STAT3 negatively regulates thyroid tumorigenesis

pY-STAT3 Is Expressed in Human Primary PTC.

The expression of activated pY-STAT3 in human thyroid tumors has been poorly characterized. We analyzed nuclear pY-STAT3 levels by immunohistochemistry (IHC) in a panel of 146 primary thyroid lesions. pY-STAT3 expression was seen in 10 of 12 (83%) benign FTAs, 63 of 110 (57%) PTCs, and 6 of 24 (25%) follicular thyroid carcinomas (FTCs) (Fig. 1 A and B and Table 1). We also investigated whether pY-STAT3 expression was associated with clinical–pathological parameters in PTC, such as tumor size, vascular invasion, and presence of distant metastases. The average PTC tumor size was ∼4 cm, and tumors were grouped into ≥4- and <4-cm size categories. We found that PTCs larger than 4 cm expressed lower levels of pY-STAT3 in tumor cells [score: 0.7 ± 0.3 (≥4 cm) vs. 2.9 ± 0.6 (<4 cm); Fisher P = 0.04]. No significant association was found between pY-STAT3 and vascular invasion. However, an inverse association between pY-STAT3 expression and the presence of distant metastases was determined (Fisher P = 0.03). Specifically, in a subset of aggressive PTC cases, only 7 of 20 (35%) were pY-STAT3–positive, whereas 56 of 90 (62%) of the remaining more indolent PTCs expressed pY-STAT3. Approximately one-half of PTCs harbor BRAFV600E mutations (17). Here, the BRAF mutational status was determined in 98 of 110 of the PTCs (17); 42% harbored the BRAFV600E mutation and expressed higher levels of pY-STAT3 relative to BRAFwt tumors (Mann–Whitney P = 0.04) (Table 1). In ∼70% of the PTCs (as well as in the FTAs and FTCs), we detected the highest levels of pY-STAT3 within tumor cells at the leading edge of the tumor in association with stromal cells (fibroblasts and endothelial and immune cells), which were also strongly positive for pY-STAT3 (Fig. 1C).

Fig. 1.

pY-STAT3 expression in human thyroid lesions. (A) Quantification of pY-STAT3 expression levels in thyroid tumors. (B) Representative pY-STAT3 staining in benign FTA and malignant PTC and FTC. Arrow points at a pY-STAT3–positive endothelial cell in PTC. (C, 1) Tumor cells (T) within the stroma are strongly pY-STAT3–positive. (C, 1) Stromal cells, namely fibroblasts (F) and endothelial cells lining up vessels (V), highly express pY-STAT3.

STAT3 Is Regulated by the IL-6/gp130/JAK Signaling in TCCs.

Aberrant IL-6/gp130/JAK signaling has been shown to be a principle regulator of STAT3 activation in many cancers (11, 12, 18). We analyzed pY-STAT3 expression in several TCCs harboring RET, BRAF, and RAS alterations (Table S1) (19, 20). As in primary tumors, pY-STAT3 was heterogeneously expressed in these cell lines (Fig. S1A). We determined the role of IL-6 signaling in mediating STAT3 activation in the pY-STAT3–expressing cell lines (8505C, TPC-1, HTH-7, and SW1736) with inhibitors of the IL-6 pathway, namely anti-human IL-6 (α-IL6), IL-6R [MRA (also known as tocilizumab)], and gp130 (clone BR3) antibodies, (Fig. 2A) and JAK inhibitors P6 and Astrazeneca (AZD) 1480 (Fig. 2B). BR3, P6, and AZD1480 effectively blocked STAT3 phosphorylation (Fig. 2 A and B). IL-6 was secreted by all of the cell lines to variable levels (Fig. S1B). In all cell lines, except HTH-7, α-IL-6 and MRA reduced pY-STAT3 (Fig. 2A). Additionally, 8505C, TPC-1, and SW1736 cells expressed mRNA for IL-6 and the membrane form of the IL-6R (mIL-6R), whereas none expressed the soluble form of the receptor (sIL-6R) (Fig. S1C). We also examined the mRNA levels of other members of the IL-6/gp130 family of cytokines, including leukemia inhibitory factor (LIF) and oncostatin M (OSM) ligands and receptors (Fig. S1C). HTH-7 did not express mIL-6R but did express OSM and OSM receptor (Fig. S1C). However, because of technical limitations, attempts to block OSM signaling were unsuccessful.

