| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Other Original Articles |
Istituto di Medicina Interna e di Malattie Endocrine e del Metabolismo, Cattedra di Endocrinologia, University of Catania, Ospedale Garibaldi (V.V., G.P., L.S., R.M., R.V., V.P.), 95123 Catania, Italy; and Dipartimento di Medicina Sperimentale e Clinica, Cattedra di Endocrinologia, University of Catanzaro, Policlinico Mater Domini (A.B.), 88100 Catanzaro, Italy
Address all correspondence and requests for reprints to: Prof. Riccardo Vigneri, Cattedra di Endocrinologia, Ospedale Garibaldi, 95123 Catania, Italy. E-mail: vigneri{at}mbox.unict.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In conclusion, malignant thyrocytes, especially when poorly differentiated, produce IGF-II and overexpress IR, predominantly as IGF-II-sensitive isoform A. A growth-promoting autocrine loop is activated, therefore, and may affect thyroid cancer biology.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We recently observed that insulin receptors (IRs) are overexpressed in most thyroid tumors as an early step in thyroid carcinogenesis (11). The role of overexpressed IRs was not clear, because insulin is not locally produced in these tumors. One possibility is that IRs may contribute to transmit the mitogenic signals of insulin homolog IGF-I and IGF-II, produced locally in thyroid cancers (12, 13). IGFs are potent mitogenic and antiapoptotic factors that play a major role in a variety of human malignancies (14). Both IGFs are believed to signal through the IGF-I receptor (IGF-I-R), because they have a low affinity for IR (15, 16). However, we recently identified new mechanisms for the interaction of IGFs with IR. One mechanism involves paracrine IGF-I secretion. IR overexpression may amplify the response of thyroid malignant cells to locally produced IGF-I by increasing the formation of IR/IGF-I-R hybrids (heterodimers formed by one IR 
- and ß-subunit complex and one IGF-I-R - and ß- subunit complex) (17). Hybrid receptors bind IGF-I with high affinity, similar to that of typical IGF-I-Rs (18). Another mechanism involves direct IR interaction with IGF-II. Although in most cells IGF-II interacts with IR with relatively low affinity, atypical IRs that bind IGF-II with high affinity have been described in human placenta and IM-9 lymphoblasts (19, 20). In addition, in IGF-I-R-deficient mouse fibroblasts that overexpress the IR, IGF-II stimulates cell proliferation via this receptor, which behaves, therefore, as an IGF-II-R (21). The molecular basis of this phenomenon relies on the fact that IGF-II binds with high affinity and activates only one of the two IR isoforms, isoform A (22). The relative abundance of IR isoforms A and B is tightly regulated in a tissue-specific manner by alternative splicing of the 36-bp exon 11 (Ex 11-) of the IR gene (23). Although the IR-B isoform is predominantly expressed in most adult tissues, the IR-A isoform, which binds IGF-II with high affinity, is preferentially expressed in fetal tissues and in certain human malignancies, including breast cancer (22, 24).
In the present study we tested the hypothesis that the IR, overexpressed in most thyroid tumors (11, 17), may behave as an IGF-II-R. We investigated, therefore, the expression of IR-A in thyroid cancer cells and its relevance in establishing an autocrine loop with IGF-II. We found that 1) malignant thyrocytes overexpress IR, predominantly as isoform A; 2) autocrine IGF-II production is activated in malignant thyrocytes; 3) IR-A may be directly activated by IGF-II and mediates its mitogenic effect; and 4) both IR-A and IGF-II expression in thyroid cancer correlate with tumor dedifferentiation.
The present data, therefore, identify a novel autocrine loop, involving IGF-II and the IR-A, which may contribute to determine thyroid cancer progression and aggressiveness, especially in undifferentiated carcinomas.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following materials were purchased: FCS, glutamine, and gentamicin were obtained from Life Technologies, Inc. (Paisley, UK); RPMI 1640 medium, BSA (RIA grade), bacitracin, phenylmethylsulfonylfluoride (PMSF), and porcine insulin were obtained from Sigma (St. Louis, MO); protein A-Sepharose was obtained from Pharmacia Biotech (Uppsala, Sweden); 125I-labeled insulin (SA, 74,000 GBq/mmol) was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK); IGF-I and IGF-II were obtained from Calbiochem (La Jolla, CA); human Del(1–6)IGF-II was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).
