A basic peptide derived from the HARP C-terminus inhibits anchorage-independent growth of DU145 prostate cancer cells

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

A basic peptide derived from the HARP C-terminus inhibits anchorage-independent growth of DU145 prostate cancer cells☆ Oya Bermek a , Zoi Diamantopoulou b , Apostolis Polykratis b , Celia Dos Santos a , Yamina Hamma-Kourbali a , Fabienne Burlina c , Jean Delbé a , Gerard Chassaing c , David G. Fernig d , Pagnagiotis Katsoris b , José Courty a,⁎ a

Laboratoire de recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires (CRRET), CNRS UMR 7149, Université Paris 12, 61 Avenue du Général de Gaulle, 94010 Créteil Cedex, France b Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, GR 26504, Greece c Laboratoire de Synthèse, Structure et Fonction des Molécules Bioactives, CNRS UMR 7613, Université Pierre et Maris Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France d School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Heparin affin regulatory peptide (HARP) is an 18 kDa heparin-binding protein that plays a key

Received 2 May 2007

role in tumor growth. We showed previously that the synthetic peptide P(111–136) composed

Revised version received

of the last 26 HARP amino acids inhibited HARP-induced mitogenesis. Here, to identify the

31 July 2007

exact molecular domain involved in HARP inhibition, we investigated the effect of the shorter

Accepted 31 July 2007

basic peptide P(122–131) on DU145 cells, which express HARP and its receptor protein

Available online 7 August 2007

tyrosine phosphatase β/ζ (RPTPβ/ζ). P(122–131) was not cytotoxic; it dose-dependently inhibited anchorage-independent growth of DU145 cells. Binding studies using biotinylated P

Keywords:

(122–131) indicated that this peptide interfered with HARP binding to DU145 cells.

Heparin affin regulatory peptide

Investigation of the mechanisms involved suggested interference, under anchorage-

Prostate cancer

independent conditions, of P(122–131) with a HARP autocrine loop in an RPTPβ/ζ-

Anchorage-independent growth

dependent fashion. Thus, P(122–131) may hold potential for the treatment of disorders

Basic peptide

involving RPTPβ/ζ.

Receptor protein tyrosine

© 2007 Elsevier Inc. All rights reserved.

phosphatase

Introduction Prostate cancer is the most commonly diagnosed malignancy in men in many industrialized countries [1]. Initially, the growth of prostate cancer cells is mainly dependent on

androgen, and androgen deprivation is widely used as a treatment [2]. However, androgen-independent growth occurs eventually [3], leading to metastasis formation in 85% to 100% of patients with advanced prostate cancer. No effective treatments are available to control the metastatic tumors, which

☆

HARP C-terminus inhibits anchorage-independent growth of DU145. ⁎ Corresponding author. Fax: +33 145 171 816. E-mail address: [email protected] (J. Courty).

0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.07.032

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are responsible for patient death. The molecular mechanisms responsible for the transition to androgen-independence are unclear, and novel therapeutic strategies are needed for patients at this stage of prostate cancer. In adults, the functional integrity of the normal prostate depends on mesenchymal–epithelial interactions, which contribute to homeostasis and repair of the glandular prostate epithelium. Disturbances in prostate epithelium homeostasis lead to the development of diseases such as cancer. Although the mechanisms that control mesenchymal-epithelial interactions are poorly understood, numerous studies suggest a crucial role for growth factors. Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a 136-amino acid secreted polypeptide. HARP is one of the heparin-binding growth differentiation factors, which induce transformation of several cell lines [4,5]. In addition, HARP is mitogenic for endothelial cells [6] and angiogenic both in vitro and in vivo [5,7,8]. The biological activities of HARP are mediated by three distinct receptors: syndecan 3 (N-syndecan), receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ), and anaplastic lymphoma kinase (ALK) receptor [9]. The mitogenic, angiogenic, and phenotypetransforming effects of HARP are mediated by the ALK receptor [10]. N-syndecan and RPTPβ/ζ are cell-surface proteoglycans involved in the neurite outgrowth-promoting activity of HARP [11,12]. RPTPβ/ζ has also been reported to mediate HARP-induced migration of tumor cells. HARP is expressed in several human tumors and tumor cell lines. Studies using an antisense and a dominant negative strategy have established that HARP is the rate-limiting factor for phenotype transformation, angiogenesis, and metastasis [7,13,14]. More specifically, the functional significance of HARP in the progression of prostate cancer has been convincingly demonstrated, and HARP expression has been documented in various prostate-derived cell lines including DU145, PC3, and LNCaP [15], for which HARP acts as an autocrine growth factor [16,17]. We showed that the C-terminus of HARP (amino acids 111 through 136, P(111–136)) plays a critical role in the interaction of HARP with its high-affinity tyrosine kinase receptor ALK [13]. Another recent study using human glioblastoma cells suggested that processing of the HARP C-terminus might produce two isoforms: the full-length 18 kDa polypeptide and a truncated 15 kDa form lacking the last 12 amino acids (124 through 136). In a study of various glioblastoma cell lines [18], the truncated form promoted cell proliferation in an ALKdependent fashion, whereas the full-length form promoted cell migration via the RPTPβ/ζ-dependent pathway. Here, we characterize a 10-amino acid peptide P(122–131) corresponding to the basic cluster of residues at the C-terminus of HARP. P (122–131) inhibited RPTPβ/ζ-dependent growth of DU145 cells in soft agar.

