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Originally published In Press as doi:10.1074/jbc.M307627200 on October 6, 2003 Originally published In Press as doi:10.1074/jbc.M307627200 on September 30, 2003

J. Biol. Chem., Vol. 278, Issue 50, 50402-50411, December 12, 2003
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Fibronectin Protects Prostate Cancer Cells from Tumor Necrosis Factor-{alpha}-induced Apoptosis via the AKT/Survivin Pathway*

Mara Fornaro{ddagger}, Janet Plescia{ddagger}§, Sophie Chheang{ddagger}, Giovanni Tallini{ddagger}, Yong-M. Zhu{ddagger}, Michael King{ddagger}§, Dario C. Altieri{ddagger}§, and Lucia R. Languino{ddagger}§

From the §Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and the {ddagger}Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510

Received for publication, July 15, 2003 , and in revised form, September 23, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84-> Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via {beta}1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-{alpha} (TNF-{alpha}). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-{alpha}-induced apoptosis. Evidence is provided that expression of {beta}1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-{alpha}-induced apoptosis. In contrast, the {beta}1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-{alpha} in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It has become increasingly clear that the interactions between extracellular matrix (ECM)1 proteins and integrins, their cognate cell surface receptors, mediate cell adhesion but also engender signals that participate in cell survival (1). It has been shown that insufficient or inappropriate cell-ECM interactions cause apoptosis, previously designated anoikis (1). This mechanism of integrin-dependent cytoprotection is operative against a variety of death-promoting stimuli, acting through the intrinsic (mitochondrial) or extrinsic (death receptor) apoptotic pathway and involving growth factor withdrawal (2–5), exposure to cytotoxic drugs (6, 7), and ligation of death receptors (8, 9). Critical signaling intermediates involved in integrin-dependent cell survival have been identified, and integrin-mediated adhesion to the ECM stimulates the production of PI(3,4)P2 and PI(3,4,5)P3 (10, 11), the association of the p85 PI 3-kinase subunit with focal adhesion kinase (12) and AKT activation (10, 11). In turn, active AKT interferes with the apoptotic machinery by phosphorylating thus sequestering the proapoptotic Bcl-2 family protein BAD, by inactivating members of the forkhead family of transcription factors, and by promoting NF-{kappa}B-dependent cytoprotection via transcriptional activation of a plethora of downstream target genes (13).

The effector molecules involved in integrin-dependent cell survival have not been completely elucidated. Engagement of fibronectin through {alpha}5{beta}1 and {alpha}v{beta}1 and of vitronectin through {alpha}v{beta}3 integrins has been associated with up-regulation of anti-apoptotic Bcl-2 and with Bcl-2-mediated protection from apoptosis induced by serum deprivation (2, 14). Whether other cytoprotective mechanisms for integrin-dependent cell survival exist in normal or tumor cells has not been determined. In addition to the Bcl-2 family of cell death regulators, a second group of inhibitor of apoptosis (IAP) proteins has been recently identified (15). Survivin, a member of the IAP family of proteins, is expressed during embryonic and fetal development but undetectable in most normal adult tissues (16). However, survivin becomes the fourth most expressed transcript in human cancer (17) where it correlates with a more aggressive and disseminated disease and reduced overall survival (18). Several studies have shown that survivin expression inhibits cell death induced by various apoptotic stimuli in vitro (19) as well as in vivo (18, 20).

Adenocarcinoma of the prostate is the most common malignancy in men and the second leading cause of cancer-related deaths in the United States (21). Evolution from a localized to an invasive fatal disease is accompanied by progression from an androgen-dependent to an androgen-independent state (22) and by the emergence of apoptosis-resistant cells within primary and/or metastatic tumors. Although several anti-cancer strategies have been shown to exert their cytotoxic effect via activation of an apoptotic program, the limited efficacy of anti-cancer drugs against epithelial neoplasms implies the ability of tumor cells to raise their anti-apoptotic threshold, thus evading cell death.

Despite growing evidence regarding the important role of integrins in tumorigenesis and in resistance to chemotherapeutic drugs, the molecular mechanisms underlying integrin-mediated survival signaling in cancer cells are not completely understood. In this study, we demonstrate that cell adhesion to fibronectin via {beta}1 integrins regulates survivin protein levels and provides protection against death-induced stimuli via AKT activation. Conversely, inhibition of survivin function reverses the cytoprotective effect of ECM proteins and results in tumor cell apoptosis. We also show that the {beta}1A integrin cytodomain is essential for cytoprotection against death-inducing stimuli in a survivin-dependent manner. We conclude that the activation of survival pathways mediated by integrins and survivin may contribute to drug resistance in cancer cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—The following antibodies were used: a rabbit antibody to survivin (Novus Biologicals, Littleton, CO) was characterized in previous studies (23); rabbit affinity-purified IgG to ERK1 and 2 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit affinity-purified IgG to XIAP (Santa Cruz Biotechnology); rabbit affinity-purified IgG to caspase-3 (BD Transduction Laboratories, San Diego, CA); rabbit affinity-purified IgG to phospho-(Ser/Thr) AKT substrates (Cell Signaling, Beverly, MA); monoclonal antibody (mAb) to survivin (23) (60.1, Novus Biologicals); mAb to Bcl-2 (BD Transduction Laboratories); mAb to Poly(ADP-ribose) polymerase (PARP, BD Biosciences); mAb to {beta}-actin (Sigma); 1C10 [PDB] , anti-human endothelial cell 140-kDa protein (Invitrogen); W1B10 mAb against the extracellular domain of chicken {beta}1 integrin (Sigma); and 12CA5 mAb to hemagglutinin (HA, Roche Applied Science). Non-immune rabbit IgG and mouse IgG were purchased from Sigma and used as controls. Fibronectin and vitronectin were purified from human plasma and serum, respectively, as described previously (24, 25). Reagents used in this study were: Z-VAD-fmk (Enzyme Systems Products, Livermore, CA); cycloheximide (CHX, Sigma); tumor necrosis factor-{alpha} (TNF-{alpha}) (R&D, Minneapolis, MN); actinomycin D (Act D, Sigma); etoposide (Sigma); propidium iodide (Sigma); RNase A (Roche Applied Science); and bovine serum albumin (BSA, Sigma).

