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Cancer Research 67, 8932, September 15, 2007. doi: 10.1158/0008-5472.CAN-06-4814
© 2007 American Association for Cancer Research
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Endocrinology

17ß-Estradiol Up-regulates the Insulin-like Growth Factor Receptor through a Nongenotropic Pathway in Prostate Cancer Cells

Giuseppe Pandini1, Marco Genua1, Francesco Frasca1, Sebastiano Squatrito1, Riccardo Vigneri1 and Antonino Belfiore2

1 Endocrinologia, Dipartimento di Medicina Interna e Medicina Specialistica, University of Catania, Ospedale Garibaldi-Nesima, Catania, Italy and 2 Endocrinologia, Dipartimento di Medicina Sperimentale e Clinica, University of Catanzaro, Catanzaro, Italy

Requests for reprints: Riccardo Vigneri, Endocrinologia, Dipartimento di Medicina Interna e Medicina Specialistica, University of Catania, Ospedale Garibaldi-Nesima, via Palermo 632, 95122 Catania, Italy. Phone: 39-095-759-8702; Fax: 39-095-715-8072; E-mail: vigneri{at}unict.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostate carcinomas frequently express estrogen receptors (ER), irrespective of androgen receptor (AR) expression; however, the role of ERs and estrogens in prostate cancer is controversial. We found that 17ß-estradiol (E2) is able to markedly up-regulate insulin-like growth factor (IGF)-I receptor (IGF-IR) mRNA and protein expression in both AR-positive (LNCaP cells) and AR-negative (PC-3 cells) prostate cancer cells. This effect occurs not only via ER{alpha} but also via ERß stimulation and is specific for IGF-IR because it does not involve the cognate insulin receptor. IGF-IR up-regulation is associated with increased IGF-IR phosphorylation and with increased mitogenic and motogenic activities in response to IGF-I. IGF-IR up-regulation by E2 does not require ER binding to DNA and is poorly sensitive to antiestrogen blockade, whereas it is associated with the activation of cytosolic kinase cascades involving Src, extracellular signal–regulated kinase (ERK)-1/2, and, to a lesser extent, phosphatidylinositol 3-kinase and is sensitive to the inhibition of these kinases. In conclusion, our data indicate that estrogens may contribute to IGF system deregulation in prostate cancer through the activation of a nongenotropic pathway. Estrogens may have a role, therefore, in tumor progression to androgen independence. Inhibition of the IGF-IR or the Src-ERK pathway should be considered, therefore, as an adjuvant therapy in prostate cancer. [Cancer Res 2007;67(18):8932–41]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate carcinomas are usually androgen dependent in their initial stages. However, although initially responsive to antiandrogen treatments, these carcinomas eventually progress to an androgen-independent phenotype, for which no efficacious treatment is currently available. Deregulated growth and survival signaling is a characteristic of androgen-independent tumors, implying that factors other than androgens contribute to prostate cancer progression. Accumulating evidence suggests that also estrogens can have an effect on prostate cancer. Most primary prostate cancers express a recently discovered subtype of the estrogen receptor (the ß subtype or ERß; ref. 1) whereas the classic {alpha} subtype (ER{alpha}), which is predominant in breast cancer, is silenced by DNA hypermethylation. Interestingly, the ERß subtype is expressed in most metastases, suggesting that this receptor may be a target in devising new treatments for late-stage prostate cancer (2, 3). Moreover, ER antagonists may inhibit growth and/or induce apoptosis in prostate cancer cell lines that express either only ERß or both ER subtypes (1, 4). Taken together, these data suggest that estrogens could have a direct effect on prostate cancer.

The insulin-like growth factor (IGF) system is another strong candidate among the factors implicated in prostate cancer progression. This system plays a key role in regulating growth, resistance to apoptosis, and invasion in a variety of human malignancies (5, 6), and various lines of evidence suggest a role for the IGF system also in prostate cancer. IGF-I may increase proliferation of prostate cancer cells and protect them from apoptosis (5, 7), whereas antisense-mediated IGF-I receptor (IGF-IR) down-regulation suppresses rat tumor growth in vivo and prevents prostate cancer cell invasiveness (8). Similarly, IGF-IR blockade by monoclonal antibodies induces growth inhibition in human prostate cancer transplanted in immunodeficient mice (9). Moreover, in human prostate cancer cell xenografts, progression to androgen independence is associated with increased expression of both IGF-IR and IGF-I (10, 11) and increased responsiveness to IGF-I (12). Transgenic mice expressing human IGF-I in the prostate basal epithelium have activated IGF-IR and are prone to spontaneous prostate tumorigenesis (13). Finally, epidemiologic studies found an association between borderline to high IGF-I serum levels and prostate cancer risk (14). These data support the hypothesis that the IGF system is involved in prostate cancer, particularly in progression to androgen independence.

A number of studies have shown that estrogens and the IGF system may functionally interact. Most of these studies regard the ER{alpha} subtype and have been carried out in breast cancer MCF-7 cells. In these cells, estrogens are able to up-regulate insulin receptor substrate-1 expression, leading to increased signaling through the insulin receptor substrate-1/phosphatidylinositol 3-kinase (PI3K)/Akt pathway in response to IGF-I (15, 16). Moreover, estrogens increase IGF-I binding and IGF-IR mRNA expression in MCF-7 cells (17), whereas the antiestrogen ICI 182,780 decreases IGF-IR mRNA levels (18). The two ER subtypes, however, affect in a partially different way the IGF system; ER{alpha}, but not ERß, transactivates the IGF-I promoter (19). Down-regulation of IGF-binding proteins is another mechanism through which estrogens may increase IGF-I response (20). The functional interactions between estrogens and the IGF system in prostate cancer are largely unknown.

