Androgens Up-regulate the Insulin-like Growth Factor-I Receptor in Prostate Cancer Cells

2005;65:1849-1857. Cancer Res Giuseppe Pandini, Rossana Mineo, Francesco Frasca, et al. Receptor in Prostate Cancer CellsAndrogens Up-regulate the Insulin-like Growth Factor-I

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Androgens Up-regulate the Insulin-like Growth Factor-I Receptor

in Prostate Cancer Cells

Giuseppe Pandini,1Rossana Mineo,

1Francesco Frasca,

1Charles T. Roberts, Jr.,

2

Marco Marcelli,3Riccardo Vigneri,

1and Antonino Belfiore

4

1Dipartimento di Medicina Interna e di Medicina Specialistica, Cattedra di Endocrinologia, University of Catania, Ospedale Garibaldi,Catania, Italy; 2Department of Pediatrics, Oregon Health and Science University, Portland, Oregon; 3Department of Medicine, Divisionof Endocrinology, Diabetes and Metabolism, Baylor College of Medicine and Veterans Affairs Medical Center, Houston, Texas; and4Dipartimento di Medicina Sperimentale e Clinica, Cattedra di Endocrinologia, Policlinico Mater Domini, University of Catanzaro‘‘Magna Graecia,’’ Catanzaro, Italy

Abstract

In this study, we show that androgens up-regulate insulin-likegrowth factor-I receptor (IGF-IR) expression and sensitizeprostate cancer cells to the biological effects of IGF-I. Bothdihydrotestosterone and the synthetic androgen R1881 inducedanf6-fold increase in IGF-IR expression in androgen receptor(AR)–positive prostate cancer cells LNCaP. In accordance withIGF-IR up-regulation, treatment with the nonmetabolizableandrogen R1881 sensitized LNCaP cells to the mitogenic andmotogenic effects of IGF-I, whereas an IGF-IR blockingantibody effectively inhibited these effects. By contrast, theseandrogens did not affect IGF-IR expression in AR-negativeprostate cancer cells PC-3. Reintroduction of AR into PC-3 cellsby stable transfection restored the androgen effect on IGF-IRup-regulation. R1881-induced IGF-IR up-regulation was par-tially inhibited by the AR antagonist Casodex (bicalutamide).Two other AR antagonists, cyproterone acetate and OH-flutamide, were much less effective. Androgen-induced IGF-IRup-regulation was not dependent on AR genomic activity,because two AR mutants, AR-C619Y and AR-C574R, devoid ofDNA binding activity and transcriptional activity were still ableto elicit IGF-IR up-regulation in HEK293 kidney cells inresponse to androgens. Moreover, androgen-induced IGF-IRup-regulation involves the activation of the Src-extracellularsignal-regulated kinase pathway, because it was inhibited byboth the Src inhibitor PP2 and the MEK-1 inhibitor PD98059.The present observations strongly suggest that AR activationmay stimulate prostate cancer progression through the alteredIGF-IR expression and IGF action. Anti-androgen therapy maybe only partially effective, or almost ineffective, in blockingimportant biological effects of androgens, such as activationof the IGF system. (Cancer Res 2005; 65(5): 1849-57)

Introduction

Prostate cancer is the most common malignancy in men.Androgen stimulation is essential for growth and resistance toapoptosis in f70% of prostate carcinomas. This is the basis oftherapeutic approaches based on androgen deprivation. Thepalliative clinical benefits of androgen deprivation are temporary,however, because although initially responsive to anti-androgen

treatments these carcinomas eventually progress to androgen-independent tumors, for which no efficacious treatment is currentlyavailable. The molecular basis of androgen stimulation of prostatecancer growth and the switch to androgen independency areincompletely understood. Recent evidence suggests that androgensmay regulate prostate cancer proliferation by up-regulating auto-crine loops involving peptide growth factors and their cognatereceptors (1). The progression to androgen independence may beexplained by the appearance of malignant cell clones that areresponsive to growth factors other than androgens. These cellstherefore are only partially responsive to androgen deprivation andcan be successfully killed only by a combined approach targeting notonly the androgen receptor (AR) but also other relevant growthregulators. Hopefully, such a combined approach could slow downthe progression of androgen-independent prostate cancer.

The insulin-like growth factor (IGF) system plays a key role inregulating growth, resistance to apoptosis, and invasion in a varietyof human malignancies (2–5). Various lines of evidence suggest arole for the IGF system in prostate cancer (6, 7). First, clinical andepidemiologic studies indicate that increased IGF-I serum levels areassociated with an increased risk of prostate cancer (6, 8). Second,IGF-I may increase in vitro proliferation of prostate cancer cells,whereas antisense-mediated inhibition of IGF-I receptor (IGF-IR)expression suppresses in vivo tumor growth and prevents prostatecancer cell invasiveness (9). Third, in human prostate cancer cellxenografts, progression to androgen independence in someexperimental models is associated with increased expression ofboth IGF-IR and IGF-I (10).

Our understanding of the mutual regulation of the androgen andIGF systems in human prostate cancer is limited, however. Differentstudies have established that IGF-I may influence AR signaling. Earlyreports have indicated that IGF-I may transactivate the AR intransfected DU-145 human prostate cancer cells (11). Other authorsfound that IGF-I enhanced androgen-mediated AR transcriptionalactivity in DU-145 cells but was unable to transactivate AR in theabsence of androgens (12). Recently, Plymate et al. showed that theIGF-I effect on AR transcriptional activity is even more complex anddepends on the cell context (13). They found that IGF-I enhanceddihydrotestosterone-stimulated, but not basal, AR transcriptionalactivity in nonmetastatic AR-transfected prostate cancer cells (PRIcells). However, IGF-I suppressed AR activity in response todihydrotestosterone in PRI-derived metastatic cells.Lin et al. have shown that IGF-I, through the activation of the

phosphatidylinositol 3-kinase/Akt serine-threonine kinase pathway,

phosphorylates the AR at Ser210 and Ser790. This AR phosphorylation

may inhibit AR-mediated apoptosis possibly by inhibiting the

interaction between AR and coregulators (14). In addition, activation

Requests for reprints: Antonino Belfiore, Dipartimento di MedicinaSperimentale e Clinica, Cattedra di Endocrinologia, Policlinico Mater Domini,University of Catanzaro ‘‘Magna Graecia,’’ via T. Campanella 115, 88100 Catanzaro,Italy. Phone: 39-961-712-423; Fax: 39-961-772-748; E-mail: [email protected].

