Abstract
Anaplastic thyroid carcinoma (ATC) is refractory to radioiodine therapy in part because of impaired iodine metabolism. We targeted the mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3′K) pathways with the intent to induce radioiodine uptake for radioiodine treatment of ATC. Methods: Human ATC cells were used to evaluate the ability of pharmacologic inhibition of the mitogen-activated protein kinase and PI3′K pathways to induce radioiodine uptake. Thyrocyte-specific double-mutant BRAFV600E PIK3CAH1047R mice were treated with a MEK inhibitor followed by radioiodine treatment, and tumor burden was monitored by ultrasound imaging. Results: ATC cell lines showed an increase in sodium-iodine symporter transcription when treated with a MEK or BRAFV600E inhibitor alone and in combination with PI3′K inhibitor. This translated into a dose-dependent elevation of iodine uptake after treatment with a MEK inhibitor alone and in combination with a PI3′K inhibitor. In vivo, MEK inhibition but not BRAF or PI3′K inhibition upregulated sodium-iodine symporter transcription. This translated into a stable reduction of tumor burden when mice were treated with a MEK inhibitor before radioiodine administration. Conclusion: This study confirms the ability of MEK inhibition to induce iodine uptake in in vitro and in vivo models of ATC. The approach of using a MEK inhibitor before radioiodine treatment could readily be translated into clinical practice and provide a much-needed therapeutic option for patients with ATC.
Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human malignancies (1,2) and is refractory to radioiodine therapy (3).
Impaired iodine metabolism can result from dysregulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3′K) pathways (Fig. 1), which regulate a variety of cellular activities and have been implicated in the development of various human cancers (4). MAPK mutations are common in ATC, with 25% of tumors harboring KRAS mutations, and depending on the study, 27%–45% are carrying BRAF mutations (5–7). The most common mutation found in BRAF codes for the protein kinase BRAFV600E, which induces constitutive activation of the downstream pathway (Fig. 1). Several important PI3′K pathway mutations in ATC include PTEN deletions (10%–20%) and PIK3CA mutations (15%–25%) (8). We have previously shown that the BRAFV600E mutation in combination with the PIK3CAH1047R mutation induces tumor progression from papillary thyroid cancer (PTC) to ATC in a mouse model (9).
The link between alterations of the MAPK and PI3′K pathways and impairment of iodine metabolism is well documented, although the underlying mechanisms not fully elucidated. BRAF mutations are associated with impairment of NIS function and subsequent increased resistance to radioiodine uptake in (10,11) thyroid cancers. Some studies suggest that BRAF inhibits the expression of PAX8 and prevents transcription of NIS (12).
The relationship between the MAPK pathway and the ability to store iodine intracellularly has been further demonstrated in mouse models of PTC, where selective MAPK pathway antagonists have been shown to increase the expression of NIS, with resultant increased uptake of iodine (13). Inhibition of the MAPK pathway via MEK is a promising strategy for treatment of iodine-refractory thyroid cancers and, specifically, ATC. Several studies suggest that PI3′K inhibition increases radioiodine uptake in thyroid cancer cells (14,15); however, this has not yet been evaluated in vivo. In this study, we aimed to investigate the consequences of pharmacologically targeting the MAPK and PI3′K pathways separately and in combination to induce radioiodine uptake in ATC cell lines and in a lethal double-mutant ATC (BRAFV600E PIK3CAH1047R) mouse model. We hypothesized that pharmacologic restoration of radioiodine uptake with subsequent radioactive iodine would be an effective means of therapy in a murine model of ATC.
MATERIALS AND METHODS
Small-Molecule Kinases Inhibitors
The inhibitors PD-325901 (MEK1/2), PLX-4032/vemurafenib (BRAFV600E), PLX-4720 (BRAFV600E), and GDC-0941 (class I PI3′K) were purchased from Abmole Bioscience. PLX-4032 was used in vitro as a BRAFV600E inhibitor, but because of poor solubility (16), PLX-4720 was used for in vivo experiments.
BRAFV600E PIK3CAH1047R Mice
Transgenic mice were bred by combining the following alleles as previously described (9): BrafCA (17), Pik3caLat (18), and Thyro::CreERT2 (19). Mutations were induced by 5 consecutive daily injections of 1 mg of tamoxifen intraperitoneally. Mice were cared for in accordance with Swiss federal guidelines, housed in isolated ventilated cages, and fed ad libitum in a 12 h/12 h cycle of light and dark. The experimental protocol was approved by the Bernese Cantonal Ethical Commission for Animal Experimentation (license number BE120/13).