We now report that estrogens markedly up-regulate the IGF-IR in prostate cancer cells. This effect is specific for IGF-IR because it does not occur for the cognate insulin receptor and can be mediated not only by ER{alpha} but also by ERß. IGF-IR up-regulation by estrogens occurs also in androgen receptor (AR)–negative prostate cancer cells and does not require ER binding to DNA but it requires the activation of the Src-extracellular signal–regulated kinase (ERK)-1/2 pathway. Estrogens may therefore enhance the biological effects of IGFs in prostate cancer cells through a "nongenotropic" pathway (i.e., by a kinase-initiated effect that does not involve steroid receptor binding to canonical steroid response elements on DNA but may eventually affect gene transcription; ref. 21).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell media and all chemicals, unless otherwise stated, were obtained from Sigma. FCS and geneticin (G418) were from Invitrogen Laboratories; IGF-I, LY294002, PD98059, and PP2 were from Calbiochem; synthetic androgen R1881 was from NEN Life Science Products; Fugene6 transfection reagent from Roche Diagnostics; luciferase assay system from Promega Corp.; anti–IGF-IR (clone {alpha}IR3) and anti–v-Src (clone 327) monoclonal antibodies from Merck Chemicals Ltd.; polyclonal anti–IGF-IR antibody and anti-AR (clone 441), anti-ER{alpha} (clone D12), and anti-ERß (H150) antibodies from Santa Cruz Biotechnology, Inc.; anti-phosphotyrosine antibody (4G10) from UBI; and anti–phospho-ERK1/2 and anti-ERK1/2 antibodies from Cell Signaling Technology, Inc. The cDNA encoding the human AR cloned into the expression vector pSV0 was provided by Dr. A.O. Brinkmann (Department of Reproduction and Development, Erasmus University, Rotterdam, the Netherlands). The cDNA encoding the mutated human AR ART877A was provided by Dr. M. Marcelli (Department of Medicine, Baylor College of Medicine and VA Medical Center, Houston, TX). The cDNA encoding the human ER{alpha} cloned into the expression vector pSG5 and the cDNA encoding the human ERß cloned into the expression vector pSG5/puromycin were provided by Prof. P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Illkirch, France). The cDNAs encoding the kinase-inactive mitogen-activated protein kinase/ERK kinase (MEK)-1 (Ser221Ala), the kinase-inactive form of Src (Lys259Met), and the Myc-tagged wild-type p85 were provided by Dr. G. Castoria (Dipartimento di Patologia Generale della II Università di Napoli, Naples, Italy). The cDNA encoding the dominant-negative CCAAT/enhancer binding protein (C/EBP) was provided by Dr. C. Vinson (Laboratory of Metabolism, National Cancer Institute, Center for Cancer Research/NIH, Bethesda, MD). Rat IGF-IR gene promoter sequences corresponding to the full-length fragment (–476/+640), the 5'-flanking fragment (–476/+41), and the 5' untranslated region (5'-UTR; +41/+640) fragment ligated upstream of the firefly luciferase reporter cDNA in the pGL3 vector (22) were provided by Dr. C.T. Roberts, Jr. (Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon). The cDNAs encoding the various mutants of the ligand binding domain of the human ER{alpha}, E-CFP (fused to nontargeted CFP), E-Mem-CFP (with a membrane localization sequence), and E-Nuc-CFP (with a nuclear localization sequence); the plasmid encoding the full-length human ER{alpha} fused to the nontargeted cyan fluorescent protein (ER{alpha}-CFP) as well as the plasmid encoding the serum response element (SRE) ligated to the secreted alkaline phosphatase (SEAP) reporter gene; and the pyrazole compound were all provided by Drs. S. Kousteni and S. Manolagas (Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR).

Cells. AR-positive LNCaP and AR-negative PC-3 human prostate cancer cells, human kidney 293 cells (HEK293; AR and ER negative), and Cos-1 cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as follows: LNCaP cells in RPMI, and PC-3, HEK293, and Cos-1 cells in DMEM, supplemented with 10% fetal bovine serum and 1% glutamine. AR-transfected PC-3 cells and PC-3-Neo cells were provided by Dr. E. Baldi (Department of Clinical Physiopathology, University of Florence, Florence, Italy).

Transient transfection and reporter assays. A transfection mixture containing 1 µg of DNA, 3 µL Fugene6 in 40 µL of medium without serum was added to each well. After 18 h, the medium was changed to serum-containing medium for 30 h. Cells were then serum starved overnight and incubated with 10 nmol/L 17ß-estradiol (E2), R1881, or vehicle for 24 h. For luciferase assay, cells were lysed and processed according to the manufacturer's instructions (Promega Corp.). Luciferase activity was normalized for transfection efficiency using a vector coding for the H2B-GFP reporter gene (pBOS H2B-GFP-N1; provided by Dr. J. Wang, Division of Biological Sciences and the Cancer Center, University of California, San Diego, CA).

For SRE-SEAP activity, supernatant was collected and SEAP activity measured using the Great EscAPe SEAP Chemiluminescence Kit (Clontech Laboratories, Inc.). The activity of each sample was measured by a multilabel counter Wallac 1420 VICTOR 3 (Perkin-Elmer).

IGF-IR and insulin receptor measurement and IGF-I binding studies. Cell lysate preparation and IGF-IR and insulin receptor measurements were carried out by Western blot analysis as previously described (23, 24). For binding studies, LNCaP cells grown to ~60% confluence were serum starved and further cultured in the presence or absence of 10 nmol/L E2, R1881, or vehicle for 24 h. Cells (3 x 106) were then incubated with 125I-IGF-I (10 pmol/L) for further 16 h at 4°C in the presence of increasing concentrations of cold IGF-I and cell-associated radioactivity was then measured in a gamma counter. Scatchard analysis was done with GraphPad Prism 4 software.

IGF-IR and ERK1/2 activation. For IGF-IR activation, cells were serum starved in medium without phenol red 24 h before stimulation with 10 nmol/L IGF-I. Cells were lysed in cold radioimmunoprecipitation assay buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 0.25% sodium deoxycolate, 10 mmol/L sodium pyrophosphate, 1 mmol/L NaF, 1 mmol/L sodium orthovanadate, 2 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL pepstatin, and 10 µg/mL leupeptin. The insoluble material was separated by centrifugation and the supernatants were incubated at 4°C for 2 h with 4 µg of the anti–IGF-IR {alpha}IR3 antibody coated with protein G-Sepharose. Immunoprecipitates were subjected to SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membranes, immunoblotted with anti-phosphotyrosine 4G10 antibody, and detected by enhanced chemiluminescence (ECL). The filter was then stripped with buffer Restore (Pierce) and reprobed with an anti–IGF-IR antibody.