The following antibodies were employed: 1) anti-IR antibodies: MA-20 and MA-51 monoclonal antibodies that recognize IR -subunit (I. D. Goldfine, San Francisco, CA) (25, 26), CT-1 monoclonal antibody that recognizes the IR ß-subunit (27) and 83-7 monoclonal antibody that recognizes the IR -subunit (K. Siddle, Cambridge UK) (28), and rabbit polyclonal antibody that recognizes the IR ß-subunit (Transduction Laboratories, Inc., Lexington, KY); 2) anti-IGF-I-R antibodies: IR3 monoclonal antibody that recognizes IGF-I-R -subunit (29) (Oncogene Research, Cambridge, MA), and chicken polyclonal antibody that recognizes IGF-I-R -subunit (Upstate Biotechnology, Inc.). Antiphosphotyrosine 4G10 and anti-IGF-II (clone S7F2) monoclonal antibodies were purchased from Upstate Biotechnology, Inc. Monoclonal antibody antimannose-6-phosphate receptor (anti-MPR) was purchased from Calbiochem.
Thyroid cell cultures and human tissue specimens
Thyroid primary cell cultures were prepared from both neoplastic (papillary, n = 7; anaplastic, n = 1) and normal thyroid tissue (n = 11) obtained at surgery. Briefly, the tissue was fragmented with a scalpel, suspended in PBS without Ca2+ and Mg2+, and digested with a solution of type V collagenase (1 mg/ml) in a 37 C shaking bath for 90 min. The cell suspension, containing intact and fragmented thyroid follicles, was centrifuged (400 x g for 10 min), and the pellet was resuspended in culture medium consisting of RPMI 1640 supplemented with 2 mM glutamine, 5 µg/ml gentamicin, and 3% FCS. Under these conditions follicles formed a monolayer after 1–2 d. The medium was routinely changed every 2 d. To score the degree of nonepithelial cells present in the cultures, epithelial thyroid cells were identified by indirect immunofluorescence staining with anti-Tg and anticytokeratin antibodies as previously described (30). Less than 5% cells were cytokeratin negative, indicating that contamination with nonepithelial cells was low.
Permanent thyroid cancer cell lines (TPC-1, B-CPAP, NPA, FRO, WRO, and ARO) (31, 32, 33) and thyroid primary cultures were grown in RPMI 1640 supplemented with 2 mM glutamine, 10% FCS, and 5 µg/ml gentamicin. In all cell lines the medium was routinely changed every 2 d.
Thyroid tissue specimens were collected at surgery, immediately frozen, and stored in liquid nitrogen until processing. Twenty-one thyroid cancer specimens (13 differentiated, 4 less differentiated, and 4 anaplastic) and 5 normal thyroid specimens were used.
Thyroid membrane fraction preparation
The crude thyroid membrane fractions were prepared as follows. Briefly, homogenized tissue specimens or confluent cells were harvested and washed twice with PBS and once with Tris buffer, pH 7.4 (50 mM Tris-HCl, 20 mM benzamidine, 200 µg/ml bacitracin, 30 µg/ml aprotinin, and 10 mM PMSF). They were resuspended in Tris buffer, sonicated three times for 10 sec each time, and centrifuged at 2500 rpm for 10 min at 4 C. Supernatants were removed and saved. The pellets were resuspended in Tris buffer, sonicated three times for 20 sec each time, and centrifuged as in the previous step. Supernatants were removed and added to the previous ones. Combined supernatants were centrifuged at 11,000 rpm for 30 min at 4 C. The pellets were resuspended in Tris buffer using a syringe with a 25-gauge needle. The suspensions were aliquoted in microfuge tubes and centrifuged for 30 min. Supernatants were removed, and pellets were stored at -80 C.
Cell monolayers, tissues, and membrane fractions were solubilized with 50 mM HEPES buffer, pH 7.6, containing 1 mM PMSF, 1 mg/ml bacitracin,150 mM NaCl, 2 mM sodium orthovanadate, and 1% Triton X-100 for 60 min at 4 C. The solubilized material was then centrifuged at 10,000 x g, and the supernatant was frozen at -80 C until assayed. The protein content in the cellular extracts was measured by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL).