Cells—RWPE-1, LNCaP, and PC3 cells were obtained from ATCC. LNCaP-LN3 cells (26) were a kind gift of Dr. C. A. Pettaway (The University of Texas, M. D. Anderson Cancer Center, Houston, TX). LNCaP, LNCaP-LN3, and PC3 cells were cultured as described previously (27). RWPE-1 cells were grown in keratinocyte serum-free medium (Invitrogen) supplemented with 5 ng/ml epidermal growth factor (EGF, Invitrogen) and 0.05 mg/ml bovine pituitary extract (Invitrogen). To obtain PC3 stable cell lines expressing either {beta}1C or {beta}1A integrins under the control of a tetracycline-regulated promoter, chimeric cDNAs consisting of the chicken {beta}1 extracellular and transmembrane domain and either human {beta}1C or human {beta}1A cytodomain sequences were generated by PCR-driven splice overlap extension (28). These chimeric constructs allow one to distinguish between endogenous {beta}1 integrin and exogenous {beta}1C and {beta}1A integrin variants in human cells. PCR reactions were performed using an automatic thermal cycler (PerkinElmer Life Sciences). The conditions used for the PCR reactions were as follows: denaturation at 95 °C for 1 min; annealing at 45 °C for 1 min; and extension at 72 °C for 1 min. 30 cycles of amplification were used. The amplification was performed in 1x Vent polymerase buffer (New England Biolabs, Beverly, MA), 200 µM each dNTP (PerkinElmer Life Science), 0.15 µM of each primer, 2 mM MgSO4 (PerkinElmer Life Science), and 0.5 units of Vent polymerase (New England Biolabs). The following synthetic nucleotides were used as primers to amplify chicken {beta}1 subunit from nucleotides (nt) 1787 to 2365 using chicken {beta}1 cDNA template (29): C1 (nt 1787–1807 of chicken {beta}1) 5'-AACTGCGATCGATCAAACGGT-3' and CH1 (nt 2357–2371 of human {beta}1 and nt 2351–2365 of chicken {beta}1) 5'-TATCATTAAAAGCTTCCAAATCAATAACAA-3'. Additional primers were designed to amplify either human {beta}1A cytodomain sequences from nucleotides 2357 to 2497 (30) or human {beta}1C cytodomain sequences from nucleotides 2357 to 2613 (31) using human {beta}1A or human {beta}1C cDNA template, respectively. These primers were: CH2 (nt 2351–2365 of chicken {beta}1 and nt 2357–2371 of human {beta}1) 5'-TTGTTATTGATTTGGAAGCTTTTAATGATA-3' and H1 (nt 2483–2505 of human {beta}1) 5'-GGACTAGTTTTTCCCTCATACTT-3'. The chimeric primers CH1 and CH2 corresponded to a region that was identical between the species and were complementary to each other. The amplified chicken and human cDNA fragments were mixed and subjected to an additional amplification using C1 as forward primer and H1 as reverse primer. The resulting chimeric cDNA fragments (718-bp chicken/human {beta}1A and the 834-bp chicken/human {beta}1C) were subcloned into pCR 2.1 vector (Invitrogen) following manufacturer's instructions. The 718-bp chicken/human {beta}1A and the 834-bp chicken/human {beta}1C chimeras were excised from pCR2.1 using ClaI and SpeI and subcloned into pTet-Splice expression vector (32). The resulting constructs were digested with SalI and ClaI to allow ligation of a DNA fragment corresponding to nucleotides 1–1787 of the chicken {beta}1 extracellular domain, which was isolated from pECE-{beta}1 (kindly provided by Dr. Hynes, Massachusetts Institutes of Technology, Boston, MA) using SalI and ClaI restriction enzymes. The chimeric constructs (pTet-chicken/human {beta}1C and pTet-chicken/human {beta}1A) were sequenced by the dideoxynucleotide method to confirm the nature of the chimeric inserts. PC3 cells were electroporated as described previously (33) using 100 µg of pTet-chicken/human {beta}1C, pTet-chicken/human {beta}1A, or pTet-Splice along with 10 µg of pTet-tTA. Neomycin-resistant cells were selected using medium containing 0.2 mg/ml G418 (Invitrogen). G418-resistant clones were isolated and screened for cell surface expression of either chicken/human {beta}1C or chicken/human {beta}1A integrin by FACS using W1B10, a monoclonal antibody against the extracellular domain of chicken {beta}1 integrin, or 12CA5 as a negative control as described previously (34).

