Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2018.6305

or-39-05-2306

Articles

Vandetanib has antineoplastic activity in anaplastic thyroid cancer, in vitro and in vivo

Ferrari

Silvia Martina

1 Bocci

Guido

1 2 Di Desidero

Teresa

1 Ruffilli

Ilaria

1 Elia

Giusy

1 Ragusa

Francesca

1 Fioravanti

Anna

1 Orlandi

Paola

1 Paparo

Sabrina Rosaria

1 Patrizio

Armando

1 Piaggi

Simona

3 Motta

Concettina La

4 Ulisse

Salvatore

5 Baldini

Enke

5 Materazzi

Gabriele

6 Miccoli

Paolo

6 Antonelli

Alessandro

1 Fallahi

Poupak

1Department of Clinical and Experimental Medicine, School of Medicine, University of Pisa, I-56126 Pisa, Italy 2Istituto Toscano Tumori, I-50139 Florence, Italy 3Department of Translational Research and of New Technologies in Medicine and Surgery, University of Pisa, I-56126 Pisa, Italy 4Department of Pharmacy, University of Pisa, I-56126 Pisa, Italy 5Department of Surgical Sciences, ‘Sapienza’ University of Rome, I-00161 Rome, Italy 6Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, I-56124 Pisa, Italy

Correspondence to: Professor Alessandro Antonelli, Department of Clinical and Experimental Medicine, School of Medicine, University of Pisa, Via Savi 10, I-56126 Pisa, Italy, E-mail: alessandro.antonelli@med.unipi.it

052018

08032018

39 5 2306 2314 01112017 28022018

2018

The antitumor activity of vandetanib [a multiple signal transduction inhibitor including the RET tyrosine kinase, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) receptor (VEGFR), ERK and with antiangiogenic activity], in primary anaplastic thyroid cancer (ATC) cells, in the human cell line 8305C [undifferentiated thyroid cancer (TC)] and in an ATC-cell line (AF), was investigated in the present study. Vandetanib (1 and 100 nM; 1, 10, 25 and 50 µM) was tested by WST-1, apoptosis, migration and invasion assays: in primary ATC cells, in the 8305C continuous cell line, and in AF cells; and in 8305C cells in CD nu/nu mice. Vandetanib significantly reduced ATC cell proliferation (P<0.01, ANOVA), induced apoptosis dose-dependently (P<0.001, ANOVA), and inhibited migration (P<0.01) and invasion (P<0.001). Furthermore, vandetanib inhibited EGFR, AKT and ERK1/2 phosphorylation and downregulated cyclin D1 in ATC cells. In 8305C and AF cells, vandetanib significantly inhibited the proliferation, inducing also apoptosis. 8305C cells were injected subcutaneously in CD nu/nu mice and tumor masses became detectable after 30 days. Vandetanib (25 mg/kg/day) significantly inhibited tumor growth and VEGF-A expression and microvessel density in 8305C tumor tissues. In conclusion, the antitumor and antiangiogenic activity of vandetanib is very auspicious in ATC, opening the way to a future clinical evaluation.

vandetanib anaplastic thyroid cancer primary anaplastic thyroid cancer cells tyrosine kinase inhibitors in vitro studies in vivo studies

Introduction

Anaplastic thyroid cancer (ATC) represents ~1% of all thyroid cancer (TC) cases, and is one of the most aggressive human tumors, accounting for 15–40% of TC-related deaths (1,2). ATC is classified as stage IV (American Joint Committee on Cancer), regardless of tumor size, or presence of lymph node or distant metastases (present in ~80% of patients at diagnosis) (3–5); median survival is 6 months. The multimodal treatment [including debulking, chemotherapy (doxorubicin, cisplatin, paclitaxel or docetaxel), and hyperfractionated accelerated external beam radiotherapy] is the most effective treatment, with a median survival of 10 months (6,7). Several genetic alterations have been shown in ATC molecular pathways, leading to tumor aggressiveness and progression [p53, BRAF, RAS, RET/PTC, vascular endothelial growth factor (VEGF) receptor (VEGFR)-1, VEGFR-2, epidermal growth factor receptor (EGFR), PDGFRα, PDGFRβ, KIT, MET, PIK3Ca, PIK3Cb and PDK1] (7,8). New drugs targeting these molecular alterations have been recently evaluated in ATC (7), but to date no significant improvement in patient survival has been observed.

Vandetanib (ZD6474, Caprelsa^®) is an oral once-daily multi-tyrosine kinase inhibitor (TKI), that inhibits the activation of RET, EGFR, VEGFR-2, VEGFR-3, and slightly VEGFR-1, and has potent antiangiogenic activity (9). Potent antineoplastic action of vandetanib was shown against transplantable medullary thyroid carcinoma (MTC) in nude mice (10). In patients with aggressive MTC, a phase III clinical study showed that vandetanib improved progression-free survival (30.5 vs. 19.3 months in the control group) (11). It was approved by the Food and Drug Administration, and the European Medicines Agency, in 2011, to treat locally advanced or metastatic MTC (12). Vandetanib has also shown promising results in aggressive differentiated TC patients not responsive to the usual therapies (13,14). In the present study, we aimed to evaluate the antineoplastic activity of vandetanib in ATC continuous cell lines, and in primary ATC cells, in vitro and in vivo.

