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Department of Biochemistry and Molecular Biology (B.S.S., M.-F.L., J.C.B., R.G.M.), University of Nebraska Medical Center, Omaha, Nebraska 68198-4525; and Division of Life Sciences (J.H.Y.P.), Hallym University, Chunchon, Korea 200-702
Address all correspondence and requests for reprints to: Richard G. MacDonald, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, Nebraska 68198-4525. E-mail: rgmacdon{at}unmc.edu.
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Abstract |
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Introduction |
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Binding of IGF-II to the IGF2R results in the internalization and subsequent degradation of the ligand in the lysosomes (12), thereby suppressing mitogenesis by reducing IGF-II availability for binding to the IGF-I receptor. Overexpression of IGF-II has been reported in breast cancer, hepatoma, smooth muscle tumors, liposarcoma, bladder carcinoma, paraganglioma, and Wilms’ tumor (13, 14). Analysis of liver metastases of colorectal carcinoma revealed increased IGF-II expression at the invasive margin, compared with the center of the tumor, suggesting that IGF-II may play a role in metastasis (15). Increased IGF-II mRNA (16), loss or relaxation of IGF-II imprinting (17), and overexpression of IGF-II (14) have also been observed in prostate cancer, implying that IGF-II is an important mitogen for and could play a role in metastasis of prostate cancer.
The IGF2R has recently been identified as a potential tumor suppressor. Loss of heterozygosity (LOH) at the IGF2R locus (chromosome 6q26-q27) coupled with mutations in the remaining allele are thought to be early events in human hepatocellular carcinoma (4). LOH at chromosome 6 has also been identified in breast cancer (4), non-Hodgkin’s lymphoma (18), malignant melanoma (19), ovarian cancer, renal cell carcinoma, and squamous cell lung carcinoma (4), suggesting that alterations of chromosome 6 are a common event in the initiation and/or progression of cancers. An analysis of genetic alterations in recurrent, androgen-insensitive prostate tumors identified loss of 6q24-qter, which encompasses the IGF2R gene, as a common, minimal loss in 27% (10/37) of the tumors studied (20), suggesting that loss of the IGF2R locus is also common in androgen-insensitive prostate cancer.
Thus, previous work suggests that increased IGF-II and/or decreased IGF2R could play a role in the growth of prostate cancer cells. In the present study, analysis of the expression and function of the IGF2R in the lymph node carcinoma of the prostate (LNCaP) cells suggested that decreased IGF2R could partially account for increased growth of late-passage LNCaP cells, relative to low-passage cells. Contrary to our expectations, expression of IGF2RMyc resulted in increased proliferation of high-passage LNCaP cells, compared with control cells. However, expression of IGF2RMyc in PC-3 cells resulted in decreased proliferation. Expression of IGF2RMyc I/T, a mutant receptor incapable of binding IGF-II, resulted in increased proliferation, when compared with control cells, in both cell lines. Our data suggest a complex role for the IGF2R in growth regulation of prostate cancer cells, including both IGF-II-dependent growth-inhibitory and Man-6-P-dependent growth-stimulatory functions.
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Materials and Methods |
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Cell culture
LNCaP and PC-3 cells were maintained in RPMI 1640 medium supplemented with 7% FBS, 2 mM L-glutamine, plus 50 µg/ml gentamicin, and incubated at 37 C in a humidified 5% CO2-95% air environment. Cells were provided fresh growth medium every 2–3 d. LNCaP cells were subcultured 1:10, once per week, using 0.05% trypsin and 0.53 mM EDTA. PC-3 cells were subcultured 1:5, before confluence, using 0.05% trypsin and 0.53 mM EDTA; 293T cells were maintained in DMEM supplemented with 5% FBS plus 5 µg/ml gentamicin, incubated at 37 C in a humidified 5% CO2-95% air environment, and subcultured 1:10, twice per week, using 0.05% trypsin and 0.53 mM EDTA.
Growth curves of LNCaP cells
For growth analysis of LNCaP cells, cells of different passage were plated in 12-well plates at 5 x 104 cells per well and provided fresh, complete medium every 2 d. On the first, second, and every other day thereafter, the medium was aspirated, and the cells were incubated for 3 h with 1 ml phenol-red free RPMI 1640 containing 1 mg/ml 3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyl tetrazolium bromide (MTT). After a 3-h incubation at 37 C in a humidified 5% CO2-95% air environment, the excess MTT was aspirated, and the dark-blue formazan product was solubilized with isopropanol. The optical density of the resulting purple solution was measured at 570 nm specific absorbance vs. 690 nm background absorbance on a UV Max Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA).