Fig. 2.

pY-STAT3 is regulated by the IL-6/gp130/JAK signaling pathway in TCCs. (A) Extracts from TCCs treated for 4 h with control IgG (at 10 μg/mL) as well as blocking antibodies targeting IL-6R (MRA; at 50 μg/mL), gp130 (BR3; at 10 μg/mL), and IL-6 ligand (α-IL-6; at 1 μg/mL) were immunoblotted with the indicated antibodies. HTH-7 lanes were noncontiguous in the same gel. (B) Extracts from TCCs were treated for 16 h with JAK inhibitors P6 (1 μM) and AZD1480 (A; 1 μM) and immunoblotted with the indicated antibodies. α-tubulin was the loading control. (C) RET/PTC and BRAFV600E induce STAT3 Y-phosphorylation and transcriptional activation in HEK293 cells. (C, 1) Cells were transiently transfected with RET/PTC3, BRAFwt, and BRAFV600E expression vectors. Protein extracts were immunoblotted with the indicated antibodies. (C, 2) HEK293 cells were transiently transfected with the indicated expression vectors combined with Interleukin-6 response element (IRE)-Luciferase (Luc) reporter plasmid. Luciferase activity was measured after 48 h (mean ± SD; n = 3). (D) IL-6/JAK signaling mediates BRAFV600E-induced pY-STAT3 in PCCl3 cells. Cells were transfected with pCMV (control vector) or pCMV-BRAFV600E. After 48 h, the medium from BRAFV600E cells was replaced with fresh medium RPMI (column 7) or fresh RPMI supplemented with 50 ng/mL IL-6 (column 8) for 30 min. Cells were lysed, and protein extracts were incubated with the indicated antibodies.

In addition to the IL-6 family of cytokines, STAT3 activation can be regulated by growth factors and nonreceptor tyrosine kinases (11, 21). In thyroid cells, RET/PTC (8) and mutant RET (22, 23) mediate STAT3 phosphorylation, but no studies have examined the role of oncogenic BRAF in STAT3 activation. Given the association between BRAFV600E and pY-STAT3 positivity in our series of human PTC (Table 1), we investigated whether oncogenic BRAF could also lead to STAT3 Y705 phosphorylation and transcriptional activation. We transfected HEK293 cells with exogenous pCMV-RET/PTC3, pCMV-BRAFwt, and pCMV-BRAFV600E expression vectors. Both RET/PTC3 and mutant BRAF (but not control CMV or BRAFwt) induced pY-STAT3 expression (Fig. 2C, 1) and transcriptional activity (Fig. 2C, 2). BRAFV600E has been shown to up-regulate IL-6 in melanoma cells (24). To determine whether BRAFV600E-mediated STAT3 activation was through increased IL-6 expression, we transfected a rat thyroid cell line, PCCl3, with either pCMV-BRAFV600E or pCMV expression vectors. The addition of exogenous IL-6 on control (pCMV) transfected PCCl3 cells did not increase pY-STAT3 levels (Fig. 2D). In contrast, BRAFV600E expression (and BRAFwt, albeit at lower levels) markedly increased pY-STAT3 levels, which were abrogated by the JAK inhibitor, AZD1480 (Fig. 2D). Similarly, removal of conditioned media from the BRAFV600E-PCCl3 cells led to a reduction in pY-STAT3 levels, which could be reversed through the addition of IL-6 (Fig. 2D). These data suggest that BRAFV600E can up-regulate downstream IL-6 signaling, resulting in STAT3 activation.

STAT3 Knockdown Increases Tumorigenicity of TCC.

We evaluated the effects of STAT3 knockdown in pY-STAT3–expressing cell lines (8505C, TPC-1, HTH7, and SW1736) by transduction of shSTAT3 and scrambled control (shCT) vectors. Decreases in pY-STAT3 protein expression were confirmed by Western blot analysis (Fig. S2A). pERK1/2 levels remained unchanged, with the exception of TPC-1, where shSTAT3 displayed lower pERK1/2 levels than their respective shCT (Fig. S2A). The proliferation of shSTAT3 cells was compared with shCT cells, and viable cell numbers were determined daily, revealing no differences in growth between matched cell lines (Fig. S2B). The in vivo tumorigenic capacity of 8505C, TPC-1, and HTH7 shCT and shSTAT3 cells was assessed by s.c. injection of cells in athymic nude mice. The SW1736 cell line in our hands did not form tumors and was not further analyzed. Tumors were allowed to grow, and mice were killed 5 (8505C and TPC-1) and 3 (HTH-7) wk after injection. For all matched xenografts, shSTAT3 tumors were significantly larger than the shCT (Fig. 3A). Down-regulation of pY-STAT3 in the shSTAT3 tumors was confirmed by IHC analysis of tumor sections (Fig. 3B). No differences were found in tumor vasculature (Meca-32) or apoptosis (TUNEL) between shCT and shSTAT3 tumors (Fig. S3). Notably, pERK1/2 levels were down-regulated in 8505C- and TPC-1–shSTAT3 tumors compared with shCT. pS6 (S235/236) and pSTAT1 (Y701) levels remained unchanged in all tumors (Fig. S3).