purchased from Senn Chemicals (Cachan, France). Peptide synthesis grade solvents and other reagents were obtained from Applied Biosystems (Courtaboeuf, France). The control peptide (5K) was purchased from Sigma (Saint-Quentin Fallavier, France). Doxorubicin hydrochloride was from Teva Classics (Paris, France). Recombinant human HARP, N, and C TSR molecules were produced and purified from bacteria as previously described [19,20]. We purchased [methyl-3H] thymidine from ICN (Orsay, France), ImmunoPure® TMB substrate kits from Pierce (Rockford, USA), and Cell Counting Kit-8 from Interchim (Montluçon, France). Antibodies to human HARP were purchased from R&D systems (Oxon, UK) and antibodies to human RPTPβ/ζ from Tebu Bio (Le Perray, France). FITCconjugated monoclonal mouse antibody to biotin and horseradish peroxidase-conjugated antibodies were obtained from Jackson ImmunoResearch (Suffolk, UK). Heparitinase I, II, and III were obtained from IBEX (San Leandro, USA) and Chondroitinase ABC from Sigma (Saint-Quentin Fallavier, France). Cell culture media were from Invitrogen (Leek, The Netherlands). Rabbit polyclonal antibodies against the recombinant extracellular domain of human ALK were a gift from Prof. M. Vigny (INSERM U706, Paris, France).

Cell culture The human prostate-cancer epithelial cell line DU145 [American Type Culture Collection (ATCC) # HTB-81] was grown in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum (FCS) and 50 μg/ml gentamicin. Cultures were maintained at 37 °C with 7% CO2 and 90% humidity. Chinese hamster ovary (CHO-K1 line), MDA-MB231, and NIH-3T3 cells were cultured as previously described [21].

Peptide synthesis and characterization The peptides were produced using stepwise solid-phase synthesis with an ABI 433A synthesizer (Applied Biosystems, Courtaboeuf, France) and standard protocols for Boc chemistry (amino acid activation with dicyclohexylcarbodiimide/1hydroxybenzotriazole or HBTU). After synthesis of the KKKKKEGKKQ peptide, the peptidyl-resin was elongated by four glycin residues and biotin to produce Biot-G4-P(122–131). Peptides were cleaved from the resin by treatment with anhydrous hydrofluoric acid (1 h, 0 °C) in the presence of anisole (1.5 ml/g peptidyl-resin) and dimethylsulfide (0.25 ml/g peptidyl-resin). Peptides with purity N95% were obtained by high-performance liquid chromatography on an RP-C8 column using a linear gradient of acetonitrile in water with 0.1% trifluoroacetic acid. Peptides were characterized using MALDITOF MS (Voyager Elite, PerSeptive Biosystems) with matrix ácyano-4-hydroxycinnamic acid. The m/z values of the protonated peptides (monoisotopic peak) were 1269 for H-P(122–131) and 1683 for Biot-P(122–131).

Experimental procedures Proliferation assay Materials Standard Boc amino acids, p-methylbenzhydrylamine-polystyrene resin (0.81 mmol NH2/g), and O-(benzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophophate (HBTU) were

DU145 cells were seeded into a 12-well culture plate at 1 104 cells/well in culture medium with or without P(122–131) or antibodies to HARP, RPTPβ/ζ, or ALK. After 48 h incubation, the cells were trypsinized and counted.