Plasma membrane preparation
Crude plasma membranes were prepared as previously described (21). Briefly, LNCaP cells or PC-3 clones were subcultured 1:10 in 150-mm dishes and grown for 6 d in complete medium. On the seventh day, cells were scraped into Tris-buffered isotonic sucrose (10 mM Tris-HCl, pH 7.4; 1 mM Na2EDTA; 0.25 M sucrose) followed by Dounce homogenization and centrifugation. Final membrane pellets were resuspended in TE (10 mM Tris-HCl, pH 7.4; 1 mM Na2EDTA) supplemented with 1 mM phenylmethylsulfonylfluoride and antiprotease cocktail (10 µg/ml leupeptin, 80 µg/ml benzamidine, 10 µg/ml antipain, and 20 µg/ml aprotinin). Protein concentrations were measured using the bicinchoninic acid assay, and plasma membrane suspensions were stored at -20 C until needed. These preparations are enriched for plasma membranes, but high-density intracellular membranes are also present, so they are operationally referred to as crude plasma membranes.
Preparation of whole-cell lysates
Cell lysates were prepared as previously described (22). Briefly, the cells were solubilized in ice-cold HEPES (10 mM, pH 7.4), 1 mM MgCl2, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, and antiprotease cocktail. After a 1-h incubation at 4 C, the suspension was centrifuged at 12,000 x g for 10 min, and the supernatant was collected and stored at -80 C until needed.
Preparation of conditioned media
LNCaP cells of different passages were subcultured 1:10 and grown to approximately 90% confluence. Cells were washed with serum-free RPMI 1640 and then incubated with fresh serum-free RPMI 1640 for 72 h before harvesting the conditioned media. To analyze accumulation of IGF2R ligands in Myc-tagged IGF2R-expressing cells, RPMI 1640 supplemented with 2% FBS was added to the cells the day after transfection and harvested 72 h later. Conditioned media were stored at -20 C until needed.
Purification and iodination of IGF2R from FBS
The soluble form of the IGF2R (sIGF2R) was purified from FBS (HyClone Laboratories, Inc., Logan, UT) as described previously (23). Briefly, the FBS was diluted with buffer, bound to a pentamannose phosphate-Sepharose affinity column, and eluted with Man-6-P. Fractions were analyzed for sIGF2R by SDS-PAGE followed by Coomassie blue staining. Densitometer analysis indicated the isolated sIGF2Rs were approximately 95% pure. The fractions containing sIGF2R were pooled, dialyzed against Man-6-P-free buffer, lyophilized, and stored at -20 C until needed. Approximately 20 µg lyophilized sIGF2R was dissolved in 0.3 M sodium phosphate, pH 7.4, and added to a pre-wet IODO-GEN-coated tube (Pierce, Rockford, IL). The Na 125I (1.5 mCi) was then added, followed by a 15-min incubation. The radiolabeled sIGF2R was purified on a Sephadex G-50 (Amersham Pharmacia Biotech) column. Radioactive fractions from the void volume were pooled and stored at -20 C until needed.
Preparation of 125I-IGF-II
Approximately 13 µg (10 µl) human IGF-II (provided by Lilly Research Laboratories) was diluted in 0.3 M sodium phosphate buffer (50 µl), pH 7.4, and incubated for 30 min with 2 mCi Na125I, 50 µl Enzymobead reagent (reconstituted in water), and 0.135% glucose (25 µl 1% stock). A 30-ml Sephadex G-50 column equilibrated with PBS (10 mM sodium phosphate, pH 7.4, 0.9% NaCl) containing 1% BSA was used to separate the free iodine from the 125I-IGF-II. Radioactive fractions containing IGF-II were pooled and stored at -20 C until needed.
