Inhibition of epithelial growth factor receptor can play an important role in reducing cell growth and survival in adrenocortical tumors

Biochemical Pharmacology 98 (2015) 639–648

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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Inhibition of epithelial growth factor receptor can play an important role in reducing cell growth and survival in adrenocortical tumors Teresa Gaglianoa , Erica Gentilina,b , Federico Tagliatia , Katiuscia Benfinia , Carmelina Di Pasqualea , Carlo Feoc , Simona Fallettaa , Eleonora Rivaa , Ettore degli Ubertia,b , Maria Chiara Zatellia,b,* a

Section of Endocrinology and Internal Medicine, Department of Medical Sciences, University of Ferrara, Ferrara, Italy Laboratorio in Rete del Tecnopolo Tecnologie delle Terapie Avanzate (LTTA), University of Ferrara, Italy c Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Via A. Moro 8, 44124 Ferrara, Italy b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 September 2015 Accepted 15 October 2015 Available online 17 October 2015

Medical treatment of adrenocortical carcinoma (ACC) is still far from optimal, since even molecular targeted therapy failed to demonstrate striking results. Clinical trials enrolling ACC patients with high tissue vascular endothelial growth factor receptor (VEGFR) expression levels showed controversial results after treatment with Sunitinib, possibly due to variability in the expression of drug targets, which include epidermal growth factor receptor (EGFR). To better clarify this issue, we evaluated whether VEGFR may play a crucial role in ACC responsiveness to Sunitinib and whether EGFR may represent an alternative target in ACC medical treatment, by employing two ACC cell lines, the NCI-H295 and SW13 cells lines, and adrenocortical tissues primary cultures. Our data show that VEGF/VEGFR system may not be crucial in modulating ACC proliferation and responsiveness to Sunitinib. In addition, by cell viability, proliferation and caspase activation assays we found that Sunitinib inhibits adrenocortical cell viability acting, at least in part, through EGFR, that, in turn, is crucial for EGF proliferative effect on adrenocortical cells. The latter depends, at least in part, on ERK 1/2 activation. An EGFR selective inhibitor was highly effective in reducing cell viability in an adrenocortical tumor primary culture and in the SW13 cells, which express high EGFR levels. Our results suggest that EGFR inhibitors could represent effective therapeutic tools in ACC patients whose tumors express high EGFR levels, that, in turn, may be considered a predictive factor of response. Accurate molecular tumor profiling is crucial to predict drug efficacy and to tailor ACC patients therapeutic approach. ã 2015 Elsevier Inc. All rights reserved.

Keywords: EGFR ACC Sunitinib Erlotinib

1. Introduction Adrenocortical carcinoma (ACC) is a rare malignancy with a general poor prognosis, despite extensive surgical approaches are employed. In addition, the disease is often metastatic at diagnosis and relapses very frequently, with consequent very low survival rates at 5 years [1,2]. The high ACC recurrence rates have prompted the use of adjuvant therapy by treatment with mitotane, an adrenolytic drug, that since late 60s has been employed in ACC medical treatment, demonstrating a significant increase in

* Corresponding author at: Section of Endocrinology and Internal Medicine, Department of Medical Sciences, University of Ferrara, Ferrara, Italy. Fax: +39 532236514. E-mail address: [email protected] (M.C. Zatelli). http://dx.doi.org/10.1016/j.bcp.2015.10.012 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.

recurrence free survival rates [3]. Multiple cytotoxic approaches have also been attempted, with limited results when used alone [2]. On the other hand, the combination of three chemotherapeutic agents (etoposide, doxorubicin and cisplatin) with mitotane provided important results on clinical grounds, with a significant improvement in 5-year survival rates [4]. However, this drug regimen has a high impact on patients’ quality of life, with a high burden in terms of side effects [2]. Therefore, it is mandatory to characterize novel factors regulating adrenocortical cell proliferation and transformation leading to the employment of new therapeutic agents capable of inhibiting tumor growth. Sunitinib is a receptor tyrosine kinase (RTK) inhibitor that targets multiple receptors, including vascular endothelial growth factor receptor (VEGFR) 1 and 2, as well as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor and