Fig. 3.

STAT3 knockdown in thyroid cancer cell lines enhances their tumorigenic capacity. (A) 8505C (10 ^{cells), TPC-1 (10 cells), and HTH-7 (10 cells) were s.c. coinjected with Matrigel in female athymic nude mice (}n = 10 tumors/cell line), and tumor volume was estimated at 5 (8505C and TPC-1) or 3 (HTH-7) wk (mean ± SEM). (B) Representative sections from shCT and shSTAT3 tumor sections stained for pY-STAT3. Quantification of tumor pY-STAT3 levels (n ≥ 3) is on the right. (Magnification: 100×.) ***P < 0.0001.

Given the recently described roles for unphosphorylated STAT3 in tumorigenesis (25, 26), several experimental controls were used. First, STAT3 levels were reduced in the K1 cell line, which expresses total STAT3 but very low levels of the phosphorylated protein (Figs. S1A and S4A, 1). Injection of these cells in nude mice generated tumors with similar sizes independently of their STAT3 status (Figs. S4A, 2). Notably, all pY-STAT3–positive cells were stromal in origin (Figs. S4A, 3). Second, we introduced either a tyrosine-mutant form of STAT3 (STAT3Y705F) or WT murine-STAT3 (to test for off-target effects of the shSTAT3) into the 8505C-shSTAT3 cells (Figs. S4B, 1). These cell lines were injected s.c., and tumor volumes were determined. The expression of the tyrosine mutant did not rescue the tumor-suppressive effects of STAT3, whereas reexpression of WT STAT3 reduced tumor growth (Figs. S4B, 2). Expression of both pY-STAT3 and total STAT3 was confirmed in xenografts by IHC (Figs. S4B, 3).

Thyrocyte-Specific Deletion of STAT3 in a Murine Model of BRAFV600E-Induced PTC Leads to Increased Thyrocyte Proliferation and Tumor Growth.

The thyroid peroxidase (TPO) -Cre/lox-stop-lox (LSL)-BRAFV600E murine model of thyroid cancer has been recently characterized (27). These tumors express high levels of pY-STAT3 throughout the tumor (Fig. 4). To examine the role of STAT3 in this model, we deleted STAT3 in BRAFV600E-expressing thyrocytes by crossing STAT3 ^{mice (}28) with TPO-Cre mice (Fig. S5A). Importantly, STAT3 deficiency in thyrocytes from BRAFwt mice (TPO-Cre/STAT3^{) did not alter the phenotype and histological appearance of the thyroids compared with STAT3 deficiency in thyrocytes from C57BL/6 WT mice (}Fig. S5B). TPO-Cre/STAT3 ^{mice were crossed with BRAF/STAT3 mice to generate mice that expressed BRAFV600E in thyrocytes with or without STAT3 (}Fig. S5A). BRAF/STAT3 ^{mice were phenotypically similar to BRAF/STAT3wt mice. However, by 5 wk of age and at later time points (6–8 and 9–13 wk), the BRAF/STAT3 mice exhibited significantly larger thyroid tumors than those tumors from BRAF/STAT3wt mice (}Fig. 4A). Histologically, by 5 wk of age, tumors from mice with either genotype had similar histological features, including signs of local invasion to the skeletal muscle and blood vessels (Fig. 4C and Fig. S5B, 2). In age-matched mice, the tissue architecture of the PTC from BRAF/STAT3wt mice was homogenous: neoplastic tissue was composed of follicles and areas of papilliferous hyperplasia (Fig. 4 B and C). In contrast, the BRAF/STAT3 ^{tumors were more heterogeneous, displaying early signs of solid growth (∼75% by 5 wk of age) that were only apparent in older BRAF/STAT3wt mice (∼24% by 8 wk of age) (}Fig. 4 B and C). IHC confirmed reduction of pY-STAT3 levels in BRAF/STAT3 ^{thyrocytes (}Fig. 4D). Protein Ki67 levels were higher in the BRAF/STAT3 ^{tumors compared with the BRAF/STAT3wt tumors (}Fig. 4D), and no differences were detected in the apoptotic index (TUNEL and cleaved-caspase 3) between the two groups (Fig. S5C). We tested whether compensatory up-regulation of several pathways, known to be important in thyroid pathogenesis [PI3K/mammalian target of rapamycin (mTOR), MAPK, and TGF-β], was occurring in BRAF/STAT3 ^{tumors. Comparative IHC for pS6, p-p38/MAPK, pJNK/MAPK, and pSmad2/3 revealed no significant differences in the expression levels of any of these markers between the tumor groups. A slight reduction in pERK1/2 levels was observed in BRAF/STAT3 thyrocytes compared with BRAF/STAT3wt, similar to what we observed in the 8505C and TPC-1 xenografts (}Figs. S3 and S5C).