Affinity cross-linking of 125I-IGF-II to the IGF2R
Crude plasma membranes (0.1 mg protein) were incubated overnight with 2 nM 125I-IGF-II, with or without 500 nM unlabeled IGF-II, on an end-over-end mixer at 4 C. The IGF2R/IGF-II complexes were cross-linked by incubating with 0.25 mM dissucinimidyl suberate for 30 min at 4 C. The reactions were quenched by adding 0.8 ml 0.1 M Tris-HCl, pH 7.4, and incubating for 15 min at 4 C. Membrane proteins were electrophoresed on 6% SDS-PAGE, and the gel was stained with Coomassie blue, destained, dried, and visualized by autoradiography. Radioactivity associated with receptor bands was quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
IGF2R immunoblot
For anti-13D antibody (1:500 dilution) detection of IGF2R on immunoblots, crude plasma membranes (100 µg protein) were reduced and alkylated under denaturing conditions, electrophoresed on 6% SDS-PAGE as previously described (24), and electroblotted to BA85 nitrocellulose (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). For detection using the anti-IGF2R antibody provided by Dr. Peter Lobel (1:4000 dilution), crude plasma membranes (100 µg protein) or whole-cell lysates (100 µg protein) were electrophoresed as above without the reduction and alkylation steps. The immunoblots were blocked with 4% nonfat dry milk in TBST (15 mM Tris-HCl, pH 7.4; 0.15 M NaCl; 0.1% Tween-20), probed with the anti-IGF2R antibody, detected with 125I-protein A, and visualized by autoradiography. Radioactivity was quantified by PhosphorImager. The IGF2R antibody from Dr. Peter Lobel was used in all IGF2R immunoblots shown in this manuscript.
Immunoblot of IGF-II secreted into medium
Aliquots of conditioned medium (1 mg protein) were concentrated using Centricon-3 (Amicon, Beverly, MA) microconcentrators (molecular weight cut-off of 3000), lyophilized, redissolved in water, electrophoresed on 15% SDS-PAGE, and electroblotted to Immobilon-P (Millipore Corp., Bedford, MA). Immunoblots were blocked with 4% nonfat dry milk in TBST for 1 h at room temperature and incubated overnight at 4 C with anti-IGF-II antibody (1:1000 dilution). The following day, the immunoblots were washed with TBST, followed by incubation with rabbit antimouse IgG (1:1000 dilution), washed with TBST, and detected by 125I-protein A followed by autoradiography. Immunoblots for pro-IGF-II were done in the same manner, except a 1:500 dilution of the primary antibody was used and the incubation with the secondary antibody was omitted. Immunoblots of IGF-II in conditioned medium from transfected LNCaP cells were done in the same manner, except aliquots of conditioned medium (0.1 mg protein) were electrophoresed without prior concentration.
Detection of phosphomannosylated ligands
To detect the phosphomannosylated ligands secreted by LNCaP cells, equal vols (5–10 ml) or equal protein (1 mg) of 72-h conditioned media were incubated overnight at 4 C with sIGF2R bound to Sepharose 4B. After incubation, the resin pellets were collected, washed twice with HBS (50 mM HEPES, pH 7.4; 150 mM NaCl) + 0.05% Triton X-100, and eluted at 100 C in SDS-PAGE sample buffer. The resin-bound proteins were then electrophoresed on 12% SDS-PAGE and electroblotted to BA85 nitrocellulose. Ligand blot analysis was done according to the method of Hossenlopp et al. (25), using 125I-sIGF2R as the probe, and visualized by autoradiography.
Transfection of high-passage LNCaP cells
LNCaP cells at passage 125 or greater were seeded at 5 x 104 or 3 x 105 cells per well in 12- or 6-well plates, respectively. The following day, cells were transfected with the indicated cDNA using the Lipofectamine Plus (Invitrogen Corp.) transfection reagent, following the manufacturer’s protocol. Briefly, 0.5 µg or 1.0 µg DNA was used to transfect each well of 12- or 6-well plates, respectively, using a ratio of 6:4:1 of Plus Reagent:Lipofectamine:DNA. The following day, transfection medium was replaced with RPMI 1640 + 2% FBS. Growth was estimated on the indicated days by MTT assay. Cells of six-well transfected plates were used to prepare 1% Triton X-100 cell lysates as previously described (22). Cell lysates (0.1 mg protein) were subjected to SDS-PAGE and immunoblot analysis as above, to show increased expression of the IGF2R. In addition, cell lysates (0.1 mg protein) were subjected to SDS-PAGE and immunoblot analysis using the 9E10 anti-Myc antibody (1:1000 dilution, provided by Dr. Robert Lewis and the University of Nebraska Medical Center Monoclonal Antibody Core Facility, Omaha, NE) to determine whether the increased IGF2R was attributable to expression of the transfected cDNA.
Transfection of PC-3 cells
PC-3 cells were seeded at 5.0 x 104 or 3.0 x 105 cells per well in 12- or 6-well plates, respectively. The next day, the cells were transfected with pCMV5, IGF2RMyc, or IGF2RMyc I/T using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) in RPMI 1640 supplemented with 2% FBS. Fresh medium was added every other day until the completion of the experiment. Lysates were prepared on the third day after transfection.