I2005 American Association for Cancer Research.

www.aacrjournals.org 1849 Cancer Res 2005; 65: (5). March 1, 2005

Research Article



of the Ras/mitogen-activated protein kinase pathway by IGF-I may

sensitize the AR transcriptional complex to subphysiologic levels of

androgen in LNCaP prostate cancer cells (15).On the contrary, data regarding androgen regulation of the IGF

system in prostate cancer are lacking. We studied whether in humanAR-positive prostate cancer cells androgens influence the expressionof receptors of the IGF system and found that androgens induce aselective and marked up-regulation of the IGF-IR. Cell proliferationand invasiveness in response to IGF-I was greatly increased byandrogens. The effect of androgens on IGF-IR involved an increase inboth mRNA and protein expression and occurred through theactivation of a nongenomic AR signaling pathway.

Materials and Methods

Cell media and all chemicals, unless otherwise stated, were obtained

from Sigma (St. Louis, MO). The following materials were also purchased:

FCS and geneticin (G418) from Invitrogen Laboratories (Paisley, United

Kingdom); IGF-I, LY294002, PD98059, and PP2 from Calbiochem (San

Diego, CA); dihydrotestosterone from Fluka (Buchs, Switzerland); synthetic

nonmetabolizable androgen R1881 from NEN Life Science Products

(Boston, MA); Fugene6 transfection reagent from Roche Diagnostics

(Mannheim, Germany); luciferase assay system from Promega Corp.

(Madison, WI); monoclonal antibody anti-IGF-IR (aIR-3) from Oncogene

Research (Cambridge, MA); polyclonal anti-IGF-IR antibody and mono-

clonal antibody anti-AR from Santa Cruz Biotechnology, Inc. (Santa Cruz,

CA); monoclonal antibody anti-phosphotyrosine (4G10) from UBI (Lake

Placid, NY); and polyclonal anti-phospho-extracellular signal-regulated

kinase1/2 (ERK1/2) and anti-ERK1/2 antibodies from New England

Biolabs (Beverly, MA). Casodex (bicalutamide), an androgen antagonist,

was kindly provided by AstraZeneca (Milan, Italy). The cDNA encoding the

human AR cloned into the expression vector pSV0 was kindly provided by

Dr. A.O. Brinkmann (Rotterdam, the Netherlands). The cDNAs encoding

the kinase-inactive MEK-1 (Ser221Ala) and the kinase-inactive form of Src

(Lys259Met) were kindly provided by Dr. G. Castoria (Naples, Italy). The

cDNAs encoding the MMTV-luc reporter gene was kindly provided by Dr.

Farsetti (Rome, Italy). The expression vectors for the human transcrip-

tionally inactive AR mutants, AR-C619Y and AR-C574R, have been

described previously (16–18). Rat IGF-IR gene promoter sequences

corresponding to the full-length fragment (�476/+640), the 5V flanking

fragment (�476/+41), and the 5V untranslated region (+41/+640) ligated

upstream of the firefly luciferase reporter cDNA in the pGL3 vector have

been described previously (19).

CellsAR-positive human prostate cancer LNCaP cells, AR-negative PC-3 cells,

and human kidney 293 cells (HEK293, AR-negative) were obtained from theAmerican Type Culture Collection (Manassas, VA) and maintained as

follows: LNCaP cells in RPMI, whereas PC-3 and HEK293 cells in DMEM

supplemented with 10% fetal bovine serum and 1% glutamine. AR-

transfected PC-3 cells (clones 6 and 13) and PC-3-NEO cells were kindlyprovided by Dr. E. Baldi (Florence, Italy) and maintained in DMEM

supplemented with 10% fetal bovine serum, 1% glutamine, and 0.6 mg/mL

geneticin.

Transient Transfection and Luciferase AssaysTo evaluate whether androgen affects IGF-IR promoter activity, HEK293

cells were transiently cotransfected with 0.3 Ag of either ARwt or AR-C619Yor AR-C574R expression vectors together with 0.3 Ag IGF-IR promoter/

luciferase vector in the presence or absence of 0.3 Ag dominant-negative Src

or MEK-1 (Src k� and A221-MEK-1 k�). Briefly, 2.5 � 104 cells were seededin 12-well plates and grown for 24 hours in medium without phenol red. To

normalize values for transfection efficiency, three different vectors were

preliminary studied to avoid spurious activity in response to androgen

as described for certain Renilla vectors: (a) a vector coding for the Renillaluciferase reporter gene (pRL-SV40), (b) a b-galactosidase reporter gene

(pSV-h-galactosidase) (Promega), and (c) a vector coding for the H2B-GFP

reporter gene (pBOS H2B-GFP-N1) provided by Dr. J. Wang (San Diego, CA).

Results of these studies indicated that androgens may spuriously affect the

activity of the first two vectors, although they do not affect the activity of

the green fluorescent protein (GFP) vector, which was therefore used for

normalizing all luciferase assays.5

To evaluate the AR transactivation activity, HEK293 cells were transientlycotransfected with 0.45 Ag of either ARwt or AR-C619Y or AR-C574R

expression vectors together with 0.45 Ag of an expression vector encoding

for MMTV-luc reporter gene and 0.1 Ag of a vector coding for the H2B-GFP

reporter gene.Transfection Technique. A transfection mixture containing 1 Ag DNA

and 4 AL Fugene6 in 40 AL of medium without serum was added to each

well. After 18 hours, the medium was changed to serum-containing mediumfor 30 hours. Cells were then serum starved overnight and incubated with

10 nmol/L R1881 for 24 hours. Cells were then lysed and processed for the

Dual Luciferase Assay (Promega). Luciferase activity was normalized for

transfection efficiency (Renilla activity, h-galactosidase activity, or GFPamount). h-galactosidase activity was measured according to the manu-

facturer’s instructions. In GFP-transfected cells, the activity of each sample

(5 AL) was measured by a spectrofluorimeter (Wallac 1420 Victor 2, Perkin-

Elmer, Boston, MA).

