Epidermal growth factor receptor activation confers resistance to lenvatinib in thyroid cancer cells

Abstract Thyroid cancer is the most common endocrine malignancy. A multitargeted tyrosine kinase inhibitor, lenvatinib, has been used for the treatment of advanced thyroid cancer. To elucidate the mechanism of resistance to lenvatinib in thyroid cancer cells, we established lenvatinib‐resistant sublines and analyzed the molecular mechanisms of resistance. Two thyroid cancer cell lines (TPC‐1 and FRO) were used, and resistant sublines for lenvatinib (TPC‐1/LR, FRO/LR) were established. In TPC‐1/LR, the phosphorylation of epidermal growth factor receptor (EGFR), extracellular signal‐regulated kinase (ERK), and Akt was enhanced whereas in FRO/LR, the phosphorylation of EGFR and downstream signal transduction molecules was not enhanced. The addition of epidermal growth factor decreased sensitivity to lenvatinib in TPC‐1 and FRO. The combination of EGFR inhibitors lapatinib and lenvatinib significantly inhibited the growth of TPC‐1/LR in both in vitro and mouse xenograft models. Short‐term exposure to lenvatinib enhanced the phosphorylation of EGFR in six thyroid cancer cell lines regardless of their histological origin or driver gene mutations; however, phosphorylation of ERK was enhanced in all cells except TPC‐1. A synergistic growth‐inhibitory effect was observed in three thyroid cancer cell lines, including intrinsically lenvatinib‐resistant cells. The results indicate that signal transduction via the EGFR pathway may be involved in the development of lenvatinib resistance in thyroid cancer cells. The inhibition of the EGFR pathway simultaneously by an EGFR inhibitor may have therapeutic potential for overcoming lenvatinib resistance in thyroid cancer.

are differentiated thyroid carcinomas (DTCs), including papillary and follicular thyroid carcinomas (PTC and FTC) originating from follicular epithelial cells and anaplastic thyroid carcinoma (ATC) accounting <2%. 3 Ten-year disease-specific survival for DTC is 96%. 4 Despite a favorable prognosis by standard treatment, such as surgery, with/ without radioactive iodine therapy, some cases exhibit refractory aggressive behavior to radioactive iodine, approximately 6%-20% relapse as distant metastases, which becomes life-threatening in the patients, dropping the 10-year overall survival rate to <50%. 5 Although mutations in oncogenes BRAF, RAS, or RET/PTC rearrangement frequently occur in thyroid cancer, the activation of the MAPK signaling pathway is observed regardless of the presence or absence of these gene mutations. 6,7 Since vascular endothelial growth factor (VEGF)-driven angiogenesis and other molecular pathways of tumor growth and maintenance, including the mutated driver genes described above, fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR), is shown to associate with metastasis and aggressiveness of thyroid cancer, [8][9][10][11][12][13] multitargeted tyrosine kinase inhibitors (MKI) have been developed for the treatment of radioiodine refractory thyroid cancer. [14][15][16][17][18][19] Sorafenib and lenvatinib-tyrosine kinase inhibitors (TKIs) have also advanced as the mainstay of treatment in these cases, improving the prognosis of advanced or metastatic DTC. 14,20 Lenvatinib is an oral TKI targeting VEGFRs 1-3, FGFRs 1-4, RET, KIT, and PDGFR. 21,22 In a phase 3 study involving patients with radioiodine refractory progressive DTC, lenvatinib demonstrated prolonged progression-free survival and a good response rate than placebo. 20 However, no therapeutic effect was observed in some cases from lenvatinib initiation. Additionally, the emergence of resistance during treatment remains a serious problem in clinical practice. Therefore, it is essential to identify biomarkers that predict lenvatinib susceptibility and develop treatment strategies after acquiring resistance to lenvatinib. To date, no study on the molecular mechanism of lenvatinib resistance in thyroid cancer is reported. In this study, to elucidate this mechanism of lenvatinib used for thyroid cancer, we established lenvatinib-resistant sublines in thyroid cancer cell lines and investigated the molecular mechanisms involved in the development of resistance to lenvatinib.