For ERK1/2 activation, cells were stimulated with 10 nmol/L E2 and lysed in Laemmli buffer containing 62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/L DTT, 0.01% bromophenol blue. Cell lysates were subjected to reducing SDS-PAGE on 10% polyacrylamide gel. The resolved proteins were transferred onto nitrocellulose membranes and immunoblotted with anti–phospho-specific ERK1/2 polyclonal antibody. The filters were then stripped and reprobed with anti-ERK1/2 polyclonal antibody.

Analysis of protein-to-protein interactions. Cells were serum starved in medium without phenol red for 24 h and stimulated with E2 or R1881 for 2 min. Cells were lysed in cold RIPA buffer without sodium deoxycolate. The insoluble material was separated by centrifugation and the supernatants were incubated at 4°C under rotation for 18 h with anti–v-Src antibody or anti-ER antibodies. At the end of the incubation, immunocomplexes were separated by adding 30 µL of mix protein A/G-Sepharose for additional 30 min. The pellets were washed with lysis buffer four times and the protein was reduced in 40 µL of Laemmli buffer and subjected to SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membranes, immunoblotted with specific antibody, and detected by ECL.

Reverse transcription-PCR. Total RNA (5 µg) was reverse transcribed with ThermoScript RT (Invitrogen) and Oligo dT primers. Synthesized cDNA (50 ng) was then combined in a PCR reaction using primers 5'-TTTCTGACAACGCCAAGGA-3' (forward) and 5'-CAGGGTAGACGGCAGTTCAA-3' (reverse) specific for AR (fragment size, 341 bp); 5'-GGCTCCGCAAATGCTACGAA-3' (forward) and 5'-AGCGCCAGACGAGACCAATC-3' (reverse) specific for ER{alpha} (fragment size, 462 bp); and 5'-ATACTTGCCCACGAATCTTT-3' (forward) and 5'-TGTGATAACTGGCGATGGAC-3' (reverse) specific for ERß (fragment size, 375 bp). ELE-1 (housekeeping gene) amplification was done with the following primers: 5'-ATTGAAGAAATTGCAGGCTC-3' (forward) and 5'-TGGAGAAGAGAGGCTGTATCT-3' (reverse; fragment size, 280 bp). PCR amplification was carried out for 35 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide.

Real-time PCR. Total RNA (5 µg) was reverse transcribed with ThermoScript RT (Invitrogen) and Oligo dT primers. Synthesized cDNA (25 ng) was then combined in a PCR reaction using primers 5'-GGGCCATCAGGATTGAGAAA-3' (forward) and 5'-CACAGGCCGTGTCGTTGTCA-3' (reverse) specific for the IGF-IR (fragment size, 330 bp). ELE-1 amplification was done as described earlier. Quantitative real-time PCR was done on an ABI Prism 7500 (PE Applied Biosystems) using SYBR Green PCR Master Mix (PE Applied Biosystems) following the manufacturer's instructions. Amplification reactions were checked for the presence of nonspecific products by dissociation curve analysis and agarose gel electrophoresis. Relative quantitative determination of target gene levels was done by comparing {Delta}Ct (25).

Cell cycle, apoptosis evaluation, and cell migration. Cells were grown to ~60% confluence, serum starved in medium without phenol red for 24 h, and further cultured in the presence or absence of 10 nmol/L E2 for 24 h. The medium was then replaced with medium containing 1% stripped serum and, after 4 h, cells were incubated in the presence or absence of 10 nmol/L IGF-I for 8 h.

For cell cycle analysis, cells were harvested and permeabilized in ethanol 70% for 18 h at –20°C, then centrifuged and resuspended in PBS containing 16 µg/mL propidium iodide plus 160 µg/mL RNase, incubated for 30 min in the dark, and then subjected to fluorescence-activated cell sorting (FACS; Coulter Elite flow cytometer, Beckman Coulter).

For apoptosis evaluation, cells were treated as indicated in Annexin V-FITC Apoptosis detection kit I (BD Biosciences). Annexin-positive cells were scored by FACS analysis. Values obtained were expressed as percent of Annexin-positive cells over the total cell population. For cell migration assay, cells were treated as previous reported (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 induces IGF-IR up-regulation in LNCaP prostate cancer cells. LNCaP cells expressed the ß, but not the {alpha}, isoform of the estrogen receptor (ERß), as evaluated by reverse transcription-PCR (RT-PCR; data not shown). When exposed to E2 for 24 h, these cells showed a dose-dependent increase in the expression of IGF-IR protein, whereas its close homologue insulin receptor was not affected (Fig. 1A ). E2 effect on IGF-IR expression was evident at a dose as low as 0.01 nmol/L and reached a maximum at 1 to 100 nmol/L (Fig. 1A). Time-course experiments indicated that IGF-IR expression started to increase after 4-h cell exposure to 10 nmol/L E2 and reached a maximum after 24 h (Fig. 1B). A 24-h incubation was used in all subsequent studies.


Figure 1
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  		Figure 1. IGF-IR up-regulation and increased function of LNCaP cells exposed to E2. A, dose-dependent up-regulation of IGF-IR protein expression by E2. Serum-starved LNCaP cells were incubated in the presence or absence of E2 at the indicated doses for 24 h. Cells lysates were separated by SDS-PAGE and immunoblotted with an anti–IGF-IR antibody. Membranes were reblotted with an anti-IR antibody and an anti–ß-actin antibody to control for protein loading. Representative of three independent experiments. B, time-course regulation of IGF-IR protein expression by E2. Serum-starved LNCaP cells were incubated in the presence or absence of 10 nmol/L E2 for the indicates times. Cells lysates were separated by SDS-PAGE and immunoblotted with an anti–IGF-IR antibody. Membranes were reblotted with an anti–ß-actin antibody. Representative of three independent experiments. C, IGF-I binding. Competition-inhibition curves of 125I-IGF-I binding were carried out in LNCaP cells preincubated with E2 (bullet; 10 nmol/L), R1881 ({blacksquare}; 10 nmol/L), or vehicle ({diamondsuit}). Inset, Scatchard plot analysis of binding data. D, IGF-IR autophosphorylation. Serum-starved LNCaP cells preincubated in the presence or absence of 10 nmol/L E2 for 24 h were exposed to 10 nmol/L IGF-I for the indicated times. IGF-IR was immunopurified with antibody {alpha}IR3 from cell lysates and IGF-IR autophosphorylation was measured by Western blot analysis with an anti-phosphotyrosine antibody. Membranes were reblotted with an anti–IGF-IR antibody.