IR, IGF-IR, and Hybrid Receptor MeasurementsCell lysates were prepared as described previously and used for receptor

measurement by both ELISA and Western blot analysis.

ELISA. The characteristics and specificity of receptor ELISAs have been

described previously (20, 21). Receptors were captured by incubating celllysates (10-60 Ag protein per well) in Maxisorp immunoplates precoated

with 2 Ag/mL specific monoclonal antibody as described previously (21, 22).

Immunocaptured receptors were incubated with the specific biotinylated

monoclonal antibodies at 0.3 Ag/mL in 50 mmol/L HEPES-buffered saline(pH 7.6) containing 0.05% Tween 20, 1% bovine serum albumin, 2 mmol/L

sodium orthovanadate, 1 mg/mL bacitracin, and 1 mmol/L phenyl-

methylsulfonyl fluoride and then with peroxidase-conjugated streptavi-

din. Peroxidase activity was determined colorimetrically by adding 100 ALof 3,3V,5,5V-tetramethylbenzidine [0.4 mg/mL in 0.1 mol/L citrate-

phosphate buffer (pH 5.0) with 0.4 mL of 30% H2O2]. The reaction was

stopped by the addition of 1.0 mol/L H3PO4 and the absorbance wasmeasured at 450 nm (21).

Western Blot. To confirm data obtained by ELISA, IR, IGF-IR, and hybrid

receptor (Hybrid-R) were also measured in the same cell lysates by Western

Blot analysis using specific antibodies as described previously (22).

IGF-I Binding StudiesLNCaP cells were grown to f60% confluence, serum starved, and

further cultured in the presence or absence of 10 nmol/L R1881 for 24

hours. Cells (3 � 106) were then incubated with 125I-IGF-I (10 pmol/L)

for a further 16 hours at 4jC in the presence of increasingconcentrations of cold IGF-I. Then, cell-associated radioactivity was

measured in a gamma counter. Scatchard analysis was done using

GraphPad Prism 4 software.

IGF-IR AutophosphorylationCell monolayers were serum starved in medium without phenol red 24

hours before stimulation with 10 nmol/L IGF-I for 10 minutes. Cells were

lysed in cold radioimmunoprecipitation assay buffer containing 50 mmol/L

Tris (pH 7.4), 150mmol/L NaCl, 1% Triton X-100, 0.25% sodiumdeoxycholate,

10 mmol/L sodium pyrophosphate, 1 mmol/L NaF, 1 mmol/L sodium

orthovanadate, 2 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mL aproti-

nin, 10 Ag/mL pepstatin, and 10 Ag/mL leupeptin, and the insoluble material

was separated by centrifugation at 10,000 � g for 10 minutes at 4jC. Celllysates were incubated at 4jC under rotation for 2 hours with 4 Ag anti-IGF-IR aIR-3 antibody coated with protein G-Sepharose. Immunoprecipitates

5 G. Pandini et al., unpublished data.

Cancer Research

Cancer Res 2005; 65: (5). March 1, 2005 1850 www.aacrjournals.org



were subjected to SDS-PAGE. The resolved proteins were transferred tonitrocellulose membranes, immunoblotted with anti-phosphotyrosine 4G10monoclonal antibody, and detected by enhanced chemiluminescence. Thenitrocellulose membrane was then stripped with buffer Restore (Pierce,Rockford, IL) and subsequently reprobed with an anti-IGF-IR rabbitpolyclonal antibody.

ERK1/2 Phosphorylation in Response to AndrogenCells were stimulated with 10 nmol/L R1881 or dihydrotestosterone for

5 minutes. Cell lysates obtained by the addition of 4� sample buffer wereheated at 95jC to 100jC for 5 minutes and subjected to reducing SDS-PAGEon 10% polyacrylamide gel. The resolved proteins were transferred tonitrocellulose membranes and immunoblotted with anti-phosphospecificERK1/2 polyclonal antibody. The nitrocellulose membranes were thenstripped with buffer Restore and subsequently reprobed with anti-ERK1/2polyclonal antibody.

Real-time PCRTotal RNA (5 Ag) was reverse transcribed by ThermoScript RT

(Invitrogen) and oligo(dT) primers. Synthesized cDNA (0.15 AL) was thencombined in a PCR reaction using primers 5V-GGGCCATCAGGATTGA-GAAA-3V ( forward) and 5V-CACAGGCCGTGTCGTTGTCA-3V (reverse)specific for the IGF-IR ( fragment size 330 bp). ELE-1 (housekeepinggene) amplification was done using the following primers: 5V-ATTGAA-GAAATTGCAGGCTC-3V ( forward) and 5V-TGGAGAAGAGGAGCTGTATCT-3V (reverse; fragment size 280 bp). Quantitative real-time PCR was done onan ABI Prism 7700 (PE Applied Biosystems, Foster City, CA) using SYBRGreen PCR Master Mix (PE Applied Biosystems) following the manufac-turer’s instructions. Amplification reactions were checked for the presenceof nonspecific products by agarose gel electrophoresis. Relative quantita-tive determination of target gene levels was done by comparing DCt asdescribed previously (23).