Materials and methods <sec> <title>Drug

The effect of vandetanib (ZD6474, Caprelsa^®; Aurogene Srl, Rome, Italy; 1 and 100 nM; 1, 10, 25 and 50 µM) was investigated in vitro in primary ATC cell cultures, in the 8305C continuous cell line, and AF cells; and in vivo in 8305C cells in CD nu/nu mice.

Reagents

RPMI-1640 medium was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA); PCR reagents for quantitative real-time were obtained from Applied Biosystems (Thermo Fisher Scientific, Inc.). The other chemicals and supplements not reported in this section were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Patient source for thyroid tissue

We obtained surgical thyroid tissue samples from: i) eight ATC patients at surgery; and ⅱ) six healthy subjects who underwent parathyroidectomy. Recognized clinical, laboratory and histological criteria were used to establish the diagnosis (15–17). The absence of thyroglobulin (Tg), thyroperoxidase (TPO), thyroid-stimulating hormone (TSH) receptor, and sodium/iodide symporter (NIS) expression was shown by immunohistochemistry, that was positive for cytokeratin (Fig. 1). DNA extraction and microdissection, and the detection of BRAF mutation were conducted by PCR Single-strand Conformation Polymorphism (by accepted protocols); such as direct DNA sequencing (15–17). All patients and controls agreed to enter the study, which was approved by the local Ethics Committee of the University of Pisa.

Primary ATC cell culture

We prepared ATC cells using protocols published previously (15–17). Cancer tissues were divided in fragments of 1–3 mm, and washed 3–5 times in M-199 media together with streptomycin (500,000 U/l), penicillin (500,000 U/l) and nystatin (1,000,000 U/l). Fragments were suspended in Dulbecco's modified Eagle's medium (DMEM) with penicillin/streptomycin (50 mg/l), glutamine (1% w/v) and fetal calf serum (FCS) (20% v/v) and then incubated at 37°C in 5% CO₂ (all were from Sigma-Aldrich; Merck KGaA).

As soon as the primary culture reached confluence, the cells were transferred into primary tissue culture flasks (Becton-Dickinson Labware, Bedford, MA, USA). To evaluate colony-forming efficacy, cells on their 3rd passage were coated in Methocel™(Dow Chemical Co., Milan, Italy) (18). The biggest colonies were spread in tissue-culture flasks (15–17). At the 4th passage, the cells were tested. The absence of Tg, TSH receptor (19), NIS (20), and TPO (21) expression was shown by immunocytochemistry. Focal positivity for cytokeratin was observed by immunocytochemistry (20). DNA fingerprinting demonstrated a pattern similar to the original cancer tissue (15–17).

Thyroid follicular cell (TFC) culture

TFCs were established as previously reported (22).

AF cells

Among the eight primary ATC cell cultures, one (the AF cell line) grew >50 passages, and was also able to grow in nu/nu mice when subcutaneously inoculated.

8305C continuous cell line

8305C cells (undifferentiated TC cell line, with papillary component; DSMZ, Braunschweig, Germany) were maintained in RPMI-1640 with 15% fetal bovine serum (FBS) with the addition of 2 mM L-glutamine.

Viability and proliferation assay

In order to investigate cell proliferation, we conducted a WST-1 (Roche Diagnostics, Almere, The Netherlands) (16,17,22). We plated TFC, ATC, AF and 8305C cells (at 35,000 cells/ml in 100 µl/well, of 96-well plates), and treated them with vandetanib or with vehicle alone for 24 h. To achieve IC₅₀, the cells were treated with a concentration range of vandetanib (in quadruplicate), and IC₅₀ was estimated by linear interpolation. Triplicate experiments were conducted for each cell preparation (16,17,22). The absorbance was estimated at 450 nm after 1 and 2 h from the beginning of tetrazolium reaction.

Cell number counting was used, as well, to estimate the proliferation in all the considered cells, as previously reported (16,17,22).

Apoptosis-Hoechst uptake

Firstly, we plated ATC, 8305C and AF cells (35,000 cells/ml in 100 µl/well; 96-well plates). Thereafter, the cells were treated for 48 h with vandetanib (37°C, 5% CO₂), and dyed with Hoechst 33342 as previously described (22). The apoptotic cells/total cells ×100 ratio (apoptosis index) was evaluated.

Annexin V binding assay for apoptosis

The assay was carried out on cells seeded in Lab-Tek II Chamber Slide System (Nalge Nunc International, Penfield, NY, USA), treated with vandetanib for 48 h, as previously reported (22).

Migration and invasion assays

A 96-well Transwell Permeable Support (Corning Life Sciences, Corning, NY, USA) was used to achieve cell migration and invasion, in agreement with the manufacturer's instructions, applying minor modifications (23,24).

To assess intracellular fluorescence, we used a 96-well plate ELISA reader (excitation at 485 nm and emission at 520 nm). For the migration assay cells were incubated for 12 h; for the invasion assay, 24 h. For the invasion experiments, the inserts were coated with a basement membrane extract solution (Trevigen, Gaithersburg, MD, USA) overnight (37°C, 5% CO₂), before plating cells. For each assay we constructed a standard curve to transform the fluorescence data obtained to the number of invasive or migrated cells. All the data were analyzed by StatView version 5.0 (SAS Institute, Inc., Cary, NC, USA).