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Thymidine incorporation assay Incorporation of [methyl-3H] thymidine by serum-starved NIH-3T3 cells was evaluated as previously described [21]. Briefly, NIH-3T3 cells were seeded in 48-well culture plates at 2·104 cells/well and incubated for 24 h in DMEM with 10% (v/v) FCS. The cells were then serum-starved for 24 h, and samples were added. The cells were incubated for 18 h in 5% CO2 at 37 °C then for 6 h with 0.5 μCi [methyl-3H] thymidine. Macromolecules were precipitated with 10% (w/v) trichloroacetic acid, washed with water, and lysed with 0.2 N NaOH. Radioactivity precipitated with trichloracetic acid was counted using a micro-beta scintillation counter (LKB, PerkinElmer Life Sciences, Courtaboeuf, France).

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body diluted 1:1000 in PBS containing 0.5% (w/v) BSA (w/v), for 1 h at room temperature; then exposed to peroxidase-conjugated anti-goat antibody diluted 1:100,000, for 1 h at room temperature. Peroxidase activity was then as described above.

Heparitinase and chondroitinase treatment of cells DU145 cells were plated in triplicate into 96-well plates at 2 104 cells/well, incubated overnight and starved for 24 h. Cells were

Cytotoxicity assay Cell viability was measured using the CCK-8 Kit (Interchim, Montluçon, France) according to the manufacturer's instructions. Briefly, CHO-K1 cells were seeded in 96-well plates at 2·103 cells/well and incubated for 24 h in HAM's F12 medium with 10% (v/v) FCS. Cells were then incubated with or without 1, 10, or 50 μM of P(122–131) for 72 h. Doxorubicin hydrochloride (0.5 μM) served as the positive control. The number of viable cells was assessed by using the cell counting reagent, and absorbance at 450 nm was measured.

Soft agar assay DU145 and MDA-MB231 cells were seeded into 12-well plates containing 0.6% (w/v) agar at 2·103 cells/well in 0.35% (w/v) agar and growth medium. After 2 weeks, colonies larger than 50 μm in diameter were counted using a phase-contrast microscope equipped with a measuring grid. The cells were treated with the peptide three times within 1 week and with the antibodies twice during the 2 weeks.

Cell ELISA binding assay DU145 cells were seeded in triplicate on 96-well plates at 2·104 cells/well, incubated overnight, and subsequently starved for 24 h. Before the binding experiment, the wells were incubated with 300 μl/well RPMI containing 3% bovine serum albumin (BSA, w/v), for 1 h at room temperature. Binding of biotinylated P(122–131) was achieved in RPMI containing 1% (w/v) BSA for 2 h at room temperature. Unbound biotinylated peptide was removed by washing the cells three times with phosphatebuffer saline (PBS) containing 1% (w/v) BSA (washing buffer), and the cells were fixed with PBS supplemented with 4% (w/v) paraformaldehyde (PFA) for 10 min at room temperature (100 μl/well). The cells were washed three times with washing buffer, and non-specific binding sites were blocked for 1 h at room temperature with PBS containing 3% (w/v) BSA (300 μl/ well). The bound peptide was characterized using a peroxidase-labeled antibody to anti-biotin antibody diluted 1:2000 in PBS containing 0.5% BSA (v/v), for 1 h at room temperature. Peroxidase activity was detected using 3,3,5,5-tetramethylbenzidine dihydrochloride substrate according to the supplier's instructions. Absorbance was measured at 450 nm. For HARP binding, cells were incubated with goat anti-HARP anti-

Fig. 1 – P(122–131) inhibits HARP-induced mitogenesis. Stimulation of [3H]thymidine incorporation in serum-starved NIH-3T3 cells treated with various concentrations of P(122–131) (A) or the control peptide P5K (B) with or without 4 nM HARP. The cytotoxicity of P(122–131) was investigated using CCK-8 kit (C). Doxorubicin hydrochloride (doxo) served as the positive control. Results are the means of three separate experiments, each carried out in triplicate, and the standard errors are indicated.

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then incubated with either heparitinase I, II, and III (7.5 mU/ml) or chondroitinase ABC (0.4 U/ml) for 2 h or 24 h at 37 °C in RPMI1640 medium. Binding experiments were performed as described according to the cell ELISA binding assay. The efficacy of enzymes treatment was confirmed using the bioassay developed by Barbosa et al. [22].