| Cell lines and reagents
The PTC cell line TPC-1 and ATC cell line FRO were donated by Dr. Yamashita from Nagasaki University. 23 The FTC cell line, FTC-133, was donated by Dr. Takeda from Shinshu University School of Medicine. 24 The ATC cell line, ACT-1, which was established by Dr.
Ohata from Tokushima University, 25 OCUT-1F, and OCUT-3 cells were donated by Dr. Onoda from Osaka City University. 26 The genetic information for these cell lines was provided by the researchers who gave us these cell lines. We outsourced the genetic analysis of FRO cells during this study. The cell lines were cultured in RPMI (Sigma-Aldrich) supplemented with 10% FBS at 37°C under 5% CO 2 .
Lenvatinib-resistant sublines were established by stepwise increase in lenvatinib for more than 6 months. We subsequently selected several resistant clones for each thyroid cancer cell line and used one representative clone in subsequent experiments. The established lenvatinib-resistant clones were maintained in lenvatinibcontaining media throughout the experiments. We maintained lenvatinib-resistant TPC-1 (TPC-1/LR) cells in a medium containing 15 nM lenvatinib and lenvatinib-resistant FRO (FRO/LR) cells in a medium with 30 nM lenvatinib. All cells were examined regularly for mycoplasma contamination. Lenvatinib and lapatinib were purchased from Selleck Biotech Co., Ltd. Human recombinant EGF was purchased from the FUJIFILM Wako Pure Chemical Corporation. In the experiments to examine the dose-dependent effects of EGF, the FBS concentration in the medium was reduced to 1% and cells were incubated for 24 h before addition of EGF. To analyze the biology of cancer cells on acquisition of sufficient resistance to lenvatinib, we removed lenvatinib 24 h before each assay to examine the response of lenvatinib-resistant sublines to drugs other than lenvatinib.

| WST assay for cell proliferation
The growth-inhibitory effects of lenvatinib and lapatinib were measured using a tetrazolium salt-based proliferation assay (WST assay; FUJIFILM Wako Pure Chemical Corporation). 27 Each experiment was independently performed and repeated in triplicate.

| Western blotting
Proteins were isolated from cells as previously described and used for western blot analyses (4.0-10 μg/lane). 27,28 The membranes were probed with various antibodies (Table S1). Each experiment was repeated at least thrice. The protein levels were quantified on ChemiDoc XRS (Bio-Rad Laboratories). Protein levels corresponding to each band were quantified based on band intensity using the ImageJ program (version 1.52u). 29

| Immunohistochemistry
Sections (3 μm) of paraffin-embedded tumor samples were used for immunohistochemical analysis. The slides were heated for antigen retrieval in 10 mM sodium citrate (pH 6.0). Sections were subsequently exposed to specific antibodies for TUNEL (#MK500; Takara Bio Inc.)

| Statistical analysis
Statistical significance was tested by performing unpaired Student's t-tests or one-way ANOVA with Tukey's multiple comparisons; a P value <0.05 was considered statistically significant, calculated using StatFlex ver. 6 (Artech Co., Ltd.).

| Evaluation of synergy of growth-inhibitory effect by two drugs
The synergy was evaluated using the Bliss independence model according to a previous study. 30 The Bliss independence model was applied to the quantification data of the WST assays. Briefly, Bliss independence was calculated as follows: when drug A at dose a inhibited Ya% and drug B at dose b inhibited Yb% of the tumor growth, the combined percentage inhibition Yab,P could be predicted using complete additivity in probability theory as Yab,P = Ya × Yb − YaYb. The observed combined percentage inhibition Yab,O was then compared with Yab,P.
If Yab,O > Yab,P, the combination effect was considered synergistic. 31