ELISA tests in ATC cells Phospho-EGFR inhibition cell-based assay

ATC cells (5×10⁴ cells/well) were plated in 1% FBS medium for 24 h, and then treated for 72 h with vandetanib at a concentration similar to the experimental IC₅₀ of cell proliferation (25 µM for ATC), and with a higher (50 µM), or with a lower (1 µM) concentration of vandetanib, or with vehicle. We collected cell lysates as previously reported (25) and we assayed them with PathScan phospo-EGFR (Tyr1173) and total EGFR sandwich ELISA kits (Cell Signaling Technology, Inc., Danvers, MA, USA). Optical density (OD) was estimated at 450 nm.

AKT (pThr308), or ERK1/2 (pTpY185/187)

ATC cells (5×10⁴ cells/well) were exposed for 72 h to vandetanib and subsequently lysed (25), and tested for human AKT or ERK1/2 phosphorylation by the PhosphoDetect AKT (pThr308) or by PhosphoDetect ERK1/2 (pThr185/pTyr187) ELISA kits (Calbiochem; EMD Millipore, Billerica, MA, USA). Data were normalized by total protein AKT, or ERK1/2 concentrations evaluated by AKT, or ERK1/2 ELISA kits, respectively. OD was estimated at 450 nm.

Cyclin D1 protein expression

The ATC cells were exposed for 72 h to vandetanib at the above considered concentrations or to vehicle, in order to evaluate the vandetanib-modulated expression of the protein cyclin D1. Cyclin D1 protein amount was measured by lysing cells with (ice-cold 1X) lysis buffer (0.5 ml), as previously reported (25). Lysates were gathered and then sonicated on ice (for 10 sec). Subsequently, the samples were microcentrifuged at 4°C for 10 min and the supernatant was gathered. Cyclin D1 was measured in cancer cell lysates by the human ELISA kit (Uscn Life Sciences, Inc., Wuhan, China). OD was measured at 450 nm, and the obtained data were reported as cyclin D1 ng/mg of total protein.

In vivo studies Animals and treatment

Six-week-old CD nu/nu male mice weighing 20–25 g, supplied by Envigo (Milan, Italy), were housed in microisolator cages on vented racks and manipulated using aseptic techniques and were allowed unrestricted access to sterile food and water. We proceeded according to the protocol approved by the Academic Organization Responsible for Animal Welfare [Organismo Preposto per il Benessere Animale (OPBA)] at the University of Pisa, in agreement with the Italian law D.lgs. 26/2014, and by the Italian Ministry of Health (authorization no. 613/2015-PR). In order to obtain statistically meaningful results, each experiment was conducted with the minimum necessary number of mice. In each mouse we inoculated, subcutaneously, 2×10⁶±5% viable 8305C cells, on day 0. Animal weights were monitored, and tumor volume (mm³) was defined as: [(w1 × w1 × w2) × (π/6)], where w1 refers to the smallest tumor diameter (mm), whereas w2 to the largest one. We started the treatment (n=5 mice/group) after 30 days from cell inoculation, when the mean volume was ~100 mm³. All mice were randomized just before initiation of treatment. Control mice received vehicle alone, or 25 or 12.5 mg/kg/day of vandetanib, injected intraperitoneally (i.p.) for 29 days. An anesthetic overdose of urethan was used to sacrifice mice, and the tumors were then excised and measured.

Microvessel density in the cancer tissue, and immunohistochemistry

Cancer tissues from the three treatment groups were weighed, and then fixed in formalin and subsequently embedded in paraffin. The sections (5 µm) were stained with hematoxylin and eosin, and immunostaining was conducted as previously described (23). An anti-VEGF rabbit polyclonal antibody (diluted at 1:50; cat. no. sc-152; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used to estimate VEGF expression, as the percentage of positive cells in ~1,000 tumor cells. Anti-FVIII polyclonal antibody (cat. no. 760-2642; Ventana Medical Systems, Inc.:Roche Group, Tucson, AZ, USA) was used to determine microvascular count (MVC), as previously described (23).

Statistical analysis

For normally distributed variables, the values are expressed as mean (±SD), or as median (and interquartile range). Experiments were conducted thrice with ATC from each subject, and here we report the mean in the eight samples obtained by different donors (for TFC and ATC). One-way ANOVA, or Mann-Whitney U or Kruskal-Wallis test, were used to compare mean group values for normally distributed variables. χ² test was applied to compare proportions. Post hoc comparisons on normally distributed variables were conducted by Bonferroni-Dunn test. Apoptosis results were analyzed by one-way ANOVA with Newman-Keuls multiple comparisons test. All the data were analyzed by StatView version 5.0 (SAS Institute, Inc.).