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others [5]. Sunitinib has been employed in clinical studies involving ACC [6], where patients were selected for treatment on the basis of high VEGFR expression levels [7–10]. The results of these studies are not clear-cut, since some patients displayed stable disease under treatment with Sunitinib, while others showed progressive disease. Therefore, treatment failure may be due to variability in drug target expression, indicating the need to better characterize the role of Sunitinib targets in ACC treatment [6]. Among others, EGFR plays a pivotal role in neoplastic transformation [11] and is over-expressed in the majority of ACC [12,13], suggesting a possible role for EGFR pathway in regulating adrenocortical cell proliferation and transformation. The aim of our study is to clarify whether VEGFR plays a crucial role in ACC responsiveness to Sunitinib and whether EGFR may represent an alternative target in ACC medical treatment.

96-well white plates, as previously described [18], and treated with the indicated compounds for 120 h. Control cells were treated with vehicle alone (0.1% DMSO). After incubation, the revealing solution was added and the luminescent output (relative luminescence units, RLU) was recorded using the Envision Multilabel Reader (PerkinElmer, Monza, Italy). Results are expressed as mean value  standard error of the mean (SEM) percent relative light units (RLU) vs. vehicle-treated control cells from 3 independent experiments in 6 replicates. 2.4. Caspase activity evaluation Caspase activity was measured using Caspase-Glo 3/7 assay (Promega, Milano, Italy), as previously described [19]. Results are expressed as mean value  SEM percent RLU vs. vehicle-treated control cells from three independent experiments in six replicates.

2. Material and methods 2.5. Measurement of DNA synthesis 2.1. Compounds Sunitinib malate was kindly provided by Pfizer Inc. (New York, NY, USA). Erlotinib and SCH772984 were purchased from Selleckchem (Boston, MA, USA). All other reagents, including EGF and VEGF, if not otherwise specified, were purchased from Sigma.

DNA synthesis was assed by evaluating the incorporation of 5bromo-2-deoxyuridine (BrdU) into DNA using the BrdU Cell Proliferation Assay kit (Cell Signaling Technology, Danvers, MA) as previously described [20]. 2.6. Western blot analysis

2.2. Cell culture and tissues The NCI-H295 and the SW-13 human ACC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). NCI-H295 and SW-13 cells were maintained as previously described [14,15]. Primary cultures from human adrenocortical tissues were obtained as previously described, with minor modifications [16]. Briefly, tissue samples were collected in accordance with the guidelines of the local committee on human research, obtained under sterile conditions, and immediately minced in RPMI 1640 medium. Tissues were dissociated using 0.35% collagenase and 1% trypsin at 37
C for 60 min. Cell suspensions were washed twice with serum-free RPMI (Euroclone Ltd., Wetherby, UK), and then passed through 18 gauge and then 20 gauge syringe needles. Tumor cells were resuspended in F-12 medium with 10% FBS and antibiotics (Euroclone Ltd., Wetherby, UK), seeded in 96-well culture plates (2  104 cells/well) and incubated at 37
C in a humidified atmosphere of 5% CO2 and 95% air, as previously described [17]. After 18 h, cells were treated with test substances, with further evaluation of cell viability and/or caspase activity. 2.3. Viable cell number assessment Variations in viable cell number were assessed by the ATPlite kit (PerkinElmer Life Sciences, MA, USA), seeding 2  104 cells/well in

For immunoblotting, cells and tissues were dissolved in RIPA Buffer (Thermolab Inc., Waltham, MA, USA), as previously described [18]. Protein concentration was measured by BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA), as previously described [21]. Proteins were fractionated on 7.5% SDS-PAGE, as previously described [22], and transferred by electrophoresis to Nitrocellulose Transfer Membrane (PROTRAN, Dassel, Germany). Membranes were incubated with anti-p44/42 MAPK (ERK 1/2), anti-phospho-p44/42 MAPK (ERK 1/2) (Thr202/Thy 204), antiEGFR, (Cell Signaling, Danvers, MA, USA). Horseradish peroxidaseconjugated antibody IgG (Dako, Cernusco sul Naviglio, Milan, Italy) was used to detect immunoreactive bands and binding was revealed using enhanced chemiluminescence (Pierce, Rockford, IL, USA). The blots were then stripped and used for further blotting with anti-GAPDH antibody (Cell Signaling, Danvers, MA, USA). Quantification of the bands was performed by the Quantity One software (Bio-Rad, Milano, Italy). 2.7. Kinase activity assay Phosphorylated levels of EGFR (Tyr1068) were measured using AlphaScreen SureFire assays (PerkinElmer). Briefly, cells were seeded at 2  104 cells/well in 96-well plates and, after overnight attachment, were incubated for 1 h with or without Sunitinib;