Incorporation of [3H]Thymidine[3H]Thymidine incorporation was carried out as described previously

(24). Briefly, LNCaP cells, preincubated or not with androgens for 24 hours,

were seeded in 24-well plates and allowed to attach for 24 hours. Complete

medium was replaced with serum-free medium without phenol red and

containing 0.1% bovine serum albumin for 48 hours and then treated for a

further 18 hours with IGF-I in the presence or absence of anti-IGF-IR

monoclonal antibody aIR-3. After incubation, 0.5 ACi/mL [3H]thymidine

was added for 4 hours. The cells were washed with ice-cold PBS and

incubated with 1 mL of 10% ice-cold trichloroacetic acid for 30 minutes.

The acid-insoluble fraction was solubilized with 0.1 N NaOH and the

incorporation of [3H]thymidine into DNA was determined by scintillation

counting in a h counter.

Migration AssaysLNCaP cells were serum starved for 24 hours in medium without phenol

red and stimulated with R1881 for a further 24 hours. Cells were then

removed from plates with HBSS containing 5 mmol/L EDTA and 25 mmol/L

HEPES (pH 7.2) and 0.01% trypsin and resuspended at 106/mL, and 100 ALwere added to the top of each migration chamber. Cell migration assays

were done as described previously (25), with minor modifications, using

modified Boyden chambers (6.5 mm diameter, 10 Am thickness, 8 Am pores,

Transwell, Costar Corp., Cambridge, MA) containing polycarbonate

membranes coated at the lower side with 250 Ag/mL collagen VI. Cells

were allowed to migrate to the underside of the top chamber for 18 hours in

response to 10 nmol/L IGF-I added to the lower chamber and in the

presence or absence of anti-IGF-IR antibody aIR-3. Cells that had migrated

to the lower side of the filter were fixed and stained with 0.1% crystal violet

in 20% ethanol for 20 minutes. After three washes with water and complete

drying, the crystal violet was solubilized by in 10% acetic acid and its

concentration was evaluated as absorbance at 590 nm.

Figure 1. Expression of IR, IGF-IR, andHybrid-R and IGF-I binding in prostatecancer cells in response to androgen. IR,IGF-IR, and Hybrid-R expression:serum-starved AR-positive LNCaP (A andB) or AR-negative PC-3 cells (C and D )were incubated in the presence or absenceof 10 nmol/L R1881 for 24 hours. Cellswere then lysed and receptor subtypeswere immunopurified by specific antibodiesand measured by ELISA (A and C ) orWestern blot (B and D ) as described inMaterials and Methods. Columns,mean ELISA data of three independentexperiments; bars, SD. RepresentativeWestern blot experiments. IGF-I binding:competition-inhibition curves of 125I-IGF-Ibinding were carried out in LNCaP cells (E)and PC-3 wt cells (F ) preincubated in thepresence (.) or absence (n) of 10 nmol/LR1881 for 24 hours. Inset, Scatchard plotanalysis of binding data.

IGF-I Receptor Up-regulation by Androgens




Results

Androgens Up-regulate the IGF-IR, but not the IR, inAR-Positive Prostate Cancer CellsThe expression of the IGF-IR and IR was determined in

serum-starved LNCaP and PC-3 cells exposed to the non-metabolizable androgen R1881 (10 nmol/L) for 24 hours. In AR-positive LNCaP cells, exposure to R1881 increased IGF-IRexpression by f5-fold (0.8-4.0 ng IGF-IR/100 Ag protein asmeasured by ELISA; Fig. 1A and B). In contrast, R1881 did notaffect LNCaP cell IR content, showing that the androgen effecton the IGF-IR was specific.We also studied IR/IGF-IR Hybrid-Rs that are formed by

random assembly of IR and IGF-IR hemidimers and behave asfunctional IGF binding sites (21, 22, 26). Hybrid-Rs were alsoincreased in LNCaP cells exposed to R1881 (Fig. 1A and B)presumably as a consequence of increased IGF-IR expression.Both ELISA measurements and Western blot analysis gave verysimilar results. Exposure to R1881 did not affect IGF-IR orHybrid-R expression in AR-negative PC-3 cells (Fig. 1C and D).The effects of testosterone or dihydrotestosterone (10 nmol/L)on IGF-IR and IR expression were almost identical to those ofR1881 in both cell lines (data not shown).To evaluate whether the increased IGF-IR expression reflected

an increased IGF-I binding capacity, LNCaP cells cultured in thepresence or absence of 10 nmol/L R1881 for 24 hours wereincubated with a tracer dose of 125I-IGF-I and with increasingconcentrations of cold IGF-I. Scatchard plot analysis of bindingdata showed a 7-fold increase of specific IGF-IRs (5.6-36.0 pmol/L/

106 cells) in androgen-exposed LNCaP cells compared withunexposed cells. The dissociation constant (Kd), however, wasvery similar (0.32 versus 0.22 nmol/L in androgen-exposed andnonexposed cells, respectively), showing that androgen increasesIGF-I binding sites but only minimally affects the receptor affinityfor the ligand (Fig. 1E). As expected, no change in IGF-I bindingwas observed in AR-negative PC-3 cells exposed or unexposed toandrogen (Fig. 1F).Dose-response experiments showed that R1881 was able to

increase IGF-IR protein expression in a dose-dependent manner.IGF-IR started to increase at a dose as low as 0.01 nmol/L andreached maximum levels at 1 to 10 nmol/L R1881 (Fig. 2A). Timecourse experiments with 10 nmol/L R1881 showed that an increaseof IGF-IR protein expression was already evident at 12 hours andincreased steadily up to 24 to 48 hours (Fig. 2B). A 24-hourincubation length was therefore used in subsequent studies.IGF-IR Phosphorylation. As shown in Fig. 2C , LNCaP cells

preincubated with R1881 exhibited increased ligand-induced IGF-IR tyrosine phosphorylation compared with untreated cells,indicating that the increase in IGF-IR expression resulted inincreased signal transduction.Androgens Up-regulate IGF-IR in AR-Transfected PC-3 Cells.