Analysis of Survivin and Bcl-2 Expression by Immunoblotting—Subconfluent RWPE-1, LNCaP, LNCaP-LN3, and PC3 cells were lysed as described previously (35). In some experiments, subconfluent PC3 and LNCaP cells were serum-starved for 24 h, detached using 0.05% trypsin, 0.53 mM EDTA (Invitrogen), and washed three times with serum-free medium. PC3 (4 x 105) and LNCaP (1 x 106) cells were then plated onto 60-mm Petri dishes coated with 10 mg/ml BSA, 5 µg/ml fibronectin, or with 10 µg/ml vitronectin for 8 and 24 h in serum-free medium in the absence or in the presence of either 20 µg/ml Act D or of 10 µg/ml CHX. Cells were then lysed as described above. Protein content in each lysate was quantitated using the BCA protein assay reagent (Pierce). Samples were heated at 100 °C for 5 min and separated by electrophoresis on a 12–15% SDS-polyacrylamide gel and transferred onto Immobilon-P membrane (Millipore, Bedford, MA). Expression of survivin, and Bcl-2 was analyzed by immunoblotting using 1 µg/ml of the polyclonal antibody to survivin and 1 µg/ml mAb to Bcl-2, respectively, as described previously (35). Rabbit affinity-purified antibody to ERK1/2 was used to control for protein loading as described previously (35).

Tissue Specimen Procurement and Immunohistochemistry—Specimens from 53 human radical prostatectomies performed for prostatic adenocarcinoma at the Yale-New Haven Hospital (New Haven, CT) were processed according to Review Board-approved protocols. Hematoxylin and eosin sections from all of the specimens were evaluated microscopically to assess tissue integrity and preservation. Sections from formalin-fixed, paraffin-embedded specimens were immunostained as described previously (33) using either 60.1 mAb to survivin (1:1000 dilution) or mAb 1C10 [PDB] as control antibody (1:1000 dilution).

Plasmids, Transient Transfections, and Determination of Apoptosis—The survivin (C84A) mutant or survivin antisense cDNA was subcloned into pEGFPc1 (36, 37, 39). Replication-deficient adenoviral constructs encoding wild-type survivin (pAd-survivin), the survivin T34A mutant (pAd-T34A), or control GFP (pAd-GFP) were generated using the pAd-Easy system as described previously (37). The pCMV6 plasmids containing HA-tagged AKT K179M mutant (dn AKT) or wild type AKT were a kind gift of Dr. Franke (Columbia University, New York, NY) (38).

PC3 cells were plated onto 12-well plates at a density of 2 x 105 cells/well. Cells were transfected with the various GFP constructs using a mixture of 2 µg of plasmid DNA and 6 µl of LipofectAMINE 2000 (Invitrogen) in 1 ml of Opti-MEM I (Invitrogen). After a 7-h culture at 37 °C, the transfection mixture was aspirated and substituted with complete growth medium. Cells (floating plus attached cells) were harvested after 1–4 days from transfection, fixed in 70% ethanol for 1 h on ice, and spun for 5 min at 1,300 rpm. Cells were incubated in phosphate-buffered saline containing 1 mg/ml RNase for 30 min at 37 °C, propidium iodide was added at a final concentration of 8 µg/ml, and samples were analyzed for DNA content by flow cytometry. Alternatively, cells were harvested 1–4 days after transfection, washed in phosphate-buffered saline, pH 7.4, and fixed in 4% paraformaldehyde containing 0.25% Triton X-100 for 10 min at 22 °C. Cell nuclei were stained with 6.5 µg/ml 4,6-diamidino-2-phenylindole (Sigma), 16% polyvinyl alcohol (Air Products and Chemicals, Allentown, PA), and 40% glycerol. GFP-expressing cells were independently scored for morphologic signs of apoptosis (chromatin condensation, DNA fragmentation) using a Zeiss Axiophot microscope. In another set of experiments, PC3 cells (1 x 106) in 100-mm plates were transfected with 15 µg of dn AKT along with 3 µg of pEGFPc1 plasmid (Invitrogen) using LipofectAMINE. Cells were then detached using 0.05% trypsin, 0.53 mM EDTA, washed three times with serum-free medium, and plated (4 x 105) onto 60-mm Petri dishes coated with either 10 mg/ml BSA or with 5 µg/ml fibronectin for 8 h in serum-free medium at 37 °C. Cells were then treated with 150 ng/ml TNF-{alpha} plus 3 µg/ml CHX for an additional 42 h at 37 °C. Survivin expression was analyzed by immunoblotting as described above.

Caspase Activation—PC3 cells transfected with the various GFP constructs in the presence or in the absence of Z-VAD-fmk (20 µM) were harvested after a 2-day culture at 37 °C and assayed for caspase-3-dependent hydrolysis of the fluorogenic substrate Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-aldehyde, BD Biosciences) as described previously (39). For analysis of caspase-3-proteolytic cleavage of PARP, PC3 cells (1 x 107/ml), transfected with the various GFP constructs in the presence or in the absence of Z-VAD-fmk (20 µM), were lysed in 40 µl of lysis buffer containing 2% SDS, 50 mM Tris-HCl, and 10% glycerol. Samples were heated at 100 °C for 3 min and separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred onto Immobilon-P membrane for 60 min at 55 V. The transfer membrane was blocked in phosphate-buffered saline, pH 7.4, containing 5% nonfat dry milk at 4 °C overnight, washed in phosphate-buffered saline, pH 7.4, and separately processed for immunoblotting with a mAb to PARP as described previously (39).

DNA Fragmentation Assay—PC3 cells (4 x 105) were plated onto 60-mm Petri dishes coated with either 10 mg/ml BSA or with 5 µg/ml fibronectin for 4 h in serum-free medium. Cells were treated with 150 ng/ml TNF-{alpha} plus 3 µg/ml CHX or with 500 µM etoposide in the presence or in the absence of Z-VAD fmk (40 µM) for an additional 42 h at 37 °C. DNA fragmentation enzyme-linked immunosorbent assay on floating plus attached cells was performed using the Cell Death Detection enzyme-linked immunosorbent assay kit (Roche Applied Science) according to the manufacturer's instructions.