Fig. 4.

STAT3 deficiency in thyrocytes of LSL-BRAFV600E mice increases thyroid tumor size and proliferation. (A) BRAF/STAT3wt and BRAF/STAT3 ^{mice were killed at distinct time points and thyroid volume was determined (mean ± SEM). Images represent thyroids from 5-wk-old mice. (}B) Distribution of mouse genotype by histological pattern. (C) Histological sections of thyroids from BRAF/STAT3wt and BRAF/STAT3 ^{mice. (}C, 1 and 2) BRAF/STAT3wt thyroids revealing hobnail PTC with follicular architecture at 5 wk and (C, 3) mixed follicular (F) and solid (S) architecture at 9 wk. (C, 4 and 5) Thyroid section of a 5-wk-old BRAF/STAT3 ^{mouse revealing predominantly solid areas. (}D) IHC analysis of pY-STAT3 and Ki67 in the thyroids of 5-wk-old BRAF/STAT3wt and BRAF/STAT3 ^{mice. *}P < 0.05, ***P < 0.0001.

STAT3 Knockdown in TCCs and Transgenic Mice Leads to IGFBP7 Down-Regulation.

We performed genome-wide expression analysis in 8505C, TPC-1, and HTH-7 shCT and shSTAT3 cell lines to identify potential molecules or signaling pathways explaining the increased growth of shSTAT3 tumors. A total of 235 genes were down-regulated, and 146 genes were up-regulated in the three shSTAT3 cell lines compared with their respective shCT (Dataset S1). We focused on those genes with differential regulation that was common to at least two of the cells lines (Table S2). Of the seven commonly down-regulated genes, IGFBP7 was decreased in 8505C and HTH-7 shSTAT3 cells compared with shCT (Table S2). IGFBP7 has been shown to have a tumor-suppressive activity in models of melanoma, colorectal cancer, and thyroid cancer (29–31). We determined that IGFBP7 RNA (Fig. 5A) [by quantitative real-time RT-PCR (Q-PCR)] and protein levels (by IHC) (Fig. 5 B and C) were decreased in 8505C-, HTH-7– and TPC-1–shSTAT3 cells, xenografts, and transgenic tumor models compared with control mice. In the xenografts and transgenic tumors, a positive association between pY-STAT3 and IGFBP7 levels was observed (Fisher exact P = 0.06) (Fig. 5 B and C). A similar relationship between pY-STAT3 and IGFBP7 was detected in 47 primary human PTC samples by IHC (Fig. 5D). IGFBP7 was heterogeneously expressed in all 47 cases: 10 cases (21%) displayed low levels of IGFBP7, 17 cases (36%) had moderate levels of IGFBP7, and 20 cases (43%) had high levels of IGFBP7. A significant positive correlation between pY-STAT3 and IGFBP7 expression was observed (Spearman correlation P = 0.03) (Fig. 5D).

Fig. 5.

IGFBP7 levels are reduced in STAT3-deficient thyroid cancer models. (A) Q-PCR of IGFBP7 RNA levels in shCT and shSTAT3 8505C, TPC-1, and HTH-7 cell lines. Bars represent average IGFBP7/HRPT expression ± SEM of three independent experiments. (B) Representative immunostaining of IGFBP7 in sections of shCT and shSTAT3 tumor xenografts and (C) BRAF/STAT3wt vs. BRAF/STAT3 ^{transgenic tumors. (}D) pY-STAT3 expression positively correlates with IGFBP7 expression in human PTC. Representative serial sections of 54 human PTC specimens were stained for nuclear pY-STAT3 and cytoplasmic IGFBP7. Examples of low, moderate, and strong pY-STAT3 and IGFBP7 staining are shown. Of 54 cases, 38 cases (70%) displayed none or low pY-STAT3 IGFBP7 (0/+), and 11 cases displayed equally moderate/high (++/+++) levels of both proteins (20%). Fisher exact P < 0.001; Spearman correlation P = 0.03. (Magnification: 200×.)

