Biochemical and Biophysical Research Communications

Vandetanib inhibits cell growth in EGFR-expressing cutaneous squamous cell carcinoma

Shinya Kitamura, Takuya Maeda, Teruki Yanagi*

Department of Dermatology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, 060-8638, Japan

Epidermal growth factor receptor (EGFR) Squamous cell carcinoma (SCC) Rearranged during transfection (RET) Keratinocyte


Advanced cutaneous squamous cell carcinoma (SCC) responds poorly to chemotherapy, leading to sig- nificant morbidity or death. Overexpression of epidermal growth factor receptor (EGFR) is frequently observed in advanced cutaneous SCC. Vandetanib is a multiple tyrosine kinase targeting vascular endothelial growth factor receptor-2 (VEGFR2), EGFR, and the rearranged during transfection (RET) proto-oncogene. Vandetanib has been reported to inhibit tumor growth in head and neck SCC. However, the efficacy of vandetanib against cutaneous SCC has not been thoroughly investigated. The aim of this study is to evaluate the efficacy of vandetanib against cutaneous SCC in vitro and in vivo. Vandetanib is found to inhibit the proliferation of cutaneous SCC cells as assessed by cell viability and clonogenic assay. Cell death analysis indicates that vandetanib induces cell death in SCC cells but not in normal human keratinocytes or fibroblasts. The in vivo anti-tumor effect of vandetanib is shown in xenograft tumor models using A431 SCC cells. Mechanistically, vandetanib suppresses the phosphorylation of EGFR in SCC cells. Clinically, EGFR expression levels are elevated in cutaneous SCC specimens, relative to normal epidermis. In conclusion, we identified vandetanib as a novel therapeutic option for cutaneous SCC, especially in tumors with high EGFR expression.

1. Introduction

Cutaneous squamous cell carcinoma (SCC) is the second most common malignant skin neoplasm, and it arises from keratinocytes in the epidermis. The main onset factors of cutaneous SCC are sun exposure and aging [1,2]. The prevalence of cutaneous SCC has been increasing for the past several decades because of accelerated de- mographic aging [3]. In most cases, tumors are resectable in sur- gery; however, advanced metastatic SCC results in significant morbidity or death. Advanced SCC patients often respond poorly to chemotherapy [4]. Recently, epidermal growth factor receptor (EGFR) has been recognized as an important molecule related to cell proliferation in several cancers, such as non-small cell lung, colon, and head and neck SCC [5e7]. Overexpression of EGFR is frequently observed in advanced cutaneous SCC; thus, cetuximab, a monoclonal EGFR-targeted inhibitor, has been clinically applied for advanced cutaneous head and neck SCC [8]. The activation of EGFR- dependent signaling pathways has been clinically validated in malignant neoplasms, including cutaneous SCC [9]. Thus, EGFR- targeted therapies are promising for advanced cutaneous SCC.

Vandetanib (Caprelsa®, AstraZeneca) is an orally administered anti-tumor agent that selectively targets multiple tyrosine kinases, such as vascular endothelial growth factor receptor-2 (VEGFR2), EGFR, and the rearranged during transfection (RET) proto- oncogene. Vandetanib can inhibit tumor angiogenesis and cell proliferation in medullary thyroid carcinoma [10]. Vandetanib has been applied as a treatment for several RET mutant cancers such as non-resectable, locally advanced or metastatic medullary thyroid cancers [11], and non-small lung cell carcinomas [12]. Also, van- detanib has been reported to inhibit tumor growth in vivo and in vitro in head and neck SCC [13e15]. However, the efficacy of vandetanib for cutaneous SCC has not been investigated in detail. In the present study, we evaluated the efficacy of vandetanib against cutaneous SCCs, and found that 1) vandetanib inhibits cell proliferation and induces the cell death of cutaneous SCC cells, 2) vandetanib inhibits in vivo tumor growth, 3) vandetanib suppresses the phosphorylation of EGFR, and 4) cutaneous SCC samples show high EGFR expression levels. These results indicate the anti-tumor effect of vandetanib in cutaneous SCC cells through the inhibition of EGFR signaling.