Results In vitro studies in primary cell cultures Cell proliferation

Vandetanib significantly reduced ATC cell proliferation vs. the control, at 1 and 2 h (P<0.01, by ANOVA, for both) (Fig. 2A). Cell counting confirmed these results: after 1 h, the cell number was 12,120±680/100 µl/well in the ATC control group; 11,998±710 (99%) with vandetanib 1 nM; 11,635±780 (96%) with vandetanib 100 nM; 10,787±690 (89%) with vandetanib 1 µM; 9,332±710 (77%) with vandetanib 10 µM; 8,610±580 (71%) with vandetanib 25 µM; and 3,878±490 (32%) with vandetanib 50 µM (P<0.01, ANOVA); after 2 h, cell number was 18,950±910/100 µl/well; 18,382±920 (97%) with vandetanib 1 nM; 17,623±990 (93%) with vandetanib 100 nM; 14,592±1,010 (77%) with vandetanib 1 µM; 10,423±1,180 (55%) with vandetanib 10 µM; 6,443±1,190 (34%) with vandetanib 25 µM; and 2,274±750 (12%) with vandetanib 50 µM; (P<0.01, ANOVA). The IC₅₀ value, obtained with linear interpolation, was 13±2.9 µM for vandetanib.

The results of WST-1 assay in TFC cells with vandetanib showed a slight but significant reduction in proliferation with respect to the control both at 1 h (P<0.01, ANOVA) with vandetanib 10 µM (94% vs. control), 25 µM (87% vs. control), and 50 µM (81% vs. control), and at 2 h (P<0.01, for both, ANOVA) with vandetanib 10 µM (87% vs. control), 25 µM (81% vs. control), and 50 µM (76% vs. control). Cell counting supported the previously reported results: after 1 h, the cell number was 11,290±730/100 µl/well in the TFC control; 10,612±1,080 (94%) with vandetanib 10 µM; 9,822±940 (87%) with vandetanib 25 µM; and 9,145±880 (81%) with vandetanib 50 µM; (P<0.01, ANOVA); after 2 h, the cell number was 17,950±910/100 µl/well; 15,615±1,090 (87%) with vandetanib 10 µM; 14,540±950 (81%) with vandetanib 25 µM; and 13,640±890 (76%) with vandetanib 50 µM (P<0.01, ANOVA).

Proliferation and BRAF

Three considered ATCs had ^V600EBRAF mutation, while RET/PTC1 and RET/PTC3, H-RAS or N-RAS mutations were not reported in primary ATC cells by real-time PCR. Proliferation was inhibited similarly in ATC from cancers with or without ^V600EBRAF mutation (data not shown).

Apoptosis

Vandetanib increased the number of apoptotic ATC cells dose-dependently (P<0.001; by ANOVA; Fig. 2B). The Annexin V assay corroborated these results (Fig. 2C and D).

Migration and invasion

The effect of vandetanib on migration and invasion was evaluated in ATC cells showing a reduction in both migration (Fig. 3A) and invasion (Fig. 3B).

EGFR inhibition in ATC cells by vandetanib

Vandetanib significantly reduced the phosphorylated (p)EGFR/total EGFR ratio in the ATC cell lysates in a dose-dependent manner (Fig. 4A).

Inhibition of AKT or ERK1/2 phosphorylation in ATC cells by vandetanib

Vandetanib significantly reduced the pAKT/total AKT and pERK1/2/total ERK1/2 protein ratios in the ATC cells (Fig. 4B and C).

Vandetanib decreases cyclin D1 protein levels in ATC cells

Lower cyclin D1 concentrations were detected in ATC cells treated with vandetanib vs. those treated with vehicle, and vandetanib inhibited cyclin D1 gene expression in a dose-dependent manner (P<0.05; Fig. 4D).

Studies in vitro in AF and 8305C cells

Vandetanib demonstrated a dose-dependent antiproliferative activity in the 8305C cell line (IC₅₀ of 9.6±3.4 µM) (Fig. 5A), and in AF cells (IC₅₀ of 4.7±1.8 µM) (Fig. 5B). Vandetanib dose-dependently induced the apoptosis of the 8305C cells: apoptotic cells were 16.8% with vandetanib 10 µM, and 20 and 33.2% with vandetanib 25 and 50 µM, respectively (P<0.001; by ANOVA; Fig. 5C). In AF cells, vandetanib dose-dependently increased apoptosis: apoptotic cells were 19.9% with vandetanib 10 µM, and 22.7 and 27.8% with vandetanib 25 and 50 µM, respectively (P<0.001; by ANOVA; Fig. 5D).

In vivo studies Vandetanib inhibits 8305C tumor growth in mice with no body weight loss

Thirty days after subcutaneous xenotransplantation of 8305C cells in CD nu/nu mice, tumor masses reached the average volume of 100 mm³ and treatment with vandetanib was initiated. In order to establish the optimal antitumor dose, two different doses were administered. At the lower dose of 12.5 mg/kg a slight, but not significant, antitumor activity was recorded (overall from days 16 to 22), and no statistical differences were obtained when tumor volumes in the treatment group were compared to the control group (Fig. 6A). In contrast, at the highest dose of 25 mg/kg/day i.p., vandetanib significantly inhibited tumor growth (e.g., at day 25, 282.1 vs. 1,086.9 mm³ of controls P<0.05; Fig. 6A). Notably, no loss of mouse body weight throughout the course of the experiment was observed at both the administered doses, suggesting that the vandetanib treatment was well tolerated even at its optimal antitumor dose (Fig. 6B).