IR and isoform measurements
IR ELISA. IRs were captured by incubating membrane fraction lysates from cells or tissues in Maxisorp immunoplates (Nunc, Roskilde, Denmark) precoated with 2 µg/ml MA-20, as previously described (24). After washing, the immunocaptured receptors were incubated with the biotinylated anti-IR CT-1 antibody (0.3 µg/ml in 50 mM HEPES-buffered saline, pH 7.6, containing 0.05% Tween 20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) and then with peroxidase-conjugated streptavidin. The peroxidase activity was determined colorimetrically by adding 100 µl 3,3',5,5'-tetramethylbenzidine (0.4 mg/ml in 0.1 M citrate-phosphate buffer, pH 5.0, with 0.4 µl/ml 30% H2O2). The reaction was stopped by the addition of 1 M H3PO4, and the absorbance was measured at 450 nm.
IR isoform relative abundance was measured by RT-PCR, as previously described (24, 34). Briefly, total cellular RNA was isolated from each cell line or specimen using TRIzol RNA isolation reagent (Life Technologies, Inc., Gaithersburg, MD). First strand cDNA synthesis was performed on 4–8 µg total RNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and random hexamer primers (Pharmacia Biotech) in a total volume of 40 µl 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, and 0.5 mM dNTPs.
cDNA synthesis reaction (5-µl volume) was combined in a 50-µl final reaction volume for PCR amplification containing 0.2 µM oligonucleotide primers spanning nucleotides 2229–2250 (5'-AAC-CAG-AGT-GAG-TAT-GAG-GAT-3') and 2844–2865 (5'-CCG-TTC-CAG-AGC-GAA-GTG-CTT-3') of the human IR and 1.25 U Taq DNA polymerase (AmpliTaq, Perkin-Elmer Corp.). PCR amplification was carried out for 30 cycles of 15 sec at 95 C, 30 sec at 60 C, and 45 sec at 72 C, using a DNA thermal cycler (Perkin-Elmer Corp.). Products of PCR amplification were resolved by electrophoresis on 5% polyacrylamide gels. The electrophoretic analysis showed the 600- and 636-bp DNA fragments representing Ex11- and Ex11+ IR isoforms, respectively. Gels were silver stained, and band density was quantified by scanning densitometry.
Ligand-binding assay for IR
Membrane fractions were solubilized as described above, and IR was immunocaptured by incubating for 22 h in Maxisorp immunoplates Break-Apart (Nunc, Roskilde, Denmark), precoated with 2 µg/ml anti-IR 83-7. After washing, the immunocaptured receptors were incubated with [125I]insulin (10 pM in 50 mM HEPES-buffered saline, pH 7.6, containing 0.05% Tween 20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) in the presence or absence of increasing concentrations of various unlabeled ligands (insulin, IGF-I, or IGF-II). After 2 h at room temperature, plates were washed, and the radioactivity in each well was counted in a 
-counter.
IR autophosphorylation: ELISA
IR autophosphorylation in intact cells.
Ligand-activated IR autophosphorylation in intact cells was measured as previously described (35). Cells in monolayer cultures were stimulated with increasing doses (0–100 nM) of insulin, IGF-II, or IGF-I for 5 min at 37 C. Cells were then solubilized, and receptors were captured by incubating cell lysates (containing 
2 ng receptor) in Maxisorp plates precoated with antireceptor antibody MA-20. After washing, a biotinylated antiphosphotyrosine antibody (4G10 PY from Upstate Biotechnology, Inc., Lake Placid, NY; 0.3 µg/ml in 50 mM HEPES-buffered saline, pH 7.6, containing 0.05% Tween 20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) was added to reveal phosphorylated receptors by the peroxidase-conjugated streptavidin method. Peroxidase activity was determined colorimetrically by adding 100 µl 3,3',5,5'-tetramethylbenzidine (0.4 mg/ml in 0.1 M citrate-phosphate buffer, pH 5.0, with 0.4 µl/ml of 30% H2O2). The reaction was stopped by the addition of 1 M H3PO4, and the absorbance was measured at 450 nm.
IR phosphorylation in isolated receptors. IR kinase activity was also measured in purified receptors. Membrane fractions were solubilized as described above and IR immunocaptured in Maxisorp plates coated with the anti-IR antibody MA-20. The immunopurified receptors were then stimulated with various concentrations of either insulin or IGFs in the presence of ATP (10 µM), MgCl2 (10 mM), and MnCl2 (2 mM). After washing, the phosphorylated proteins were incubated with an antiphosphotyrosine-biotin-conjugated antibody, and the reaction was detected as described above.