2. Materials and methods

2.1. Reagents and antibodies

The cell viability assay kit Cell Titer-Glo® was obtained from Promega. The following items were purchased from the indicated sources: antibodies against RET (rabbit, #A97064, Sigma), VEGFR2 (rabbit monoclonal, #2479, Cell Signaling), EGFR (mouse mono- clonal, #4267, Cell Signaling), phospho-EGFR (rabbit, Y1068, #3777, Cell Signaling), beta-actin (mouse monoclonal, #A5441, Sigma), PARP (rabbit, #9542, Cell Singling), Ki-67 (mouse, #ab8191, Abcam), TUNEL (TdT-mediated dUTP nick end labeling; #11684817910, Roche), HRP-linked horse anti-mouse IgG second- ary antibody (#7076, Cell Signaling), and HRP-linked goat anti- rabbit IgG secondary antibody (#7074, Cell Signaling). Matrigel was purchased from BD Biosciences (#356234). Vandetanib was purchased from Adooq Bioscience (#A10963). The vandetanib was dissolved in dimethylsulfoxide (DMSO) and aliquoted. Small aliquots were stored at 30◦ until use to avoid repeated freezing/ thawing. Recombinant human epidermal growth factor (EGF) was purchased from Sigma (E9644).

2.2. Cell lines and cell culture

A431 human cutaneous SCC cells and human normal dermal fibroblast (NHDF) cells were purchased from the American Type Culture Collection. The human cutaneous SCC DJM1 cells were isolated from human skin SCC [16]. The human immortalized ker- atinocyte cell line (HaCaT) was purchased from Cell Lines Service. The human primary epidermal keratinocyte progenitor cells (HPEKp) were purchased from CELLnTEC. These cells were cultured at 37◦ in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS, Life Technologies) or CnT-PR (CELLnTEC).

2.3. Cell viability assays using ATP measurement

Cells were plated in 96-well solid white plates at a density of 5000e10,000 cells per well in 100 ml of complete medium and cultured for 48 h. The cells were then treated with various con- centrations of compounds for 24 h. Cell Titer Glo solution was added at 100 ml per well, and the plates were kept in the dark for 15 min before luminescence was measured with a luminometer (Spectra Max Paradigm; Molecular Devices) as in our previous report [17].

2.4. Clonogenic assay

Cells were seeded at 5.0 102 cells per 3.5-mm well and cultured for 14 days. After fixation by methanol, cells were washed with PBS and incubated with 0.5% crystal violet dye for 20 min. A colony was defined as consisting of at least 50 cells according to the procedures of our previous report [18].

2.5. Cell death analysis

Cell death was evaluated as in our previous study [17]. Cells were spread in a 60-mm dish at 40% confluence and treated with vandetanib (1.0 mM for 24 h). Dead cells were counted, and the dead cell ratio (dead cells/all cells) was calculated using an automated cell counter (TC10™, Bio-Rad).

2.6. SDS-PAGE and immunoblotting

Cells were lysed in RIPA Buffer (50 mM Tris pH 7.5, 150 mM NaCl, NP40 1%, sodium deoxycholate 0.5%, SDS 0.1%, NaF, final concen- tration 1 nM, NaV3O4, final concentration 10 mM) supplemented with a protease inhibitor cocktail (Roche). The lysates were sepa- rated using SDS PAGE (5e10% gradient gel) and transferred to polivinylidene difluoride membranes (Invitrogen). Blocking and incubation with antibodies were carried out in Tris-buffered saline with 5% non-fat dry skim milk or 5% BSA. Signals were detected with chemiluminescence reagents. The immunoblotting was quantified using the Image J software application (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at

2.7. In vivo xenograft tumor models

In vivo xenograft experiments were performed according to the procedures of our previous reports [17]. BALB/cAJcl-nu/nu mice (5 weeks old, female) were purchased from CLEA Japan, Inc. The mice were given sterile distilled water and standard chow ad libitum and held in a 12-h light-dark cycle. The animal experiments in this study were approved by the Hokkaido University Animal Care Committee. 5.0 106 cells were suspended in 200 ml of phosphate- buffered saline (PBS) and injected subcutaneously into the bilateral flanks. For the vandetanib treatment, when the tumors reached 5 mm in diameter (usually 10 days after xenotransplantation), vandetanib was injected into the tail vein (200 mg in 100 ml of 0.1% DMSO in PBS, equivalent to 20 mg/kg) and re-injected every two days. The control xenograft tumors were treated with 100 ml of 0.1% DMSO in PBS. Tumor volume was calculated by an approximation formula: ((1/2) (major axis) (minor axis)2). On post-operative day 20, the tumors were extracted and the tumor weights were recorded.