Table 1 PCR primers and conditions for EGFR exons 18–21 amplification. Exon

Primers

Amplicon

Cycles

Denaturation

Annealing

Extension

18

For: 50 -GTGAGGGCTGAGGTGACCCTTGTCTC-30 Rev: 50 -CAGTGGTCCTGTGAGACCAA-30 For: 50 -GCAGCATGTGGCACCATCTCACAATTGCC-30 Rev: 50 -TCTAGACCCTGCTCATCTCCACATCC-30 For: 50 -GTCTTCACCTGGAAGGGGT-30 Rev: 50 -GGAGGGGAGATAAGGAGCCAGGA-30 For: 50 -CTCAGA GCCTGGCATGAA-30 Rev: 50 -GGCAAAGTAAGGAGGTGGCT-30

368 bp

35

265 bp

35

314 bp

35

255 bp

35

94
C 3000 94
C 3000 94
C 3000 94
C 3000

58
C 10 57
C 10 56
C 10 56
C 10

72
C 3000 72
C 3000 72
C 3000 72
C 3000

19 20 21

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AlphaScreen SureFire assays were performed as previously described [18]. 2.8. Genetic analysis DNA was isolated from cell lines and tissues by using the QIAamp DNA Micro Kit (QIAGEN, Milano, Italy) on the QIAcube automated system (QIAGEN). At least 100 ng of DNA were used for each application. Polymerase Chain Reactions for EGFR (GenBank accession no NG_007726) exons 18–21 were performed as described in Table 1 by using the Invitrogen Taq DNA polymerase following the manufacturer’s instructions, with the Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The amplified products were purified with the QiaQuick PCR purification kit (QIAGEN); sequencing reactions were performed by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems), applying the following cycle profile: 96
C for 10 s and 60
C for 4 min (45 cycles). The samples were then purified as described previously [23,24]. Direct DNA sequencing of the EGFR from exon 18 to exon 21 using sequence-specific primers (see Table 1) was performed as described previously [23,24].

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both in NCI-H295 and SW13 cells. On the contrary, treatment with Sunitinib significantly induced this parameter in both NCI-H295 (+29%; P < 0.01 vs. untreated cells) and SW13 cells (+25%; P < 0.01 vs. untreated cells). Treatment with VEGF did not affect Sunitinib action in both cell lines. In order to explore whether the effects exerted by Sunitinib could affect DNA synthesis, NCI-H295 and SW13 cells were treated

2.9. Statistical analysis Results are expressed as the mean  SEM. Statistical analyses were carried out using ANOVA after proof of homogeneity of variances and normality tests. Data were analyzed using GraphPad (Prism v-5.0); P values <0.05 were considered significant. 3. Results 3.1. Effects of VEGF and Sunitinib on ACC cell lines viability, caspase activation and DNA synthesis In order to explore whether the effects exerted by Sunitinib on ACC cells might be mediated by VEGFR inhibition, NCI-H295 and SW13 cells were treated without or with VEGF 1 mM, alone or in combination with Sunitinib 2.5 mM. VEGF concentration was chosen on the basis of previous published studies [25]. Table 2A shows Sunitinib IC50 for cell viability in both cell lines, as obtained in preliminary experiments. As shown in Fig. 1A, treatment with VEGF did not affect viability of both NCI-H295 and SW13 cells. On the contrary, treatment with Sunitinib significantly reduced viability both in NCI-H295 (22%; P < 0.01 vs. untreated cells) and in SW13 cells (24%; P < 0.01 vs. untreated cells). VEGF did not affect Sunitinib action in both cell lines. These results were mirrored by those obtained with caspase 3/7 assay. Table 2A shows Sunitinib IC50 for caspase activation in both cell lines, as obtained in preliminary experiments. As shown in Fig. 1B, treatment with VEGF did not affect basal caspase activity Table 2 IC50 of Sunitinib (A) and Erlotinib (B) on cell viability, caspase activation, BrdU in corporation in NCI-H295 and SW13 cells. A IC50 Sunitinib (mM)