Promoter methylation has been described as the most frequent event accounting for IGFBP7 down-regulation in cancer models (30, 32). We analyzed the −237- to +10-bp region of the IGFBP7 promoter, corresponding to a CpG island associated with variations in IGFBP7 expression (30, 32), in shCT and shSTAT3 8505C and TPC-1 cell lines. We detected additional partially methylated CpG sites in 8505C and TPC-1 shSTAT3 cell lines at positions −100, −169 (8505C), and −100 (TPC-1) bp (Fig. S6).

Increased Glycolysis of STAT3-Deficient TCCs.

STAT3 down-regulation in 8505C, TPC-1, and HTH-7 cell lines led to increased tumor growth with no apparent effects in vitro (Fig. S2). We investigated whether the tumor microenvironment might explain such differences in cellular behavior. IHC characterization of xenografts and transgenic mice revealed no differences in tumor vasculature (Figs. S3 and S5C). Moreover, no significant differences were detected in T-cell numbers (CD3 staining) and activated macrophages (Iba-1) in BRAF/STAT3 ^{tumors compared with STAT3wt tumors from transgenic mice (}Fig. S5C).

The metabolic switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis is a hallmark of many cancers (33). STAT3 has been shown to mediate metabolic changes in cells through the regulation of energy metabolism and oxidative stress through canonical (transcriptional) and noncanonical activities (34, 35). We hypothesized that STAT3 may function primarily as a positive regulator of OXPHOS in thyroid cancer. Thus, a reduction in STAT3 levels might shift the balance to enhanced glycolysis for energy production, leading to a selective growth advantage in a hypoxic in vivo tumor microenvironment. To test this hypothesis, we determined the growth of 8505C and TPC-1 shCT and shSTAT3 cell lines under different concentrations of cobalt chloride (CoCl₂), a commonly used hypoxia-mimetic; 8505C and TPC-1 shSTAT3 cells grew more efficiently under CoCl₂ treatment than their respective shCT cells (P < 0.0001) (Fig. 6A, 1). CoCl₂ stabilizes the HIF1α in normoxia, impeding its proteasomal-dependent degradation (36). STAT3 has been shown to both transcriptionally regulate HIF1α and impede its degradation through the sequestration of the von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase (VHL-E3) (37, 38). We observed that CoCl₂ induced HIF1α accumulation at similar levels in both shCT and shSTAT3 cells (Fig. 6A, 2). Surprisingly, HIF1α protein levels were higher in shSTAT3 cells compared with shCT at basal levels (Fig. 6A, 2). Notably, HIF1a mRNA levels were lower in shSTAT3 compared with shCT cells (Fig. S7A). Finally, CoCl₂ treatment led to a reduction in pY-STAT3 levels (Fig. 6A, 2). These observations suggest that STAT3 is a negative regulator of HIF1α protein expression/stability in these TCCs. Response to hypoxia through HIF1α leads to the up-regulation of glycolytic enzymes, increased glucose consumption and lactate production, and negative regulation of OXPHOS (33). Both under normoxic conditions and after treatment with CoCl₂, shSTAT3 cells consumed larger amounts of glucose and produced more lactate than their respective shCT cells (Fig. 6B and Fig. S7B). Consistently, in shSTAT3 cells, significant drops in oxygen consumption rate (P < 0.001) as well as mitochondrial membrane potential (mtΔψ), which reflects the pumping of hydrogen ions across the inner membrane during OXPHOS (P = 0.03), were detected (Fig. 6C).

Fig. 6.

