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Angiogenesis Laboratory (V.P.), Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114; Department of Medical Oncology (C.S.M., C.M., D.S., G.F., N.M.), Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115; Department of Pathology (V.K.), School of Medicine, Aristotle University of Thessaloniki, Thessaloniki 54621, Greece; and Endocrine Unit (S.T.-B., D.A.K., N.M.), Evgenidion Hospital, Athens 11527, Greece
Address all correspondence and requests for reprints to: Nicholas Mitsiades, M.D., Ph.D., Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Mayer Building, M555, Boston, Massachusetts 02115. E-mail: mitsiades{at}netscape.net.
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(HIF-1) and activator protein-1 (AP-1). Yet, the paracrine signaling pathways regulating VEGF production and angiogenesis in thyroid cancer have not been fully elucidated. In this study, we, therefore, investigated the regulation of VEGF production by the thyroid carcinoma cell line SW579. We found that IGF-I up-regulated VEGF mRNA expression and protein secretion. Furthermore, transfection of SW579 cells with vector expressing a constitutively active form of Akt, a major mediator of IGF-I signaling, also stimulated VEGF expression. The IGF-I-induced up-regulation of VEGF production was associated with activation of AP-1 and HIF-1 and was abrogated by phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002); Jun kinase inhibitor (SP600125); HIF-1 antisense oligonucleotide; or geldanamycin, an inhibitor of the heat shock protein 90 molecular chaperone, which regulates the three-dimensional conformation and function of IGF-I-receptor and Akt. These data indicate that IGF-I stimulates VEGF synthesis in thyroid carcinomas in an Akt-dependent pathway via AP-1 and HIF-1 and provide the framework for clinical use of small-molecule inhibitors, including geldanamycin analogs, to abrogate proangiogenic cascades in thyroid cancer. |
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and the activator protein-1 (AP-1) transcription factors (4, 5). This leads to transcriptional activation of the VEGF gene, increased stability of VEGF mRNA, and increased production of VEGF protein. VEGF participates in the pathogenesis and progression of a wide range of angiogenesis-dependent diseases, including proliferative diabetic retinopathy (6), certain inflammatory disorders (1, 7), and cancer (1, 7). In thyroid disorders, overexpression of VEGF has been demonstrated in subacute thyroiditis (8, 9), chronic lymphocytic thyroiditis (8, 10), and Graves’ disease (8). Insulin has been reported to up-regulate VEGF mRNA in nonneoplastic thyrocytes (11). Moreover, differentiated thyroid carcinomas strongly express VEGF, in larger amounts than normal thyroid tissue (10, 12, 13). In addition, samples of metastatic thyroid cancer lesions express higher levels of VEGF mRNA and protein than their primary counterparts (10, 13), whereas serum VEGF levels are significantly elevated in patients with metastatic differentiated thyroid cancer (14). Higher contents of VEGF mRNA and VEGF protein are associated with more intense mitogenic activity (10) and higher tumorigenic potential in thyroid carcinomas (15). Furthermore, endothelial cells have higher mitogenic activity in neoplastic tissues in contrast to the quiescent endothelium of nontumoral tissues (10). Finally, a neutralizing anti-VEGF monoclonal antibody has been demonstrated to inhibit thyroid cancer growth in vivo (16), and the farnesyltransferase inhibitor manumycin has been reported to decrease VEGF production from thyroid carcinoma cells and inhibit angiogenesis in thyroid carcinoma xenografts (17).
Although these data support a role for VEGF as a regulator of tumor progression in thyroid carcinomas as well as a promising marker of tumor aggressiveness and predictor of metastatic potential (18), the molecular mechanisms regulating VEGF production by thyroid carcinoma cells are still unknown. We have, therefore, investigated the effect of IGF-I, a pivotal growth/survival factor for thyroid carcinoma cells, on VEGF expression. We found that IGF-I stimulates VEGF expression in thyroid carcinoma cells via the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway and the activation of the transcription factors AP-1 and HIF-1. These data provide the framework for therapeutic strategies to suppress thyroid cancer-associated neoangiogenesis by targeting the regulation of VEGF production by thyroid carcinoma cells.