| Establishment of lenvatinib-resistant cells in TPC-1 and FRO cells, and protein expression in parental and lenvatinib-resistant TPC-1 and FRO cells
Initially, we examined the expression and phosphorylation of molecules in the signal transduction pathway at steady state in six thyroid cancer cell lines ( Figure 1A). The phosphorylated molecules in the signal transduction pathway in steady state were different among cell lines. The phosphorylation of either or both MEK-ERK and Akt-mTOR pathways was observed in five cell lines except for TPC-1. In contrast, phosphorylation of EGFR was elevated in TPC-1 relative to the other cell lines.   Thereafter, we tested the growth-inhibitory effects of lenvatinib in these cell lines ( Figure 1B and Table 1). TPC-1 showed the highest sensitivity to lenvatinib, while ACT-1 and OCUT-1F showed little lenvatinib sensitivity. As the cell lines exhibited intrinsic resistance to lenvatinib were not considered suitable for developing resistant sublines, we established lenvatinib-resistant sublines in TPC-1 and FRO cells with a stepwise increase in lenvatinib concentration for more than 6 months. The lenvatinib-resistant cell lines were designated as TPC-1/LR and FRO/LR, respectively ( Figure 1C). As demonstrated in Table 2 We measured the EGF in serum-free conditioned media in TPC-1 and TPC-1/LR to determine whether an increase in autocrine EGF was involved in the enhancement of EGFR phosphorylation in TPC-1/LR ( Figure S1). Although EGF in the conditioned media was slightly increased in TPC-1/LR compared to TPC-1, no significant difference was observed.

| Enhanced EGFR phosphorylation by EGF confers lenvatinib resistance in TPC-1 and FRO cells
Since enhanced EGFR phosphorylation was observed in lenvatinibresistant TPC-1/LR cells, we tested whether lenvatinib resistance is induced by activation of the EGFR pathway in the presence of 3 or 10 ng/ml of EGF in TPC-1 and FRO cells. Addition of 10 ng/ml exogenous EGF significantly increased the IC 50 for lenvatinib in parental TPC-1 and FRO cells (Figure 2A and Table 3). The enhancement of lenvatinib resistance by 10 ng/ml EGF was more prominent in TPC-1 than in FRO while not affecting the sensitivity to lenvatinib with addition of exogenous EGF to TPC-1/LR and FRO/LR.
Next, the effect of exogenous EGF on cell proliferation was investigated in these cells ( Figure 2B). Although cell growth was not

| Effect of EGFR inhibitor, lapatinib on growth, and protein expression in parental and lenvatinib-resistant TPC-1 and FRO cells
To investigate whether the EGFR signaling pathway was involved in the acquisition of resistance to lenvatinib in TPC-1 cells, we examined the inhibitory effect of the EGFR signaling pathway using lapatinib in TPC-1 and FRO cells. The IC 50 values for lapatinib in each cell line are shown in Table 5. As the IC 50 for lapatinib in TPC-1/LR (1.6 μM) was less than half of that in the parental TPC-1 cells, TPC-1/LR showed a significantly higher sensitivity to lapatinib than parental TPC-1 cells ( Figure 3A). However, sensitivity to lapatinib was slightly decreased in FRO/LR cells compared to that in the parental FRO cells.

| Effect of combination of lenvatinib and lapatinib on lenvatinib-resistant thyroid cancer cells
As we observed increased sensitivity to lapatinib in lenvatinibresistant TPC-1 (TPC-1/LR) cells, we next tested the combined effects of lenvatinib and lapatinib in TPC-1/LR cells. First, we confirmed that 0.1-10 μM of lenvatinib enhanced EGFR phosphorylation in TPC-1/LR cells 6 h after treatment with lenvatinib, while 30 μM lenvatinib reduced EGFR phosphorylation ( Figure 4A).
When lapatinib and lenvatinib were used in combination, 0.1 μM of lapatinib significantly increased the growth-inhibitory effect ( Figure 4B and Table 6). Additionally, when the combined effect of lapatinib and lenvatinib was evaluated using the Bliss independence model, a synergistic growth-inhibitory effect was confirmed in the presence of 0.3 μM of lapatinib, for which the concentration was <20% of IC 50 of lapatinib monotherapy for TPC-1/LR ( Figure 4C and Table 5).
Although 1 and 3 μM of lapatinib inhibited the phosphorylation of EGFR at 6 h after treatment, the inhibition of phosphorylation of ERK was observed only in the presence of lenvatinib ( Figure 4D).