IR autophosphorylation: Western blot analysis
Confluent cells were incubated in serum-free medium for 48 h, stimulated with 10 nM insulin, IGF-II, or IGF-I for 5 min at 37 C, and solubilized in RIPA buffer [50 mM Tris (pH 7.4), 1% NP-40, 0.25% sodium deoxycolate, 150 mM NaCl, 1 mM EGTA (pH 8.0), 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 2 mM Na orthovanadate, and 1 mM NaF]. Cell lysates were immunoprecipitated with either anti-IR (MA-20 monoclonal), or anti-IGF-I-R (-IR3 monoclonal) antibodies. Immunoblottings were performed using 4G10 antiphosphotyrosine antibody. To show the amounts of receptors loaded in each lane, filters were first subjected to stripping in Tris-HCl buffer (62.5 mM, pH 6.7) containing 2% SDS and 100 mM ß-mercaptoethanol for 30 min at 50 C. Next, the half-filter containing IRs was reprobed with an anti-IR ß-subunit polyclonal antibody, and the half-filter containing IGF-I-Rs was reprobed with an anti-IGF-I-R -subunit polyclonal antibody. All immunoblots were revealed by an ECL method (Amersham Pharmacia Biotech), autoradiographed, and subjected to densitometric analysis.
Measurement of IGF-I/II production
IGF-I and IGF-II mRNA content in human thyroid cancer cell lines and tissue specimens was evaluated by PCR. Total RNA was prepared from cells and tissue specimens using TRIzol RNA isolation reagent (Life Technologies, Inc.). First strand cDNA synthesis was performed on 4–8 µg total RNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and random hexamer primers (Amersham Pharmacia Biotech) in a total volume of 40 µl 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, and 0.5 mM dNTPs. PCR amplification was performed with 5 µl cDNA, adding 1.25 U Taq polymerase (Perkin-Elmer Corp.), 2.5 mM specific primers, 2.5 mM MgCl2, and 0.2 mM dNTPs. The following primer sequences were used: 5'-3', GAA-GTC-GAT-GCT-GGT-GCT-TC; and 3'-5', CTT-CCG-ATT-GCT-GGC-CAT-CT. Conditions for PCR were: 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec for 30 cycles. PCR products were resolved by electrophoresis on 5% polyacrylamide gels and silver stained.
IGF-II protein secreted by cultured thyroid cancer cells in the conditioned medium was measured by a commercial immunoradiometric assay that detects only the IGF-II mature form (BIOS, Naples, Italy) and was expressed as nanograms of IGF-II secreted per h/106 cells.
Cell growth studies
To evaluate to what extent the mitogenic effect of IGF-II on thyroid cancer cells occurred via the IR, the growth of B-CPAP and ARO cells was measured in the presence or absence of the anti-IR blocking antibody MA-51 as previously described (36). Briefly, cells (1.5 x 103) were seeded in 96-well plates. After 24 h the medium was removed and replaced with medium containing 1% charcoal-stripped FCS. After an additional 24 h various concentrations of insulin, IGF-II, or IGF-I (0–10 nM) were added in fresh medium in the presence of a 5-fold molar excess of MA-51, aIR-3, or both antibodies. Normal mouse IgGs were used in control wells. Cell growth was measured after 4 d by measuring the rate of tetrazolium salts reduction to formazan (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Amersham Pharmacia Biotech), which is proportional to the number of living cells (37). At the end of the incubation the absorbance was read at 540 nm.
To investigate the autocrine effect of IGF-II two different methods were used: 1) B-CPAP or ARO cells were cultured in serum-free medium, and their growth rate was measured in the presence of a blocking anti-IGF-II monoclonal antibody at 2 µg/ml concentration or in the presence of an unrelated monoclonal antibody (control cultures); 2) in serum-starved cells growth was also evaluated at 2, 4, and 6 d in the presence or absence of 10 nM exogenous IGF-II with or without the addition to the culture medium of 1 µg/ml hydroxy-2-naphthalenylmethyl phosphoric acid triacetoxymethyl ester (HNMPA), a specific IR tyrosine kinase inhibitor (38).
MPR Western blot analysis
To measure IGF type II receptor (MPR), membrane proteins extracted from thyroid cells were separated by SDS-PAGE (7.5% polyacrylamide) under nonreducing conditions. Immunoblotting was performed using an anti-MPR antibody. The immunoblot was revealed by an ECL method (Amersham Pharmacia Biotech), autoradiographed, and subjected to densitometric analysis.