NCI-H295

SW13

Viability Caspase activation BrdU incorporation

3.3 2.8 20.2

3.0 3.0 9.3

IC50 Erlotinib (mM)

NCI-H295

SW13

Viability Caspase activation BrdU incorporation

24.7 24.6 147

2.2 2.5 8

B

Fig. 1. Effects of VEGF and Sunitinib on ACC cell lines viability, caspase activation and DNA synthesis. (A) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with VEGF 1 mM and/or Sunitinib 2.5 mM. Cell viability was measured by ATPlite assay after 120 h. **P < 0.01 vs. untreated cells. (B) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with VEGF 1 mM and/or Sunitinib 2.5 mM. Apoptosis activation was measured by Caspase 3/7 activity assay after 120 h. *P < 0.05 vs. untreated cells. (C) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with VEGF 1 mM and/or Sunitinib 5 mM. DNA synthesis was measured by BrdU incorporation assay after 120 h. **P < 0.01 vs. untreated cells; (D) VEGFR expression in NCI-H295 and SW13 was evaluated by Western blot, with GAPDH as loading control.

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without or with VEGF 1 mM alone or in combination with Sunitinib 5 mM. Table 2A shows Sunitinib IC50 for DNA synthesis inhibition, measured as variations in BrdU incorporation, in both cell lines, as obtained in preliminary experiments. As shown in Fig. 1C, treatment with VEGF did not affect BrdU incorporation in both NCI-H295 and SW13 cells. Treatment with Sunitinib did not significantly affect DNA synthesis in NCI-H295, while a significant reduction was observed in SW13 cells (46%; P < 0.01 vs. untreated cells). Treatment with VEGF did not affect Sunitinib action in both cell lines. We then evaluated VEGFR expression levels in NCI-H295 and SW13 cells and found that NCI-H295 cells express higher VEGFR levels as compared with SW13 cells (Fig. 1D). These data indicate that VEGF is not capable of rescuing NCIH295 and SW13 cells from the antiproliferative and pro-apoptotic effects of Sunitinib, suggesting that Sunitinib exerts antiproliferative effects on ACC cell lines independently of VEGFR.

untreated cells). Erlotinib did not modify NCI-H295 cell viability, while it significantly reduced this parameter in SW13 (47%; P < 0.01 vs. untreated cells). Furthermore, Erlotinib was capable of completely blocking the stimulatory effects of EGF on SW13 cell viability. These results were mirrored by those obtained with caspase 3/7 assay. Table 2B shows Erlotinib IC50 for caspase activation in both