Confocal microscopy DU145 cells (5·104 cells/well) were plated in 4-well glass slides (Lab-Tek Brand, Nalge Nunc International, Naperville, IL) in complete medium and then serum-starved for 24 h in RPMI medium. The cells were incubated with 100 μM biot-P(122–131) peptide for 2 h at room temperature with or without 1 mM P (122–131). After extensive washing with PBS, the cells were fixed with 4% PFA (w/v). Non-specific sites were saturated in PBS containing 1% BSA (w/v), and bound biotinylated peptide was revealed using anti-biotin antibody linked to FITC and diluted 1:100 in PBS containing 1% (w/v) BSA. Nuclei were stained with DAPI according to standard procedures. Slides were examined using a Zeiss LSM 510 META confocal laser microscope (Zeiss, Iena, Germany) with a Plan Apochromat 63× N.A.1.4 objective.

Immunoprecipitation and Western blot analysis The medium of DU145 cells grown in 60-mm plastic Petri dishes was removed. The cells were washed twice with icecold PBS and lysed with 1 ml of cell lysis buffer (50 mM HEPES, pH 7, 150 mM NaCl, 10 mM EDTA, 1% TritonX-100, 1% Nonidet P-40 (both v/v), 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 5 μg/ml aprotinin, and 5 μg/ml leupeptin). Cells were scraped from the plates, transferred to

microfuge tubes, sonicated for 4 min while kept on ice, and then centrifuged at 20,000×g for 10 min at 4 °C. Approximately 400 μg of the supernatant was precleared by incubation with 30 μl of protein A-Sepharose or streptavidin–agarose beads for 60 min at room temperature, followed by centrifugation at 10,000×g for 5 min. The beads were collected by centrifugation and the supernatants were transferred to new microfuge tubes. After this precleaning step, supernatants were incubated overnight at 4 °C with RPTPβ/ζ primary antibodies diluted 1:200 or with biotinylated P(122–131). A suspension of protein A-Sepharose or streptavidin–agarose beads in a volume of 80 μl was added. After 3 h incubation at 4 °C, beads and bound proteins were collected by centrifugation (10,000×g, 4 °C) and washed by centrifugation three times with ice-cold cell lysis buffer. The pellet was resuspended in 60 μl of 2× SDS loading buffer (100 mM Tris–HCl, pH 6.8, 4% w/v) sodium dodecyl sulfate (SDS), 0.2% (w/v) bromophenol blue, 20% glycerol, 0.1 M dithiothreitol), and kept at 4 °C until use. Before electrophoresis, samples were heated at 95 °C for 5 min then centrifuged; 50 μl of the supernatant was subjected to SDS-polyacrylamide gel electrophoresis, after which the separated polypeptides were electrotransferred to an Immobilon P membrane (Millipore, St. Quentin en Yvelines, France). After 3 h incubation in 48 mM Tris, pH 8.3, 39 mM glycin, 0.037% (w/v) SDS, and 20% (v/v) methanol, the membrane was blocked for 1 h at 37 °C in 5% (w/v) non-fat milk, 0.1 M Tris-buffered saline (TBS), Tween 20. The membrane was then probed overnight at 4 °C under continuous agitation using anti-RPTPβ/ζ antibodies diluted 1:500. The blot was incubated with the secondary antibody coupled to horseradish peroxidase, and bands were detected using the ChemiLucent Detection System Kit (Chemicon International Inc., Temecula, CA), according to the manufacturer's instructions.

Fig. 2 – P(122–131) inhibits proliferation of DU145 and MDA-MB231 cells. (A) Inhibition of DU145-cell colony formation on soft agar by anti-HARP polyclonal antibodies toHARP and (B) by P(122–131). Similar experiments were performed with MBA-MB231 cells. Inhibition of colony formation was tested with or without polyclonal antibodies to HARP (C) or P(122–131) (D) Results are the means of three separate experiments, each carried out in triplicate, and the standard errors are indicated.

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(Perbio, Chester, UK), and captured on streptavidin-derivatized planar aminosilane cuvettes (NeoSensors), as described [24– 26]. Neither HARP [24] nor peptides bound non-specifically to streptavidin-derivatized surfaces (data not shown).

Statistical analysis

Fig. 3 – Expression by DU145 cells of mRNA encoding receptors RPTPβ/ζ and ALK. Expression of mRNA encoding RPTPβ/ζ, ALK, and TFIID in DU145 cells cultured on agar (lane 2) or plastic at medium (lane 3) or high confluence (lane 4). Control RT-PCRs without RNA (lane 1) or with RNA from the positive U87MG cell line (lane 5) are also shown. PCR fragments for RPTPβ/ζ, ALK, and TFIID were 344 bp, 236 bp, and 194 bp, respectively.

The significance of differences between results from the various groups was evaluated using unpaired t-tests. Each experiment included at least triplicate measurements for each test condition. All results are expressed as mean ± S.E.M. from at least three independent experiments.