| Effect of simultaneous administration of lenvatinib and lapatinib in lenvatinib-resistant TPC-1 in vivo
Next, we tested the effects of lenvatinib and lapatinib combination

| Effects of lenvatinib on the signal transduction pathway in various thyroid cancer cell lines
Results of lenvatinib-resistant TPC-1 cells suggest that lenvatinib might activate the EGFR pathway in thyroid cancer cells. To investigate whether short-term exposure to lenvatinib alters the EGFR pathway in thyroid cancer cells, we exposed six different thyroid

| Effect of simultaneous administration of lenvatinib and lapatinib in various thyroid cancer cell lines
Enhanced phosphorylation of EGFR by lenvatinib was observed in six thyroid cancer cell lines. We analyzed whether the growthinhibitory effect of lenvatinib is enhanced during simultaneous administration with lapatinib in OCUT-1F cells. This showed least sensitivity to lenvatinib monotherapy ( Table 1). The combined use of lapatinib ≥0.1 μM significantly reduced the IC 50 of lenvatinib ( Figure 7A and Table 6).
In the analyses of signal transduction pathway by western blotting in OCUT-1F, lapatinib monotherapy suppressed EGFR phosphorylation (not Akt), however, the combined use suppressed

| DISCUSS ION
Since the favorable results of the phase 3 trial for radioiodine refractory DTC were reported in 2015, 20 lenvatinib has been widely used for advanced or recurrent thyroid cancer. However, no precise molecular mechanism of lenvatinib resistance in thyroid cancer has been reported so far. Here, we show that short-term administration of lenvatinib induces increased EGFR phosphorylation in multiple thyroid cancer cell lines and activation of the EGFR pathway contributes to lenvatinib resistance in a subset of thyroid cancer cells.
Furthermore, we have shown that the combined administration of an EGFR inhibitor and lenvatinib could restore lenvatinib susceptibility to these cells that acquired resistance to lenvatinib in in vitro and in vivo mouse model systems. To our knowledge, this is a novel study analyzing the molecular mechanisms of lenvatinib resistance in thyroid cancer cells.
Lenvatinib acts as a multi-tyrosine kinase inhibitor and has a strong anti-angiogenic effect. Its pharmacological action has made it a potent therapeutic agent for various malignancies that share similar biological characteristics. 22 Currently, lenvatinib is used worldwide for thyroid cancer, hepatocellular carcinoma (HCC), and renal cell carcinoma. 32,33 Furthermore, it has recently been approved for unresectable thymic cancer in Japan. 34 However, few reports analyze the molecular mechanisms of lenvatinib resistance development. Recently, the inhibition of receptor tyrosine kinases, such as those belonging to the VEGFR and FGFR families, by lenvatinib has been shown to induce activation of signaling through the receptor tyrosine kinases that are not targeted by lenvatinib, thereby conferring lenvatinib-resistance in both HCC and RCC cells. 30,33,35 Based on this, the therapeutic potential of a simultaneous dual inhibition strategy of administration of lenvatinib and another moleculartargeted agent that suppresses the alternate pathway activated by lenvatinib while overcoming lenvatinib resistance has been demonstrated in these cancers. EGFR has been reported to be expressed at higher levels in thyroid cancer tissues than in normal thyroid tissue with a poor prognostic factor for thyroid cancer. [36][37][38][39][40] Previous preclinical studies in vitro have demonstrated that EGF stimulates thyroid follicular cell proliferation and enhances migration and invasion of PTC cells, 36,41,42 playing an important role in thyroid tumor development. 43 These findings suggest that the molecular blockade of EGFR activation may have therapeutic potential in advanced thyroid cancer. In a study using ATC cell lines, Schiff et al. 44 showed the growth-inhibitory effect of the EGFR inhibitor gefitinib on the ATC cell line with enhanced EGFR phosphorylation in vitro and in vivo. However, they also showed that EGFR activation occurred only in a subset of ATC cell lines. Subsequently, in a phase 2 trial for gefitinib against ATC, although reductions in tumor volume were observed in one-third of patients, none met the criteria for a partial response. 