To evaluate whether the effect of androgens on IGF-IR wasrestricted to LNCaP cells, we carried out studies in PC-3 cellstransfected with the wild-type AR cDNA. Two cell clones (PC-3-AR6and PC-3-AR13) with different AR expression levels were used. Asshown in Fig. 2D , the PC-3-AR6 clone had an AR expression level,as measured by Western blot, which was only slightly lower than

Figure 2. IGF-IR up-regulation and increased autophosphorylation by R1881 in AR-positive prostate cells. IGF-IR expression: dose-response experiments(A) were carried out by incubating serum-starved LNCaP cells in the presence or absence of the indicated doses of R1881 for 24 hours. Time course experiments(B) were carried out by incubating serum-starved LNCaP cells with or without 10 nmol/L R1881 for the indicated times. Whole-cell lysates containing equal amountsof protein were separated by SDS-PAGE and immunoblotted with an anti-IGF-IR antibody (top ). Membranes were reblotted with an anti-h-actin antibody (bottom ).C, IGF-IR autophosphorylation: serum-starved LNCaP cells preincubated in the presence or absence of 10 nmol/L R1881 for 24 hours were exposed to 10 nmol/LIGF-I for 5 minutes. IGF-IR was immunopurified with antibody aIR-3 from cell lysates and IGF-IR autophosphorylation was measured by Western blot analysis withan anti-phosphotyrosine antibody as described in Materials and Methods. Top, anti-phosphotyrosine blot; bottom, reblotting with anti-IGF-IR antibody. IGF-IR regulationby R1881 in LNCaP cells and transfected PC-3 cell clones: D, Western blot showing AR expression in different PC-3 cell clones stably transfected with AR.LNCaP cells were used as positive control and PC-3wt and PC-3-NEO cells were used as negative controls (top ). Membranes were reblotted with an anti-h-actinantibody to control for protein loading (bottom ). E, IGF-IR regulation by androgen was then studied after incubation of serum-starved cells in the presenceor absence of 10 nmol/L R1881 for 24 hours. Whole-cell lysates containing equal amounts of protein were separated on SDS-PAGE and immunoblotted withan antibody to IGF-IR (top ). Membranes were reblotted with an anti-h-actin antibody to control for protein loading (bottom ). Representative of three independentexperiments.

Cancer Research




LNCaP cells, whereas the PC-3-AR13 clone expressed the AR atf6-

to 8-fold lower levels than LNCaP cells.R1881 markedly up-regulated IGF-IR expression in AR-positive

but not in AR-negative transfected cells, and the IGF-IR response

was proportional to the AR expression level. In LNCaP and PC-3-

AR6 cells that exhibited the highest level of AR expression, IGF-IR

content increased 6- to 8-fold and 4- to 6-fold, respectively. Only

a slight increase was observed in PC-3 AR13 cells (Fig. 2E), which

had a lower AR content (Fig. 2D), and no effect of R1881 on IGF-IR

expression was observed in control PC-3-NEO cells and PC-3wt

cells not expressing AR (Fig. 2D and E).

Androgens Induce IGF-IR mRNA ExpressionThe increase in IGF-IR expression induced by R1881 in LNCaP

cells was completely inhibited by either actinomycin D or

cycloheximide, suggesting that both de novo mRNA and protein

synthesis are required for this effect (data not shown). IGF-IR

mRNA expression was then studied in LNCaP cells by

quantitative real-time PCR after cell exposure to 10 nmol/L

R1881. Dose-response experiments carried out in cells exposed to

R1881 for 24 hours showed that IGF-IR mRNA increased at

a R1881 dose of 0.001 nmol/L and reached a plateau at 1 to 10

nmol/L (Fig. 3A). Time course experiments carried out with

10 nmol/L R1881 indicated that mRNA started to increase at

4 hours and reached levels f15-fold higher after 16- to 24-hour

exposure to R1881 (Fig. 3B). The increase in IGF-IR mRNA in

response to androgen treatment was partially inhibited by

preincubation with cycloheximide, suggesting that new protein

synthesis was required for androgen stimulation of IGF-IR gene

expression (Fig. 3C).

Androgen-Induced IGF-IR Up-regulation Is PartiallyInhibited by the AR Antagonist Casodex and IsSensitive to c-Src and MEK-1 InhibitionWe next evaluated whether AR antagonists could block IGF-IR

up-regulation in response to androgens. LNCaP cells were

incubated with 10 nmol/L R1881 (or dihydrotestosterone) in

the presence of 50 nmol/L cyproterone acetate or 3 Amol/L OH-

flutamide or 10 Amol/L Casodex. Cells were then lysed and IGF-

IR expression was studied by Western blot. The results showed

that R1881-induced IGF-IR up-regulation was partially inhibited

by Casodex (�40 F 6%), whereas cyproterone acetate and OH-

flutamide were almost ineffective (about �13% for both; Fig. 4A).

Dose-response experiments with these anti-androgens showed

that cyproterone acetate at concentrations >50 nmol/L was

actually stimulatory and that OH-flutamide at concentrations

>3 Amol/L was cytotoxic (data not shown).These results suggested that IGF-IR up-regulation might not

be mediated by classic AR transactivation pathway, which are

sensitive to AR antagonists. An alternative AR signaling pathway

has been described, which involves the activation of a Src/Raf-1/

ERK pathway that in turn elicits important biological effects,

such as cell proliferation (27).To evaluate the involvement of the Src/Raf-1/ERK pathway in

IGF-IR up-regulation, LNCaP cells were incubated with R1881 in

the presence or absence of various kinase inhibitors, including

PP2, a Src inhibitor, PD98059, a MEK-1 inhibitor, and LY294002, a

phosphatidylinositol 3-kinase inhibitor. IGF-IR expression was

then measured by Western blot analysis. The R1881 effect was

blocked almost completely by both PP2 (10 Amol/L) and PD98059

(50 Amol/L). LY294002 (up to 20 Amol/L) was ineffective (Fig. 4B).