 
Functional characteristics of IGF-IR in LNCaP cells preincubated with E2. To evaluate possible changes in ligand binding characteristics associated with IGF-IR up-regulation, we carried out a Scatchard plot analysis of 125I-IGF-I binding to LNCaP cells cultured in the presence or absence of 10 nmol/L E2 for 24 h. Cells treated with E2 showed a 4-fold increase of specific IGF-I binding (from 5.5 to 22.7 pmol/L/106 cells) as compared with untreated cells. The dissociation constant (Kd), however, remained similar (0.32 versus 0.40 nmol/L in estrogen-treated or untreated cells, respectively) showing that E2 increases IGF-I binding sites but only minimally affects the receptor affinity for IGF-I (Fig. 1C). In the same experiment, R1881 increased specific IGF-I binding by 7-fold (from 5.5 to 38.7 pmol/L/106 cells; Fig. 1C). To evaluate whether the increased IGF-I binding capacity caused an enhanced IGF-I signaling, we measured IGF-IR autophosphorylation in response to IGF-I in LNCaP cells either preincubated or not with 10 nmol/L E2 for 24 h. Time-course experiments indicated that IGF-IR autophosphorylation in response to IGF-I was markedly enhanced by preincubation with E2 (Fig. 1D).

IGF-IR up-regulation is not due to E2 binding to AR-T877A of LNCaP cells and can be mediated by both ER{alpha} and ERß. The AR expressed by LNCaP cells bears a point mutation, AR-T877A, which affects AR binding specificity (27). To ascertain whether IGF-IR up-regulation could be mediated, at least in part, by E2 binding to AR-T877A, we stably transfected HEK293 cells with either AR-T877A or wild-type AR. As shown in Fig. 2A , E2 was unable to up-regulate IGF-IR in HEK293 cells transfected with either AR-T877A or wild-type AR, whereas R1881 was effective in both cases.


Figure 2
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  		Figure 2. ER{alpha} and ERß, but not the LNCaP mutated AR (AR-T877A), mediate the E2 effect on IGF-IR up-regulation. A, IGF-IR protein expression in AR-transfected cells. HEK293 cells were transfected with plasmids coding for either the wild-type AR (ARwt) or the AR-T877A mutant. Control cells were transfected with an empty vector. Transfected cells were then exposed to 10 nmol/L R1881 or E2 for 24 h and IGF-IR expression was measured by Western blot analysis. Filters were reblotted with an anti–ß-actin antibody. B, IGF-IR up-regulation in ER{alpha}- and ERß-transfected HEK293 cells. Transfected HEK293 cells were exposed to R1881 or E2 (both at 10 nmol/L) for 24 h and IGF-IR expression was measured by Western blot analysis. Filters were reblotted with an anti-ER{alpha} antibody, with an anti-ERß antibody, and then with an anti–ß-actin antibody. C, IGF-IR protein expression. PC-3 cells were stably transfected with plasmids coding for the wild-type AR, and two cell clones (PC-3AR6 and PC-3AR13), expressing AR to different degrees, were selected for further analysis. Serum-starved LNCaP cells, wild-type PC-3 (PC-3wt) cells, and PC-3 selected clones were then exposed to 10 nmol/L R1881 or E2 for 24 h and IGF-IR was detected by Western blot. Membranes were reblotted with an anti–ß-actin antibody. AR, ER{alpha}, and ERß protein expression was also detected by Western blot. D, IGF-I binding. Competition-inhibition curves of 125I-IGF-I binding were carried out in wild-type PC-3 cells preincubated in the presence of E2 (bullet; 10 nmol/L), R1881 ({blacksquare}; 10 nmol/L), or vehicle ({diamondsuit}). Inset, Scatchard plot analysis of binding data.

 
To evaluate whether, in addition to ERß (the only ER subtype expressed by LNCaP cells), ER{alpha} is also able to up-regulate IGF-IR, we transfected HEK293 cells with either ER{alpha} or ERß cDNA (Fig. 2B). Exposure of these cells to E2 resulted in IGF-IR up-regulation in all cases (Fig. 2B), whereas R1881 was ineffective. Taken together, these data indicate that E2 is able to up-regulate IGF-IR by binding to both ER{alpha} and ERß and not by cross-reaction with AR-T877A. We then evaluated whether E2 is able to up-regulate IGF-IR also in AR-negative prostate cancer cells and whether ectopic AR expression has any influence on the effect of E2 in transfected cells. To this aim, we used androgen-independent PC-3 prostate cancer cells that express both ER subtypes ({alpha} and ß) but not AR (confirmed by RT-PCR; data not shown). In addition to wild-type PC-3, we also studied two PC-3 cell clones transfected with AR cDNA and expressing AR to different degrees: the PC-3-AR6 clone, with an AR expression only slightly lower than LNCaP cells, and the PC-3-AR13 clone, with an AR content ~6- to 8-fold lower than LNCaP cells (Fig. 2C). IGF-IR up-regulation by R1881 was strictly related to the AR content in these cell clones and it did not occur in wild-type PC-3. In contrast, E2 up-regulated IGF-IR also in wild-type PC-3. The presence of AR, however, slightly enhanced the effect of E2 (Fig. 2C). To confirm immunoblotting data, we carried out Scatchard plot analysis of 125I-IGF-I binding data in PC-3 cells cultured in the presence or absence of 10 nmol/L E2 for 24 h. E2 treatment increased specific IGF-I binding from 13.1 to 26.3 pmol/L/106 cells. The Kd was similar (0.30 versus 0.22 nmol/L in estrogen-treated or untreated cells, respectively; Fig. 2D). As expected, R1881 was ineffective (Fig. 2D).