2.8. Immunohistochemical analysis

Immunohistochemical analysis was carried out on 4-mm formalin-fixed, paraffin-embedded sections. Immunostaining was evaluated by the same observer; the immunostaining score for EGFR was calculated as follows; 1 : weak intensity, faint brown membranous; 2 : moderate intensity, brown membranous of in- termediate darkness producing a complete or incomplete circular outline of the neoplastic cell; and 3 : strong intensity, dark brown or black membranous producing a thick outline, complete or incomplete of the neoplastic cell [19]. The immunostaining score for RET was calculated as follows; 0: no staining; 1 : faint cyto- plasmic staining; 2 , moderately, smooth cytoplasmic staining;
3 : intense, granular cytoplasmic staining [20]. The proportion of immunoreactive cells was determined by counting 100 cells in three randomly chosen fields. Concerning on the Ki-67 index, positive cells were based on a count of at least 100 tumor cells in the peripheral area including the hot spot and the lower level of 3% was regarded as ‘considerably lower Ki-67 index values’ in this study.

2.9. Patient selection

30 skin samples from the SCC patients were immunohis- tochemically assessed. All the patients were under treatment by the Department of Dermatology of Hokkaido University Hospital. SCC samples were obtained from patients whose ages ranged from 51 to 103 years (average 77.2 years, male: female ratio 10:20). The present study included 15 patients without metastasis and 15 with lymph node or distant metastasis. This study was approved by the institutional review board of Hokkaido University Hospital (017- 0438). Tumor volume, initial clinical stage, and observed time were retrieved from clinical data. The clinical information is summarized in Supplemental Table S1.

