicated. Activity elicited by vector control was set to 1. At 2 days after transfection, cell extracts were prepared and analyzed for luciferase activity. All activities were normalized on the basis of an internal transfection control (thymidine kinase promoter-driven Renilla luciferase reporter). b and c, 578T is resistant to DNA damage-induced apoptosis. b, flow cytometric analysis of annexin-V and actinomycin D staining of 578T cells after a 24-h treatment with the indicated drugs or carrier control. c, immunoblot analysis of extracts from 578T cells treated with different chemotherapeutic drugs as indicated for 48 h to examine RKIP expression and PARP cleavage with specific antibodies. d, RKIP induces apoptosis in 578T cells. 578T cells were transfected with the indicated expression vectors or empty vector control. Forty to 48 h after transfection, the cells were harvested and analyzed for PARP cleavage by immunoblotting. MCF7 breast cancer cells are resistant to DNA damage-induced apoptosis. e and f, resistance of MCF7 breast cancer cells to DNA damage-induced apoptosis. e, flow cytometric analysis of annexin-V and 7-AAD staining of MCF7 cells after 24 h of treatment with the indicated drugs or carrier control. f, immunoblot analysis of extracts from MCF7 cells treated with different chemotherapeutic drugs as indicated for 24 h to examine RKIP expression and PARP cleavage with specific antibodies. RKIP induces apoptosis in MCF7 cells. g and h, RKIP induces apoptosis in MCF7 cells. g, MCF7 cells were transfected with the indicated expression vectors or empty vector control. Forty to 48 h after transfection, the cells were harvested and analyzed for PARP as well as pro-caspase-8 cleavage by immunoblotting. h, MCF7 cells were transfected with pCMV-LacZ and the indicated expression vectors or empty vector control. Forty to 48 h after transfection, the cells were fixed and stained with X-gal. Viable blue-stained cells were determined as described in Fig. 3d.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The underlying mechanism determining whether a cell will die after DNA damage is complex (32). However, it is known that the threshold for the activation of apoptosis in response to DNA damage is set lower in cancer cells than in their normal counterparts (1–3). Although it is axiomatic that the pro-apoptotic trait of cancer cells results from activation of oncogenes, the effectors through which oncoproteins generate sensitivity to apoptosis remain elusive (4). Here we show that RKIP expression levels are low in prostate cancer cells and increase after DNA damage. The direct correlation of RKIP expression with the extent of apoptosis raises the possibility that up-regulation of RKIP is one of the mechanisms that sensitizes cancer cells to apoptotic signals in response to DNA damage. Four lines of evidence support a causal role for RKIP. First, we observed a direct correlation among the expression levels of RKIP, the tumorigenic ability of cells when xenografted in thymus-less (nude) mice, and the sensitivity of cancer cells to DNA damage-mediated apoptosis. Both DU145 and PC3 cells are tumorigenic in nude mice and have a low level of RKIP expression, which can be significantly induced to high levels after DNA damage. In contrast, non-tumorigenic LNCaP cells have a comparatively high level of RKIP expression. After DNA damage, the RKIP expression levels in LNCaP cells remained unchanged, and the cells went into growth arrest, whereas RKIP expression levels in PC3 and DU145 cells robustly increased, and the cells traversed rapidly from G1 to S, followed by extensive cell death. Second, 9NC-resistant prostate cancer RC1 cells, whose RKIP expression levels did not change upon DNA damage, were re-sensitized to undergo 9NC-triggered apoptosis after RKIP overexpression. This raises the possibility of using RKIP overexpression as a mean to overcome resistance to 9NC in RC1 cells (33). Third, the ectopic expression of RKIP-induced apoptosis in the absence of apoptotic signals in DU145 cells. Fourth, down-regulation of RKIP expression protected DU145 cells from 9NC-induced apoptosis.

Mechanistically, sensitization could be a direct consequence of increased death signaling. Alternatively, RKIP could sensitize cancer cells to apoptosis by inhibiting the Raf/MEK/ERK and NF-{kappa}B survival signaling pathways. Although the two mechanisms are not mutually exclusive, several lines of evidence presented in this communication strongly suggest the indirect model. We have shown that both Raf/MEK/ERK and NF-{kappa}B are important survival signaling pathways in DU145 cells. Down-regulation of either pathway can induce apoptosis. Conversely, up-regulation of both pathways completely protected DU145 from 9NC-induced apoptosis. Also, up-regulation of either pathway diminished the extent of apoptosis induced by RKIP. It remains to be determined whether RKIP also plays a direct role in inducing apoptosis in DU145 cells.