3.2. Effects of EGF and Sunitinib on ACC cell lines viability, caspase activation and DNA synthesis To explore whether EGFR could mediate the effects of Sunitinib on ACC cells, NCI-H295 and SW13 cells were treated without or with EGF 30 nM, alone or in combination with Sunitinib 2.5 mM. EGF concentration was chosen on the basis of previous published studies [26]. As shown in Fig. 2A, treatment with EGF did not affect viability of NCI-H295 cells, while it significantly induced SW13 cell viability (+19%; P < 0.01 vs. untreated cells). On the contrary, as described above, treatment with Sunitinib significantly reduced viability in both NCI-H295 (22%; P < 0.01 vs. untreated cells) and SW13 cells (24%; P < 0.01 vs. untreated cells). Furthermore, Sunitinib was capable of completely blocking the stimulatory effects of EGF on SW13 cell viability. These results were mirrored by those obtained with caspase 3/7 assay. As shown in Fig. 2B, treatment with EGF did not affect basal apoptosis in NCI-H295 cells, while it significantly reduced this phenomenon in SW13 cells (34%; P < 0.01 vs. untreated cells). On the contrary, treatment with Sunitinib significantly induced caspase activation in both NCI-H295 (+29%; P < 0.01 vs. untreated cells) and SW13 cells (+25%; P < 0.01 vs. untreated cells). Furthermore, Sunitinib was capable of completely blocking the inhibitory effects of EGF on caspase activation in SW13 cells. In order to explore whether EGF could influence DNA synthesis, BrdU incorporation was measured. As shown in Fig. 2C, treatment with EGF did not affect DNA synthesis in NCI-H295 cells, while it significantly increased this parameter in SW13 cells (+80%; P < 0.01 vs. untreated cells). Treatment with Sunitinib did not significantly affect DNA synthesis in NCI-H295, while a significant reduction was observed in SW13 cells (46%; P < 0.01 vs. untreated cells). Moreover, Sunitinib was capable of completely blocking the effects of EGF on DNA synthesis in SW13 cells. We then evaluated EGFR expression levels in NCI-H295 and SW13 cells. As shown in Fig. 2D, EGFR protein levels were higher in SW13 as compared to NCI-H295 cells. 3.3. Effects of an EGFR inhibitor on ACC cell lines viability, apoptosis and DNA synthesis In order to evaluate whether a selective EGFR inhibitor, such as Erlotinib, may affect ACC cell proliferation, NCI-H295 and SW13 cells were treated without or with EGF 30 nM alone or in combination with Erlotinib 2.5 mM. Table 2B shows Erlotinib IC50 for cell viability in both cell lines, as obtained in preliminary experiments. As shown in Fig. 3A, treatment with EGF did not affect NCI-H295 cell viability, while it significantly induced this parameter in SW13, as previously described (+28%; P < 0.01 vs.

Fig. 2. Effects of EGF and Sunitinib on ACC cell lines viability, caspase activation and DNA synthesis. (A) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and /or Sunitinib 2.5 mM. Cell viability was measured by ATPlite assay after 120 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (B) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and /or Sunitinib 2.5 mM. Apoptosis activation was measured by Caspase 3/7 activity assay after 120 h. *P < 0.05 and **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (C) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or Sunitinib 5 mM. DNA synthesis was measured by BrdU incorporation assay after 120 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (D) EGFR expression in NCI-H295 and SW13 was evaluated by Western blot, with GAPDH as loading control.

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cell lines, as obtained in preliminary experiments. As shown in Fig. 3B, treatment with EGF did not affect basal apoptosis in NCIH295 cells, while it significantly reduced this phenomenon in SW13 cells (32%; P < 0.01 vs. untreated cells). Erlotinib did not modify NCI-H295 basal apoptosis, while it significantly induced caspase activation in SW13 cells (+170%; P < 0.01 vs. untreated cells). Furthermore, Erlotinib was capable of completely blocking the inhibitory effects of EGF on caspase activation in SW13 cells. In order to explore whether the effects exerted by Erlotinib could affect DNA synthesis, NCI-H295 and SW13 cells were treated without or with EGF 30 nM alone or in combination with Erlotinib 5 mM. Table 2B shows Erlotinib IC50 for DNA synthesis inhibition, measured as variations in BrdU incorporation, in both cell lines, as obtained in preliminary experiments. As shown in Fig. 3C, EGF did