Statistical analysis
Differences between means were evaluated using paired t test, Mann-Whitney test, or one-way ANOVA, as indicated in Results. The ability of IGF-II to activate the IR and the relative abundance of IR-A were correlated by linear regression analysis. The statistical program PRISM (GraphPad Software, Inc., San Diego, CA) was used.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IRs were measured by ELISA in cell membrane preparations from both thyroid cell primary cultures and a variety of thyroid malignant cells in permanent culture. Primary cultures were obtained from either normal thyroid (n = 11) or thyroid carcinomas (papillary, n = 7; anaplastic, n = 1). The median IR content was 8.7- to 37.6-fold higher in malignant thyrocytes compared with normal thyrocytes, which expressed the IR at 1.2–1.7 ng/100 µg cell membrane protein (Table 1
).
|
and Fig. 1).
|
). In particular, the IR content reached levels up to 23 times higher than normal (in B-CPAP cells), and IR-A relative abundance increased up to 80% (in the anaplastic cells ARO; Table 2).
|
IGF-I/II secretion in cultured thyrocytes. We have previously shown that IR-A, but not IR-B, binds IGF-II with high affinity (22). As thyroid cancer cells overexpress IR-A, we next evaluated whether these cells produce IGF-II, thus raising the possibility of an autocrine loop by which IGF-II stimulates cell growth via the overexpressed IR-A.
We first evaluated IGF-I and IGF-II mRNA in both primary cultures and permanent cell lines by RT-PCR. IGF-I mRNA was absent in all thyroid cells, both normal and malignant, although it was present in control osteosarcoma cells (data not shown). At variance with IGF-I, IGF-II mRNA was found in all cancer cell lines and in 7 of 11 primary cultures from normal thyroid (data not shown). IGF-II protein secretion was also measured in cell-conditioned medium by an immunoradiometric assay. Median values of IGF-II protein secretion were approximately 5- to 7-fold higher in differentiated thyroid cancer cells (from either papillary or follicular cancer) than in normal thyroid cells. Again, as for the relative abundance of IR-A, IGF-II secretion was most elevated in undifferentiated cancer cells (Table 3).
|
30%). To avoid the possible interference of IGF-binding proteins (IGF-BPs), cell membrane fractions were extracted and solubilized, and IRs were immunocaptured onto Maxisorp plates. 125I-Labeled insulin binding was displaced with insulin, IGF-I, or IGF-II. Competition for labeled insulin binding revealed a low affinity of IGF-II for the IR obtained from normal thyrocytes and containing only approximately 30% IR-A. In contrast, labeled insulin binding was displaced by IGF-II with a high affinity in IRs extracted from B-CPAP and ARO cells (containing the IR-A isoform up to 70–80%; Fig. 2).
|
).
|
). In contrast, IR autophosphorylation in response to IGF-II differed in malignant vs. normal thyrocytes. In normal thyrocytes IGF-II induced IR autophosphorylation with an affinity that was always less than 1% that of insulin (ED50 = >100 nM), whereas in all thyroid cancer cells IGF-II activated IR autophosphorylation with an ED50 much closer to that of insulin. Median ED50 values were 4.8 nM in papillary cancer cells, 13.7 nM IGF-II in follicular cancer cells, and 1.6 nM in anaplastic cancer cells (Table 3).
In the 25 cell cultures studied (Table 3), the relative affinity of IGF-II for the IR (calculated as the percentage of insulin ED50/IGF-II ED50 for stimulation of IR autophosphorylation) was correlated with the relative abundance of IR-A expression (r = 0.628; P = 0.0001; Fig. 4). IGF-I was always less than 1% as effective as insulin in stimulating IR autophosphorylation (data not shown).
|
IGF-II stimulation of thyroid cancer cell growth: role of the IR-A.
To evaluate whether IR-A overexpression may have a role in mediating a mitogenic response to IGF-II in thyroid cancer cells, we studied B-CPAP (papillary cancer) and ARO cells (undifferentiated cancer), which predominantly express IR-A (approximately 70% and 80%, respectively). The biological effects of IGF-II in target cells may be regulated by mechanisms that influence IGF-II bioavailability, including the expression of IGF-II/MPR, which decreases IGF-II bioavailability, and the presence of IGF-BPs, which may either increase or decrease IGF-II bioavailability. Therefore, we first studied the expression of IGF-II/MPR in these cell lines. Western blot analysis showed that IGF-II/MPR was undetectable in normal thyroid cells and expressed at low levels in B-CPAP and ARO cell lines (Fig. 5). To evaluate the possible effect of IGF-BPs, serum-starved cells were exposed for 4 d to different doses of IGF-II or del(1–6)IGF-II, a truncated IGF-II analog that does not bind to IGF-BPs. In B-CPAP and ARO cell lines both ligands stimulated 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide incorporation in a dose-dependent manner. The maximal growth effect was observed at 10 nM for each ligand and ranged from 152–169% in B-CPAP cells and from 174–207% in ARO cells, with no difference between the two ligands. The very similar growth response obtained with both IGF-II and del(1–6)IGF-II suggests that IGF-BPs do not have a major role in regulating the IGF-II growth response in this system.