STAT deficiency induces a glycolytic metabolism in TCCs. (A, 1) shCT and shSTAT3 TCCs (8505C and TPC-1) were grown for 96 and 48 h, respectively, with RPMI (1% serum) and different concentrations of the hypoxia-mimetic CoCl₂. Growth was determined by the sulpharhodamine B assay. (A, 2) shCT and shSTAT3 cell lines (8505C and TPC-1) were treated with CoCl₂ (100 μM) for 24 h. Total protein was probed with the indicated antibodies. (B and C) Cells were grown in plain RPMI for 48 h with or without CoCl₂ (100 μM). (B) Glucose uptake was determined by subtracting the glucose concentration in the supernatant from the initial concentration in fresh RPMI. Lactate was measured in the supernatant of the cell. Results were normalized to protein amount (milligrams). Bars represent mean ± SEM (n = 3). (C) The oxygen consumption rate and mitochondrial membrane potential (JC-1) were measured in equal numbers of 8505C shCT and shSTAT3 cells (mean ± SEM; n = 3). (D) 8505C shCT and shSTAT3 cell lines were treated with CoCl₂ (100 μM) for 24 h, and the RNA levels of the indicated glycolytic enzymes (PDK1 and GLUT1/3) were determined and normalized to β-actin. Bars are mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.0001.

The glycolysis regulator, pyruvate dehydrogenase kinase (PDK), inactivates the oxidation of pyruvate by pyruvate dehydrogenase in the mitochondria, resulting in increased lactate production. CoCl₂ induced an expected increase in PDK1 mRNA levels in shCT cells (1.4-fold for 8505C and twofold for TPC-1), which was more pronounced in the 8505shSTAT3 (twofold) and TPC-1shSTAT3 (sixfold) cell lines (Fig. 6D and Fig. S7C). In parallel, glucose transporter 3 (GLUT3) and hexokinase II (HK2) RNA levels were up-regulated in 8505C shSTAT3 cells compared with shCT. GLUT3, HK2, and GLUT1 levels were all increased to a greater degree in the shSTAT3 cells after CoCl₂ treatment (Fig. 6D). Altogether, these data suggest that glycolysis is increased in STAT3-deficient TCCs.

Human Tumor Samples.

Human PTC samples (formalin-fixed and paraffin-embedded) were retrieved from Hospital de S. João/Medical Faculty of the University of Porto, Porto, Portugal; Institute of Pathology and Molecular Immunology of the University of Porto, Porto, Portugal; Portuguese Institute of Oncology (IPO), Porto, Portugal; Hospital de S. Marcos, Braga, Portugal; and Hospital Clínico Universitario, Santiago de Compostela, Spain. All of the procedures were in accordance with institutional ethical standards. The histological classification of the samples was done by an experienced thyroid pathologist (M.S.-S.).

IHC, Reagents, and Antibodies.

IHC was carried out using the streptavidin-biotin-HRP technique. The results were independently evaluated by two observers and scored either semiquantitatively (pY-STAT3, STAT3, pY-STAT1, pS6, pERK1/2, p-p38, p-JNK, pSmad2/3, vimentin, E-cadherin, and IGFBP7) or quantitatively (Ki67, cyclin D1, Meca-32, TUNEL, cleaved-caspase 3, iba1, and CD3). Semiquantitative analysis was performed by considering both the intensity (0, negative; 1, low; 2, moderate; 3, strong) and the percentage of cells displaying identical staining (0, negative; 1, 0–5%; 2, 5–25%; 3, 25–50%; 4, 50–75%; 5, 75–100%), and the respective indexes were multiplied by each other to calculate a score value. The score values of pY-STAT3 and IGFBP7 were grouped into categories: low reactivity (score < 4), moderate reactivity (score = 4–7), and high reactivity (score ≥ 8).

Cell Lines and Reagents.

8505C, TPC-1, HTH-7, and SW1736, as well as their derived shCT and shSTAT3 cell lines, were maintained in RPMI, 10% FBS, and 1% PenStrep. PCCl3 was maintained as described (48). 293T and HEK293T cells were maintained in DMEM, 10% FBS, and 1% PenStrep. STAT3 shRNA lentiviral construct and scrambled control construct were previously described (63). Retroviral vectors pBabe-STAT3 and pBabe-Y705F are described in ref. 21.

Protein Extracts and Western Blotting.

Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) buffer supplemented with proteinase and phosphatase inhibitors. For subcellular protein fractionation, the Standard Fractionation Kit from Mitosciences (Abcam) was used according to the manufacturer’s instructions. Protein extracts were run in SDS gels and transferred to Hybond ECL membrane (Amersham Biosciences). The primary antibodies were the same as used for IHC at different dilutions. Secondary antibodies (anti-rabbit, anti-mouse, and anti-goat) conjugated with peroxidase were from Santa Cruz BT, and bands were visualized by the ECL detection system (Perkin-Elmer).

ELISA.