Adenoviral Targeting of Survivin in PC3 Cells—The experimental procedures for the generation and expansion in human embryonic kidney 293 cells of replication-defective adenovirus constructs encoding survivin (pAd-survivin), the phosphorylation-deficient survivin Thr-34-> Ala dn mutant (pAd-T34A), or control pAd-GFP have been described previously (37). With this protocol, there was no generation of replication competent adenoviral particles. PC3 cells (1.5 x 106) in 100-mm plates were infected with pAd-GFP, pAd-survivin, or pAd-T34A at m.o.i of 50–100 for 24 h at 37 °C. Cells were then detached using 0.05% trypsin, 0.53 mM EDTA, washed three times with serum-free medium, and plated (4 x 105) onto 60-mm Petri dishes coated with either 10 mg/ml BSA or with 5 µg/ml fibronectin for 8 h in serum-free medium. Cells were then treated with 150 ng/ml TNF-{alpha} plus 3 µg/ml CHX for an additional 42 h at 37 °C. Cells (floating plus attached cells) were fixed in 70% ethanol, and GFP-expressing cells were analyzed for DNA content as described above. PC3-{beta}1C and PC3-{beta}1A stable cell lines were cultured for 72 h in growth medium either in the absence or in the presence of 1 µg/ml tetracycline. Cells were infected with pAd-GFP or pAd-survivin at m.o.i. of 50–100 for 24 h at 37 °C during the last 24 h of the 72-h culture. Cells were harvested using 0.05% trypsin, 0.53 mM EDTA and then plated (4 x 105) onto 60-mm Petri dishes coated with 10 mg/ml BSA for 8 h in serum-free medium. Cells were then treated with 150 ng/ml TNF{alpha} plus 3 µg/ml CHX for an additional 20–24 h at 37 °C and then analyzed for DNA content as described above.

Statistical Analysis—Group differences were compared using Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Survivin Targeting Causes Spontaneous Apoptosis in PC3 Cells—The relative levels of survivin expression among different prostate cell lines were compared by immunoblotting. Consistent with previous observations (19), survivin expression was detected in all of the prostate cancer cell lines analyzed. The highest levels of survivin were present in highly aggressive prostate cancer cells such as PC3 and LNCaP-LN3 (Fig. 1A). At variance with the results obtained in malignant epithelial cells, survivin expression was very low in non-tumorigenic and noninvasive LNCaP prostate cancer cells or in RWPE-1 normal prostate cells (Fig. 1A). Immunohistochemical analysis of 53 human prostatic adenocarcinoma tissue specimens showed that survivin was expressed in glandular epithelium as well as in fibromuscular stromal cells (Fig. 1B and 1C). Transfection of PC3 cells with GFP constructs expressing the dn survivin, C84A mutant (40), or survivin antisense cDNA resulted in the appearance of PC3 cells with hypodiploid (apoptotic) DNA content, as shown by propidium iodide staining (respectively, Figs. 2A and 3B), and caused spontaneous DNA fragmentation, as indicated by 4',6-diamidino-2-phenylindole staining of GFP-expressing cells (Fig. 3A). In time course experiments, expression of GFP-survivin C84A mutant or GFP-survivin antisense cDNA resulted in progressively increased apoptosis with 60–65% of GFP-expressing cells exhibiting hypodiploid (apoptotic) DNA content 4 days after transfection (respectively, see Figs. 2B and 3C). Transfection of PC3 cells with survivin C84A mutant resulted in a 2–3-fold increase in caspase-3 activity as determined by DEVD hydrolysis (Fig. 2C) (40). Similarly, transfection of PC3 cells with survivin C84A mutant resulted in proteolytic cleavage of the 116-kDa caspase-dependent substrate, PARP, with the generation of a 85-kDa product (Fig. 2D). Preincubation of PC3 cells with Z-VAD-fmk entirely suppressed caspase-3 activity and PARP cleavage induced by survivin targeting (Fig. 2, C and D). These findings indicate that inhibition of survivin function in prostate cancer cells is sufficient to cause all of the biochemical hallmarks of apoptosis including hypodiploid DNA content, caspase-3 activity, and cleavage of caspase substrate(s).



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  		FIG. 1.
Survivin expression in prostatic adenocarcinoma. A, subconfluent RWPE-1, LNCaP, LNCaP-LN3, and PC3 prostate cells were lysed as described under "Experimental Procedures" and the levels of expression of survivin were analyzed by immunoblotting (IB) using 1 µg/ml rabbit affinity-purified antibody to survivin (top panel). Rabbit affinity-purified antibody to ERK1/2 was used as control for protein loading (bottom panel). In A, proteins were visualized by ECL. B and C, the expression of survivin in human prostate carcinoma was evaluated by immunohistochemistry using either 60.1 mAb to survivin (B, 1:1000 dilution) or mAb 1C10 [PDB] as negative control antibody (C, 1:1000 dilution). A representative case of human prostate adenocarcinoma is shown.

 



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  		FIG. 2.
Effect of survivin targeting on prostate cancer cell survival. A–D, PC3 cells were transiently transfected with GFP-vector or the GFP-survivin (C84A) mutant and cultured in growth medium for 48 h. A, hypodiploid DNA content was evaluated by propidium iodide staining and flow cytometry. Data are representative of one of three independent experiments. B, time course of apoptosis. GFP and GFP-survivin (C84A)-transfected cells were harvested at the indicated time points, and DNA content was analyzed as described in A. C, cell extracts were analyzed for caspase-3 activity by hydrolysis of the fluorogenic substrate DEVD-AMC. Data are the mean ± S.D. of replicates of a representative experiment of at least two independent determinations. D, the experimental conditions are the same as in A with the exception that SDS-extract of PC3 cells transfected with the indicated constructs with or without 20 µM Z-VAD-fmk, a caspase inhibitor, was immunoblotted with an antibody to PARP. The 116-kDa form of PARP and 85-kDa caspase-cleaved product are indicated. Proteins were visualized by ECL.