Cell Lines
The human cell lines 8505c, Sw1736, and OCUT-2 were obtained and maintained as described previously (20).
In Vitro Evaluation of ATC NIS Messenger RNA (mRNA) Expression
Cells were seeded in 10-cm Petri dishes at 25% confluency overnight and then treated with PD-325901 at 10 nM, GDC-0941 at 100 nM, PLX-4032 at 100 nM, and PD-325901 at 10 nM; with GDC-0941 at 100 nM or PLX-4032 at 100 nM; and with GDC-0941 at 100 nM at 37°C for 48 h. Cells were washed twice with 5 mL of cold phosphate-buffered saline and then collected in 1 mL by scraping the dish surface. A centrifugation for 10 min at 3,000g was performed, and the cell pellets were frozen in liquid nitrogen then stored at −80°C. NIS mRNA levels were evaluated by quantitative polymerase chain reaction.
In Vivo Evaluation of Thyroid-Specific Gene Expression After Kinase Inhibition
Thyroids from 8 single-mutant BRAFV600E mice (BrafCA/+; Thyro::CreERT2) that had been treated by oral gavage for 21 d with a 12.5 mg/kg dose of PD-325901 (n = 4) or with 0.5% hydroxypropyl methylcellulose and 0.2% polysorbate-80 (n = 4) were used as a control. These thyroid tissues were fixed in formalin and embedded in paraffin blocks (21), from which RNA was extracted with High Pure FFPET RNA isolation kit (Roche) according to the manufacturer’s protocol.
Twenty-three BRAFV600E PIK3CAH1047R double-mutant mice (BrafCA/+; Pik3caLat/+; Thyro::CreERT2) were treated 2 mo after tumor induction with PD-325901 at 5 mg/kg (n = 4), PLX-4720 at 30 mg/kg (n = 5), or GDC-0941 at 50 mg/kg (n = 5) as single treatments, or with PLX/GDC (n = 5) and PD/GDC (n = 4) in combination, by oral gavage daily for 10 d. Additionally, thyroid glands from 12 Cre-negative mice (BrafCA/+; Pik3caLat/+) were combined for a total of n = 3 and used as nontumoral controls. Hydroxypropyl methylcellulose at 0.5% and polysorbate-80 at 0.2% were used as a control. Mice were then sacrificed, thyroid glands were dissected and snap-frozen using liquid nitrogen, and total RNA was purified with the QIAzol reagent from Qiagen according to the manufacturer’s instructions with a TissueLyser LT using iron beads. Nis, Tshr, Tg, Pax8, Tpo, and Ttf1 mRNA levels were evaluated.
Quantitative Polymerase Chain Reaction
A 500-ng quantity of RNA underwent reverse transcription using Oligo(dT)12-18 Primer and the Super Script II Reverse Transcriptase from Invitrogen following the manufacturer’s suggested protocol. Samples were run on ViiA 7 real-time polymerase chain reaction system (Applied Biosystems, Life Technologies) using the TaqMan gene expression Master Mix and primers from Applied Biosystems. GAPDH was used for normalization using the 2∆∆Ct calculation method. All values were expressed as percentage of the baseline transcription levels found in nontumoral Cre-negative thyroid tissues. The following primers were purchased from Applied Biosystems, Life Technologies: NIS (Hs00166567_m1), Nis (Mm01351811_m1), Tg (Mm01200340_m1), Ttf1 (Mm00657018_m1), Pax8 (Mm00440623_m1), Tpo (Mm00456355_m1), Tshr (Mm00442027_m1), GAPDH (Hs02758991_g1), and Gapdh (Mm99999915_g1).
In Vitro 123I Uptake Assay
Cells were seeded in Corning 96-well plates at 30% confluency, incubated overnight at 37°C and 5% CO2, and treated for 48 h with various concentrations of PD-325901 (0, 0.1, 1, 10, 100, and 1,000 nM) with or without 200 nM GDC-0941. All except one of the PD-325901 concentrations used were below the Cmax value (200 nM), which corresponds to the maximal concentration in human plasma without toxicity recorded (22). Cells were washed with phosphate-buffered saline, and cell viability was assessed with the AlamarBlue Cell Viability Reagent (no. 765506, Invitrogen) by measuring fluorescence at 634 nm (SpectraMax M4 plate reader; Molecular Devices). After removal of AlamarBlue, cells were washed with phosphate-buffered saline and incubated with culture medium containing 18.5 kBq of 123I (GE Healthcare) for 1 h at 37°C. The medium was removed, and the cells were washed 3 times with phosphate-buffered saline, which was then replaced by 50 μL of scintillation reagent (MicroScint-20; PerkinElmer) per well. Activity was measured with a microplate scintillation counter (Packard Topcount NXT).