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Materials and Methods |
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The SW579 cell line, derived from a poorly differentiated human thyroid adenocarcinoma (poorly differentiated carcinoma with nuclear features of papillary carcinoma and squamous differentiation), was purchased from American Type Culture Collection (Manassas, VA) and grown in DMEM (BioWhittaker, Walkersville, MD) with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS; GIBCO/BRL, Gaithersburg, MD). The NPA (papillary), FRO (anaplastic), and WRO (follicular) cells lines (generous gifts from Dr. James A. Fagin, University of Cincinnati School of Medicine, Cincinnati, OH) have been previously described (19). The heat shock protein (Hsp) 90 chaperone inhibitor geldanamycin, the Jun kinase (JNK) inhibitor SP600125, and the PI-3K inhibitors wortmannin and LY294002 were purchased from Calbiochem (La Jolla, CA) and used at concentrations that did not affect cell viability within the time period of our experiments. IGF-I was purchased from R&D Systems (Minneapolis, MN).
Determination of IGF-I receptor status by flow cytometric analysis
The cell surface expression of the IGF-I receptor (IGF-IR) (CD221) was characterized in SW579 cells by flow cytometry. Staining was performed as described previously (20). Briefly, 106 cells were incubated with a phycoerythrin (PE)-conjugated mouse monoclonal antihuman IGF-IR antibody (R&D Systems) or an isotype-matched control (Ms-IgG1-PE) (5.0 µg) for 45 min. Cells were then washed, fixed with 1% formaldehyde PBS, and analyzed on an EPICS-XL-MCL flow cytometer (Coulter, Hialeah, FL). Results were confirmed with indirect staining of SW579 cells, using the mouse monoclonal human anti-IGF-IR antibody IR3 (Oncogene Research, La Jolla, CA) or isotype-matched control. Cells were then washed with PBS and incubated for 45 min with 2.0 µg goat antimouse IgG fluorescein isothiocyanate-conjugated F(ab')2 fragment, washed, fixed, and analyzed as described previously.
Quantification of VEGF mRNA presence in thyroid carcinoma cells
SW579 cells were plated in 25-cm2 flasks in medium containing 10% FCS and grown to 70–80% confluency. Subsequently, the cells were washed extensively with serum-free DMEM medium and incubated for 24 h with fresh serum-free medium at 37 C. The cells were then treated with or without IGF-I (200 ng/ml) for the indicated time periods, in fresh serum-free medium and harvested by scraping in cold PBS. Total RNA was extracted and purified with the RNeasy kit (Qiagen, San Diego, CA). One microgram of total RNA per specimen was used to quantify the amount of VEGF mRNA, using Quantikine colorimetric mRNA assay and a human VEGF probe that detects all known human VEGF mRNA splice variants (both from R&D Systems), according to the manufacturer’s instructions. Results were expressed as percentage of control (untreated) cells.
Determination of VEGF secretion by thyroid carcinoma cells
SW579 cells were plated in 24-well plates in medium containing 10% FCS and grown to 70–80% confluency. Subsequently, the cells were washed extensively with serum-free DMEM medium and incubated with fresh serum-free medium at 37 C for the time periods indicated in each experiment. The cells were then treated with the indicated concentrations of IGF-I for the indicated periods of time, in fresh serum-free medium, in the presence or absence of wortmannin (5 µM), LY294002 (20 µM), geldanamycin (1 µM), or SP600125 (25 µM). These inhibitors did not have any negative impact on cell viability within the time frame of our experiments. VEGF levels in the conditioned media were determined using a sandwich ELISA (R&D Systems), according to the manufacturer’s instructions. Results were expressed as percentages of the value of the respective control (untreated) cells. All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Representative data from one among three equivalent experiments are shown.
Transfection in thyroid carcinoma cells of a vector expressing constitutively active Akt
SW579 cells were plated on 24-well plates and 24 h later were washed with serum-free DMEM medium and transfected with a construct encoding the myc-tagged, myristoylated, constitutively active form of Akt (1 µg DNA/well; Upstate Biotechnologies, Lake Placid, NY) or the empty vector, with the help of Superfect (Qiagen, Valencia, CA) according to the instructions of the manufacturer. Subsequently, the cells were washed extensively with serum-free DMEM medium and incubated with fresh serum-free medium at 37 C. Twenty-four hours later, the cells were washed and incubated with fresh serum-free medium for additional 8 h. The conditioned media were collected and VEGF levels were quantified by ELISA as above. Successful transfection of the constitutively active Akt construct into the SW579 cells was verified by immunoblotting for Akt as well as for the myc tag that the transfected construct carries.