Vandetanib decreases VEGF-A expression and microvessel density in 8305C tumor tissues. 8305C cells led to the formation of a tumor histologically similar to ATC. Vandetanib significantly reduced VEGF-A and FVIII immunostaining. A localized immunoreactivity for VEGF-A was present in cells of the control cancer mass, and vandetanib reduced it (51±9 vs. 37±7; P<0.05), with a concurrent reduction in microvessel density (15±4 vs. controls 26±7; P<0.05).

Discussion

Vandetanib belongs to the TKIs, that are under evaluation for ATC treatment (26).

With the present study, we contributed to the understanding of the anticancer activity of vandetanib, demonstrating that: i) it inhibited primary ATC cell proliferation in vitro, through increased apoptosis, and suppressed the migration and invasion abilities as well; and ⅱ) it blocked 8305C cell proliferation in vitro, increasing apoptosis and reducing 8305C tumor growth in CD nu/nu mice as well, with no toxicity.

Our results are in line with those of another study that reported how vandetanib is able to inhibit 8305C cell growth in vivo, and to block angiogenesis, decreasing vascular permeability (27). We also observed the important antiangiogenic activity of vandetanib in 8305C xenotransplants.

The antiproliferative action of vandetanib was observed in all the used primary ATC cells, independently from the absence/presence of ^V600EBRAF mutation.

Our data support the concept that vandetanib could be used for a multiple signal inhibition (involving RET, VEGFR, EGFR, ERK, AKT, and others), and it also exhibits antiangiogenic activity (28).

It is suggested that AKT plays a crucial role in ATC oncogenesis, and it has been demonstrated that pharmacological and molecular inhibition of PI3K or AKT isoforms are able to reduce the in vitro growth and motility of human TC cell lines (29,30). Moreover, both RAS/RAF/MAPK, ERK and PI3K pathways are implicated in the carcinogenesis of TCs, and mutations in such genes have been reported in ATC (31). Since AKT and ERK proteins are activated, once phosphorylated, in ATC, these proteins have been suggested as potential targets of therapy. In the present study in ATC cells, vandetanib significantly inhibited ERK1/2 and AKT phosphorylation.

Furthermore, EGFR phosphorylation in ATC cells was significantly reduced by vandetanib treatment, according to the results obtained by Di Desidero et al (32) and our previous results (33), reporting that TKI suppressed EGFR phosphorylation in ATC.

Cyclin D1 regulates cell cycle progression (34), and its expression was observed at different levels in ~67% of ATC cells by Lee et al (35). In addition, overexpression of cyclin D1 was reported in 77% of ATC by Wiseman et al (36). Vandetanib, a TKI of both VEGFR-2, and EGFR, inhibited cell growth downregulating cyclin E and D1 expression (37). Notably, we showed that vandetanib downregulated the cyclin D1 protein in ATC cells.

We found a significant 8305C cell-derived tumor growth inhibition in CD nu/nu mice by vandetanib, without body weight loss, suggesting a minimal toxicity profile, whereas other compounds are known to provoke different adverse effects in humans and animals (38). Nevertheless, we did not collect data concerning the kidney, liver, or other biochemical tests, that will be provided in future studies.

Antineoplastic activity of vandetanib in ATC is the result of different effects on tumoral cells, that include: i) antiproliferative activity; ii) increased apoptosis; ⅲ) inhibition of both migration, and invasion; and ⅳ) inhibition of cancer neovascularization.

New therapeutic attempts for the treatment of ATC are ongoing, even if various limitations are still present for the selective use of new molecules. Even if neoplastic tissue has a potential target (as BRAF), the tumor response is present in only a few patients. As we achieve target inhibition, any response may occur owing to the increased activity of other compensatory pathways, that rescue cancer cell growth. The efficacy of treatment could be increased by assessing the sensitivity of primary ATC cells from each subject to different TKIs. In fact, in vitro chemosensitivity tests are able to predict in vivo effectiveness in 60% of cases (39), while a negative chemosensitivity test in vitro is associated with a 90% of ineffectiveness of the treatment in vivo (39,40), thus avoiding the administration of inactive chemotherapeutics to these patients (15,16,24,26).

In the present study, we first showed an antitumoral activity of vandetanib (a multi-targeted kinase inhibitor, with antiangiogenic effect) in human primary ATC cell cultures established directly from patients, paving the way for personalized TKI and for future clinical trials.

Acknowledgements

Not applicable.