FLAG and Myc immunoblots
For analysis of expression of FLAG- or Myc-tagged proteins, cell lysates (0.1 mg) were subjected to SDS-PAGE under reducing conditions, transferred to BA85 nitrocellulose, incubated with anti-FLAG antibody M2 (1:1000 dilution, Sigma) or anti-Myc antibody 9E10, washed, and incubated with a rabbit antimouse IgG before detection with 125I-protein A.
Preparation of IGF2RMyc retroviral construct
Subcloning of a full-length, Myc-tagged IGF2R (IGF2RMyc) into pCMV5 has been previously described (23). A two-step subcloning approach was used to transfer the IGF2RMyc into the pSR
MSVtkneo retroviral vector. In the first step, pCMV5/IGF2RMyc was digested with EcoRI and XbaI, and two fragments (approximately 500 bp and 6.9 kb) were purified from a 1% agarose gel using the QIAquick method. The larger fragment was then directionally subcloned into the pSRMSVtkneo vector that had been digested with EcoRI and XbaI. The resulting pSRMSVtkneo vector and large IGF2RMyc fragment were then digested with EcoRI and treated with calf intestinal alkaline phosphatase, and the smaller IGF2RMyc fragment (
500 bp) was subcloned into the construct. The correct orientation of the EcoRI fragment was confirmed by restriction enzyme digestion.
Generation of IGF2RMyc viral stock
To generate viral stock for infections, 100-mm dishes were seeded with 2.5 x 106 293T cells and transfected with the pSRMSVtkneo/IGF2RMyc and the amphotropic packaging vectors, using the calcium phosphate transfection method as described previously (26). The following day, the transfection medium was aspirated, and 6 ml serum-free DMEM was added to the dishes. For the next 2 d, the medium containing the viral particles was collected, and 6 ml fresh serum-free DMEM was again added to the dishes. After the final aliquot of viral-containing medium was collected, the collected media were filtered through a 0.22-µm filter and stored at -80 C until needed.
Infection of PC-3 cells with retroviral IGF2RMyc and selection of stable clones
PC-3 cells were seeded at 3 x 106 per well in six-well plates. The following day, 2 ml viral stock plus 40 µg/ml Polybrene was added to each well of cells. After 3 h incubation at 37 C, the infection medium was aspirated, and 4 ml standard growth medium was added to each well. The next day, selection medium containing 500 µg/ml G418 was added to the infected cells. Selection medium was replaced every 2 d for 2 wk. Single colonies were isolated, using cloning cylinders, and expanded for analysis of IGF2RMyc expression. A single PC-3 clone (IGF2R#26) that stably expressed the IGF2RMyc and PC-3 clones expressing vector alone (pSR#1-#5) were isolated for further analysis.
Rapid endocytosis assay for internalization of 125I-IGF-II
The ability of the IGF2RMyc to internalize IGF-II was measured using a previously described protocol (27). Briefly, IGF2R#26, pSR#3, and pSR#5 were seeded in 12-well plates and allowed to grow to confluency. The wells were rinsed twice with ice-cold PBS plus 1% BSA. Then, 2 nM 125I-IGF-II in 0.5 ml ice-cold RPMI 1640 plus 2% BSA was added to each well, and the plate was incubated on ice for 30 min. The wells were washed three times with ice-cold PBS plus 1% BSA and incubated, for the indicated times, in a 37 C water bath with 0.5 ml prewarmed RPMI 1640. The plates were then placed on ice, the medium was aspirated, and 0.5 ml ice-cold stop solution (0.2 M acetic acid, pH 3.5; 0.5 M NaCl) was added to each well, incubated for 5 min (twice), and collected for later analysis. The two aliquots of stop solution were combined and counted to determine the surface-associated 125I-IGF-II. The cells were solubilized in ice-cold 0.1-N NaOH (1 ml, twice), and the solubilized cells were counted to determine the amount of 125I-IGF-II internalized. Data were graphed as a ratio of surface-associated or internalized 125I-IGF-II vs. total 125I-IGF-II.
Coexpression of 1–15F or 1–15F I/T and 1–15Myc constructs
Generation of the 1–15F, 1–15F I/T, and 1–15Myc constructs has been previously described (23). Coexpression of the 1–15F or 1–15F I/T and 1–15Myc was accomplished by mixing 1–15F or 1–15F I/T and 1–15Myc constructs in a 1:2 ratio (10 µg 1–15F or 1–15F I/T and 20 µg 1–15Myc) before transfection. The 1–15F/1–15Myc DNA mixture was used to transfect 293T cells (2.5 x 106 cells in 100-mm dishes) using the calcium phosphate method as previously described (26). Lysates were prepared on the 5th day after transfection (22) and stored at -80 C until needed.