Quantification of AP-1 and HIF-1 DNA binding activity
The DNA binding activity of the transcription factors AP-1 and HIF-1 was evaluated using the Trans-AM AP-1 (c-jun) and trans-AM HIF-1 transcription factor assay kits, respectively (Active Motif North America, Carlsbad, CA), according to the manufacturer’s instructions. Briefly, nuclear extracts from SW579 cells, treated with or without IGF-I for the indicated time periods, were prepared as previously described (21) and incubated in 96-well plates coated with immobilized double-stranded oligonucleotides (5'-CGCTTGATGAGTCAGCCGGAA-3' or 5'-GATCGCCCTACGTGCTGTCTCAGATC-3') containing a consensus binding site (underlined sequences) for AP-1 or HIF-1, respectively. Transcription factor binding to the target oligonucleotide was detected by incubation with primary antibody specific for the activated form of c-Jun or HIF-1, respectively (Active Motif North America), visualized by anti-IgG horseradish peroxidase conjugate and developing solution, and quantified at 450 nm with a reference wavelength of 655 nm. To monitor the specificity of the assay, background binding was calculated by adding in selected wells the respective consensus oligonucleotides in excess (20 pmol/well) as soluble competitors that prevented transcription factor binding to the probe immobilized on the plate. The resulting values were subtracted from the values obtained in wells with immobilized oligonucleotides alone. This methodology has significantly higher sensitivity than EMSA and allows high-throughput automated quantification of transcription factor activity in our model in a format amenable to repeated measurements and statistical analysis (21, 22, 23, 24).
Transfection of HIF-1 antisense oligonucleotide
To delineate the role of HIF-1 as a mediator of IGF-I-induced VEGF expression in thyroid carcinoma cells, we transfected SW579 cells with a fully phosphorothioated single-stranded antisense oligonucleotide directed against the human HIF-1 translation initiation site (sequence, 5'-GCCGGCGCCCTCCAT-3') or control phosphorothioate oligonucleotide. SW579 cells were plated in 24-well plates and transfected with the help of oligofectamine (Life Technologies, Inc., Gaithersburg, MD) using 200 nM of each oligonucleotide (50 pmol/well), according to the instructions of the manufacturer. Subsequently, the cells were washed extensively with serum-free DMEM and incubated with fresh serum-free medium at 37 C. Twenty-four hours later, the cells were incubated with fresh serum-free medium, and IGF-I (200 ng/ml) was added to appropriate wells for additional 8 h. VEGF levels in the conditioned media were quantified by ELISA as above.
Statistical analysis
Quantitative comparisons were examined with the ANOVA method, followed by the Duncan’s test. Statistical significance was set at P < 0.05.
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Flow cytometric analysis of SW579 cells demonstrated cell surface expression of IGF-IR (CD221). Data obtained with a PE-conjugated anti-IGF-IR antibody are presented in Fig. 1
and were confirmed using the aIR3 antibody in indirect staining (data not shown).
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We investigated the effect of IGF-I, a well known growth/survival factor, in the regulation of VEGF expression in SW579 thyroid carcinoma cells. We found that IGF-I (200 ng/ml) up-regulated VEGF mRNA levels (P < 0.05; Fig. 2A). We also evaluated the effect of IGF-I on VEGF secretion. We found that IGF-I (100, 200, or 300 ng/ml for 8 h) stimulated the release of VEGF in the supernatant of SW579 cells that had been incubated previously in serum-free medium for 24 h (Fig. 2B). We then performed a time-course analysis of the stimulatory effect of IGF-I. SW579 cells were serum starved for 24 h and then cultured for 8, 16, or 24 h, either without cytokine stimulation (control cells) or in the presence of IGF-I (200 ng/ml). For each time point of the analysis, the VEGF levels secreted by control or IGF-I-stimulated cells were compared with the VEGF levels in unstimulated SW579 cells cultured for 8 h (Fig. 2C). When we calculated, for each time point of the experiment, the percentage of increase of VEGF secretion in IGF-I-stimulated cells over their respective control (SW579 cells cultured for the same duration, in the absence of IGF-I stimulation), we again confirmed that IGF-I induces an increase in VEGF secretion. We did not observe significant differences in the magnitude of VEGF increase between time points (52 ± 2%, 54 ± 22%, and 43 ± 10%, at 8, 16, or 24 h, respectively) (Fig. 2C). We subsequently evaluated the effect of the preincubation in serum-free medium on IGF-stimulated inducibility of VEGF secretion. We found that SW579 cells, serum starved for 0, 24, or 48 h and then treated with IGF-I (200 ng/ml for 8 h), demonstrated similar percentage increases in VEGF secretion over their respective controls (i.e. cells serum starved for the same period of time but not treated with IGF-I) (51 ± 8%, 47 ± 2%, and 55 ± 16%, respectively), although, as expected, the baseline VEGF concentration in supernatants from unstimulated cells decreased with prolonged preincubation in serum-free medium (Fig. 2D). Taken together, these data demonstrate that IGF-I produces significant and sustained increases in the secretion of VEGF from thyroid carcinoma cells, irrespective of the baseline status of the cancer cells.