Funding

GB was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (IG-17672).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

SMF, GB, PM, AA and PF made substantial contributions to conception and design, and to the acquisition of data; TDD, IR, GE, FR, AF, PO, SRP, AP, SP, CLM, SU, EB and GM analysed the data; SMF, GB, TDD, AA and PF have been involved in drafting the manuscript; AA revised it critically for important intellectual content. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the study are appropriately investigated and resolved. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All patients and controls agreed to enter the study, which was approved by the local Ethics Committee of the University of Pisa (see Materials and methods section).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

References 1

Hundahl

Fleming

Fremgen

Menck

A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985–1995 [see comments]

Cancer83263826481998

10.1002/(SICI)1097-0142(19981215)83:12<2638::AID-CNCR31>3.0.CO;2-1

9874472

Kitamura

Shimizu

Nagahama

Sugino

Ozaki

Mimura

Ito

Tanaka

Immediate causes of death in thyroid carcinoma: Clinicopathological analysis of 161 fatal cases

J Clin Endocrinol Metab84404340491999

10.1210/jcem.84.11.6115

10566647

Greene

Page

Fleming

Fritz

Balch

Haller

Morrow

Thyroid

American Joint Committee on Cancer: AJCC Cancer Staging Manual6th

Springer-Verlag

New York, NY

772002

Miccoli

Materazzi

Antonelli

Panicucci

Frustaci

Berti

New trends in the treatment of undifferentiated carcinomas of the thyroid

Langenbecks Arch Surg3923974042007

10.1007/s00423-006-0115-8

17131154

Kebebew

Anaplastic thyroid cancer: Rare, fatal, and neglected

Surgery152108810892012

10.1016/j.surg.2012.08.059

23158179

De Crevoisier

Baudin

Bachelot

Leboulleux

Travagli

Caillou

Schlumberger

Combined treatment of anaplastic thyroid carcinoma with surgery, chemotherapy, and hyperfractionated accelerated external radiotherapy

Int J Radiat Oncol Biol Phys60113711432004

10.1016/j.ijrobp.2004.05.032

15519785

Smallridge

Ain

Asa

Bible

Brierley

Burman

Kebebew

Lee

Nikiforov

Rosenthal

American Thyroid Association Anaplastic Thyroid Cancer Guidelines Taskforce: American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer

Thyroid22110411392012

10.1089/thy.2012.0302

23130564

Antonelli

Fallahi

Ferrari

Ruffilli

Santini

Minuto

Galleri

Miccoli

New targeted therapies for thyroid cancer

Curr Genomics126266312011

10.2174/138920211798120808

22654562

Jasim

Ozsari

Habra

Multikinase inhibitors use in differentiated thyroid carcinoma

Biologics82812912014

25506209

Johanson

Ahlman

Bernhardt

Jansson

Kölby

Persson

Stenman

Swärd

Wängberg

Stridsberg

A transplantable human medullary thyroid carcinoma as a model for RET tyrosine kinase-driven tumorigenesis

Endocr Relat Cancer144334442007

10.1677/ERC-06-0033

17639056

Wells

JrRobinson

Gagel

Dralle

Fagin

Santoro

Baudin

Elisei

Jarzab

Vasselli

Vandetanib in patients with locally advanced or metastatic medullary thyroid cancer: A randomized, double-blind phase III trial

J Clin Oncol301341412012

10.1200/JCO.2011.35.5040

22025146

Cooper

Alghamdi

Shaheen

Steinberg

Vandetanib for the treatment of medullary thyroid carcinoma

Ann Pharmacother483873942014

10.1177/1060028013512791

24259657

Leboulleux

Bastholt

Krause

de la Fouchardiere

Tennvall

Awada

Gómez

Bonichon

Leenhardt

Soufflet

Vandetanib in locally advanced or metastatic differentiated thyroid cancer: A randomised, double-blind, phase 2 trial

Lancet Oncol138979052012

10.1016/S1470-2045(12)70335-2

22898678

AstraZeneca

Evaluation of Efficacy, Safety of Vandetanib in Patients With Differentiated Thyroid Cancer (VERIFY) [ClinicalTrials.gov Identifier: NCT01876784]

2016Available fromhttps://www.clinicaltrials.gov/ct2/show/NCT01876784Last Update Posted: September 6, 2017

Antonelli

Ferrari

Fallahi

Berti

Materazzi

Barani

Marchetti

Ferrannini

Miccoli

Primary cell cultures from anaplastic thyroid cancer obtained by fine-needle aspiration used for chemosensitivity tests

Clin Endocrinol (Oxf)691481522008

10.1111/j.1365-2265.2008.03182.x

18194485

Antonelli

Ferrari

Fallahi

Berti

Materazzi

Marchetti

Ugolini

Basolo

Miccoli

Ferrannini

Evaluation of the sensitivity to chemotherapeutics or thiazolidinediones of primary anaplastic thyroid cancer cells obtained by fine-needle aspiration

Eur J Endocrinol1592832912008

10.1530/EJE-08-0190

18583391

Antonelli

Ferrari

Fallahi

Berti

Materazzi

Minuto

Giannini

Marchetti

Barani

Basolo

Thiazolidinediones and antiblastics in primary human anaplastic thyroid cancer cells

Clin Endocrinol (Oxf)709469532009

10.1111/j.1365-2265.2008.03415.x

18785992

Fiore

Pollina

Fontanini

Casalone

Berlingieri

Giannini

Pacini

Miccoli

Toniolo

Fusco

Cytokine production by a new undifferentiated human thyroid carcinoma cell line, FB-1