|
IR3 (IGF-I-R-blocking antibody), or a combination of these two antibodies. An unrelated monoclonal antibody was used as control.
In B-CPAP cells, insulin-induced cell growth was effectively blocked by MA-51 (P < 0.001), but not by IR-3 (Table 4). A combination of the two antibodies was as effective as MA-51 alone. In contrast, IGF-II-induced growth was significantly inhibited by either MA-51 or IR-3 to a similar extent (P = 0.025), suggesting that both IR-A and IGF-I-R are similarly important in mediating IGF-II effects in B-CPAP cells (Table 4). The two antibodies together were more effective than either antibody alone and almost completely blocked the growth response to IGF-II. As expected, IGF-I-induced growth was significantly decreased by IR-3 (P < 0.001), but not by MA-51. A combination of the two antibodies was as effective as IR-3 alone (Table 4). Similar results were obtained in ARO cells (data not shown).
|
Next, we evaluated the effect of HNMPA, a specific IR tyrosine kinase inhibitor. HNMPA (1.0 nM) markedly inhibited the growth of serum-starved B-CPAP cells in response to 10 nM exogenous IGF-II (Fig. 6). Spontaneous cell growth was also significantly reduced by the presence of 1.0 nM HNMPA (Fig. 6), suggesting that IR tyrosine kinase inhibition blocks cell growth induced by autocrine IGF-II production. Very similar results were obtained with ARO cells (data not shown).
|
The total IR content was elevated in cancer specimens of all histotypes (range, 1.8–26.7 ng/100 µg cell membrane protein) compared with normal thyroid specimens (range, 0.7–2.1 ng/100 µg cell membrane protein; Table 5). Median IR values were increased approximately 4-fold in differentiated cancers and 8- to 10-fold in poorly differentiated and undifferentiated cancers. The IR-A transcript accounted for 40.0–50.5% (median, 44.3%) of total IR in normal thyroid tissues, whereas it ranged from 40.5–73% (median, 55.7%) in cancer specimens. This difference is highly significant (P = 0.006). Poorly differentiated and undifferentiated carcinomas expressed both higher total IR content and relative abundance of IR isoform A compared with well differentiated papillary carcinomas (P = 0.005; Table 5).
|
).
|
Taken together these data indicate that IR-A, but not IGF-I-R, overexpression is the key factor of IGF system activation in both poorly differentiated and undifferentiated thyroid cancers. Even in differentiated cancers the increase in IR-A is more marked than that in IGF-I-R.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We previously observed that IGF-I is overproduced in human thyroid cancer in a paracrine fashion and that the IGF-I-R homolog IR plays a role in mediating the IGF-I response. In fact, IR is overexpressed in most thyroid cancer cells (11) and thyroid cancer tissue specimens and amplifies the biological effects of IGF-I by forming hybrid IR/IGF-I-R receptors that bind IGF-I with high affinity (17). By this mechanism IR overexpression also causes a marked increase in IGF-I-binding sites in those cancers that do not overexpress IGF-I-Rs.
Here we demonstrate that IR overexpression may also directly increase the thyroid cancer cell response to IGF-II by activating a newly recognized autocrine loop involving IR-A, which behaves as a high affinity IGF-II-R. Differential splicing of IR mRNA is tightly regulated in a tissue-specific manner, generating various proportions of IR-A and IR-B isoforms (23). We recently demonstrated that isoform A, but not isoform B, is a physiological receptor for IGF-II in fetal tissues (22), where IR-A is predominantly expressed, whereas IR-B is predominantly expressed in adult tissues. These findings provide a mechanistic basis to studies suggesting that during fetal development the growth-promoting effect of IGF-II is partially mediated by signaling through the IR (22). We also observed that IR-A might be the predominant isoform expressed in major human carcinomas (22, 24). In breast cancer, however, IGF-II is mainly produced in a paracrine manner, and the relative abundance of IR-A appears not to be related to tumor stage, grading, or ER status (24). Further studies, however, are needed to assess the clinical relevance of IR-A in breast cancer.