Cells were plated at similar densities and grown until subconfluence in serum-free medium. The supernatants were collected, and IL-6 levels were assessed using an IL-6 ELISA kit (Cell Sciences) according to the manufacturer’s instructions. IL-6 levels were normalized to total protein extracted from the cells.

Plasmids and Luciferase Reporter Assay.

The pIRE-ti-LUC and pDM2-LacZ reporter plasmids were provided by Robert Hofstra (University of Groningen, Groningen, The Netherlands) (23). The BRAF and RET/PTC expression vectors are described in ref. 64.

In Vitro Cell Growth.

Cell growth was assessed by Calcein AM (calcein acetoxymethylester; Invitrogen) or sulforhodamine (SRB) staining as previously described (65).

In Vivo Tumorigenicity Studies.

All of the procedures were included in a protocol approved by the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee. Cells were harvested, mixed with an equal volume of matrigel (BD Biosciences), and injected s.c. into the flank of female homozygous athymic nu/nu mice (Harlan Laboratories). Tumor dimensions length, width, and height were determined, and tumor volume was calculated by the expression (1/2 × length × width × height) (66).

Transgenics.

All of the procedures were included in a protocol approved by the MSKCC Institutional Animal Care and Use Committee. All of the genotypes were in C57BL/6 background. LSL-BRAFV600E mice are described in ref. 27. STAT3 ^{mice were a gift from Shizuo Akira (Hyogo College of Medicine, Nishinomiya, Japan) (}28). These mice were backcrossed to C57BL/6 (a gift from Hua Yu, City of Hope, El Duarte, CA).

Gene Expression Profiling Analysis.

RNA samples were hybridized to a Human HT12 Illumina array according to the manufacturer’s instructions. Data analysis was done in Partek. A gene was considered differentially expressed according to an arbitrary cutoff of twofold change with P < 0.05.

IGFBP7 Promoter Methylation Analysis.

Genomic DNA was extracted using phenol–chloroform and converted using sodium bisulfite with the EpiTect Bisulfite Kit (Qiagen). The converted DNA was amplified using primers specific for the −237- to +40-bp region of the IGFBP7 promoter, and the amplification products were subjected to direct sequencing.

RT-PCR/Q-PCR.

Total RNA was extracted from cells using the TRIzol method (Invitrogen) followed by cDNA conversion (BioRad) using the manufacturer’s instructions. For Q-PCR, the Quantitect Sybr Green (Qiagen) was used for human IGFBP7 RNA quantification, and hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as the housekeeping control. Primer sequences are available on request. For human HIF1a, PDK1, GLUT1 (SLC2A1), GLUT3 (SLC2A3), and HK2 RNA quantification, Taqman gene expression assays (Applied Biosystems) were used.

Glucose and Lactate Quantification.

Identical cell numbers were plated, and after 24 h, the medium was replaced with fresh RPMI supplemented with 1% serum and the indicated drugs. After 24 h, the medium was collected. Glucose present in the conditioned medium was quantified using the Glucose GOD/PAP Kit (Roche) and subtracted from the initial concentration present in fresh RPMI. Lactate was quantified with the LO-POD enzymatic colorimetric assay (Spinreact).

Oxygen Consumption.

Oxygen consumption in 8505C-derived cells was measured using the Seahorse 24XF instrument (Seahorse Biosciences) according to the manufacturer’s protocols.

Membrane Potential.

Mitochondrial membrane potential was measured using the JC1 Mitochondrial Membrane Potential Assay Kit (Mitosciences; Abcam) following the manufacturer’s instructions.

Statistical Analysis.

Statistical analysis was done in StatView (SAS Institute) using Student t, Mann–Whitney u, χ^{, and Fisher correlation tests. Results were considered statistically significant when} P < 0.05 (*P < 0.05, **P < 0.01, and ***P < 0.001).

Supplementary Material

Author Summary

Members of the IL-6 cytokine family play a critical role in normal immune cell functions and diseases, such as rheumatoid arthritis and cancer. These cytokines act by binding to a specific cell surface receptor complexed with the signal-transducing molecule glycoprotein (gp) 130. This interaction induces phosphorylation of gp130-associated protein kinases called JAKs, which in turn, phosphorylate tyrosine residues on gp130, resulting in the recruitment and subsequent phosphorylation of the transcription factor STAT3. Activated STAT3 dimerizes, enters the nucleus, and binds to promoter sequences of target genes involved in controlling normal cellular functions and regulating many aspects of tumorigenesis (proliferation, survival, differentiation, metabolism, immune cell response, angiogenesis, migration, and invasion) (1). Normally, STAT3 is activated transiently by phosphorylation of a specific tyrosine residue. However, in many cancers, STAT3 is persistently activated by aberrant IL-6 signaling (2), and many lines of evidence indicate that STAT3 promotes tumorigenesis. In contrast, recent evidence indicates that STAT3 inhibits tumor growth and progression (3). Here, we determined that STAT3 suppressed the growth of thyroid cancer cell lines in vivo. This finding suggests that a careful analysis of the genetic, molecular, and cellular components of the tumor should be determined before targeting this transcription factor.