Results We previously reported that the biological effects of HARP were inhibited by the truncated mutant HARPΔ111–136 and

Reverse transcriptase-polymerase chain reaction (RT-PCR) for ALK and RPTPβ/ζ Total RNA was extracted from cells using the RNA Instapure Kit (Eurogentec, Seraing, Belgium) according to the manufacturer's instructions. To obtain DU145 cells growing under anchorage-independent conditions, cells were plated and cultured for 3 weeks over a layer of 0.5% agar in complete medium, as described by Dong and Cmarik [23]. The growth medium and unattached cells were then collected, and the recovered cells were pelleted by centrifugation and subjected to RNA extraction. cDNAs were synthesized from 1 μg of total RNA using random hexamer primers and Superscript II™ reverse transcriptase (Invitrogen, Cergy Pontoise, France). Then, 1/5 or 1/2 (v/v) of the reaction products were subjected to PCR amplification using the GenAmp9600 system (PE Applied Biosystems, Les Ulis, France) for detection of RPTPβ/ζ and ALK, respectively. Primers were as follows: RPTPβ/ζ detection, 5′-CTAAAGCGTTTCCTCGCTTG-3′ (forward) and 5′-TCTGAAACTCCTCCGCTGAC (reverse); ALK detection, 5′-CAACGAGGCTGCAAGAGAGAT-3′ (forward) and 5′-GTCCCATTCCAACAAGTGAAGGA-3′ (reverse); and TFIID detection, 5′-AGTGAAGAACAGTCCAGACTG-3′ (forward) and 5′-CCAGGAAATAACTCGGCTCAT-3′ (reverse). After 5 min at 94 °C, the following number of cycles was used: 30 cycles for TFIID, 35 cycles for RPTPβ/ζ and ALK. Each cycle included denaturation at 94 °C for 1 min, annealing at 60 °C (for TFIID) or 57 °C (for RPTPβ/ζ or ALK) for 1 min, and primer extension at 72 °C for 1 min. RT-PCR products were subjected to electrophoresis on 2% (w/v) agarose gels containing 0.5 mg/ml ethidium bromide. Gels were photographed using a ChimiGenius system (Syngene, Cambridge, UK).

Optical biosensor binding assays Binding assays were performed in an IAsys optical biosensor (NeoSensors, Sedgefield, UK) at 20 °C in which the response is measured in arc s (1 arc s = 1/3600°; 600 arc s = 1 ng protein/mm2 sensor surface). Pig mucosal heparin (15–20 kDa, Sigma, Poole, UK) was biotinylated on free amino groups with NHS biotin

Fig. 4 – Involvement of RPTPβ/ζ in the growth of DU145 cells. Representative phase-contrast microphotography of colony formation of DU145 cells treated with anti-RPTPβ/ζ (A), anti-ALK (B), control IgG (C); in (D), no treatment was used. Magnification, ×50; scale bar, 250 μm. Colonies larger than 50 μm in diameter were counted and presented in (E). Results are the means of three separate experiments, each carried out in triplicate, and the standard errors are indicated.

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corresponding synthetic peptide P(111–136) (KLTKPKPQAESKKKKKEGKKQEKMLD) [13]. Here, we sought to identify the minimum sequence responsible for the inhibition of HARP activity. Since an obvious feature of P(111–136) is the stretch of basic residues, which may be involved in ionically driven molecular interactions, we investigated whether the basic sequence P(122–131) (KKKKKEGKKQ) mediated the proliferation-inhibiting effect of P(111–136).

Effect of P(122–131) on inhibition of HARP-induced mitogenesis The mitogenic activity of HARP in the presence of P(122–131) was investigated using serum-starved NIH-3T3 cells with or without HARP stimulation, as described in Experimental procedures. As shown in Fig. 1A, stimulation with 4 nM HARP induced a 5.3-fold increase in [3H]-thymidine incorporation and 1, and 10 μM of peptide P(122–131) inhibited this effect by 30% (Fig. 1A). Since P(122–131) contains a cluster of five lysine residues, we performed a control experiment with a five-lysine peptide (P5K) to evaluate the specificity of the inhibitory effect of P(122–131). P5K at concentrations of up to 1 μM failed to inhibit HARP-induced mitogenesis (Fig. 1B) whereas 10 μM P5K increased slightly HARP-induced mitogenesis. On NIH-3T3 cells, no significant effect was observed when P(122–131) or P5K was tested alone (Figs. 1A and B). To confirm the inhibitory effect of P(122–131) on thymidine incorporation, we performed a cytotoxic assay using CHO-K1 cells. In contrast to doxorubicin used as the positive control, increasing concentrations of P(122– 131) ranging from 1 to 50 μM did not inhibit CHO-K1 cell growth (Fig. 1C). Results were similar with NIT 3T3 cells (data not shown). Taken together, these results demonstrated that P(122–131) was not cytotoxic and inhibited HARP-induced cell proliferation.