In contrast, in the thyroid model, the relative abundance of IR-A progressively increases from normal thyrocytes (that predominantly express IR isoform B) to differentiated papillary thyroid cancer cells, to undifferentiated thyroid cancer cells that express a very high (>70%) IR-A proportion. Autocrine IGF-II production is also activated in thyroid cancer cells and follows the same expression pattern as IR-A, being especially elevated in poorly differentiated tumors. In contrast, primary cultures from normal thyroid produced IGF-II in only in 7 of 11 cases and only in small amounts. IR activation by IGF-II is directly and significantly related to the relative abundance of IR-A.
Therefore, in thyroid cancer this newly identified autocrine loop involving IGF-II and IR-A appears particularly relevant in poorly differentiated thyroid cancers. The biological relevance of the IGF-II/IR-A loop in thyroid cancer is confirmed by the observation that blockade of either IGF-II or IR, by multiple approaches, significantly reduces thyroid cancer cell growth induced by either exogenous or autocrine IGF-II.
In contrast to IRs, IGF-I-Rs are overexpressed only in well differentiated thyroid papillary tumors where paracrine IGF-I secretion is also increased (17), but not in poorly differentiated or undifferentiated tumors, where paracrine IGF-I secretion is rarely increased (17).
Taken together these studies indicate that IR-A overexpression is a major determinant for the overactivation of the IGF system in poorly differentiated thyroid cancer, both by activating the autocrine loop involving IGF-II and by contributing to the paracrine loop involving IGF-I, via the formation of IR/IGF-I-R hybrids (17). In differentiated thyroid cancer, both the paracrine IGF-I/IGF-I-R loop and, to a lesser extent, the autocrine IGF-II/IR-A loop are activated.
In addition to the specific growth factor (TSH), therefore, a variety of growth factors (IGF-I and IGF-II, hepatocyte growth factor, epidermal growth factor, and fibroblast growth factor) and their tyrosine kinase receptors (4, 5, 6, 7, 8, 9, 10) may play an important role in thyroid cancer growth and progression. These mechanisms should be taken into account in thyroid tumors that progress despite TSH suppression, as therapeutic strategies that involve the interruption of these autocrine/paracrine loops may positively influence cancer biology and patient outcome.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
1 Recipient of a fellowship from the Giuseppe Alazio Foundation for cancer research. 
2 Recipient of a Federazione Italiana per la Ricerca sul Cancro fellowship.
Abbreviations: HNMPA, Hydroxy-2-naphthalenylmethyl phosphoric acid triacetoxymethyl ester; IGF-BP, IGF-binding protein; IR, insulin receptor; IGF-I-R, IGF-I receptor; MPR, mannose-6-phosphate receptor; PMSF, phenylmethylsulfonylfluoride.
Received March 28, 2001.
Accepted September 24, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. Belfiore, F. Frasca, G. Pandini, L. Sciacca, and R. Vigneri Insulin Receptor Isoforms and Insulin Receptor/Insulin-Like Growth Factor Receptor Hybrids in Physiology and Disease Endocr. Rev., October 1, 2009; 30(6): 586 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Chakravarty, A. A. Santillan, C. Galer, H. P. Adams, A. K. El-Naggar, S. A. Jasser, S. Mohsin, D. Mondal, G. L. Clayman, and J. N. Myers Phosphorylated Insulin Like Growth Factor-I Receptor Expression and Its Clinico-Pathological Significance in Histologic Subtypes of Human Thyroid Cancer Experimental Biology and Medicine, April 1, 2009; 234(4): 372 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. H. Surinya, B. E. Forbes, F. Occhiodoro, G. W. Booker, G. L. Francis, K. Siddle, J. C. Wallace, and L. J. Cosgrove An Investigation of the Ligand Binding Properties and Negative Cooperativity of Soluble Insulin-like Growth Factor Receptors J. Biol. Chem., February 29, 2008; 283(9): 5355 - 5363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q.