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We investigated further the intracellular signaling pathway of IGF-induced up-regulation of VEGF secretion. IGF-I signaling has been reported to proceed through PI-3K and Akt in many models, including thyroid carcinoma (22, 25). We found that the PI-3K inhibitors LY294002 and wortmannin suppressed IGF-induced up-regulation of VEGF secretion (Fig. 4A; P < 0.005 in both cases). Moreover, transfection of a vector encoding a constitutively active form of Akt potently stimulated VEGF secretion (Fig. 4B; P < 0.001), thus mimicking the effect of IGF-I. Our data support the role of PI-3K and Akt in IGF-induced stimulation of VEGF secretion.
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in thyroid carcinoma cells
Mapping of the VEGF promoter has identified response elements for the transcription factors AP-1 and HIF-1 that cooperate for optimal activation in response to hypoxia (4, 5). We, therefore, investigated the effect of IGF-I on the DNA binding activity of AP-1 and HIF-1. We found that IGF-I stimulated the ability of both transcription factors to bind respective consensus DNA sequences (Fig. 5A; P < 0.05).
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To further investigate the role of the AP-1 pathway in the IGF-I-induced stimulation of VEGF, in our model, we evaluated the effect conferred on this stimulation by SP600125, a small-molecule inhibitor of JNK, the kinase responsible for phosphorylation and activation of the AP-1 subunit c-Jun. We found that SP600125 (25 µM) suppressed IGF-I-induced stimulation of VEGF expression (P < 0.05; Fig. 5B
). SP600125 alone did not suppress VEGF secretion. These data support a role for JNK and AP-1 as mediators of the effect of IGF-I in our model.
Inhibition of HIF-1 suppresses IGF-I-induced up-regulation of VEGF production in thyroid carcinoma cells
To further characterize the role of HIF-1 for IGF-I-induced stimulation of VEGF expression by thyroid cancer cells, we used a HIF-1 antisense oligonucleotide to specifically inhibit the activity of this transcription factor. We found that the HIF-1 antisense oligonucleotide attenuated the IGF-I-induced stimulation of VEGF expression (IGF-I induced only a 41 ± 4% increase of VEGF production in the presence of the HIF-1 antisense oligonucleotide, compared with a 68 ± 1% increase in the presence of the control oligonucleotide; P < 0.05) (Fig. 5C). Neither oligonucleotide had any effect on VEGF secretion in unstimulated cells. These data support a role for HIF-1 as a mediator of at least part of the stimulatory effect of IGF-I on VEGF secretion in our model.
Geldanamycin suppresses IGF-I-induced up-regulation of VEGF production in thyroid carcinoma cells
We have previously reported that IGF-I activates the kinase Akt in SW579 cells and that the ansamycin antibiotic geldanamycin, which inhibits the chaperoning activity of Hsp90 (26, 27), reduces the intracellular levels and activity of Akt (25) and attenuates the anti-apoptotic effect of IGF-I (25). We now found that geldanamycin (1 µM) suppressed both baseline and IGF-I-induced VEGF production by SW579 cells (Fig. 6). These data are in agreement with similar findings in another model (28) and support the role of the PI-3K/Akt pathway and the hsp90 chaperone in the regulation of VEGF production in thyroid carcinoma cells.
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. The IGF/IGF receptor pathway plays an important role in the pathogenesis of thyroid cancer. In the local microenvironment of thyroid carcinomas, IGF-I is produced by stromal cells, in significantly higher amounts than in normal thyroid tissue (29), and promotes proliferation (30) and suppresses apoptosis of neoplastic cells (25, 31) through the action of the IGF-IR, which is expressed on the surface of thyroid carcinoma cells. Our present study suggests an additional role for IGF-I in thyroid cancer, namely the regulation of angiogenic activity via the stimulation of VEGF expression. Most human tumors initially develop without significant intrinsic proangiogenic activity and exist in situ as microscopic lesions of 0.2–2-mm diameter for months to years. Without a major increase in their blood supply, only a small percentage of those microscopic lesions develop beyond that size (1, 7). Therefore, progression from microscopic carcinoma to clinically detectable tumor with metastatic potential requires that cancer cells not only evade the immune system (32) and degrade the extracellular matrix (33) but also stimulate the development of their own vasculature (1, 7). Before such an angiogenic switch occurs, tumor cells may be detectable by careful histologic examination or sensitive molecular techniques, but they generally do not lead to the full-blown clinical manifestations of neoplasia. Autopsy studies in accident victims who had never been diagnosed with cancer during their lifetime revealed microscopic thyroid carcinomas in more than 98% of individuals in the age group of 50–70 yr, whereas thyroid cancer is clinically diagnosed in only 0.1% of the population in this age range (1). This leads to the hypothesis that the angiogenic switch could be an important step in the progression of thyroid carcinomas.