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not affect DNA synthesis in NCI-H295 cells, while it significantly induced this parameter in SW13 cells (+80%; P < 0.01 vs. untreated cells). Treatment with Erlotinib did not affect DNA synthesis in NCI-H295 cells, while it significantly reduced this parameter in SW13 cells (38%; P < 0.01 vs. untreated cells). Moreover, Erlotinib was capable of completely blocking the effects of EGF on DNA synthesis in SW13 cells. 3.4. EGFR and ERK phosphorylation To explore whether EGFR activation is involved in the effects of Sunitinib on ACC cells, NCI-H295 and SW13 cells were treated without or with EGF 30 nM, alone or in combination with Sunitinib 5 mM for 1 h. Then, EGFR phosphorylation (pEGFR) was assessed as described in Section 2. We found that pEGFR basal levels in SW13 cells were 2-fold higher as compared to NCI-H295 cells. As shown in Fig. 4A, treatment with EGF did not significantly affect basal pEGFR in both cell lines. Treatment with Sunitinib did not affect pEGFR in NCI-H295 cells, while it significantly reduced pEGFR in SW13 cells (37%; P < 0.05 vs. control), an effect completely counteracted by co-treatment with EGF. We then evaluated ERK phosphorylation (pERK), which is activated by EGFR downstream signaling. As shown in Fig. 4B and C, treatment with EGF markedly induced pERK in both cells lines, while Sunitinib did not affect this parameter. However, the latter was capable of counteracting the stimulatory effects of EGF on pERK in SW13 cells. To explore whether EGFR activation is involved in the effects of Erlotinib on ACC cells, NCI-H295 and SW13 cells were treated without or with EGF 30 nM, alone or in combination with Erlotinib 2.5 mM for 1 h. Then, pEGFR was assessed as described in Section 2. As shown in Fig. 4D, treatment with EGF did not significantly induce basal pEGFR in both cell lines. Treatment with Erlotinib did not affect pEGFR in NCI-H295 cells, while it significantly reduced pEGFR in SW13 cells (36%; P < 0.05 vs. control), an effect counteracted by co-treatment with EGF. We then evaluated pERK. As shown in Fig. 4E and F, treatment with EGF induced pERK in both cells lines. Erlotinib did not affect this parameter in NCI-H295 cells, while in SW13 cells Erlotinib reduced pERK levels and counteracted the stimulatory effects of EGF. 3.5. Effects of an ERK inhibitor on ACC cell lines viability In order to evaluate whether ERK activation may mediate EGFinduced proliferative effects, we assessed the effects of an ERK inhibitor, SCH772984 [27], on NCI-H295 and SW13 cell viability in the absence or in the presence of EGF 30 nM. As shown in Fig. 4G, in keeping with previous results, NCI-H295 cell viability was not affected by treatment with EGF, but was significantly reduced by treatment with SCH772984 10 nM, alone or in combination with EGF. On the contrary, SW13 cell viability was induced by EGF, as already reported, while it was not significantly affected by treatment with SCH772984 10 nM. However, the latter completely blocked the proliferative effects of EGF.

Fig. 3. Effects of an EGFR inhibitor on ACC cell lines viability, apoptosis and DNA synthesis. (A) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or Erlotinib 2.5 mM. Cell viability was measured by ATPlite assay after 120 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (B) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or Sunitinib 2.5 mM. Apoptosis activation was measured by Caspase 3/7 activity assay after 120 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (C) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or Erlotinib 5 mM. DNA synthesis was measured by BrdU incorporation assay after 120 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF.

3.6. Effects of Sunitinib and Erlotinib in an adrenocortical tissues primary culture We had the opportunity to evaluate cell viability and caspase 3/ 7 activation in an adrenocortical tumor primary culture and its normal counterpart under Sunitinib or Erlotinib treatment. As shown in Fig. 5A, EGF did not affect cell viability in the normal counterpart, while it induced a significant increase in this parameter in the neoplastic tissue sample (+50%; P < 0.05 vs. untreated cells). Treatment with Sunitinib did not significantly

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decrease cell viability in the normal counterpart, while it significantly reduced this parameter in the neoplastic tissue sample (41%; P < 0.01 vs. untreated cells). Furthermore, Sunitinib was capable of completely blocking the stimulatory effects of EGF on neoplastic tissue cell viability. These results were mirrored by those obtained with the caspase 3/7 assay. As shown in Fig. 5B, EGF

did not affect apoptosis activation in the normal counterpart, while it significantly reduced this phenomenon in the neoplastic tissue sample (60%; P < 0.01 vs. untreated cells). Treatment with Sunitinib induced caspase activation in both the normal counterpart and in the neoplastic tissue sample (+70% and +500%; P < 0.01 vs. untreated cells). Furthermore, Sunitinib was capable of