As expected, incubation with R1881 for 30 minutes induced a

marked ERK1/2 activation, and both PP2 and PD98059, but not

LY294002, at the doses used, completely blocked this effect (Fig.4C). Taken together, these data strongly suggest that IGF-IR up-regulation by R1881 involves the activation of the Src/Raf-1/ERKpathway as reported previously for other nongenomic, androgen-mediated effects. This effect was, however, specific of androgens,as a potent stimulator of the ERK1/2 activity, such as epidermalgrowth factor (10 nmol/L), was ineffective in inducing IGF-IR up-regulation (data not shown).

Two Transcriptionally Inactive AR Mutants Are Ableto Increase IGF-IR Promoter Activity and IGF-IRProtein LevelsTo further evaluate whether IGF-IR up-regulation can occur

independently of the transcriptional activity of the AR, we usedtwo different AR mutants, AR-C619Y and AR-C574R, bothreported to be unable to bind DNA and activate transcription(16–18). In particular, AR-C574R is additionally unable totranslocate into the nucleus (17, 18). AR-negative HEK293 cellswere then transfected with either the ARwt or the AR-C619Y orAR-C574R cDNAs, and IGF-IR protein expression was measured,after exposure to androgen, by Western blot analysis. Cellstransfected with each of the two transcriptionally inactive AR

Figure 3. IGF-IR mRNA up-regulation by androgen in LNCaP cells.Dose-response experiments (A) were carried out by incubating serum-starvedLNCaP cells in the presence or absence of increasing concentrations ofR1881 for 24 hours. For time course experiments (B), LNCaP cellswere incubated with or without 10 nmol/L R1881 for the indicated times.Total RNA prepared from LNCaP cells was used as template for real-timereverse transcription-PCR as described in Materials and Methods.Relative mRNA amounts were normalized to the abundance of the ELE-1mRNA. Actinomycin D (1 Ag/mL) completely blocked IGF-IR mRNAup-regulation by R1881, whereas cycloheximide (10 Ag/mL) was only partiallyeffective (C ). Columns, mean of three separate experiments; bars, SD.





mutants showed an up-regulation of IGF-IR protein expression ata level comparable with that observed in cells transfected withthe ARwt, thus indicating that AR DNA binding and transcrip-tional activity were unnecessary for this effect (Fig. 5A).To confirm the absence of transcriptional activity of the two AR

mutants in our system, HEK293 cells were transiently transfectedwith either the ARwt or each of the two AR mutants along withthe androgen-responsive MMTV-luc reporter. To avoid possiblespurious Renilla luciferase activation by androgen described incertain systems (28), transfection efficiency was measured by usinga GFP vector that is completely insensitive to androgens asdescribed in Materials and Methods. As shown in Fig. 5B , R1881was able to induce increased activity of the MMTV-luc reporter inthe presence of the ARwt but not in the presence of AR mutantsAR-C619Y or AR-C574R. R1881 also caused an increase of theMMTV-luc activity in LNCaP cells.HEK293 cells were then transiently cotransfected with

plasmids encoding either the ARwt or the AR-C619Y or AR-C574R and luciferase constructs containing each of the threeIGF-IR promoter sequences ( full-length, 5V flanking fragment,and 5V untranslated region fragment). Cotransfection with theGFP vector was used to normalize for transfection efficiency.The promoter activity of the full-length fragment (bp �476/+640) was 180% to 200% higher in R1881-stimulated cells incomparison with unstimulated cells. This effect was seen not

only in cells transfected with ARwt-transfected cells but also incells transfected with each of the two mutants (Fig. 5C-E).Cotransfection with plasmids encoding either a dominant-negative MEK-1 or a dominant-negative c-Src was able toabolish this increase in promoter activity in all cases (Fig. 5C-E).Control plasmids were ineffective (data not shown).A similar increase in luciferase activity (+154% increase) was

observed with the 5V untranslated region promoter fragment (bp�41/+640), whereas no increase was seen with the 5V flankingfragment (bp �476/+41), indicating that the sequences responsiblefor androgen stimulation are between bp �41 and +640 of the IGF-IR promoter region (data not shown).Results similar to those obtained in HEK293 cells were also

obtained in AR-transfected PC-3 cells (data not shown).

Biological Effects of Androgen-Induced IGF-IRUp-regulationCell Growth. We evaluated whether androgen-induced IGF-

IR up-regulation in LNCaP prostate cancer cells may result inincreased mitogenic effect of IGF-I. Serum-starved cellspreincubated with or without R1881 for 24 hours wereexposed to IGF-I for 18 hours. IGF-IR levels were f7-foldhigher in cells incubated with R1881 (Fig. 6A). In these cells,IGF-I increased [3H]thymidine incorporation by 3-fold, where-as no effect was observed in control cells (Fig. 6B). Most ofthis effect was blocked by the anti-IGF-IR monoclonal

Figure 4. Inhibition of androgen-induced IGF-IR expression byanti-androgens or kinase inhibitors. A, inhibition of androgen-inducedIGF-IR expression by anti-androgen: serum-starved LNCaP cellswere treated with 10 nmol/L R1881 for 24 hours in the presenceor absence of either 50 nmol/L cyproterone acetate, 3 Amol/LOH-flutamide, or 10 Amol/L Casodex. Whole-cell lysatescontaining equal amounts of protein were separated bySDS-PAGE and immunoblotted with an antibody to the IGF-IR.Representative of four independent experiments (top ). MeanColumns, mean densitometric values of IGF-IR/h-actin ratios fromfour independent experiments; bars, SD (bottom ). B, inhibition ofandrogen-induced IGF-IR up-regulation by kinase inhibitors:LNCaP cells were incubated with 10 nmol/L R1881 for 24 hoursand in the presence or absence of either 10 Amol/L PP2 (c-Srcinhibitor), 50 Amol/L PD98059 (MEK-1 inhibitor), or 20 Amol/LLY294002 (phosphatidylinositol 3-kinase inhibitor). IGF-IRexpression was then measured by Western blot analysis (top ),as described in Materials and Methods, using an antibody to theIGF-IR. Membranes were reblotted with an anti-h-actin antibodyto control for protein loading (bottom ). Representative of threeindependent experiments (top ). Columns , mean densitometricvalues of IGF-IR/h-actin ratios from three independentexperiments; bars, SD (bottom ). C, androgen activation ofERK1/2 and inhibition by both PD98059 and PP2: serum-starvedLNCaP cells were incubated with 10 nmol/L R1881 for 5 minutesin the presence or absence of either 10 Amol/L PP2 (c-Srcinhibitor), 50 Amol/L PD98059 (MEK-1 inhibitor), or 20 Amol/LLY294002 (phosphatidylinositol 3-kinase inhibitor). ERK1/2activation was then evaluated by Western blot analysis using aphosphospecific antibody (top ). Membranes were stripped andreblotted with an anti-EPK1/2 antibody (bottom ).