Coimmunoprecipitation of truncated, FLAG- and Myc-tagged receptors from cell lysates
Immunoblot analysis of cell lysates (30 µl) was performed to assay for coexpression of the constructs, and equimolar amounts of the 1–15F or 1–15F I/T proteins were incubated with 12 µl M2 -FLAG resin in HBS with 1.0% BSA, at 4 C overnight. The next day, the resins were collected by centrifugation at 14,000 x g for 10 sec, washed twice with 1 ml HBS, and subjected to SDS-PAGE followed by immunoblot analysis for FLAG- and Myc-tagged, truncated receptors.
IGF-II binding analysis of 1–15F- or 1–15F I/T-15Myc dimers
Equimolar amounts of the 1–15F or 1–15F I/T proteins were incubated with 12 µl M2 -FLAG resin in HBS plus 1% BSA as described above. The following day, the resins were collected by centrifugation at 14,000 x g for 10 sec and washed twice with 1 ml HBS. Immunoprecipitated proteins were then incubated with 2 nM 125I-IGF-II, with or without 500 nM IGF-II, at 4 C for 4 h. After two washes with 1 ml HBS plus 0.05% Triton X-100, the resin pellet was counted in a 
-counter to quantify bound 125I-IGF-II.
Statistical analysis
All analyses were done using GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA). Data from three to six experiments were combined so that the average and SE of the mean were used to generate graphs. Statistical comparisons were made by using one-way ANOVA followed by Tukey’s honestly-significant-difference post hoc test.
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Results |
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). This behavior implies that additional changes, which provide further growth advantage, take place after attainment of the androgen-insensitive phenotype. Hence, our analysis of the IGF2R function in the LNCaP model used androgen-sensitive low-passage (passages < 35) and androgen-insensitive mid-passage (80–105) as well as high-passage (>125) LNCaP cells.
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). Immunoblot analysis of IGF2R in whole-cell lysates of low-, mid-, and high-passage LNCaP cells showed a similar decrease in IGF2R (Fig. 2B). PhosphorImager analysis of immunoblots of crude plasma membrane protein from low- and mid-passage LNCaP cells indicated approximately the same level of IGF2R protein, but there was a significant decrease (47.7 ± 2.9%) in IGF2R protein between the low- and high-passage LNCaP plasma membranes (Fig. 2C). A corresponding decrease in 125I-IGF-II affinity cross-linking was also observed in plasma membranes of low- and high-passage LNCaP cells (Fig. 2, D and E). Although mid-passage LNCaP cells showed no decrease in IGF2R protein on the immunoblot, a significant decrease (33.5 ± 6.0%) in 125I-IGF-II affinity cross-linking was observed in plasma membranes of mid-passage LNCaP cells, compared with low-passage LNCaP cells (Fig. 2E).
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show that LNCaP cells secrete increasing amounts of the mature, 7.5K form of IGF-II, and a higher molecular weight form of IGF-II, as a function of increasing passage. PhosphorImager analysis of IGF-II immunoblots indicated that the high-passage LNCaP cells accumulate significantly increased amounts of total IGF-II in conditioned medium, compared with low- or mid-passage LNCaP cells (Fig. 3B).
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).
Expression of full-length, Myc-tagged IGF2R (IGF2RMyc) in high-passage LNCaP cells
To determine whether the decreased IGF2R level contributed to the increased proliferation of LNCaP cells, a full-length, Myc-tagged IGF2R (IGF2RMyc) or an IGF2RMyc incompetent to bind IGF-II [I1572T (22) termed IGF2RMyc I/T] were expressed in high-passage LNCaP cells. Immunoblot analysis, using an anti-IGF2R antibody, of crude plasma membrane proteins derived from high-passage LNCaP cells indicated increased expression of the IGF2R in IGF2R-transfected cells, compared with vector-only transfected cells (Fig. 4A). Immunoblot analysis, using an anti-Myc antibody, showed expression of the Myc-tagged IGF2R proteins, suggesting that increased expression of the IGF2R observed in the anti-IGF2R immunoblot was attributable to forced expression of the Myc-tagged IGF2Rs (Fig. 4A).