P(122–136) inhibits DU145 cell proliferation The effect of P(122–131) on HARP-induced tumorigenesis was evaluated by measuring DU145 growth on agar. This prostate cancer cell line produces HARP [15], which in turn stimulates the growth of the cells [17]. We studied DU145 growth under anchoring-independent conditions in the presence of an antiHARP polyclonal antibody. The number of cell colonies decreased by 30% in the presence of 5 μg/ml of HARP antibodies (Fig. 2A). Thus, endogenous HARP may act as an autocrine growth factor for DU145 cells. We then investigated inhibition of DUI45 growth by P(122–131) by seeding cells on agar and adding various amounts of P(122–131) to the culture medium. The number of cell colonies decreased by 50% in the presence of 10 μM P(122–131) (Fig. 2B). To confirm these results, we looked for inhibitory effects of P(122–131) on the growth of MDA-MB231 tumor cells, previously shown to be dependent on a HARP autocrine loop [21]. Anti-HARP antibodies caused a dose-dependent decrease in the number of colonies to a maximum of −55% with 0.8 μM of anti-HARP (Fig. 2C). When increasing concentrations of P(122–131) were added to the culture medium, significant inhibition was also observed, confirming that P(122–131) inhibited the tumorigenic effect of HARP (Fig. 2D). We used RT-PCR to investigate the expression of RPTPβ/ζ and ALK receptors, two of the receptors involved in mediating the biological activities of HARP. The human glioblastoma cell line U87MG served as the positive control for the expression of the two receptors, as previously described [18]. mRNA corresponding to RPTPβ/ζ was the only receptor expressed in DU145 cells (Fig. 3). Therefore, we investigated whether RPTPβ/ζ was involved in the HARP-dependent autocrine stimulation of DU145 seeded on soft agar by using a polyclonal antibody against the extracellular domain of RPTPβ/

Fig. 5 – P(122–131) binding to DU145 cells. Binding of HARP and biot-P(122–131) was investigated as described in Experimental procedures. (A) Inhibition of HARP binding to DU145 cells by increasing concentrations of P(122–131). (B) Kinetics of biot-P(122–131) binding to DU145 cells. Saturation binding curve (C). Binding of biot-P(122–131) to DU145 cells with (black triangle) or without (black square) excess P(122–131) (1 mM). The specific resulting binding is shown (inverted black triangle).

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dose-dependent manner up to 30% at 5 μM (Fig. 5A). We then evaluated P(122–131) binding to DU145 using biotinylated-P (122–131) (biot-P(122–131), which displays similar biological effects to the non-biotinylated peptide (not shown). When DU145 cells were incubated for 30 to 180 min with 20 μM biot-P (122–131) and binding was evaluated as described in Experimental procedures, binding reached a maximum after 120 min (Fig. 5B). The specific binding of biot-P(122–131) to DU145 was specific and saturable (Fig. 5C), with an estimated Kd value of 25 μM. This value is in the same range of concentration in which P(122–131) acts on cell proliferation. To strengthen these data, competition experiments were performed using biot-P (122–131) as ligand and either HARP or P(122–131) as competitor. As shown in Fig. 5D, HARP and P(122–131) inhibited the

Fig. 6 – Confocal immunohistological analysis of biot-P(122–131) binding to DU145. DU145 cells were incubated with biot-P(122–131) with (B) or without (A) a 10-fold P(122–131) excess. Bound peptide was revealed as described in Experimental procedures. Scale bar, 80 μm. (C) Whole DU145 cell lysates were incubated (1) with anti-RPTPβ/ζ immobilized on protein A sepharose beads, (2) with biot-P(122–131) immobilized on streptavidin agarose beads, or (3) with streptavidin immobilized on agarose beads. The precipitates were analyzed by 5% SDS-PAGE and electroblotted; and the membrane was probed against RPTPβ/ζ using specific antibodies.

ζ idiotypic immunoglobulins and antibody to ALK served as controls. Anti-RPTPβ/ζ decreased the anchorage-independent growth of DUI45, compared to untreated cells (Fig. 4). No growth inhibition occurred with control immunoglobulin oranti ALK.