In Vivo 125I Uptake Assay
Eleven BRAFV600E PIK3CAH1047R double-mutant mice were treated daily by oral gavage for 2 wk with control (n = 4) or with PD-325901 at 5 mg/kg alone (n = 4) or in combination with GDC-0941 at 30 mg/kg (n = 3). Hydroxypropyl methylcellulose with 0.5% polysorbate-80 was used as a control. Then, mice were injected via tail vein with 1.11 MBq of 125I (ANAWA; Biomedical Services and Products) and euthanized 1 h later with CO2. Thyroid glands, salivary glands, liver, and blood were harvested and weighed. Radioactivity was quantified for 30 s per sample using a PerkinElmer 2470 automatic γ-counter.
In Vivo Tumor Treatment Experiment
16 BRAFV600E PIK3CAH1047R double-mutant mice were treated daily by oral gavage for 10 d with control (n = 8) or PD-325901 at 5 mg/kg (n = 8). Afterward, half the mice in each group were injected with 18.5 MBq of 131I (PerkinElmer) via tail vein. Tumor volume was assessed weekly for 9 wk with the following previously described protocol (20). Tumor burden was determined by measuring the surface of the largest thyroid cross section normalized to the size of the gland at the beginning of the study. In this model of thyroid cancer, all thyrocytes are undergoing molecular changes leading to cancerization of the entire thyroid gland. Therefore, the assessment of whole thyroid size for tumor burden is a reliable and well-accepted approximation (9,21,23).
Statistical Analysis
A 2-way ANOVA test was performed to evaluate the difference in tumor burden measurement between treatment groups. A 1-way ANOVA test was used to evaluate the difference in NIS mRNA levels in vitro, the difference in radioiodine uptake in vivo, and the difference between treatments on mRNA levels of different genes in vivo (followed by the Tukey multiple-comparisons test). All statistical analyses were performed using GraphPad Prism. P values equal to or less than 0.05 were considered significant.
RESULTS
MEK Inhibition Alone or in Combination with PI3′K Inhibition Enhances NIS mRNA Transcription and Increases Intracellular Iodine Transport In Vitro
The effect of the MEK inhibitor PD-325901 and the BRAF inhibitor PLX-4032 (vemurafenib) alone or combined with the PI3′K inhibitor GDC-0941 was evaluated on mRNA transcription levels of NIS in 8505c and OCUT-2, 2 ATC cell lines harboring a BRAFV600E mutation (Fig. 2A). In 8505c, PD-325901 treatment alone induced a 4-fold increase in NIS mRNA level after 48 h, and the addition of GDC-0941 did not further augment NIS mRNA expression. GDC-0941 alone did not have an effect on NIS transcription, and PLX-4032 did not lead to a significant elevation of NIS mRNA expression when it was applied alone or in combination with GDC-0941. The OCUT-2 cell line, which has a PIK3CAH1047R mutation in addition to a BRAFV600E mutation, showed similar upregulation of NIS mRNA transcription after treatment with PD-325901, with an additional response to GDC-0941 alone (3-fold increase in NIS mRNA level) and with the combination of PD-325901 and GDC-0941 (9-fold increase). In OCUT-2, PLX-4032 alone or in combination with GDC-0941 resulted in a 5-fold increase in NIS mRNA levels.
After 48 h of treatment with PD-325901 at varying concentrations ranging from 0 to 1 μM, cellular iodine uptake increased by more than double in OCUT-2 and 8505c, as well as in SW1736, a third ATC cell line also harboring a BRAFV600E mutation. Addition of 200 nM GDC-0941 with PD-325901 further increased uptake of 123I in the OCUT-2 and SW1736 cell lines, but not in 8505c (Fig. 2B).