 



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  		FIG. 3.
Effect of survivin antisense on prostate cancer cell survival. PC3 cells were transiently transfected with GFP-vector or GFP-survivin antisense and cultured in growth medium for 48 h (A and B) or for the indicated times (C). A, nuclear morphology of PC3 cells transfected with GFP-vector (left panels) or GFP-survivin antisense (right panels) analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining (bottom panels). Top panels, phase contrast. Middle panels, GFP-positive cells. The arrows indicate apoptotic cells. B, GFP-survivin antisense transfected cells were cultured in growth medium for 48 h with or without 20 µM Z-VAD-fmk. Hypodiploid DNA content was evaluated by propidium iodide staining and flow cytometry. The percentage of cells with hypodiploid DNA content is indicated in parenthesis. C, time course of apoptosis. Cells transfected with GFP-vector or GFP-survivin antisense were harvested at the indicated time points, and DNA content was analyzed as described in B.

 
Fibronectin Adhesion Increases Survivin Protein Levels in PC3 but Not in LNCaP Cells—To test whether matrix attachment modulates survivin expression, PC3 and LNCaP cells were kept in suspension or plated on Petri dishes coated with fibronectin and survivin expression was analyzed by immunoblotting. As shown in Fig. 4, PC3 cell adhesion to fibronectin for 24 h resulted in increased survivin expression levels compared with cells kept in suspension (panel A, lanes 3 and 4, and panel B, 3-fold increase in three experiments). PC3 cell adhesion to fibronectin is predominantly mediated by {beta}1 integrins, known to be expressed at high levels in these cells (27, 41). In contrast, survivin levels in LNCaP cells were not modulated by adhesion to fibronectin (Fig. 4A, right panel, lanes 6 and 8). We found that fibronectin did not increase survivin levels in PC3 cells in the presence of either Act D, a transcription inhibitor, or CHX, a protein synthesis inhibitor (Fig. 4C). These results suggest that regulation of survivin levels by fibronectin occurs at transcriptional and translational levels. In addition, no changes in expression of anti-apoptotic Bcl-2 or XIAP were observed following cell adhesion to fibronectin at all of the time points examined (Fig. 4D, top panel, and data not shown). However, the adhesion of PC3 cells to vitronectin resulted in increased levels of Bcl-2 by Western blotting (data not shown), in agreement with previous observations (7, 14), whereas survivin expression was unchanged (Fig. 4E, top panel). In conclusion, survivin levels in PC3 cells are up-regulated in response to cell adhesion to fibronectin.



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  		FIG. 4.
Adhesion to fibronectin increases survivin protein levels in prostate cancer cells. A, PC3 and LNCaP cells were detached and plated on Petri dishes coated with 10 mg/ml BSA (S, lanes 1, 3, 5, and 7) or with 5 µg/ml fibronectin (Fn, lanes 2, 4, 6, and 8) for either 8 h (lanes 1, 2, 5, and 6) or 24 h (lanes 3, 4, 7, and 8) at 37 °C. Cells were lysed, and survivin expression was detected by immunoblotting using 1 µg/ml rabbit affinity-purified antibody to survivin (top panels). B, graph represents means of relative levels of survivin expression ± S.E. The increase in survivin expression in cells plated on fibronectin is significantly higher than that of cells held in suspension (p = 0.04). The bar graph shows the results of three independent experiments. C, PC3 cells were detached and cultured on Petri dishes coated with 10 mg/ml BSA (S, lanes 1, 3, and 5) or with 5 µg/ml fibronectin (Fn, lanes 2, 4, and 6) for 24 h in serum-free medium in the absence (lanes 1 and 2) or in the presence of either 20 µg/ml Act D (lanes 3 and 4) or 10 µg/ml CHX (lanes 5 and 6) at 37 °C. Survivin expression was analyzed by immunoblotting as described in A. D, PC3 cells were treated as described in A, and Bcl-2 expression was analyzed by immunoblotting using 1 µg/ml mAb to Bcl-2 (top panel). E, PC3 cells were detached and plated on Petri dishes coated with 10 mg/ml BSA (S, lanes 1 and 3) or with 10 µg/ml vitronectin (Vn, lanes 2 and 4) for either 8 h (lanes 1 and 2) or 24 h (lanes 3 and 4) at 37 °C. Cells were lysed, and survivin expression was detected as described above. Rabbit affinity-purified antibody to ERK1/2 was used as control for protein loading (A, C, D, and E, bottom panels). Proteins were visualized by ECL.

 
Fibronectin Adhesion Protects PC3 Cells from TNF-{alpha}-induced Apoptosis—To determine whether survivin is downstream of fibronectin in a novel survival pathway that protects cells from TNF-{alpha}-induced apoptosis, PC3 cells were either held in suspension or plated on Petri dishes that had been coated with fibronectin. Cells were treated with either TNF-{alpha} plus CHX or etoposide and analyzed for apoptosis by DNA fragmentation assay (Fig. 5A) or by propidium iodide staining and flow cytometry (Fig. 5, B and C). As shown in Fig. 5, TNF-{alpha} treatment induced a significant increase in DNA fragmentation (panel A) and in hypodiploid (apoptotic) DNA content (panel B) in cells held in suspension. In contrast, PC3 cell adhesion to fibronectin nearly completely suppressed DNA fragmentation and appearance of cells with hypodiploid (apoptotic) DNA content in response to TNF-{alpha} treatment (Fig. 5, A and B). At variance with these data, apoptosis induced by etoposide was not affected by cell adhesion to fibronectin (Fig. 5, A and C). Pretreatment with the caspase inhibitor Z-VAD-fmk abrogated apoptosis induced by TNF-{alpha} or etoposide (Fig. 5, A and B), thus confirming the involvement of caspases in this apoptotic response. These data show that fibronectin-induced survival signaling results in protection from specific apoptotic stimuli.