VEGF is a pivotal regulator of angiogenesis and has been demonstrated to play a role in thyroid cancer (12, 13, 17, 34). Our study shows that IGF-I, a growth/survival factor produced in the local microenvironment of thyroid tumors, stimulates VEGF transcription and secretion via a PI-3K/Akt-dependent signaling pathway. Moreover, we found that inhibition of the hsp90 molecular chaperone, e.g. by geldanamycin (the prototypic ansamycin inhibitor of the hsp90 family), suppresses baseline and IGF-I-induced VEGF production. Hsp90 function is required for the proper three-dimensional conformation and, thus, the function of kinases such as IGF-IR and Akt (26, 27). Hsp90 inhibitors lower Akt protein levels and enzymatic activity in SW579 cells (25). Because hsp90 regulates the function of several signaling proteins, even its specific inhibitors of the ansamycin family (geldanamycin and its analogs) cannot be considered as specific Akt inhibitors and cannot, on their own, support mechanistic conclusions. However, our data on the effect of geldanamycin on IGF-I-induced VEGF secretion have direct clinical implications because geldanamycin analogs, such as 17-AAG, have been tested in phase I clinical trials for solid tumors, with favorable profile of side effects and preliminary evidence of antitumor activity (35, 36), and provide the rationale for future clinical use of such agents to specifically block the proangiogenic sequelae of IGF-I in thyroid cancer. The significance of using hsp90 inhibitors to inhibit the activity of the Akt pathway is further underlined by previous data both from our group and others, which have demonstrated the PI-3K/Akt-dependent up-regulation of VEGF by insulin in retinal pigment epithelial cells (21) and thyrocytes (11) and by IGF-I in osteoblasts (28).
Moreover, we have further characterized the downstream effectors of IGF-I/Akt signaling in the regulation of VEGF expression in our model. We found that the transcription factors AP-1 and HIF-1 are activated by IGF-I treatment. Because the VEGF promoter contains binding elements for these factors (4, 5), which cooperate for optimal transcriptional activity in response to hypoxia, we hypothesized that they also mediate the stimulatory effect of IGF-I. Indeed, we found that the effect of IGF-I was attenuated by a JNK inhibitor, which blocks c-jun phosphorylation, as well as by a HIF-1 antisense oligonucleotide, thus confirming the role of these two transcription factors in our model and providing additional molecular targets for the abrogation of angiogenic signaling in thyroid cancer.
In conclusion, we have demonstrated that IGF-I stimulates VEGF production in thyroid carcinoma cells via the transcriptional factors AP-1 and HIF-1. Agents that abrogate the activity of these factors and/or IGF-I signaling, such as the novel agent geldanamycin, could exert antineoplastic activity clinically, in part by inhibiting the production of angiogenic stimuli.
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Footnotes |
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Abbreviations: AP-1, Activator protein-1; FCS, fetal calf serum; HIF-1, hypoxia-inducible factor-1; Hsp, heat shock protein; IGF-IR, IGF-I receptor; JNK, Jun kinase; PE, phycoerythrin; PI-3K, phosphatidylinositol 3-kinase; VEGF, vascular endothelial growth factor.
Received March 6, 2003.
Accepted July 30, 2003.
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and VEGF. J Clin Invest 109:805–815[CrossRef][Medline]
B and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications. Oncogene 21:5673–5683[CrossRef][Medline]
B blockade in multiple myeloma: therapeutic applications. Blood 99:4079–4086. Endocrinology 143:420–425This article has been cited by other articles:
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| 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 |
) or IGF-I-stimulated cells (
) were expressed as percentage of VEGF secretion in unstimulated SW579 cells cultured for 8 h. By calculating, for each time point of the experiment, the percentage increase of VEGF secretion in IGF-I-stimulated cells over their respective controls (SW579 cells cultured for the same duration, in the absence of IGF-I stimulation), we again confirmed that IGF-I stimulates VEGF secretion, with comparable percentage increases in VEGF secretion at all time points (52 ± 2%, 54 ± 22%, and 43 ± 10%, at 8, 16, or 24 h, respectively). D, We evaluated the effect of the preincubation in serum-free medium on IGF-induced inducibility of VEGF secretion. SW579 cells were serum starved for 0, 24, or 48 h and then cultured for 8 h either with IGF-I stimulation (200 ng/ml for 8 h; 
) and HIF-1
)
or control oligonucleotide (