Estrogen-induced IGF-IR up-regulation is mediated by a nongenotropic signaling pathway and requires Src/ERK1/2 activity. In previous studies, we found that androgen-mediated IGF-IR up-regulation is due to the activation of a nongenotropic pathway (26). We evaluated, therefore, whether E2-induced IGF-IR up-regulation also depends on a similar mechanism. We first evaluated the effect of two synthetic estrogen-like compounds, estren (4-estren-3{alpha},17ß-diol), which induces only nongenotropic activities of ER, and a pyrazole compound, which induces the transcriptional activities of ER with minimal effects on the nongenotropic pathway (28). To confirm the specific activity of these two ligands in our system, we used a construct encoding for SRE-SEAP that is efficiently induced by both ER{alpha} and ERß when located in the cytoplasm but is repressed by ERs present in the nucleus (21). As expected, estren, but not pyrazole, stimulated SEAP activity (Fig. 3A ). In LNCaP cells, estren stimulated IGF-IR up-regulation to a similar degree as E2, whereas pyrazole was ineffective (Fig. 3A), suggesting that nongenotropic pathways mediate IGF-IR up-regulation.


Figure 3
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  		Figure 3. IGF-IR up-regulation by E2 occurs via a nongenotropic pathway. A, IGF-IR protein expression was up-regulated by E2 or estren but not by pyrazole through a nongenotropic pathway. After cell exposure to E2, pyrazole, or estren (10 nmol/L) for 24 h, E2 and estren (as well as R1881 used as control), but not pyrazole, stimulated IGF-IR up-regulation in LNCaP cells. To confirm the nongenotropic activity induced by E2 and estren, LNCaP cells were transiently transfected with a plasmid coding for a reporter construct in which SRE drives the expression of SEAP. Again, E2 and estren, but not pyrazole, stimulated SEAP activity (top). B, inhibition of estrogen-induced IGF-IR up-regulation by kinase inhibitors. In LNCaP cells, E2 activated ERK1/2 phosphorylation, as evaluated by Western blot analysis with a phosphospecific anti–phospho ERK1/2 antibody (top). LNCaP cells were then incubated with 10 nmol/L E2 for 24 h in the presence or absence of 50 µmol/L PD98059 (MEK-1 inhibitor), 10 µmol/L PP2 (Src inhibitor), or 20 µmol/L LY294002 (PI3K inhibitor; middle). Parallel experiments were carried out in HEK293 cells transfected with either ER{alpha} or ERß cDNAs (bottom). IGF-IR expression was then measured by Western blot analysis. Membranes were reblotted with an anti–ß-actin antibody to control for protein loading. C, the specific antiestrogen ICI 182,780 does not affect IGF-IR up-regulation. Serum-starved LNCaP cells were treated with R1881 or E2 (both 10 nmol/L) in the presence or absence of ICI 182,780 (1 or 10 µmol/L) for 24 h. IGF-IR expression was then measured by Western blot analysis. Membranes were reblotted with an anti–ß-actin antibody to control for protein loading. D, E2 and R1881 induce the association of ER with Src, p85, and AR. Left, serum-starved LNCaP cells were treated with R1881 or E2 (both 10 nmol/L) for 2 min. Cell lysates were immunoprecipitated with an anti-Src antibody or with control antibody and the association with ERß, p85 of PI3K, or AR was evaluated by immunoblotting with specific antibodies. In the same cell lysates, ERß was also immunoprecipitated with an anti-ERß antibody and association with AR and p85 was measured by immunoblotting. Right, HEK293 cells were cotransfected with Myc-tagged wild-type p85 and either ER{alpha} or ERß cDNAs or the empty vector. Cell lysates were then immunoprecipitated with an anti-ER{alpha} or an anti-ERß antibody and immunoblotted with specific antibodies to Myc-tag or Src. Representative of three independent experiments.

 
We next evaluated the possible involvement of the Src/Raf-1/ERK pathway. Indeed, exposure of LNCaP cells to E2 increased ERK1/2 phosphorylation (Fig. 3B, top). LNCaP cells were then incubated with E2 in the presence or absence of various kinase inhibitors, including PD98059, a MEK-1 inhibitor, PP2, a Src inhibitor, and LY294002, a PI3K inhibitor. IGF-IR up-regulation by E2 was almost completely blocked by both PD98059 (50 µmol/L) and PP2 (10 µmol/L), whereas LY294002 (20 µmol/L) was less effective (Fig. 3B, middle). Similar results were obtained in HEK293 cells transfected with either ER{alpha} or ERß, although the effect of LY294002 was only evident in cells transfected with ER{alpha} (Fig. 3B, bottom). The antiestrogen ICI 182,780, which is an inhibitor of ER transcription, only slightly inhibited E2-induced IGF-IR up-regulation in LNCaP cells (Fig. 3C).

To confirm the involvement of Src, LNCaP cells were stimulated with either E2 or R1881 and then lysed and immunoprecipitated with anti-Src antibody. Immunoblotting with anti-ERß antibodies confirmed that E2 stimulation and, to a much lower extent, R1881 induced ERß association to Src. R1881, but not E2, induced AR association to Src (Fig. 3D). Both R1881 and E2 induced recruitment to Src of p85, the regulatory subunit of PI3K. Moreover, immunoprecipitation with anti-ERß antibody showed that ERß associates with AR following stimulation with either E2 or R1881 (Fig. 3D). Exposure to E2 also stimulated the association of ERß with the p85 subunit of PI3K (Fig. 3D). We also studied the ability of E2 to induce association of either ER{alpha} or ERß with p85 and Src in the absence of AR. To this aim, we cotransfected HEK293 cells with a myc-tagged p85 construct together with either ER{alpha} or ERß cDNA. Cells incubated in the presence or absence of E2 were immunoprecipitated with anti-ER antibodies and blotted with anti-myc or anti-Src antibodies. Cell exposure to E2 induced association of both ER{alpha} and ERß to p85 and Src (Fig. 3D).