J Clin Endocrinol Metab82409441001997

10.1210/jcem.82.12.4444

9398720

Agretti

De Marco

De Servi

Marcocci

Vitti

Pinchera

Tonacchera

Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation

Eur J Endocrinol1527777842005

10.1530/eje.1.01900

15879364

Copland

Marlow

Williams

Grebe

Gumz

Maples

Silverman

Smallridge

Molecular diagnosis of a BRAF papillary thyroid carcinoma with multiple chromosome abnormalities and rare adrenal and hypothalamic metastases

Thyroid16129313022006

10.1089/thy.2006.16.1293

17199440

Christensen

Blichert-Toft

Brandt

Lange

Sneppen

Ravnsbaek

Mollerup

Strange

Jensen

Kirkegaard

Thyroperoxidase (TPO) immunostaining of the solitary cold thyroid nodule

Clin Endocrinol (Oxf)531611692000

10.1046/j.1365-2265.2000.01035.x

10931096

Antonelli

Ferrari

Fallahi

Frascerra

Piaggi

Gelmini

Lupi

Minuto

Berti

Benvenga

Dysregulation of secretion of CXC alpha-chemokine CXCL10 in papillary thyroid cancer: Modulation by peroxisome proliferator-activated receptor-gamma agonists

Endocr Relat Cancer16129913112009

10.1677/ERC-08-0337

19755523

Antonelli

Bocci

La Motta

Ferrari

Fallahi

Fioravanti

Sartini

Minuto

Piaggi

Corti

Novel pyrazolopyrimidine derivatives as tyrosine kinase inhibitors with antitumoral activity in vitro and in vivo in papillary dedifferentiated thyroid cancer

J Clin Endocrinol Metab96E288E2962011

10.1210/jc.2010-1905

21147882

Antonelli

Bocci

La Motta

Ferrari

Fallahi

Ruffilli

Di Domenicantonio

Fioravanti

Sartini

Minuto

CLM94, a novel cyclic amide with anti-VEGFR-2 and antiangiogenic properties, is active against primary anaplastic thyroid cancer in vitro and in vivo

J Clin Endocrinol Metab97E528E5362012

10.1210/jc.2011-1987

22278419

Bocci

Fioravanti

La Motta

Orlandi

Canu

Di Desidero

Mugnaini

Sartini

Cosconati

Frati

Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells

Biochem Pharmacol81130913162011

10.1016/j.bcp.2011.03.022

21459081

Antonelli

Fallahi

Ulisse

Ferrari

Minuto

Saraceno

Santini

Mazzi

D'Armiento

Miccoli

New targeted therapies for anaplastic thyroid cancer

Anticancer Agents Med Chem1287932012

10.2174/187152012798764732

22043992

Gule

Chen

Sano

Frederick

Zhou

Zhao

Milas

Galer

Henderson

Jasser

Targeted therapy of VEGFR2 and EGFR significantly inhibits growth of anaplastic thyroid cancer in an orthotopic murine model

Clin Cancer Res17228122912011

10.1158/1078-0432.CCR-10-2762

21220477

La Motta

Mugnaini

Sartini

Da Settimo

Bocci

Fioravanti

Del Tacca

Martini

2007 Derivati a nucleo pirazolo (3,4-d) pirimidinico quali inibitori di proteina tirosina chinasi

RM2007A000480 del 14/9/2007

Liu

Hou

Guan

Studeman

Jensen

Vasko

El-Naggar

Xing

Highly prevalent genetic alterations in receptor tyrosine kinases and phosphatidylinositol 3-kinase/akt and mitogen-activated protein kinase pathways in anaplastic and follicular thyroid cancers

J Clin Endocrinol Metab93310631162008

10.1210/jc.2008-0273

18492751

Shinohara

Chung

Saji

Ringel

AKT in thyroid tumorigenesis and progression

Endocrinology1489429472007

10.1210/en.2006-0937

16946008

Santarpia

El-Naggar

Cote

Myers

Sherman

Phosphatidylinositol 3-kinase/akt and ras/raf-mitogen-activated protein kinase pathway mutations in anaplastic thyroid cancer

J Clin Endocrinol Metab932782842008

10.1210/jc.2007-1076

17989125

Di Desidero

Fioravanti

Orlandi

Canu

Giannini

Borrelli

Man

Fontanini

Basolo

Antiproliferative and proapoptotic activity of sunitinib on endothelial and anaplastic thyroid cancer cells via inhibition of Akt and ERK1/2 phosphorylation and by down-regulation of cyclin-D1

J Clin Endocrinol Metab98E1465E14732013

10.1210/jc.2013-1364

23969186

Antonelli

Bocci

Fallahi

La Motta

Ferrari

Mancusi

Fioravanti

Di Desidero

Sartini

Corti

CLM3, a multitarget tyrosine kinase inhibitor with antiangiogenic properties, is active against primary anaplastic thyroid cancer in vitro and in vivo

J Clin Endocrinol Metab99E572E5812014

10.1210/jc.2013-2321

24423321

Klein

Assoian

Transcriptional regulation of the cyclin D1 gene at a glance

J Cell Sci121385338572008

10.1242/jcs.039131

19020303

Lee

Foukakis

Barbaro

Kiss

Clifton-Bligh

Staaf

Borg

Delbridge

Robinson

Array-CGH identifies cyclin D1 and UBCH10 amplicons in anaplastic thyroid carcinoma