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  		FIG. 5.
Integrin-mediated cell adhesion to fibronectin attenuates TNF-{alpha}-induced apoptosis in prostate cancer cells. A, PC3 cells were detached and plated on Petri dishes coated with 10 mg/ml BSA or 5 µg/ml fibronectin (Fn) for 4 h at 37 °C in serum-free medium. Cells were treated with 500 µM etoposide or with 150 ng/ml TNF-{alpha} plus 3 µg/ml CHX either in the absence or in the presence of 40 µM Z-VAD-fmk for an additional 42 h at 37 °C or were not treated (control). Apoptosis was determined by DNA fragmentation analysis. B, the experimental conditions are the same as in A with the exception that hypodiploid (apoptotic) DNA content was evaluated by propidium iodide staining and flow cytometry. The percentage of hypodiploid cells with sub-G1 DNA content is indicated. One representative experiment of three is shown. C, summary of three independent experiments performed as described in A in which hypodiploid (apoptotic) DNA content was evaluated by propidium iodide staining and flow cytometry. Data represent means of percentage of apoptosis ± S.E. The percentage of cells in suspension that are undergoing apoptosis is significantly higher than that of cells plated on fibronectin (p = 0.007).

 
Survivin Mediates the Anti-Apoptotic Effect of Fibronectin and Increases Survival of non-Adherent Cells—To examine whether survivin is required for fibronectin protection against TNF-{alpha}-induced apoptosis, we used a second survivin dn mutant Thr34-> Ala (37, 42) that abolishes a phosphorylation site for p34cdc2 kinase. Infection of PC3 cells with pAd-T34A resulted in increased apoptosis as determined by DNA content analysis and flow cytometry compared with cells infected with pAd-GFP or pAd-survivin (Fig. 6A). Conversely, in cells held in suspension, expression of pAd-survivin decreased the percentage of cells with hypodiploid DNA content induced by TNF-{alpha} compared with cells infected with pAd-GFP or pAd-T34A (Fig. 6B).



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  		FIG. 6.
Survivin-mediated survival in TNF-{alpha}-induced apoptosis in adherent and suspended prostate cancer cells. PC3 cells were infected with pAd-GFP, pAd-survivin, or pAd-T34A survivin for 24 h at 37 °C in growth medium. Cells were detached and plated on Petri dishes coated with 10 mg/ml BSA or 5 µg/ml fibronectin for 8 h at 37 °C in serum-free medium. Cells were treated with 150 ng/ml TNF-{alpha} plus 3 µg/ml CHX for an additional 42 h at 37 °C in growth medium. Hypodiploid (apoptotic) DNA content was evaluated by propidium iodide staining and flow cytometry. The graphs show the results of two (A) and three (B) experiments. Apoptosis is represented as fold increase or decrease compared with pAd-GFP apoptosis (A, p = 0.04; B, p = 0.01). Bars, mean ± S.E.

 
The {beta}1A Integrin Cytoplasmic Domain Is Essential for Survivin-mediated Cell Survival—It is well established that the cytoplasmic domain of the {beta}1 subunit is required to modulate many cellular functions as well as to trigger integrin signaling pathways (43). Five different {beta}1 isoforms containing alternatively spliced cytoplasmic domains have been identified ({beta}1A, {beta}1B, {beta}1C, {beta}1C-2, and {beta}1D) and have been shown to differentially affect receptor localization, cell proliferation, cell adhesion and migration, interactions with intracellular proteins, and ultimately, phosphorylation and activation of signaling molecules (44). {beta}1C expression inhibits cell proliferation and is down-regulated in prostate cancer (43). In PC3 cells, the {beta}1C integrin variant is absent, whereas the {beta}1A isoform is abundantly expressed.2 To investigate the role of the {beta}1 integrin cytodomain in protection against TNF-{alpha}, we generated PC3 stable cell lines expressing chimeric {beta}1C or {beta}1A integrins consisting of the chicken {beta}1 extracellular and transmembrane domain and either human {beta}1C or human {beta}1A cytodomain sequences under the control of a tetracycline-regulated promoter. These chimeric constructs were used to distinguish between endogenous {beta}1 integrin and exogenous {beta}1C and {beta}1A integrin variants in human cells. Expression of the chimeric constructs was analyzed by FACS using W1B10 mAb to chicken {beta}1 integrin. Comparable levels of surface expression of {beta}1C and {beta}1A were consistently obtained in all of the experiments 24, 48, 72, and 96 h upon tetracycline removal (Fig. 7A and data not shown). To examine the role of different {beta}1 cytodomain splice variants in survivin-mediated protection from TNF-{alpha}-induced apoptosis, we infected PC3-{beta}1C and PC3-{beta}1A cell transfectants with pAd-GFP or pAd-survivin. Expression of pAd-survivin in PC3-{beta}1A cells held in suspension decreased the percentage of cells with hypodiploid DNA content induced by TNF-{alpha} compared with cells infected with pAd-GFP (Fig. 7, B and C). In contrast, expression of pAd-survivin in PC3-{beta}1C cells held in suspension was not able to rescue apoptosis triggered by TNF-{alpha} (Fig. 7, B and C). These data suggest that the {beta}1A cytodomain is necessary to protect cells from TNF-{alpha}-induced apoptosis in a survivin-dependent manner.