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antibody aIR-3, thus demonstrating the specificity of IGF-Ieffect (Fig. 6B).Cell Invasiveness. Chemoinvasion, measured as the cell ability to

migrate in response to IGF-I in Boyden chambers, was studied inLNCaP cells preincubated with or without R1881. IGF-I effectivelystimulated chemoinvasion in cells preincubated with R1881. Thiseffect was inhibited by aIR-3 and was not observed in control cells(Fig. 6C).

Discussion

The main findings of the present work can be summarized asfollows: (a) androgens are able to induce up-regulation of IGF-IR,Hybrid-Rs, and IGF-I binding capacity in AR-positive prostatecancer cells; (b) this effect cannot be blocked by the mostfrequently used AR antagonists, such as cyproterone acetate andOH-flutamide (almost ineffective) and Casodex (only partialinhibition); (c) IGF-IR up-regulation by androgens does not involveAR binding to DNA and classic transcriptional activity but ratheroccurs via the activation of a Src-ERK pathway; and (d) IGF-IR up-

regulation by androgens sensitizes cells to the mitogenic andmotogenic effects of IGF-I.Recent work has clearly showed a role of the IGF system in

prostate cancer (3, 6–8, 19, 29–34). The IGF-IR may be overex-pressed in prostate cancer at initial stages (35). Diseaseprogression may be associated with IGF-IR down-regulation (36),although recent evidence indicates that IGF-IR is overexpressed atmetastatic sites (37). The molecular mechanisms underlying IGF-IR regulation at the different stages of prostate cancer are still

unclear. Our data show that LNCaP cells are unresponsive to IGF-I,

unless they are preincubated with androgens, which induce a

marked up-regulation of the IGF-IR and sensitize cells to the

mitogenic and motogenic effects of IGF-I. This effect is quite rapid,

as it is already detectable 12 hours after exposure to androgens,

and occurs at an androgen dose as low as 0.01 nmol/L R1881. This

effect is not restricted to LNCaP cells and is also observed in

transfected PC-3 cells. Scatchard plot analysis of binding data

confirmed a marked increase in IGF-I binding sites after androgen

exposure with a minimal increase in binding affinity. These data

Figure 5. IGF-IR expression and transcription is up-regulated by transcriptionally inactive AR mutants. A, IGF-I protein expression is up-regulated by each of thetwo different transcriptionally inactive AR mutants: HEK293 cells were stably transfected with expression plasmids coding for either the wild-type AR (ARwt) or atranscriptionally inactive AR mutant (AR-C619Y or AR-C574R). Control cells were transfected with an empty vector (EV). Transfected cells were then exposedto 10 nmol/L R1881 for 24 hours and IGF-IR expression was measured by Western blot analysis (top ) as described in Materials and Methods. Filters were reblotted withan anti-AR antibody (middle ) and then with an anti-h-actin antibody (bottom ). Representative of three independent experiments. B, androgen-induced activity of aMMTV-luc reporter: HEK293 cells were cotransfected with either the ARwt or the AR-C619Y or AR-C574R mutant and the androgen-responsive MMTV-luc reporter.R1881 induced a marked increase of MMTV activity in cells transfected with the ARwt but not in those transfected with the AR mutants. R1881 stimulation of theendogenous AR in LNCaP cells was also able to induce MMTV-luc reporter activity. C -E, androgen induces IGF-IR promoter activity in AR-transfected cells: HEK293cells were transiently cotransfected with expression plasmids coding for either the wild-type AR (ARwt; C ) or each of the two transcriptionally inactive AR variants(AR-C619Y or AR-C574R; D and E) together with the IGF-IR promoter/luciferase vector containing the full-length promoter fragment (bp �476/+641) of the ratIGF-IR gene. In androgen-stimulated cells, the IGF-IR promoter activity was then assayed in the presence or absence of plasmids coding for either a Src k� dominant-negative or a MEK-1 k� dominant-negative. Relative luciferase activity is expressed as fold activation with respect to unstimulated cells. Columns, mean of threeindependent experiments normalized for transfection efficiency with a GFP vector as described in Materials and Methods; bars, SD.





are in partial agreement with the findings of Iwamura et al. (38),

who showed previously that IGF-I is mitogenic in LNCaP cells only

after pretreatment with dihydrotestosterone. These authors,

however, could not explain the mechanism of this finding, as they

were unable to show an increase in cell IGF-I binding sites after

dihydrotestosterone exposure. The reason for the discrepancy

between our study and the study of Iwamura et al. (38), as far as

the increase of IGF-I binding is concerned, is unclear and may

involve cell variability. Moreover, in our study, the direct

measurement of IGF-IR protein or mRNA expression confirmed

the increase of IGF-I binding sites after exposure to androgens.

These measurements were not undertaken in the Iwamura et al.

study (38).The effect of androgens on IGF-IR is specific, as androgens did

not affect the expression of the closely related IR (39–42).