45  Herein, some thyroid cancer cell lines showed a strong intrinsic resistance to lenvatinib. However, a synergistic enhancement in growth inhibition was observed when lapatinib was used in combination with lenvatinib in these cell lines. In clinical practice, some thyroid cancers show resistance to lenvatinib from the initial treatment or gain resistance within a short period. Among these F I G U R E 7 Effect of combination of lenvatinib and lapatinib on thyroid cancer cell lines. (A) Sensitivity to lenvatinib in combination with lapatinib (0, 0.1, 1, and 5 μM) in OCUT-1F cells was evaluated by WST assay. (B) Expression and phosphorylation of EGFR, ERK, and Akt in OCUT-1F cells simultaneously treated with lenvatinib and lapatinib for 24 h. α-tubulin was used as a loading control and 6 μg/lane proteins were loaded. The relative phosphorylation levels are disclosed below each protein band. (C) Bliss independence model evaluating the synergistic effect between lenvatinib with lapatinib in thyroid cancer cells. The green background indicates the synergistic effect with P < 0.05 and when the reduction in viability is >10% lenvatinib-resistant thyroid cancer cells, EGFR may have a crucial function in survival and simultaneous use of EGFR inhibitors may be a promising therapeutic strategy against these lenvatinib-resistant thyroid cancers.  [47][48][49] Hence, molecular-targeted therapies based on gene alterations will be introduced earlier than MKIs such as lenvatinib or sorafenib to treat advanced thyroid cancer. 50,51 However, approximately 20%-30% of thyroid cancers do not have driver gene mutations, thus MKIs such as lenvatinib will still be in use. Additionally, lenvatinib is anticipated as a second-line treatment for cases that have acquired resistance to BRAF, RET, or NTRK inhibitors, therefore it is crucial to develop strategies to enhance the antitumor effects of lenvatinib on thyroid cancer cells.
A synergistic effect can be achieved using multiple moleculartargeted drugs to inhibit signaling molecules from being activated by one drug. 52 For BRAFV600E mutation-positive ATC, a simultaneous inhibition of MEK, which is activated in the presence of BRAF inhibitor, shows a higher antitumor effect which has not been obtained with any other conventional treatment strategies. 47 Although the mechanisms of activation of EGFR in the presence of lenvatinib observed in the present study require further elucidation, we think that the synergistic growth inhibitory effect obtained with lenvatinib in combination with lapatinib may be associated with an increased signaling transduction through EGFR, as has been proven by the combination of BRAF and MEK inhibitors.
One of the limitations of this study is that we did not analyze the effects of lenvatinib on the tumor microenvironment in vivo.
Lenvatinib is a potent inhibitor of VEGFRs 1-3 and FGFRs 1-4, and inhibition of angiogenesis in the tumor microenvironment is reportedly responsible for its antitumor effects in vivo. 53 In this study, we did not evaluate whether there were differences in the expression of angiogenic factors such as VEGF and FGF between parental and lenvatinib-resistant TPC-1 cells. To elucidate the mechanism underlying lenvatinib resistance in thyroid cancer in vivo, it is necessary to analyze the effects of lenvatinib not only on cancer cells but also on the tumor microenvironment, including angiogenesis inhibition and immunomodulation. Therefore, we are currently attempting to construct an experimental system to evaluate the effects of lenvatinib in vivo.
In conclusion, we demonstrated the possibility of the involvement of activation of the EGFR pathway in the development of lenvatinib resistance in thyroid cancer cells. We also showed that a simultaneous inhibition of the EGFR pathway may have therapeutic potential for overcoming lenvatinib resistance in thyroid cancer in vitro and in vivo.

ACK N OWLED G M ENTS
We thank Ms. Yukiko Kanai and Ms. Hiromi Fujita for their technical assistance.

D I SCLOS U R E
The authors have no conflict of interest.