Taken together, these data indicate that E2-induced IGF-IR up-regulation occurs through the activation of nongenotropic signaling pathways involving Src and ERK1/2 activation, and that E2 stimulates the association of both ER{alpha} and ERß with Src and the p85 subunit of PI3K. In LNCaP cells, E2 also induces AR association with Src and ER.

E2 stimulates IGF-IR mRNA expression and IGF-IR promoter activity. To evaluate whether IGF-IR protein up-regulation after cell exposure to E2 was due to reduced degradation or increased synthesis, LNCaP cells were incubated for 24 h with 10 nmol/L E2 in the absence or presence of either actinomycin D or cycloheximide. Both compounds completely inhibited IGF-IR up-regulation by E2, suggesting that both de novo mRNA and protein synthesis are required for this effect (Fig. 4A ). IGF-IR mRNA expression, measured by quantitative real-time PCR, increased by ~4-fold after 10 nmol/L E2 for 24 h; this increase was also blocked by either actinomycin D or cycloheximide (Fig. 4B). Dose-response experiments indicated that IGF-IR mRNA started to increase with cell exposure to 0.01 nmol/L E2 and reached maximum levels with 10 nmol/L E2 (Fig. 4C). IGF-IR mRNA started to increase at 2 to 4 h after exposure to 10 nmol/L E2 and reached maximum levels at 24 h (Fig. 4D).


Figure 4
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  		Figure 4. IGF-IR mRNA expression is up-regulated by E2 in LNCaP cells. A, E2-induced IGF-IR up-regulation requires both de novo mRNA and protein synthesis. Serum-starved LNCaP cells were incubated with 10 nmol/L E2 in the presence or absence of either actinomycin D (1 µg/mL) or cyclohexemide (10 µg/mL). IGF-IR protein was detected by immunoblotting and ß-actin was used to control for protein load. B, total RNA prepared from LNCaP cells was used as template for real-time quantitative PCR for IGF-IR mRNA. Relative mRNA amounts were normalized to the abundance of the ELE-1 mRNA. R1881 (10 nmol/L) is shown as positive control. Columns, means of three separate experiments; bars, SD. C, dose-dependent increase of IGF-IR mRNA by E2. Serum-starved LNCaP cells were incubated in the presence or absence of the indicated doses of E2 for 24 h; total RNA prepared from LNCaP cells was used as template for quantitative real-time PCR. Relative mRNA amounts were normalized to the abundance of the ELE-1 mRNA. Columns, mean of three separate experiments; bars, SD. D, time-course regulation of IGF-IR mRNA expression. Serum-starved LNCaP cells were incubated with or without 10 nmol/L E2 for the indicated times. Total RNA prepared from LNCaP cells was used as template for quantitative real-time PCR. Relative mRNA amounts were normalized to the abundance of the ELE-1 mRNA. Columns, mean of three separate experiments; bars, SD.

 
To evaluate whether the increased mRNA expression in response to E2 was reflected by an increased activity of the IGF-IR promoter, we transiently transfected HEK293 cells with a plasmid encoding the wild-type ER together with one of the three following luciferase constructs, each containing sequences of the IGF-IR promoter: the full-length promoter region (bp –476/+640), the 5'-flanking fragment (bp –41/+640), and the 5'-UTR fragment (–476/+41). Cotransfection with a green fluorescent protein (GFP) vector was used to normalize for transfection efficiency. After exposure to E2, an increase in luciferase activity was observed in cells transfected with either the full-length fragment or the 5'-UTR promoter fragment (142 ± 12% and 138 ± 16% of unstimulated cells, respectively). In contrast, no luciferase increase was seen in cells transfected with the 5'-flanking fragment, indicating that the sequences responsible for E2 stimulation are between bp –41 and +640 of the IGF-IR promoter region (not shown). We then evaluated the full-length IGF-IR promoter activity after cotransfection with either ER{alpha} or ERß or AR-T877A. As expected, the promoter activity was increased by E2 in the presence of ER{alpha} or ERß but not AR-T877A (Fig. 5A ).


Figure 5
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  		Figure 5. IGF-IR promoter activity is enhanced by E2 via ER nongenotropic activity. A, IGF-IR promoter activity is up-regulated by E2 binding to ER{alpha} or ERß and not to AR-T877A. HEK293 cells were transiently cotransfected with plasmids coding for ER{alpha}, ERß, or the mutant receptor AR-T877A and the IGF-IR/luciferase vector containing the full-length IGF-IR promoter fragment. Transfected cells were serum starved for 16 h and then exposed to 10 nmol/L of either E2 or R1881 for 24 h, and IGF-IR promoter activity was assayed. Columns, mean of three separate experiments, normalized for transfection efficiency with a GFP vector; bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, basal versus stimulated (two-tailed Student's t test for paired values). B, subcellular distribution of ER-targeted mutants and nongenotropic activity induced by E2 binding to ER mutants. Photomicrographs obtained by epifluorescence microscopy of Cos-1 cells transfected with either expression vectors coding for the full-length ER{alpha} fused to the nontargeted CFP (ER{alpha}-CFP) or with expression vectors coding for the E domain of ER{alpha} fused to the nontargeted CFP (E-CFP), membrane-targeted CFP (E-Mem-CFP), or nuclear-targeted CFP (E-Nuc-CFP; top). SEAP activity in HEK293 cells transiently transfected with a plasmid coding for ER{alpha}-CFP, E-CFP, E-Mem-CFP, or E-Nuc-CFP together with a SRE-SEAP reporter (bottom). Serum-starved cells were exposed or not to E2 10 nmol/L for 24 h. Supernatants were collected and SEAP activity was assayed as described in Materials and Methods. SEAP activity was not observed when using nuclear-targeted (E-Nuc-CFP) plasmid. C, IGF-IR promoter activity induced by E2 binding to ER mutants. HEK293 cells were transiently cotransfected with a plasmid coding for either the ER{alpha}-CFP or each of the ER mutants (E, E-Mem, and E-Nuc) together with the IGF-IR/luciferase vector containing the full-length IGF-IR promoter fragment. Transfected cells were serum starved for 16 h and then exposed to 10 nmol/L E2 or estren for 24 h; cellular lysates were collected and IGF-IR promoter activity was assayed. No effect of estrogen on the IGF-IR promoter is observed when using nuclear-targeted (E-Nuc-CFP) plasmid. Columns, means of three separate experiments, normalized for transfection efficiency with a GFP vector; bars, SD. *, P < 0.05, basal versus stimulated (two-tailed Student's t test for paired values). D, E2-induced IGF-IR promoter activation requires MEK-1 and C/EBPß. HEK293 cells transfected with either ER{alpha} or ERß were transfected with dominant-negative plasmids for MEK-1 or C/EBP or the corresponding empty vector. Both dominant-negatives completely blocked E2-induced promoter activity.