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  		FIG. 7.
Survivin rescues PC3-{beta}1A, but not PC3-{beta}1C, cells from TNF-{alpha}-induced apoptosis. A, PC3-{beta}1C (left panels) or PC3-{beta}1A stable cell lines (right panels) were cultured for 96 h in growth medium either in the absence or in the presence of 1 µg/ml tetracycline and analyzed by FACS using W1B10 mAb to chicken {beta}1 integrin or 12CA5 as a negative control followed by fluorescein isothiocyanate-conjugated antibody. Fluorescence intensity is expressed in arbitrary units. FACS analysis of a representative clone for each {beta}1 variant is shown. Thick line, W1B10; dotted line, 12CA5. B and C, PC3 cell transfectants cultured for 96 h in growth medium in the absence of tetracycline were infected with pAd-GFP or pAd-survivin during the last 24 h of the 72-h culture. Cells were held in suspension and treated with 150 ng/ml TNF-{alpha} plus 3 µg/ml CHX for an additional 20–24hat37 °C. Hypodiploid (apoptotic) DNA content was evaluated by propidium iodide staining and flow cytometry. B, one representative experiment is shown. TNF-{alpha}-induced apoptosis is represented as percentage of apoptosis of pAd-GFP-infected cells. C, data represent means of percentage of apoptosis ± S.E. of 3–4 independent experiments. TNF-{alpha}-induced apoptosis is represented as fold decrease compared with pAd-GFP-infected cells (p = 0.004).

 
Fibronectin Induces Survivin Expression via AKT—In epithelial cells, matrix attachment results in activation of AKT and mediates integrin survival signaling (10, 11, 45). This pathway acts as a general cell viability signal that may also involve increased survivin expression (46). To investigate the role of AKT in fibronectin-mediated increase in survivin expression, we transfected PC3 cells with a plasmid encoding for a dn AKT. As shown in Fig. 8, expression of dn AKT, efficiently achieved in these cells, but not of control vector, completely reversed the ability of fibronectin to up-regulate survivin expression in PC3 cells as evaluated by immunoblotting (Fig. 8, lane 4). Expression of wt AKT did not further increase survivin levels (not shown). The expression of dn AKT inhibited the phosphorylation of AKT substrates, as determined by immunoblotting using a phospho-(Ser/Thr) AKT-substrate antibody (47), thus confirming that the dn AKT mutant is functional (not shown). These results suggest that integrin-mediated induction of survivin expression through the AKT pathway is responsible for the protective effects against TNF{alpha}-induced apoptosis.



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  		FIG. 8.
A dominant negative form of AKT prevents fibronectin-mediated increased survivin levels. PC3 cells were transfected with vector alone or dn AKT for 24 h at 37 °C in growth medium. Cells were detached and plated on Petri dishes coated with 10 mg/ml BSA or 5 µg/ml fibronectin (Fn) for 48 h at 37 °C in serum-free medium. Survivin expression was determined by immunoblotting as described in Fig. 1. Expression of HA-tagged dn AKT was analyzed using 12CA5 mAb to HA before plating on BSA or fibronectin. Rabbit affinity-purified antibody to ERK1/2 was used as control for protein loading. Proteins were visualized by ECL.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that integrin ligation by fibronectin stimulates specific survival signals that counteract apoptosis in prostate carcinoma PC3 cells. Relevant elements of this pathway included the identification of the IAP protein survivin, but not Bcl-2 as a critical downstream effector of fibronectin-dependent cytoprotection, and the dependence of this mechanism on AKT. We also show that survivin-dependent cytoprotection of non-adherent cells by TNF-{alpha} requires the {beta}1A integrin subunit cytodomain.

Although it has long been proposed that integrins participate in cell survival and either detachment of adherent cells from their matrices or lack of appropriate engagement by their ligands is typically followed by a form of programmed cell death designated anoikis (1), discrete survival pathways that link integrin ligation to the cellular apoptotic machinery have not been completely elucidated. Here, integrin engagement in PC3 cells with fibronectin was associated with up-regulation of survivin expression and protection from apoptosis, whereas interference with survivin function using two independent dn mutations induced apoptosis and reversed the cytoprotective effect of fibronectin. Taken together with a previous report (48), which has shown that integrin engagement by fibronectin results in increased survivin expression in pre-B ALL cells, our results demonstrate that integrin signaling functions as a broad cell viability pathway in cancer cells using regulated expression of survivin as a critical downstream mediator. Consistent with its known overwhelming expression in tumors (18), we found that the highest levels of survivin were present in highly aggressive prostate cancer cells such as PC3 and LNCaP-LN3 (26, 41). Whereas the molecular basis for the overexpression of survivin in cancer has not been fully elucidated, current evidence suggest a global deregulation of survivin gene expression in transformed cells (18) that may involve the loss of p53 (49) and de-repression of survivin gene transcription. This mechanism may be consistent with the findings presented here in which fibronectin-induced survivin expression is observed exclusively in p53-mutated PC3 cells but not in non-tumorigenic wild type p53 LNCaP cells (50) and also with the reported ability of p53 to counteract integrin-dependent survival signals (51). Furthermore, previous reports have shown that cell adhesion to fibronectin through {alpha}5{beta}1 and {alpha}v{beta}1 and to vitronectin through {alpha}v{beta}3 integrins resulted in up-regulation of Bcl-2 in Chinese hamster ovary cells (2, 14) and that Bcl-2 prevents anoikis in prostate cancer cells (52), whereas the levels of Bcl-2 were unchanged in response to fibronectin adhesion in myeloma cells (53). We have found that PC3 cell adhesion to vitronectin up-regulates the levels of Bcl-2 (data not shown); however, we could not detect a significant change in the levels of Bcl-2 in response to fibronectin adhesion. The apparent discrepancy between our results and the aforementioned studies could be ascribed to the different cell types used.