We examined the mechanism of STAT3 activation and its role in the most common endocrine cancer, papillary thyroid carcinoma (PTC). PTC results from oncogenic activation of the receptor protein tyrosine kinase rearranged in transformation (RET) by genomic rearrangements (RET/PTC) or mutations that activate the downstream effectors Rat Sarcoma (SRC) and BRAF, which in turn, activate the ERK/MAPK signaling pathway. Moreover, PTC frequently arises in association with chronic inflammation (thyroiditis). This study determines the mechanism of STAT3 activation and its functional role in PTC.

We analyzed STAT3 phosphorylation (pY-STAT3) in 146 primary human thyroid lesions and detected pY-STAT3 in a small fraction of tumor cells in most benign tumors (83%) and a subset of PTC (57%). In contrast, pY-STAT3 was present in only 25% of follicular thyroid carcinomas. In most lesions, cells in tumors positive for pY-STAT3 were associated with stromal cells (endothelial cells, fibroblasts, and immune cells were also positive for pSTAT3) found on the periphery of tumors. This result is consistent with published data, and it may reflect paracrine release of growth factors by the stroma.

Growth factor receptor protein tyrosine kinases, such as oncogenic RET, have been described to mediate STAT3 activation (4). Here, we extended these observations using a nontransformed rat thyroid line (PCCl3) transfected with constructs expressing BRAFwt and BRAFV600E. We determined that BRAFV600E can also induce tyrosine phosphorylation and activation of STAT3 and that such activation is mediated by autocrine IL-6 expression, reinforcing a principle role for the gp130/JAK signaling in STAT3 activation in thyroid models.

We then examined the functional role of STAT3 in thyroid cancer by using in vitro and in vivo human (xenograft) and murine transgenic models, respectively. STAT3 knockdown in pY-STAT3–positive human cell lines using an interfering shRNA (shSTAT3) did not alter their growth in vitro, whereas short hairpin STAT3 generated significantly larger tumors in vivo (xenografts) compared with controls (Fig. P1). Similar results were obtained using a transgenic murine model of BRAFV600E-induced PTC, where thyrocyte-targeted deletion of STAT3 led to enhanced tumor size and proliferation (Fig. P1). These data suggest that, in vivo, STAT3 restrained thyroid tumor growth rather than promoting it.

Fig. P1.

(A) STAT3 deficiency in thyrocytes of BRAFV600E mice increases thyroid tumor size and tumor cell proliferation (*P < 0.05). (B) The role of STAT3 during thyroid tumor growth. Small thyroid tumors are positive for pY-STAT3, which is regulated by stromal paracrine and oncogene-induced autocrine signaling. STAT3 may prevent excessive proliferation by promoting secretion of growth inhibitory factors, including insulin-like growth factor binding protein 7. As thyroid cancer cells grow farther from the periphery and blood supply, the oxygen levels drop, inducing stabilization of hypoxia-inducible transcription factor 1α and a decrease in the level of pY-STAT3. Such a decrease in activated STAT3 enhances aerobic glycolysis (increased glucose consumption, lactate production, and decreased oxygen consumption), conferring a growth advantage on these tumors.

Given the hypoxic nature of tumors (5), we investigated whether STAT3 deficiency could alter the metabolic function of thyroid cell carcinomas. Inhibition of STAT3 expression in these cell lines led to increased glucose consumption and lactate production. Consistent with this phenotype, down-regulation of STAT3 increased the expression of hypoxia-inducible transcription factor 1α and genes encoding glycolytic enzymes. These results suggest that the absence of STAT3 confers a growth advantage under hypoxic stress caused by metabolic reprogramming.

Our data presented here show that STAT3 is a suppressor of thyroid tumor growth in preclinical models, suggesting that targeting this transcription factor should be used with caution. We conclude that a better understanding of the mechanisms and contexts that predict the dual-edged function of STAT3 in tumorigenesis must be obtained.