Endocr Relat Cancer158018152008

10.1677/ERC-08-0018

18753363

Wiseman

Masoudi

Niblock

Turbin

Rajput

Hay

Bugis

Filipenko

Huntsman

Gilks

Anaplastic thyroid carcinoma: Expression profile of targets for therapy offers new insights for disease treatment

Ann Surg Oncol147197292007

10.1245/s10434-006-9178-6

17115102

Sarkar

Mazumdar

Dash

Sarkar

Fisher

Mandal

ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR-2, inhibits MAPK/ERK and AKT/PI3-K and induces apoptosis in breast cancer cells

Cancer Biol Ther95926032010

10.4161/cbt.9.8.11103

20139705

Santarpia

Gagel

The evolving field of tyrosine kinase inhibitors in the treatment of endocrine tumors

Endocr Rev315785992010

10.1210/er.2009-0031

20605972

Blumenthal

Goldenberg

Methods and goals for the use of in vitro and in vivo chemosensitivity testing

Mol Biotechnol351851972007

10.1007/BF02686104

17435285

Antonelli

Ferri

Ferrari

Sebastiani

Colaci

Ruffilli

Fallahi

New targeted molecular therapies for dedifferentiated thyroid cancer

J Oncol20109216822010

10.1155/2010/921682

20628483

Figure 1.

Immunohistochemistry for cytokeratin of anaplastic thyroid cancer (ATC) tissue. The presence of cytokeratin expression is shown in ATC cells (magnification, ×400).

Figure 2.

Proliferation (WST-1), and apoptosis assays in primary human anaplastic thyroid cancer (ATC) cells. (A) WST-1 in ATC cells treated with vandetanib for 24 h. Vandetanib significantly reduced ATC cell proliferation vs. control. Bars represent mean (±SD). *P<0.05 vs. control with Bonferroni-Dunn test. (B) ATC cell apoptosis after treatment with vandetanib for 48 h. Apoptosis index was determined by Hoechst staining. Vandetanib dose-dependently induced the percentage of apoptotic cells. Data are presented as mean (±SD) (n=8), and were analyzed by one-way ANOVA (with Newman-Keuls multiple comparisons test, and with a linear trend test) (*P<0.001 vs. control). (C and D) Representative images of the Annexin V binding assay in control and treated ATC cells (with vandetanib 50 µM), respectively.

Figure 3.

Migration and invasion tests in anaplastic thyroid cancer (ATC) cells. ATC cells were incubated for (A) 12 h for migration and (B) 24 h for invasion. For comparison, the inhibition of proliferation (at 12 h) (% with respect to control) and the inhibition of migration are reported in the Table in (A), and the inhibition of proliferation (at 24 h) (% with respect to control) and the inhibition of invasion are shown in (B). Bars represent the mean (±SD). *P<0.05 vs. control (control = medium + FCS 10%) by Newman-Keuls test.

Figure 4.

Inhibition of epidermal growth factor receptor (EGFR), AKT, ERK1/2 phosphorylation, or cyclin D1 protein expression in anaplastic thyroid cancer (ATC) cells. (A) Inhibition of EGFR phosphorylation by vandetanib in ATC cells after 72 h of treatment. Experiments were repeated thrice, with eight samples (for each dose). Mean (±SE); *P<0.05 vs. control. (B and C) Inhibition of ERK1/2 (pThr185/pTyr187) and AKT (pThr308) phosphorylation by vandetanib in ATC cells after 72 h of treatment. Experiments were repeated, independently, thrice with eight samples (for each dose). Mean (±SE); *P<0.05 vs. control. (D) Cyclin D1 concentrations in ATC cells exposed to vandetanib or vehicle for 72 h. Cyclin D1 results are expressed as ng/mg of total protein. Experiments were repeated 6 times with eight samples (for each dose). Mean (±SE); *P<0.05 vs. control.

Figure 5.

WST-1 and apoptosis assays in 8305C or AF cells. WST-1 assay results in (A) 8305C and (B) AF cells treated with vandetanib for 24 h. A significant inhibition of proliferation vs. control is shown with vandetanib. Bars are mean (± SD). *P<0.05 vs. control with Bonferroni-Dunn test. Apoptosis in (C) 8305C or (D) AF cells exposed to vandetanib for 48 h [mean (±SD) of the samples]. Apoptosis index (ratio between apoptotic and total cells) ×100 was determined by Hoechst staining. One-way ANOVA was used to analyze the data (with Newman-Keuls multiple comparisons test, and with a test for linear trend) (*P<0.001 vs. control). IC₅₀ vs. controls were estimated by non-linear regression fit (see Results); IC₅₀ was 9.6±3.4 µM for 8305C cells, and 4.7±1.8 µM in AF cells.

Figure 6.

In vivo experiments. (A) Dose-dependent antitumor in vivo effect of vandetanib at the dose of 12.5 and 25 mg/kg/day intraperitoneally (i.p.), on 8305C tumors xenotransplanted in CD nu/nu mice. (B) Weights of mice monitored during the treatment with the two doses of the drug and vehicle alone. Symbols and bars, mean (±SE); *P<0.05 vs. vehicle-treated controls.