 
We then evaluated whether IGF-IR promoter activity stimulation by E2 also involved a nongenotropic signal. To this aim, we transfected HEK293 cells with plasmids encoding either an ER mutant targeted to the plasma membrane (E-Mem-CFP) or an ER mutant targeted to the nucleus (E-Nuc-CFP). The first ER form is unable to bind to DNA but retains the ability to activate the ERK pathway, whereas the second ER form is entrapped in the nucleus and unable to activate the ERK pathway. Receptor localization in transfected cells was confirmed by immunofluorescence (Fig. 5B, top). To confirm the specific activity of the two mutant receptors, we used a SRE-SEAP construct that is induced by the ERs located in the cytoplasm but is repressed by ERs present in the nucleus (21). As expected, E2 induced SRE-SEAP activity in the presence of E-Mem-CFP but not in the presence of E-Nuc-CFP (Fig. 5B, bottom). HEK293 cells were then transiently cotransfected with plasmids encoding each of the mutant ER constructs and luciferase constructs containing the full-length IGF-IR promoter. Both E2 and estren were able to stimulate the IGF-IR promoter activity in cells transfected with the DNA binding–defective E-Mem-CFP but not in cells transfected with the ER mutant targeted to the nucleus (E-Nuc-CFP; Fig. 5C). To gain additional insight into IGF-IR promoter regulation by E2, we cotransfected HEK293 cells with a luciferase construct containing the full-length IGF-IR promoter together with either ER{alpha} or ERß cDNA and with plasmids encoding either a dominant-negative MEK-1 or a dominant-negative C/EBP transcription factor. Both dominant-negatives completely blocked IGF-IR promoter activity in response to E2 (Fig. 5D).

Biological effects of estrogen-induced IGF-IR up-regulation. We aimed to evaluate whether E2-induced IGF-IR up-regulation enhanced the biological effects of IGF-I. To ascertain whether AR was required for E2 effects, we carried out parallel experiments in both LNCaP and PC-3 cells. IGF-I was a weak stimulator of LNCaP and PC-3 cell proliferation. However, preincubation with E2 significantly enhanced cell proliferation in response to IGF-I. The proportion of cells in S phase increased, on average, from ~8% to 14% in LNCaP cells and from ~34% to 40% in PC-3 cells (Fig. 6A and B ). These differences were statistically significant (P < 0.01). A similar enhancing effect of E2 preincubation was observed with regard to the antiapoptotic effect of IGF-I. IGF-I was barely protective from starvation-induced apoptosis (Annexin staining) in untreated LNCaP cells and virtually ineffective in PC-3 cells, which were less sensitive to serum starvation. However, IGF-I significantly reduced apoptosis in E2-preincubated cells both in LNCaP and PC-3 (Fig. 6C). We also studied migration in cells seeded in Boyden chambers, coated at the lower side with 250 µg/mL collagen, after stimulation with 10 nmol/L IGF-I. Also in this case, the effect of IGF-I was very small or absent in cells not preincubated with E2. In contrast, IGF-I consistently stimulated migration in E2-preincubated cells, although this effect did not reach statistical significance (data not shown).


Figure 6
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  		Figure 6. Biological responses to IGF-I of prostate cancer cells pre-exposed to E2. Serum-starved LNCaP and PC-3 cells preincubated with or without 10 nmol/L E2 for 24 h were cultured for 4 h in the presence of 1% charcoal-stripped FCS and then incubated in the presence or absence of IGF-I 10 nmol/L for 8 h. S-phase and apoptosis rates were then scored. A, for cell cycle analysis, cells were subjected to FACS analysis as described in Materials and Methods. Cell populations positive to propidium iodide staining were evaluated and G0-G1, S, and G2-M rates were scored. The histogram plots were obtained after gating (inset) and represent typical experiments with LNCaP (left) and PC-3 (right) cells, respectively. B, columns, mean proportion of cells in S phase from five separate experiments in LNCaP (left) and PC-3 (right) cells; bars, SE. **, P < 0.01, IGF-I versus basal in E2-preincubated cells (two-tailed Student's t test for paired values). C, apoptotic cells were evaluated by Annexin staining and expressed as percent of Annexin-positive cells over the total scored cell population. Columns, mean of five separate experiments in LNCaP (left) and PC-3 (right) cells; bars, SE. **, P < 0.01; ***, P < 0.001, IGF-I versus basal (E2-treated) cells (two-tailed Student's t test for paired values).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IGF system plays a key role in the development and maintenance of the transformed phenotype in a variety of experimental models (5, 6, 29) and human malignancies, including prostate cancer (5, 22, 30–33). In this tumor, deregulation of various components of the IGF system has been reported at different tumor stages (11, 34, 35) and may play a role in prostate cancer progression to androgen-independence (10). We previously reported up-regulation of IGF-IR in LNCaP prostate cancer cells by androgens (26). In other models, such as breast cancer, several components of the IGF system may be up-regulated by estrogens through the classic ER{alpha} (17, 36, 37).