P(122–131) binds to a DU145 cell-surface component Since P(122–131) inhibited the biological activity of HARP, we investigated whether it prevented HARP from binding to DU145. P(122–131) inhibited HARP binding to DU145 in a

Fig. 7 – Chondroitinase treatment of DU145 cells and binding of P(122–131) to heparin immobilized on a streptavidin-derived IAsys sensor surface. (A) Binding of biot-P(122–131) (20 μM) was performed on DU145 cells treated with heparitinase I, II, or III (hatched bar) or with chondroitinase (grey bar). The binding of biot-P(122–131) performed without enzymatic treatment and control performed without biotinylated peptide were shown by white and black bar, respectively. (B) The binding of HARP (30 nM), C-TSR (3 μM), N-TSR (3 μM), and P(122–131) (80 μM) to immobilized heparin was measured in an IAsys optical biosensor, as described under Experimental procedures. Results are the mean of three experiments carried out in triplicate.

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binding of biot-P(122–131) to DU145 in a dose-dependent manner with EC50 of 0.87 ± 0.08 μM and 20 ± 0.4 μM, respectively. These data indicated that the binding affinity of HARP is 22fold more potent than P(122–131). As previously suggested by Maeda et al. [27], this result suggests that other domain(s) of HARP are involved in the binding of HARP to strengthen the interaction. Confocal microscopy showed that biot-P(122–131) bound to a cell-surface component of cultured DU145 cells (Fig. 6A). With a 10-fold excess of P(122–131), fluorescence associated with cell surfaces was effectively competed, confirming that binding was specific (Fig. 6B).

Characterization of the P(122–131) binding site Since RPTPβ/ζ was expressed by DU145, these results suggested an interaction of P(122–131) with RPTPβ/ζ?. These observations prompted us to conduct immunoprecipitation experiments using whole DU145 cell lysates with either biotP(122–131) or anti-RPTPβ/ζ. RPTPβ/ζ was detected in biotP(122–131) immunoprecipitates (lane 2), indicating that this HARP receptor also interacted with P(122–131) (Fig. 6C). No band was detected when biot-P(122–131) was omitted from the assay (lane 3). Since the RPTPβ/ζ-derived chondroitin sulfate was involved in the binding to HARP, we investigated the effect of chondroitinase ABC digestion to the P(122–131) binding to the cell surface. As shown in Fig. 7A, treatment of cells with chondroitinase or heparitinase I, II or III as control had no effect on the binding of P(122–131) to DU145. These results suggest that P(122–131) did not bind to the RPTPβ/ζderived glycoaminoglycans, in spite of its basicity. To directly confirm the hypothesis that P(122–131) does not bind to glycoaminoglycans, the interactions analyzed in a optical biosensor. P(122–131) failed to bind to a heparin surface even at concentration as high as 80 μM. Similar results were obtained as control using the N-TSR domain of HARP. In contrast, HARP and its heparin domain C-TSR bound to the heparin surface (Fig. 7B).

Discussion A role for HARP in prostate cancer progression was first suggested by Vacherot et al. [17], who showed that HARP was associated with epithelial cells in human prostate cancer but not in normal prostate or benign prostate hyperplasia. Thus, HARP may contribute to unregulated prostate cancer growth. Using an antisense strategy directed against HARP mRNA, Hatziapostolou et al. demonstrated that HARP was essential for the migration and anchorage-dependant and -independent growth of the human prostate tumor cell line LnCap [16]. The same group established HARP as a key mediator of FGF-2induced stimulation of LnCap growth and migration in vitro [28]. The importance of HARP in prostate tumor growth prompted us to evaluate the effect on DU145 cell growth of the synthetic basic peptide P(122–131) derived from the HARP C-terminus. HARP was found to act as an autocrine growth factor on DU145 cell growth [17]. Our data demonstrate clearly that P(122–131) specifically and dose-dependently inhibits anchorage-independent