2.10. Statistical analysis

Quantitative data are shown as means ± SD (standard devia- tion). All statistical analyses were calculated using Microsoft® Excel 2013 (Microsoft Corporation., Washington, U.S.A.). The Student’s t- test was used to estimate statistical significance between cate- gories. At least three independent experiments were carried out for statistical comparison. All analyses were performed with a P < 0.05 level of significance unless otherwise indicated. 3. Results 3.1. Vandetanib inhibits the proliferation of cutaneous SCC cells First, we assessed the expression levels of EGFR, which is one of the main target molecules of vandetanib. Immunoblot analysis revealed the expression levels of EGFR in SCC A431, DJM1 and immortalized HaCaT cells to be higher than those in HPEKp or NHDF cells (Fig. 1A). To assess the anti-tumor efficacy of vandeta- nib, these cells above were treated with vandetanib (1.0 mM) for 24 h, and cell viability was assessed. A431, DJM1 and HaCaT cells displayed significantly lower cell viability ratios than those of the HPEKp cells or the NHDF cells (Fig. 1B). In the clonogenic assay, the number of cell colonies was significantly lower with the adminis- tration of 1-mM vandetanib in the A431 SCC cells, DJM1 SCC cells, HaCaT cells, but not in the HPEKp cells or the NHDF cells (Fig.1C and D). These results suggest that sensitivity to vandetanib may corre- late with EGFR expression levels. 3.2. Vandetanib induces SCC cell death and inhibits the phosphorylation of EGFR in SCC cells We performed cell death analysis by calculating the dead cell ratio under vandetanib administration. A431 cells treated with vandetanib (1 mM) for 1 h showed significantly higher dead cell ratios than the control groups showed. A similar tendency (P 0.07) was observed in the experiments using DJM1 cells, whereas there was no significant difference between the two groups for the HPEKp, HaCaT and NHDF cells (Fig. 2A). Immuno- blotting also showed that the administration of vandetanib induces the cleavage of PARP in A431 (Fig. 2B). These results also suggest that vandetanib correlate with EGFR expression levels. Next, to examine the molecular mechanisms behind the suppression of tumor cell growth, we assessed the phosphorylation of EGFR in SCC cells treated with or without vandetanib. A431 SCC cells were starved for 12 h with DMEM media supplemented with 0.1% FBS, and were stimulated with recombinant human EGF (0, 1, 10 ng/ml) for 10 min. There was no significant difference in total EGFR expression levels between the control group and the vandetanib- treated group, whereas the phosphorylation of EGFR (Tyr 1068) was partially suppressed in the vandetanib-treated cells (Fig. 2CeE). These findings indicate that vandetanib can inhibit the phosphorylation of EGFR in cutaneous SCC cells. 3.3. Administration of vandetanib inhibits tumor growth in vivo To extend the studies into an in vivo context, we used A431 SCC cells in a tumor xenograft model. 12 immunodeficient nu/nu mice were injected subcutaneously with A431 cells (5 106 cells), and tumors were allowed to grow for 10 days. The 12 mice were divided into two groups: a vandetanib group (N 6), and a control group (N 6). The administration of vandetanib (20 mg/kg, every other day) significantly suppressed tumor growth compared to the con- trol (Fig. 3A and B). The weights of extracted tumors in the vandetanib-treated group were significantly lower than those in the control group (Fig. 3C). Histopathologically, the vandetanib- treated tumor showed considerably lower ki-67 index values, suggesting that vandetanib inhibits tumor cell division in vivo (Fig. 3D and E). Also, the TdT-mediated dUTP nick end labeling (TUNEL) index values were significantly higher in the vandetanib- treated cells than in the control cells (Fig. 3F). Taken together, the above findings show that the systemic administration of vandeta- nib inhibits tumor growth in cutaneous SCC in vivo. 3.4. Expression levels of EGFR are elevated in cutaneous SCCs Finally, we examined the protein expression levels of EGFR in primary cutaneous SCC specimens (N 30). Immunohistochemi- cally, the expression levels of EGFR were higher in cutaneous SCC tissue than in normal epidermis (Fig. 4A and B). Based on the im- munostaining pattern, EGFR in primary SCC tumors localized mainly at the cell membrane, whereas EGFR in normal epidermis localized mainly at the that vandetanib targets with a high affinity [21]. The expression levels of RET were lower in the cutaneous SCC specimens than in normal epidermis (Fig. 4C and D). These results suggest that the main molecule targeted by vandetanib in cutaneous SCC cells is not RET but EGFR. 4. Discussion The present study demonstrates that vandetanib inhibits cell proliferation in cutaneous SCC and induces tumor cell death. Clin- ically, EGFR was found to be more highly expressed in SCC than in normal epidermis, whereas RET expression levels were lower in SCC. These results indicate that vandetanib is effective against cutaneous SCC probably via the inhibition of EGFR activation. To date, preclinical evidence has shown vandetanib to be a po- tential therapeutic candidate for head and neck SCC. For example, Gustafson et al. described vandetanib as eliciting antitumor effects by the inhibition of both EGFR and VEGFR signaling in a head and neck SCC xenograft model [13]. Sano et al. used a murine model to show that vandetanib treatment combined with cisplatin and ra- diation therapy prolongs overall survival and decreases lymph node metastases [14]. Clinically, Papadimitrakopoulou et al. demonstrated the feasibility of vandetanib therapy as a chemo- radiation therapy in a phase I study [15]. However, there have been no detailed studies analyzing the efficacy of vandetanib against cutaneous SCC; thus, the present study is the first preclin- ical data on vandetanib as a therapy for cutaneous SCC. EGFR is a transmembrane protein which transduces growth factor signaling from the extracellular milieu to the cell [22]. In some malignant neoplasms such as non-small cell lung cancer and colorectal cancer, EGFR is known as a key mediator of cell prolif- eration [23,24]. In cutaneous SCC, EGFR is overexpressed and correlated with clinical stages and poor prognosis [25]. Cetuximab, a monoclonal antibody to EGFR, has been already approved for the treatment of advanced mucosal head and neck SCC. Surgical treatment after neo-adjuvant cetuximab monotherapy has shown to improve the outcome of high-risk cutaneous SCC patients [26]. However, the efficacy of cetuximab against advanced cutaneous SCC is limited. A phase II clinical trial of cetuximab as a first-line treatment in unresectable cutaneous SCC patients reported that 58% of the patients achieved stable disease, only 8% had a partial response and 3% had complete remission [27]. Therefore, other options besides cetuximab should be considered in cutaneous SCC, especially in EGFR-overexpressing tumors. The present data sug- gest that vandetanib exerts anti-tumor effects via the inhibition of EGFR signaling; thus, vandetanib could be an option for advanced SCC with EGFR expression. However, vandetanib monotherapy has limited efficacy, as shown in Fig. 3B, where we see that vandetanib monotherapy can suppress tumor growth but not eliminate tu- mors. Further studies should be conducted, including attempts at combination therapy. In conclusion, we identified vandetanib as a novel therapeutic option against cutaneous SCC, especially in highly EGFR-expressing tumors. Declaration of competing interest The authors declare that we have no conflicts of interest. Acknowledgments This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grants Number #16K1970106 and #18K08259. Appendix A. Supplementary data Supplementary data to this article can be found online at References [1] G.W. Jung, A.I. Metelitsa, D.C. Dover, T.G. Salopek, Trends in incidence of nonmelanoma skin cancers in Alberta, Canada, 1988e2007, Br. J. Dermatol. 163 (2010) 146e154. [2] J. Ramos, J. Villa, A. Ruiz, R. Armstrong, J. Matta, UV dose determines key characteristics of nonmelanoma skin cancer, Cancer Epidemiol. Biomarkers Prev. 13 (2004) 2006e2011. [3] M.G. Kosmadaki, B.A. Gilchrest, The demographics of aging in the United States: implications for dermatology, Arch. Dermatol. 138 (2002) 1427e1428. [4] J.Y.S. Kim, J.H. 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