In the present study, therefore, we investigated whether E2 up-regulates IGF-IR also in prostate cancer cells, and found that both IGF-IR content and autophosphorylation are increased after exposure to E2 in both AR-positive and AR-negative prostate cancer cells. Moreover, E2 sensitizes prostate cancer cells to the biological effects of IGF-I. These E2 effects do not require ER binding to specific DNA sequences (estrogen response elements) but rather involve the activation of cytosolic kinases such as Src and ERK1/2 that subsequently induce an increase of IGF-IR promoter activity and gene transcription. This E2 effect on IGF-IR expression must be considered, therefore, as a nongenotropic action of estrogens. Various lines of experimental evidence support this conclusion. First, the Src inhibitor PP2 and the MEK-1 inhibitor PD98059 completely block the IGF-IR protein up-regulation by E2, both in LNCaP cells and in HEK293 transfected with either ER{alpha} or ERß. The PI3K inhibitor LY294002 partially blocks the E2 effect in LNCaP cells, suggesting also an involvement of PI3K. Moreover, E2 exposure stimulates the association of both ER{alpha} and ERß with Src and p85, the regulatory subunit of PI3K. Second, estren, a synthetic ER ligand that induces ERK1/2 and Elk-1 activation without affecting ER binding to DNA (38), reproduces this E2 effect. In contrast, pyrazole, a compound that activates only the genotropic actions of E2, is ineffective. Third, E2 activates the IGF-IR promoter in cells transfected with a mutant ER devoid of DNA binding activity while retaining the nongenotropic activity; in contrast, a mutant ER that localizes in the nucleus and is devoid of nongenotropic activity is without effect.

The present results showing a major role of Src in mediating E2 effects are supported by previous studies indicating that E2 activates a Src-dependent pathway by inducing an interaction between the ER phosphotyrosine 537 and the SH2 domain of Src (39, 40). Other studies have also shown that ER, Src, and p85 form a ternary complex, whose assembly is stimulated by E2 and which induces the activation of both the Src and the PI3K/Akt pathways (39). Activation of these kinase cascades will eventually affect gene expression by affecting multiple transcription factors, including Elk-1, which in turn activates expression of c-fos and down-regulation of C/EBPß and c-Jun (21). Our preliminary results show that inhibition of C/EBPß actually blocks E2-induced IGF-IR promoter activity. This is a novel observation, and we are currently investigating the exact mechanism involving C/EBPß in this regulation.

Because in LNCaP cells, both DHT and the synthetic androgen R1881 are potent inducers of IGF-IR (26) via the mutated AR (AR-T877A) expressed in these cells, we evaluated the possibility that the effect of E2 could occur by cross-reaction with AR-T877A. Experiments in transfected HEK293 cells indicated that IGF-IR up-regulation after E2 occurred via both ER subtypes but neither via wild-type AR nor via AR-T877A.

Data about ER subtype expression and functions in prostate epithelial cancer cells are limited. The classic ER{alpha} subtype is not expressed in normal prostate epithelium but is expressed in prostate stromal cells (1, 41), suggesting that estrogen effects on the prostate were mediated by factors produced by stromal cells. Then the ERß subtype was discovered (42) and found expressed by the prostate malignant epithelium as well as ER{alpha} (41). However, the precise roles of ER subtypes in prostate cancer are unknown. In breast cancer, ER{alpha} is the major modulator of estrogen tumor-promoting effects whereas the role of ERß is still controversial (43–46). In the prostate, ERß is frequently expressed in dysplastic and cancerous prostate tissues and also in metastases (3, 47) whereas the ER{alpha} gene is often silenced. When expressed as the only ER subtype, ERß may stimulate cell proliferation (47). Therefore, the relative abundance of the two ER subtypes may be important in determining the final biological effect. Our study suggests that, as far as nongenotropic effects are concerned, both receptor subtypes behave similarly.

In summary, our data show that E2 specifically up-regulates IGF-IR in prostate cancer cells and sensitizes cancer cells to the biological effects of IGF-I. The E2 effect can occur through both ER{alpha} and ERß and does not involve ER binding to DNA but rather the activation of kinase cascades initiated by the association between ER-Src and PI3K and followed by ERK1/2 phosphorylation. AR expression is not required, although AR coexpression may potentiate the E2 effect by associating with ER. These data raise several potential implications in prostate cancer development and treatment. First, estrogens may enhance the biological effects of IGFs by up-regulating IGF-IR both in AR-positive and AR-negative prostate cancers. Second, because ERß is expressed in both normal and precancerous prostate epithelium, it is possible that environmental xenoestrogens may mimic the nongenotropic effects of E2 and represent a risk factor for prostate cancer. Third, the modest results observed with the antiestrogen tamoxifen in prostate cancer (48) may be partially explained by the fact that tamoxifen is a partial ER agonist. Fourth, we now show that also the pure antiestrogen ICI 182,780 is only partially effective in antagonizing E2-induced IGF-IR up-regulation, whereas Src and ERK1/2 inhibitors can better block this effect. In light of these observations, inhibitors of the IGF-IR or the Src-ERK pathway should be considered as an adjuvant therapy in prostate cancer.


   Acknowledgments
 
Grant support: Associazione Italiana per la Ricerca sul Cancro (A. Belfiore and R. Vigneri) and PRIN-MIUR (Ministero Italiano Università e Ricerca) grants 2005055874-002 and 2005063915-004. G. Pandini is a recipient of a fellowship from the American Italian Cancer Foundation.

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.

We thank all researchers who have provided materials that have made this work possible: Dr. P. Chambon for the ER{alpha} and ERß expression plasmids, Dr. G. Castoria for MEK-1 and Src mutant and the Myc-tagged wild-type p85 constructs, Dr. C.T. Roberts, Jr. for IGF-IR gene promoter constructs, Drs. S. Kousteni and S. Manolagas for targeted and nontargeted constructs encoding the ER ligand binding domain, Dr. M. Marcelli for the ART877A expression vector, Dr. A.O. Brinkmann for the AR expression vector, Dr. E. Baldi for AR-transfected PC-3 cells, and Dr. C. Vinson for the dominant-negative C/EBP expression vector.


   Footnotes
 
Note: G. Pandini and M. Genua contributed equally to this work.

Received 12/31/06. Revised 6/11/07. Accepted 7/10/07.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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