Is the sensitization effect of RKIP toward DNA damage-mediated apoptosis specific to prostate carcinoma cells? Our studies with the breast cancer cell lines 578T and MCF7 showed that the expression level of RKIP was low and underwent minimal changes upon DNA damage. In agreement with the role of RKIP in inducing apoptosis, very little cell death occurred after 2 days of incubation with DNA-damaging drugs at concentrations that caused extensive apoptosis in DU145 cells. Both 578T and MCF7 cells expeditiously succumbed to apoptosis after incubation with Taxol, an anti-cancer drug with a mode of action quite different from those described for DNA-damaging drugs. Therefore, our observations in breast cancer cells are consistent with our hypothesis that RKIP plays an important role in apoptosis and suggest that the expression of RKIP can be regulated by more than one pathway after apoptotic insults. We also demonstrated that restoring normal RKIP levels in breast cancer cells by ectopic expression triggers spontaneous apoptosis. The capacity to suppress apoptosis is one of the essential acquired attributes in cancer cells. Up-regulation of survival signaling is one of the common strategies a cancer cell exploits. Previous studies by others (30, 31), together with the findings described in this communication, show that NF-{kappa}B and the activated Raf are two of the major determinants of survival in breast cancer cells. In view of its previously demonstrated inhibitory activity toward NF-{kappa}B and Raf signaling, we speculate that down-regulation of RKIP is one of the strategies breast cancer cells employ to evade oncogene-induced apoptosis.

In this study, we have shown a correlation between the expression levels of RKIP and tumorigenicity in prostate and breast cancer cells. Consistent with studies of cancer cell lines in vitro, we have also shown that the expression levels of RKIP decrease in primary prostate tumors compared with normal prostate tissue. In line with our studies, Fu et al. (34) have recently reported that RKIP is a clinically relevant and novel suppressor of prostate cancer cell metastasis. Their study demonstrates that low levels of RKIP expression correlate with increased metastatic potential. Taken together with our data, it is tempting to speculate that the metastatic potential of tumor cells could be prevented by chemotherapy-triggered induction of RKIP, which would lead to apoptosis in target cells. The expression of RKIP could represent a useful marker of response to relevant chemotherapeutic agents and eventually be implemented in a molecular-targeted therapeutic strategy for prostate and other cancers. Finally, we have shown that some cancer cells, when subjected to an apoptotic insult, have the ability to up-regulate RKIP expression and become sensitive to apoptosis. Under these conditions of intact RKIP expression, only the further acquisition of genetic lesions, as observed in RC1 cells and some breast cancer cells, will allow survival and further malignant progression. Unlike other classical tumor suppressor genes, which usually have complete loss-of-function mutations in tumor cells, the expression levels of RKIP are decreased or become non-inducible, but they are always detectable. The presence of active RKIP in cancer cells will therefore allow the isolation of agonist drugs that increase the interaction between RKIP and its targets. The identification of signaling pathways that regulate RKIP expression will also expedite the design of effective therapy for patients with cancers refractory to chemotherapeutic treatments. In summary, we hypothesize that RKIP may function as a tumor suppressor gene in the development of human prostate and breast cancer. The expression of RKIP may represent a novel prognostic marker in the evolution of cancer and provides a therapeutic target for the treatment of cancer.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R01 GM64767 (to K. C. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

b Support was provided by the T. J. Martell Foundation (to D. C. and J. D.) and a Lifespan Developmental Grant (to D. C.). Back

h Supported in part by awards from the Stuart and Barbara Padnos Endowed Research Fund of the University of Michigan Comprehensive Cancer Center and the Association for the Cure of Cancer of the Prostate. Back

j To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. E-mail: kyeung{at}mco.edu.

1 The abbreviations used are: NF-{kappa}B, nuclear factor {kappa}B; 9NC, 9-nitrocamptothecin; PARP, poly(ADP-ribose) polymerase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; 7-AAD, 7-amino-actinomycin D; X-gal, 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside; Z-VAD-FMK, benzyloxycarbonyl-VAD-fluoromethyl ketone; ERK, extracellular signal-regulated kinase. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Albert Baldwin, Mark Marshall, Han-Fei Ding, and Junying Yuan for plasmid constructs, Dr. Ray Frackelton for MCF7 breast cancer cell line, and Miranda Yeung for excellent technical assistance. K. C. Y. also thanks Drs. Han-Fei Ding, Walter Kolch, John Nagle, and the members of the Yeung laboratory for critical reading of the manuscript and helpful comments.



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