growth of DU145 cells. This effect is likely to involve binding of P(122–131) to cell-surface RPTPβ/ζ, which is the only HARP receptor expressed in DU145 cells?. Early studies showed that RPTPβ/ζ was linked to neural cell migration. Subsequently, RPTPβ/ζ was found to be overexpressed in tumor cells such as astrocytomas and to correlate with malignancy [29]. RPTPβ/ζ overexpression was documented in melanoma [30] and in carcinomas of the lung, colon, breast, and prostate, compared with normal tissue [9], in which expression was very low. These observations suggested that RPTPβ/ζ might be an excellent tumor marker and therapeutic target. The growth factor HARP exerts potent biological effects. It increases cell proliferation, migration, and differentiation; tumor growth; and angiogenesis. These effects are mediated by interactions with two receptors, ALK and RPTPβ/ζ? HARP, expressed in several tumors and can be a rate-limiting factor for tumor growth and metastasis in vivo. Thus, HARP holds promise as a treatment target in patients with cancer. We recently reported that the HARP C-terminus (amino acids 111 through 136) and a corresponding synthetic peptide P(111–136) inhibited the biological effects of HARP [13]. In this previous study, P(111–136) was found to bind to the extracellular domain of ALK and to inhibit HARP-induced cell proliferation. In addition, P(111–136) was active in vivo and inhibited tumor growth when injected daily around the tumor (manuscript in preparation). To better characterize this peptide and to identify the likely minimum amino acid sequence involved in its biological activity, we evaluated the effects of the peptide P (122–131) comprising the most basic tract of P(111–136). We studied the effects of P(122–131) on HARP-induced cell proliferation using the prostate tumor-derived cell line DU145, known to undergo HARP stimulation via an autocrine loop. We found that P(122–131) dose-dependently inhibited HARP-induced cell proliferation and anchorage-independent DU145 growth. Since P(111–136) was previously reported to bind to the ALK receptor, and P(122–131) was derived from P(111–136), we hypothesized that P(122–131) might inhibit DU145 growth by interacting with ALK and further blocking the HARP-activating pathways. However, DU145 cells expressed the RPTPβ/ζ receptor but not the ALK receptor, suggesting mediation of the HARP autocrine loop in DU145 cells by the RPTPβ/ζ receptor. Previous studies showed that the extracellular RPTPβ/ζ domain 6B4 proteoglycan/phosphacan bound to HARP at both high- and low-affinity binding sites [12]. Chondroitinase ABC treatment decreased the affinity of 6B4 proteoglycan/phosphacan for HARP without significantly affecting the number of binding sites. These results indicated that both the chondroitin sulfate chain and the core 6B4 proteoglycan protein were involved in binding to HARP. It is noteworthy that we have shown that chondroitin sulfate was not involved in the binding of P(122–131) suggesting that it does not bind to the glycoaminoglycan chains of RPTPβ/ζ-derived, but instead to the RPTPβ/ζ core protein. This possibility has received support from another study demonstrating that the HARP domain corresponding to the 122–131 region is not involved in HARP interactions with glycosaminoglycans [31]. A recent study using various human glioblastoma cell lines established that extracellular processing of the HARP C-terminus produced two isoforms: a full-length molecule whose binding to RPTPβ/ζinduced cell migration and a truncated form lacking the last 12

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amino acids (124–136) whose binding to the ALK receptor led to a proliferative response [18]. Our earlier data on P(111–136) and those reported here suggest that P(122–131), which includes most of the last 12 HARP amino acids present in the full length, but not the truncated isoform, may bind to RPTPβ/ζ and that the binding site of the HARP C-terminus to ALK may include the amino acids 111–121. Further experiments are needed to investigate this possibility. In tumors such as gliomas, HARP binding to RPRPβ/ζ inactivated the intracellular catalytic domain of the receptor, leading to tyrosine phosphorylation of the downstream effectors Fyn, β-catenin, and β-adducin; as well as to cytoskeleton disruption, increased cell plasticity, and loss of cell-cell adhesion [32–34]. However, RPTPβ/ζ may also be associated with tumor cell proliferation in vitro and in vivo, since interfering molecules such as siRNA or monoclonal antiRPRPβ/ζ antibodies suppressed tumor cell growth [35,36]. In addition, we have shown that RPRPβ/ζ has a direct and positive signaling role acting as phosphatase and activating Src kinase [37]. Similar results have been obtained using P(122–131) (manuscript in preparation). Therefore, a peptide that antagonizes the interaction of HARP with RPRPβ/ζ may hold promise as a therapeutic tool for cancer.

Acknowledgments This work was supported by grants from the CNRS, ANR-06-567 RIB-016-02, INCA, and Association pour la Recherche sur le Cancer (#3242), the Cancer and Polio Research Fund, the North West Cancer Research Fund, the Human Frontiers Science Programme and a short-term Marie Curie early stage training fellowship (MolFun) to OB. We thank Nicolas Setterblad at the Imagery Department of the Institut Universitaire d'Hématologie IFR105 for the confocal microscopy studies. The imaging department is supported by grants from the Conseil Regional d'Ile-de-France and the Ministère de la Recherche.

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