MEK Inhibition but Not BRAFV600E Inhibition Increases Nis, Tshr, Tg, Pax8, and Tpo mRNA Transcription In Vivo, Which Correlates with Histologic Renormalization of Thyroid Follicles
A 4-fold increase in Nis mRNA levels extracted from formalin-fixed, paraffin-embedded tumor sections in BRAFV600E single-mutant mice (21) treated with the MEK inhibitor PD-325091, as compared with control mice (Fig. 3A), was observed. Additionally, the treated group showed restoration of normal thyroid follicular structure (near-spheroid follicles containing colloid substance) on hematoxylin–eosin staining (Fig. 3B), whereas the control group displayed a papillary structure and absence of normal thyroid follicles.
We then switched to a more aggressive murine BRAFV600E PIK3CAH1047R double-mutant model. In animals treated with the MEK inhibitor PD-325901, there was upregulation of Nis mRNA by almost 20-fold. The selective BRAFV600E inhibitor PLX-4720 did not increase Nis mRNA transcription, nor did the PI3′K inhibitor GDC-0941 alone or in combination with PD-325901 or PLX-4720 (Fig. 3C).
Evaluation of mRNA transcription of additional genes known to be important in thyroid gland function was performed, including Tshr, Pax8, Tg, Tpo, and Ttf1. For all genes except Ttf-1, the same pattern as Nis was observed, with a strong response to PD-325901, no further elevation in combination with GDC-0941, and little response to any of the other drugs alone (Fig. 3C). Elevation of protein expression could be confirmed in immunohistochemistry staining for TG, PAX8, and TTF-1 (Supplemental Fig. 1; supplemental materials are available at http://jnm.snmjournals.org) (24).
Increased Nis, Tshr, Tg, Pax8, and Tpo transcription correlated with histologic near-normalization in the appearance of thyroid follicles after 10 d of PD-325901 treatment on hematoxylin–eosin, a finding that was not replicated in control mice or mice treated with PLX-4720 and GDC-0941 (Fig. 3D).
MEK Inhibition Leads to a Significant Increase in Thyroid Radioiodine Uptake in BRAFV600E PIK3CAH1047R Double-Mutant Mice
After gene expression analyses, a validation of functional changes in radioiodine uptake in BRAFV600E PIK3CAH1047R double-mutant mice using PD-325901 alone and in combination with GDC-0941 was attempted. After 14 d of daily treatment with PD-325901 with or without GDC-0941, there was a 2.5-fold increase in 125I thyroid uptake in BRAFV600E PIK3CAH1047R double-mutant mice compared with controls. There was no significant difference in thyroid radioiodine uptake between mice treated with PD-325901 or those treated with a combination of PD-325901 and GDC-0941. Liver uptake, used to assess background uptake because of passive diffusion through the apical membrane, remained stable across the different treatment regimens. The level of 125I in the blood was approximately 15% injected dose per gram and did not significantly change with the different treatments. The salivary glands, which express NIS but are not able to store iodine because of lack of TG expression, showed slightly decreased uptake with combination treatment (Fig. 4).
MEK Inhibitor Treatment Before 131I Therapy Reduces Tumor Burden in BRAFV600E PIK3CAH1047R Double-Mutant Mice
An evaluation of the translation from the observed increase in radioiodine uptake in BRAFV600E PIK3CAH1047R double-mutant mice treated with MEK inhibition via PD-325901 into reduced tumor burden when injected with therapeutic radioiodine (131I) was performed. The treatment study design, including tumor induction, treatment with PD-325901, and radioiodine therapy, is depicted in Figure 5A. The control group displayed progressive tumor growth of approximately 15% each week. Mice receiving a 10-d course of treatment with PD-325901 experienced only an initial reduction in tumor burden, with the therapeutic effect disappearing immediately after treatment cessation. The group of mice injected with radioiodine alone demonstrated a stable tumor burden for 6 wk before tumors eventually increased in size. Mice treated with a 10-d course of PD-325901 followed by injection with 131I displayed a reduction in the tumor burden by 60% of the initial size 6 wk after iodine injection and remained stable for 3 wk (Fig. 5B).
DISCUSSION
Important genes in thyroid hormone synthesis such as NIS, TSHR, TG, and TPO are frequently downregulated in advanced thyroid cancer. The loss of this machinery is progressive, beginning with NIS and then involving TPO, TG, and TSHR (25–27).
In the present study, we showed in vitro evidence that MEK inhibition induced robust NIS mRNA transcription in human ATC cell lines harboring a BRAFV600E mutation, which resulted in elevated cellular 123I uptake. We also showed that this result could be translated in vivo in a mouse model of lethally aggressive thyroid cancer by significantly elevating the radioiodine uptake, resulting in stable reduction of tumor burden.