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). In addition, expression of IGF2RMyc I/T significantly increased the number of high-passage LNCaP cells (by 42.5 ± 8.1% and 74.4 ± 8.3% on the third and fifth days after transfection, respectively), when compared with vector-only transfected cells. Growth of high-passage LNCaP cells expressing IGF2RMyc I/T was also significantly increased (by 22.3 ± 8.4% and 35.6 ± 4.6% on the third and fifth days following transfection, respectively), when compared with IGF2RMyc-expressing cells (Fig. 4B).
Analysis of IGF2R ligands in conditioned medium of high-passage LNCaP cells transiently expressing IGF2RMyc
To determine what effect the expression of the IGF2RMyc constructs had on availability of IGF-II and Man-6-P-bearing ligands, high-passage LNCaP cells were transfected as above. The next day, the transfection media were replaced with RPMI 1640 supplemented with 2% serum, and the media were conditioned by the cells for 72 h before harvesting. IGF-II immunoblot analysis of conditioned media showed no change in IGF-II present in the media supplemented with 2% serum, comparing high-passage LNCaP cells transiently expressing the IGF2RMyc proteins, relative to vector-only control cells (Fig. 4C). Ligand blot analysis revealed increased accumulation of Man-6-P-bearing proteins in transfected cells, compared with RPMI 1640 supplemented with 2% serum (data not shown). However, media conditioned by cells expressing either IGF2RMyc or IGF2RMyc I/T showed decreased accumulation of Man-6-P-bearing proteins, compared with vector-only control cells (Fig. 4D).
Expression of IGF2RMyc and IGF2RMyc I/T in PC-3 cells
Previous studies have suggested that LNCaP cells do not respond to exogenous IGF-II with a marked increase in proliferation (29, 30). To determine the effect of forced expression of the IGF2R in a prostate cancer cell line that is well-documented to be IGF-II-responsive (29, 30), PC-3 cells were transfected with either vector alone, the IGF2RMyc, or IGF2RMyc I/T. On the third day after transfection, the cells were lysed, and immunoblot analysis was performed to verify expression of IGF2RMyc (Fig. 5A). In longer-term growth curves, IGF2R expression in IGF2RMyc-expressing cells was highest on the third day after transfection, showing approximately a 2-fold overexpression, compared with control, and slowly decreased thereafter until the final day, which showed only an approximately 20% increase in IGF2R expression.
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). A similar change in growth was also documented in two additional short-term (5-d) experiments (data not shown).
Analysis of the functionality of the Myc-tagged IGF2R
Because high-passage LNCaP and PC-3 cells responded differently to overexpression of the IGF2RMyc, PC-3 cells stably expressing IGF2RMyc (IGF2R#26) or vector-only (pSR#3 and pSR#5) were used to verify the functionality of the IGF2RMyc, compared with endogenous IGF2R. As shown in Fig. 6A, IGF2R#26 expressed increased levels of the IGF2R, compared with pSR#3 and pSR#5, because of expression of the Myc-tagged IGF2R. Consistent with results from the transient transfections, growth assays indicated that the proliferation of the IGF2R#26 was significantly decreased, compared with both pSR#3 and pSR#5 (data not shown). Crude plasma membranes and microsomes prepared from IGF2R#26 and pSR#3 and pSR#5 clones were subjected to immunoblotting and PhosphorImager analysis to determine the subcellular distribution of endogenous and Myc-tagged IGF2R. Analysis of two separate experiments (n = 4) indicated there was no difference in the overall distribution of IGF2R or the Myc-tagged IGF2R in the plasma membranes vs. microsomes of the IGF2R#26 clone, compared with vector-only clones (Fig. 6, B–D). To determine whether the IGF2RMyc was competent to internalize ligands at the cell surface, a rapid endocytosis assay (27) was performed using pSR#3, pSR#5, and IGF2R#26 clones. Data from five different experiments were combined, and linear regression analysis was used to compare the internalization of pSR#3, pSR#5, and IGF2R#26 clones (Fig. 6E). Statistical analysis indicated there was no significant difference between the internalization of IGF-II by IGF2R#26 and either pSR#3 or pSR#5.
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-FLAG resin. The bound proteins were subjected to immunoblotting using anti-FLAG or anti-Myc antibodies (Fig. 7A) or incubated with 125I-IGF-II in the presence or absence of unlabeled IGF-II and the 125I-IGF-II bound to the resin counted in a -counter. If the I/T mutation acted as a dominant-negative when dimerizing with wild-type IGF2R, then neither immunoabsorbed 1–15F I/T homodimers nor heterodimers of 1–15F I/T and 1–15Myc from cotransfected 293T cells should bind 125I-IGF-II. However, dimers immunoprecipitated from lysates of 293T cells cotransfected with 1–15F I/T and 1–15Myc bound approximately 26% of the 125I-IGF-II bound by dimers immunoprecipitated from lysates of 293T cells cotransfected with 1–15F and 1–15Myc (Fig. 7B). These data are consistent with the amount of 125I-IGF-II binding predicted to occur if the I/T mutation does not function in a dominant-negative fashion when dimerizing with wild-type IGF2R.