-s. Ji, M. J. Mulvihill, M. Rosenfeld-Franklin, A. Cooke, L. Feng, G. Mak, M. O'Connor, Y. Yao, C. Pirritt, E. Buck, et al. A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor dependent tumor growth in vivo Mol. Cancer Ther., August 1, 2007; 6(8): 2158 - 2167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Blanquart, C. Gonzalez-Yanes, and T. Issad Monitoring the Activation State of Insulin/Insulin-Like Growth Factor-1 Hybrid Receptors Using Bioluminescence Resonance Energy Transfer Mol. Pharmacol., November 1, 2006; 70(5): 1802 - 1811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Aiello, G. Pandini, F. Frasca, E. Conte, A. Murabito, A. Sacco, M. Genua, R. Vigneri, and A. Belfiore Peroxisomal Proliferator-Activated Receptor-{gamma} Agonists Induce Partial Reversion of Epithelial-Mesenchymal Transition in Anaplastic Thyroid Cancer Cells Endocrinology, September 1, 2006; 147(9): 4463 - 4475. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lu, D. Katsaros, A. Wiley, I. A. Rigault de la Longrais, H. A. Risch, M. Puopolo, and H. Yu The Relationship of Insulin-Like Growth Factor-II, Insulin-Like Growth Factor Binding Protein-3, and Estrogen Receptor-{alpha} Expression to Disease Progression in Epithelial Ovarian Cancer Clin. Cancer Res., February 15, 2006; 12(4): 1208 - 1214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Denley, G. V. Brierley, J. M. Carroll, A. Lindenberg, G. W. Booker, L. J. Cosgrove, J. C. Wallace, B. E. Forbes, and C. T. Roberts Jr. Differential Activation of Insulin Receptor Isoforms by Insulin-Like Growth Factors Is Determined by the C Domain Endocrinology, February 1, 2006; 147(2): 1029 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Haluska, J. M. Carboni, D. A. Loegering, F. Y. Lee, M. Wittman, M. G. Saulnier, D. B. Frennesson, K. R. Kalli, C. A. Conover, R. M. Attar, et al. In vitro and In vivo Antitumor Effects of the Dual Insulin-Like Growth Factor-I/Insulin Receptor Inhibitor, BMS-554417 Cancer Res., January 1, 2006; 66(1): 362 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Cabanas, T Garcia-Caballero, J Barreiro, L Castro-Feijoo, R Gallego, T Arevalo, R Canete, and M Pombo Papillary thyroid carcinoma after recombinant GH therapy for Turner syndrome Eur. J. Endocrinol., October 1, 2005; 153(4): 499 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D Fuhrer, M Eszlinger, S Karger, K Krause, C Engelhardt, D Hasenclever, H Dralle, and R Paschke Evaluation of insulin-like growth factor II, cyclooxygenase-2, ets-1 and thyroid-specific thyroglobulin mRNA expression in benign and malignant thyroid tumours Eur. J. Endocrinol., May 1, 2005; 152(5): 785 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Venables Aberrant and Alternative Splicing in Cancer Cancer Res., November 1, 2004; 64(21): 7647 - 7654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Denley, E. R. Bonython, G. W. Booker, L. J. Cosgrove, B. E. Forbes, C. W. Ward, and J. C. Wallace Structural Determinants for High-Affinity Binding of Insulin-Like Growth Factor II to Insulin Receptor (IR)-A, the Exon 11 Minus Isoform of the IR Mol. Endocrinol., October 1, 2004; 18(10): 2502 - 2512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Vella, R. Mineo, F. Frasca, E. Mazzon, G. Pandini, R. Vigneri, and A. Belfiore Interleukin-4 Stimulates Papillary Thyroid Cancer Cell Survival: Implications in Patients with Thyroid Cancer and Concomitant Graves' Disease J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2880 - 2889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang, J. Grunler, and M. S. Lewitt Gender-Specific Pattern of Insulin-Like Growth Factor-Binding Protein-3 Protease Activity in Mouse Thyroid Endocrinology, March 1, 2004; 145(3): 1137 - 1143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pandini, E. Medico, E. Conte, L. Sciacca, R. Vigneri, and A. Belfiore Differential Gene Expression Induced by Insulin and Insulin-like Growth Factor-II through the Insulin Receptor Isoform A J. Biol. Chem., October 24, 2003; 278(43): 42178 - 42189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Sciacca, M. Prisco, A. Wu, A. Belfiore, R. Vigneri, and R. Baserga Signaling Differences from the A and B Isoforms of the Insulin Receptor (IR) in 32D Cells in the Presence or Absence of IR Substrate-1 Endocrinology, June 1, 2003; 144(6): 2650 - 2658. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