To causally link the increased survivin expression by fibronectin adhesion to protection from apoptosis, we used a dominant negative survivin mutant, i.e. Thr-34-> Ala, that has shown promising anti-tumor efficacy for its ability to induce apoptosis and to enhance cell death-inducing stimuli in vitro and in a number of cancer models in vivo (37, 42). Consistent with these observations, overexpression of the Thr-34-> Ala mutant reverted the cytoprotective effect of fibronectin against TNF-{alpha}-induced apoptosis. Although the molecular mechanisms of apoptosis induced by survivin mutants have not been completely elucidated, a critical lowering in endogenous survivin levels appears likely attributed to the ability of these mutants to dimerize with wild type survivin and to exhibit accelerated clearance. This is reminiscent of the apoptotic response observed after the loss of endogenous survivin levels by antisense oligonucleotide treatment and enhancement of chemotherapy-induced cell death (40, 54). It was recently reported that expression of survivin Thr-34-> Ala resulted in inhibition of cell proliferation, induction of apoptosis, and catastrophic mitotic defects in 100% of expressing cells, thus potentially in agreement with the data presented here (55). Surprisingly, however, the same authors argued that survivin Thr-34-> Ala mutant functioned to protect against various apoptotic stimuli, which contradicts their own data, and potentially reflects the highly artificial overexpression system used in these experiments.

Here, fibronectin-dependent adhesion effectively counteracted death receptor-induced (i.e. TNF-{alpha}-induced) apoptosis. The phenotype of PC3 cells transduced to express wild type survivin mirrored this cytoprotection profile. Although the precise mechanism(s) of survivin inhibition of apoptosis have not been conclusively elucidated, recent data suggest a preferential involvement in the upstream initiation of the intrinsic (i.e. mitochondrial) apoptotic pathway. Clear evidence for a role of survivin in counteracting the extrinsic, death receptor-mediated apoptotic response has also been provided, and survivin effectively blocked TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis in renal carcinoma cells and suppressed TNF-{alpha}-induced apoptosis in endothelial cells (20, 56). Although this finding may signal a potential interference of survivin in the extrinsic apoptotic cascade, it has been recently reported that prostate cancer cells efficiently couple death receptor signaling to the mitochondrial initiation of cell death via BID cleavage and cytochrome c release from mitochondria (Fig. 9) (57).



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  		FIG. 9.
Model for fibronectin-mediated protection from TNF-{alpha}-induced apoptosis. TNF-{alpha} binding to the TNF receptor (TNFR) results in DISC formation, activation of procaspase-8, BID cleavage, and release of mitochondrial cytochrome c in the cytoplasm and results in pro-caspase-9 activation. Adhesion to fibronectin increases survivin expression levels in an AKT-dependent manner. Increased survivin levels counteract the processing/function of caspase-9 and decrease apoptosis.

 
An interesting result from the current study is the finding that expression of the {beta}1A but not of the {beta}1C cytoplasmic integrin variant conferred protection against apoptosis to nonadherent cells transduced with survivin. These observations indicate that the {beta}1A cytoplasmic domain is required for the anti-apoptotic effect exerted by survivin in non-adherent cells. Although we have not examined the expression of {beta}1B and {beta}1D in PC3 cells, it is very unlikely that these variants contribute to the protective effect exerted by survivin because their expression has been shown to be restricted to skin and liver and to skeletal and cardiac muscle, respectively (43).

Our data indicate that expression of dominant negative AKT blocked integrin-mediated regulation of survivin. In agreement with previous studies that have shown that integrin ligation conveys survival signals and protect cells from drug-induced apoptosis by activating AKT (6), our results demonstrate that this pathway also inhibits apoptotic signals elicited by death receptor ligation and may therefore contribute to the development of apoptosis resistance in prostate cancer cells. Altogether, our findings suggest that survivin may be required to maintain a critical anti-apoptotic threshold in prostate cancer cells and that the ECM is an important factor that might contribute to tumor resistance mechanisms by regulating survivin levels. Understanding the molecular mechanisms underlying integrin-mediated survival signals in cancer cells may provide new therapeutic strategies for cancer treatment.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants R29 CA71870 and RO1 CA89720 (to L. R. L.) and RO1 CA78810, CA90917, and HL54131 (to D. C. A.) and by Department of Defense, Prostate Cancer Research Program, Grant DAMD17-98-1-8506 (to L. R. L.). 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

To whom correspondence should be addressed: Dept. of Cancer Biology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605. Tel.: 508-856-1606; Fax: 508-856-3845; E-mail: lucia.languino{at}umassmed.edu.

1 The abbreviations used are: ECM, extracellular matrix; IAP, inhibitor of apoptosis; ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; TNF-{alpha}, tumor necrosis factor-{alpha}; PARP, poly(ADP ribose) polymerase; CHX, cycloheximide; Act D, actinomycin D; HA, hemagglutinin; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; BSA, bovine serum albumin; nt, nucleotide; EGF, epidermal growth factor; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; dn, dominant negative. Back

2 M. Fornaro and L. R. Languino, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Hynes and Franke for cDNAs and Dr. N. Wall.



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