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Discussion |
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These data suggest that the IGF2R may play a role in prostate cancer growth suppression, possibly through internalization and degradation of IGF-II, which is a potent mitogen for many cells. Loss of imprinting of the IGF-II gene, which may be responsible for increasing IGF-II mRNA and protein production, has been shown in sporadic Wilms’ tumors, lung carcinomas (4), and human prostate tissue (17). IGF-II overexpression has also been found in cancers of the breast (37), liver (38), smooth muscle (39), and colon (4). Increased levels of high-molecular-weight forms of IGF-II, relative to the mature form of IGF-II, have been associated with Wilms’ tumors (40) and cancers of the prostate, breast, and bladder (41). Additionally, positive staining for IGF-II has been shown to correlate with increased tumor progression, increased proliferating cell nuclear antigen staining, and decreased patient survival in colorectal cancers (13). These findings indicate that IGF-II and/or pro-IGF-II may play an important role in tumor proliferation and progression. Our data showing increased levels of both the mature, 7.5-kDa IGF-II and a high-molecular-weight IGF-II are consistent with previous work and provide additional support for a role for IGF-II in the growth and progression of prostate cancer cells. Previous research also indicated that clones overexpressing pro-IGF-II exhibited increased secretion of both pro-IGF-II and phosphomannosylated ligands (42). Although our data are also consistent with these results, binding analysis of the IGF2R in mid-passage LNCaP cells, which exhibited no decrease in IGF2R expression, suggested that increased secretion of both IGF-II and Man-6-P-bearing ligands could also be attributable to decreased functionality of the IGF2R. These data suggest that quantifying the amount of IGF2R present may not be sufficient to understand the impact of the IGF2R on cellular functions; the ability of the IGF2R to bind ligand is also important.
Decreased expression of the IGF2R and increased growth of high-passage LNCaP cells are in agreement with data on the enhanced tumorigenicity of JEG-3 cells, which exhibit decreased IGF2R because of stable expression of an antisense IGF2R cDNA (43). In that study, clones that exhibited more than a 50% decrease in sIGF2R grew significantly faster in vitro and resulted in a greater tumor burden in vivo. Consistent with a role for the IGF2R in growth suppression, expression of a zinc-inducible IGF2R in SW48 colon cancer cells resulted in decreased growth in vitro. This decreased growth was attributable, at least in part, to increased apoptosis of the IGF2R-expressing cells (44).
It was very surprising, therefore, to find that forced expression of the IGF2RMyc in high-passage LNCaP cells resulted in increased cell number. These data are consistent with the increased proliferation in both regenerating rat liver and propylthioluracil-induced thyroid hyperplasia that corresponds to increased IGF2R expression (45, 46), and suggest that the increased cell number in IGF2R-transfected LNCaP cells is most likely caused by increased proliferation. However, the possibility that increased cell number may also have resulted from increased cell survival cannot be excluded. Analysis of a PC-3 clone stably expressing the IGF2RMyc showed similar distribution of endogenous and IGF2RMyc in PC-3 cells. These data indicated that approximately 50% of the IGF2R was plasma-membrane associated, significantly greater than the expected 10–20%, and suggested that the crude plasma membranes used in these studies were contaminated with internal membranes. However, the percentage of internalized 125I-IGF-II, which corresponds to the amount of plasma-membrane-associated IGF2R, was similar in the three PC-3 clones, suggesting similar trafficking of endogenous IGF2R and IGF2RMyc. In addition, internalized 125I-IGF-II at the zero time point suggested that approximately 25% of the total IGF2R is normally associated with the plasma membrane in these cells, a value close to that expected. The Myc-tagged receptor also internalized 125I-IGF-II at approximately the same rate as endogenous receptor, indicating that the Myc tag does not interfere with internalization of ligands at the cell surface. These data suggest the Myc tag does not interfere with either trafficking or internalization of ligands at the cell surface and indicate that the Myc-tagged receptor is a fully active IGF2R that is functionally indistinguishable from endogenous IGF2R.