Fig. 4. EGFR and ERK1/2 phosphorylation analysis and effects of an ERK inhibitor on ACC cell lines viability. (A) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or Sunitinib 5 mM for 1 h; EGFR phosphorylation was than measured by Alphascreen sure fire assay. *P < 0.05 vs. untreated cells; x P < 0.05 vs. cells treated with Sunitinib. (B) e (C) Western blot analysis for phosphorylated (p-ERK1/2) and total ERK 1/2 (ERK 1/2) in NCI-H295 (B) and SW13 (C) cells after 48 h treatment without or with EGF 30 nM and/or Sunitinib 5 mM, with GAPDH as loading control. (D) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or Erlotinib 2.5 mM for 1 h; EGFR phosphorylation was than measured by Alphascreen sure fire assay. *P < 0.05 vs. untreated cells. (E) e (F) Western blot analysis for phosphorylated (p-ERK1/2) and total ERK 1/2 (ERK 1/2) in NCI-H295 (E) and SW13 (F) cells after 48 h treatment without or with EGF 30 nM and/or Erlotinib 2.5 mM, with GAPDH as loading control. (G) NCI-H295 (black bars) and SW13 (white bars) cells were treated without or with EGF 30 nM and/or SCH772984 10 nM for 120 h. Cell viability was measured by ATPlite assay. *P < 0.05 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF.

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completely blocking the inhibitory effects of EGF on caspase 3/7 activity in the neoplastic tissue sample. As shown in Fig. 5C, EGF did not affect cell viability in the normal counterpart, while it induced a significant increase in this parameter in the neoplastic tissue sample, as already described (+50%; P < 0.05 vs. untreated cells). Treatment with Erlotinib did not affect cell viability in the normal counterpart, while it significantly reduced this parameter in the neoplastic tissue

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sample (40%; P < 0.01 vs. untreated cells). Furthermore, Erlotinib was capable of completely blocking the stimulatory effects of EGF on neoplastic tissue cell viability. These results were mirrored by those obtained with the caspase 3/7 assay. As shown in Fig. 5D, EGF did not affect apoptosis activation in the normal counterpart, while it significantly reduced this phenomenon in the neoplastic tissue sample, as already described (60%; P < 0.01 vs. untreated cells). Treatment with Erlotinib did not affect caspase activation in the

Fig. 5. Effects of Sunitinib and Erlotinib on adrenocortical tissues primary cultures. (A) Adrenocortical tumor primary culture (striped bars) and its normal counterpart (grey bars) were incubated without or with EGF 30 nM and/or Sunitinib 5 mM. Cell viability was measured by ATPlite assay after 96 h. *P < 0.05 and **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (B) Adrenocortical tumor primary culture (striped bars) and its normal counterpart (grey bars) were incubated without or with EGF 30 nM and/or Sunitinib 5 mM. Apoptosis activation was measured by Caspase 3/7 activity assay after 96 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. EGF treated cells. (C) Adrenocortical tumor primary culture (striped bars) and its normal counterpart (grey bars) were incubated without or with EGF 30 nM and/or Erlotinib 2.5 mM. Cell viability was measured by ATPlite assay after 96 h. *P < 0.05 and **P < 0.01 vs. untreated cells; $ P < 0.05 vs. cells treated with EGF. (D) Adrenocortical tumor primary culture (striped bars) and its normal counterpart (grey bars) were incubated without or with EGF 30 nM and/or Erlotinib 2.5 mM. Apoptosis activation was measured by Caspase 3/7 activity assay after 96 h. **P < 0.01 vs. untreated cells; $ P < 0.05 vs. EGF treated cells. (E) EGFR expression in adrenocortical tumor (T) and its normal counterpart (N) evaluated by Western blot, with GAPDH as loading control. The bars represent quantification of EGFR bands normalized against corresponding GAPDH bands.