We have previously shown that MEK inhibition in combination with PI3′K inhibition could lead to a profound reduction driven by apoptosis (28). This treatment, although promising, has the disadvantage of synergistic side effects associated with both drugs. Moreover, after treatment cessation, a rapid regrowth of tumors occurred. In the present study, a short period of drug treatment (10 d) enabled a potentially curative treatment with radioiodine.
In vivo, MEK inhibition in a BRAFV600E single-mutant mouse model resulted in a Nis 4-fold transcription elevation (Fig. 3A). This iodine induction in a PTC model is consistent with the current literature (13,29,30). In a mouse model of more advanced thyroid cancer harboring BRAFV600E and PIK3CAH1047R mutations (Fig. 3C), we demonstrated that a MEK inhibition induced a strong elevation of mRNA levels of genes important in thyroid hormone synthesis, such as Nis (6-fold), Tshr, Tg, Pax8, and Tpo. Interestingly, the combination of MEK inhibition with PI3′K inhibition did not provide further upregulation of the tested genes. Specific BRAFV600E inhibition did not induce an increase in any of these transcripts (Fig. 3C). This is concordant with Nagarajah et al., who showed that the more profound the MEK inhibition is, the stronger is the NIS reexpression (30). It was previously demonstrated that BRAF-specific inhibition can induce only transient ERK dephosphorylation and that the signal is restored with time through the wild-type allele of BRAF (31). Additionally, our recent findings show that BRAFV600E-specific inhibition led to a paradoxic activation of ERK in the double-mutant mouse model (20). Furthermore, there are very few data reporting vemurafenib treatment in ATC, and one study showed mitigated results, including one case of progression under BRAFV600E inhibitor (32).
On a functional level, a nearly 2.5-fold increase in radioiodine uptake in the thyroids of mice treated with a MEK inhibitor as compared with mice receiving the control treatment was observed (Fig. 4) after only 10 d of PD-325901 treatment. Increased radioiodine uptake translated into a stable, profound reduction in tumor burden (Fig. 5). Mice treated with radioiodine showed only an initial stabilization of the tumor burden followed by a rapid tumor growth. This short-lasting response to radioiodine could be explained by tumor heterogeneity, with regions of untransformed cells and PTC. As there was no added benefit to using a drug combination with a PI3′K inhibitor even in tumors harboring a PIK3CAH1047R mutation (Fig. 4), this was not tested in the context of curative radioiodine.
CONCLUSION
Our study shows that MEK inhibition as a single-drug treatment is the most efficient regimen to induce the expression of protein machinery required for iodine uptake in human ATC cell lines and in our aggressive murine model of thyroid cancer. Until now, most studies have focused mainly on PTC mouse models driven by a single BRAFV600E mutation (13). Here, we provide new evidence that the RAS-MEK-ERK axis is essential for NIS expression even in ATC cell lines and in a clinically relevant mouse model carrying the 2 mutations BRAFV600E and PIK3CAH1047R. We performed a metaanalysis of the 3 available studies conducting genetic profiling in ATC patients and found that the occurrence of both BRAF and PIK3CA mutations is found in 12% of ATC patients (Supplemental Table 1 (5–7)). Thus, we believe that the present results represent an encouraging initial step toward the successful treatment of non–radioiodine-responsive thyroid cancers.
DISCLOSURE
Funding support was provided by Swiss National Science Foundation grant 31003A_149824/1 and Swiss National Science Foundation grant NCCR-TransCure. No other potential conflict of interest relevant to this article was reported.
Acknowledgments
We thank Prof. Martin McMahon for his mentorship and for providing the BRAF mutant mice. We thank Prof. Engelhard, Dr. Deutsch, and Dr. Benarafa for permission to use the vivarium at the Theodor Kocher Institute. We thank Dr. Didier Colin for his help with Figure 1. Also, we acknowledge the Microscopy Imaging Center of the University of Bern (MIC) for their contributions to this study. Oussama ElMokh and Matthias Roelli were enrolled in the Graduate School for Cellular and Biomedical Research (GCB) of the University of Bern during this study.
Footnotes
Published online Nov. 21, 2018.
- © 2019 by the Society of Nuclear Medicine and Molecular Imaging.
REFERENCES
- Received for publication June 27, 2018.
- Accepted for publication November 15, 2018.