Although expression of both IGF2RMyc and IGF2RMyc I/T resulted in increased growth in high-passage LNCaP cells, cells expressing IGF2RMyc showed less of a growth stimulation than IGF2RMyc I/T-expressing cells. This behavior is consistent with growth inhibition through enhanced degradation of IGF-II arising from increased availability of IGF-II binding sites at the cell surface or within the Golgi. Analysis of media conditioned by LNCaP cells transfected with vector-only, wild-type IGF2RMyc, or IGF2RMyc I/T, indicated that IGF-II levels in the media remained approximately the same. It is possible that the decreased growth of the IGF2RMyc-expressing cells, compared with the IGF2RMyc I/T-expressing cells, is the result of a decrease in the availability of IGF-II to cells adjacent to the IGF2RMyc-expressing cells that is not reflected in the overall availability of IGF-II. If this was true, then the initial decrease in growth caused by decreased availability of IGF-II would be slight, but should be sustained if the decreased availability of IGF-II was extended over a period of time. Consistent with this, our data show a difference in the growth of LNCaP cells expressing IGF2RMyc, compared with those expressing IGF2RMyc I/T, that increases quantitatively as a function of time after transfection. Alternatively, the IGF-II autocrine loop in LNCaP cells may operate, at least partially, through binding of newly synthesized IGF-II to the IGF-I receptor in the Golgi or other intracellular compartments. Such an intracrine mechanism would not be affected by changes in the extracellular milieu but would be sensitive to changes in IGF-II binding activity arising from altered IGF2R expression at the Golgi. Analysis of pSR#3, pSR#5, and IGF2R#26 also showed no significant difference in the amount of IGF-II secreted (data not shown), suggesting that an intracrine mechanism is responsible for the decreased growth of IGF2R#26.
Recent research indicates that activation of TGF-ß1 can be growth stimulatory for some prostate cancer cells (47, 48, 49), suggesting that the increased growth of high-passage LNCaP cells expressing IGF2RMyc proteins could be attributable to increased activation of TGF-ß in these cells. However, growth of LNCaP cells transiently expressing IGF2RMyc I/T was not inhibited by treatment with a TGF-ß1 neutralizing antibody, nor did treatment of high-passage LNCaP cells with TGF-ß1 elicit an increase in growth (data not shown), suggesting that TGF-ß1 is not mitogenic for these cells.
The IGF2R has been described recently as a growth suppressor because of its ability to internalize IGF-II and activate the inhibitory activity of TGF-ß1. Paradoxically, regenerating rat liver is associated with increased expression of IGF2R during the early, rapid proliferative stage (45). IGF2R expression is also increased during propylthiouracil-induced thyroid hyperplasia in rats (46). In both instances, the increase in IGF2R expression is transitory and declines to normal levels within days of treatment. In regenerating rat liver, this decrease in IGF2R corresponds to cessation of regeneration, suggesting that the early increase in IGF2R has a role in the rapid proliferation associated with the liver regeneration. Whereas increased expression of IGF2R corresponded with increased growth in these studies, there was no indication whether the increased growth was attributable to the IGF-II- or Man-6-P-binding function of the IGF2R.
Our data showing increased growth with forced expression of IGF2RMyc in LNCaP cells are consistent with the data from regenerating rat liver and induced thyroid hyperplasia. IGF2R bearing the Ile1572Thr mutation, originally characterized in our laboratory, is incompetent to bind IGF-II (22) but retains high-affinity binding to Man-6-P-containing ligands (26). Our data suggest that the significantly increased growth of both LNCaP and PC-3 cells expressing IGF2RMyc I/T is attributable primarily to the Man-6-P-binding function of the IGF2R. The ligand or ligands responsible for this growth-stimulatory effect have not yet been identified. In addition, our data suggest that the IGF-II- and Man-6-P-binding functions of the IGF2R have opposing effects in the growth of LNCaP and PC-3 prostate cancer cells and that the relative magnitudes of inhibitory vs. proliferative responses determine whether the net effect of the IGF2R on growth is inhibitory or stimulatory. Additional research is required to determine whether other cell types are stimulated by the Man-6-P-binding activity of the IGF2R and to identify the Man-6-P ligands involved in the IGF2R proliferative response.
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Abbreviations: FBS, Fetal bovine serum; IGF2R, IGF-II/Man-6-P receptor; LNCaP, lymph node carcinoma of the prostate; LOH, loss of heterozygosity; Man-6-P, mannose 6-phosphate; MTT, 3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyl tetrazolium bromide; sIGF2R, soluble form of the IGF2R; uPAR, urokinase-type plasminogen activator receptor.
Received July 23, 2002.
Accepted for publication November 20, 2002.
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