However, because these two receptors form hybrids (Hybrid-R) by

random assembly of a/h-subunit hemireceptors (21, 22, 26, 43),

androgen-induced IGF-IR up-regulation resulted in an increase of

Hybrid-Rs. These Hybrid-Rs bind IGF-I with high affinity and

could contribute therefore to the increased IGF-I binding and to

the increased cell sensitivity to IGF-I.In some cell models, prostate cancer progression to androgen

independence is associated with increased expression of IGF-IR

(10). Among the multiple mechanisms hypothesized for prostate

cancer progression to androgen independence is the emergence ofcell clones with activating AR mutations (44, 45). We hypothesizethat these activating AR mutations may contribute to IGF-IR up-regulation in metastatic cancer. It is noteworthy that the mostfrequently used AR antagonists are either almost ineffective inblocking AR-induced IGF-IR up-regulation (cyproterone acetate andOH-flutamide) or only minimally effective (Casodex). As cyproter-one acetate is concerned, 50 nmol/L was the most effectiveinhibitory dose and higher doses were stimulatory (data not shown)according to previous reports (46). These observations stronglysuggest that in clinical practice most anti-androgen treatmentstargeting the AR are ineffective in blocking androgen-inducedactivation of the IGF system, which may play an important role incancer progression to androgen independence. The mecha-nism(s) by which androgens up-regulate the IGF-IR do notinvolve binding and transactivation of androgen responseelements in DNA, because two different AR mutants (AR-C619Y and AR-C574R), both devoid of DNA binding capacity andtranscriptional activity, are still able to elicit this effect. AR-C619Y has been described recently as a naturally occurringmutated AR in prostate cancer that is unable to bind DNA invivo and in vitro and to activate transcription (16). The AR-C574R mutant is characterized by a mutation of the DNAbinding domain of the AR and is unable to translocate into thenucleus and to bind DNA (17, 18). Nevertheless, both mutantsinduced IGF-IR up-regulation with an activity similar to theARwt.The existence of AR signaling pathways other than the

classic one involving DNA binding and transactivation hasbeen the focus of numerous recent studies (27, 47–50). Inparticular, it has been shown that dihydrotestosterone leads tothe activation of the ERK pathway, which is insensitive toandrogen blockade (49). This effect was associated to anincreased activity of the transcription factor Elk-1, which inturn activates c-fos expression and may elicit gene transcrip-tion independently of AR binding to DNA response elements(49).According to this model, androgens may induce/activateprimary-response genes via pathways that do not include ARbinding to DNA; these genes will then code for transcriptionalfactors that in turn influence the regulation of secondary-response genes.Interestingly, both the AR-C619Y and the AR-C574R variants,

although transcriptionally inactive, maintain the ability toactivate the ERK pathway. These findings further corroborateour data, indicating that the ERK pathway is involved in theandrogen-induced increase of IGF-IR in prostate cancer cells.Recently, Migliaccio et al. reported that ERK activation byandrogens requires the activation of c-Src (27). In agreementwith these findings, we observed that IGF-IR up-regulation byandrogens was blocked by the MEK-1 inhibitor PD98059 and bythe c-Src inhibitor PP2. Moreover, in cells containing either theARwt or each of the two transcriptionally inactive AR mutants,the increase of luciferase activity driven by the IGF-IR promoterin response to androgen was completely inhibited by cotrans-fection with either a dominant-negative Src or a dominant-negative MEK-1.Taken together, these data strongly suggest that the IGF-IR up-

regulation by androgens involves the activation of a Src-ERKpathway, although it does not involve AR binding to specific DNAresponse elements. The present study therefore also suggests thatnaturally occurring AR mutants in prostate cancer unable to bind

Figure 6. Enhanced biological responses to IGF-I in LNCaP cells exposedto R1881. R1881-pretreated LNCaP cells with up-regulated IGF-IR levels(A) were studied for the biological effects of IGF-I. B, [3H]thymidine incorporationassay: R1881-pretreated LNCaP cells were seeded in 24-multiwell plates,serum starved for 48 hours, and then exposed to 10 nmol/L IGF-I in the presenceor absence of an IGF-IR blocking antibody (aIR-3) for a further 18 hours.After stimulation, [3H]thymidine (0.5 ACi/well) was added for 4 hours andthymidine incorporation into nuclei was measured as described in Materialsand Methods. Columns, means of three independent experiments; bars, SE. **,P = 0.003, IGF-I versus basal; P = 0.007, IGF-I + aIR-3 versus IGF-I(two-tailed Student’s t test for paired values). C, cell migration assay:R1881-pretreated cells were seeded at the top of Boyden chambers coatedat the lower side with 250 Ag/mL collagen VI and stimulated with 10 nmol/L IGF-Iin the presence or absence of an IGF-IR blocking antibody (aIR-3) for18 hours. Migrated cells were stained as described in Materials and Methods.Columns, means of three independent experiments; bars, SE. **,P = 0.001, IGF-I versus basal; *, P = 0.02, IGF-I + aIR-3 versus IGF-I(two-tailed Student’s t test for paired values).

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DNA, such as AR-C619Y, may still sensitize cells to IGF-I and playa role in cancer progression.Agents targeting these signaling pathways or the IGF-IR

itself should therefore be considered as a complement of

standard anti-androgen therapy to induce tumor regression

and possibly reduce the chance or delay the time of tumor

progression to androgen independence. Alternatively, new anti-

androgen compounds able to completely block IGF-IR up-

regulation should be developed.

AcknowledgmentsReceived 5/25/2004; revised 12/20/2004; accepted 12/23/2004.

Grant support: Associazione Italiana per la Ricerca sul Cancro (A. Belfiore andR. Vigneri) and Ministero Italiano Universita e Ricerca, Cofin2002 and Cofin2003(A. Belfiore).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. A.O. Brinkmann for the pSV0-AR expressing plasmid, Dr. G.Castoria for the plasmids encoding for A221-MEK-1 k� and Src k�, Dr. Farsetti forthe plasmid encoding the MMTV-luc reporter gene, Dr. E. Baldi for the AR-transfected PC-3 cells and PC-3-NEO.

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Androgens Up-regulate the Insulin-like Growth Factor-I Receptor in Prostate Cancer Cells

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