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normal counterpart, while it significantly induced this parameter in the neoplastic tissue sample (+350%; P < 0.01 vs. untreated cells). Furthermore, Erlotinib was capable of completely blocking the inhibitory effects of EGF on caspase 3/7 activity in the neoplastic tissue sample. We then evaluated EGFR expression levels the adrenocortical tumor and its normal counterpart. As shown in Fig. 5E, EGFR protein levels were slightly higher in the adrenocortical tumor as compared to its normal counterpart. 3.7. EGFR gene (exons 18–21) sequencing results In order to verify whether the different responses to EGF were due to EGFR mutations, we assessed EGFR gene mutational status (exons 18–21, corresponding to the tyrosine kinase catalytic domain) in both cell lines, as well as in the adrenocortical tumor tissue and its normal counterpart. We did not identify any mutation in all the analyzed samples, that were found to display a wild type sequence in the catalytic domain of EGFR (data not shown). 4. Discussion We here show that VEGFR may not play a crucial role in ACC responsiveness to Sunitinib and that EGFR may represent an alternative target in ACC medical treatment. Indeed, our data demonstrate that VEGF does not affect ACC cell lines proliferation and apoptosis, which are influenced by Sunitinib, independently of VEGFR expression. Therefore, VEGF/ VEGFR system does not seem to play a crucial role in ACC cell line models and in transducing Sunitinib effects. In addition, our results suggest that VEGFR expression may not represent a reliable predictor of the efficacy of Sunitinib, since the latter was capable of reducing proliferation also in cells lacking VEGR expression. Our data may in part explain the uncertain and controversial results obtained in clinical trials where patients were selected for Sunitinib treatment on the basis of high VEGFR expression levels [8,28,29,10]. Sunitinib was indeed described and employed as a VEGFR inhibitor [30–32], but this activity is unlikely to be successful in ACC, since a study employing a selective VEGFR inhibitor, Axitinib, failed to achieve significant results in terms of tumor response [33]. In addition, Bevacizumab, an anti-VEGFR antibody, in combination with capecitabine, failed to demonstrate any objective response or stable disease in very advanced ACC patients [34]. Furthermore, clinical studies employing Sorafenib, which also acts on VEGFR, failed to demonstrate an objective response in ACC [35]. These studies indicate that VEGFR inhibition might not be a successful strategy to control adrenocortical cell proliferation, ruling out this receptor from a possible role as a useful molecular target for ACC medical therapy. In addition, these results indicate that other targets should be explored in order to identify possible active agents in ACC. Since Sunitinib may also act through EGFR inhibition, we explored the EGF/EGFR system in our experimental model and found that the capability of EGF to modulate adrenocortical cell proliferation depends on EGFR expression. However, our data also show that, in this system, Sunitinib inhibits cell proliferation independently of EGFR expression, indicating that this drug exerts its antiproliferative effects also by interacting with RTKs different from both EGFR and VEGFR, such as PDGFR or IGF-I R. This hypothesis, which is beyond the aim of our study, remains to be tested. In order to better characterise the role of EGFR in regulating ACC cell line proliferation, we employed Erlotinib, a selective EGFR inhibitor [36]. This drug, which we used at a concentration in the lower range of the therapeutic plasma levels (1.7–6.4 mM) [37], was effective in reducing cell viability in SW13 cells that express

higher EGFR levels as compared to NCI-H295 cells, where Erlotinib was ineffective, demonstrating a selective drug activity. In a clinical trial enrolling highly pre-treated ACC patients, Erlotinib plus gemcitabine obtained a tumor response associated with a prolonged progression free survival in one patient [38]. These data support our hypothesis that selective EGFR inhibition may represent a successful strategy to inhibit adrenocortical cell proliferation, deserving further investigation on clinical grounds. Indeed, both Sunitinib and Erlotinib antiproliferative effects in our model are mirrored by a reduction in EGFR phosphorylation, which is restored by co-treatment with EGF. This evidence demonstrates that both drugs act, at least in part, though EGFR. On the other hand, the lack of EGFR phosphorylation induction by EGF alone may be due to a sustained basal EGFR activation, especially in SW13 cells. The different sensitivity to EGFR inhibitors in the two ACC cell lines may be due to the different EGFR expression and phosphorylation levels, rather than to receptor genetic alterations. This hypothesis is supported by the evidence that the coding sequence of EGFR catalytic domain in the analyzed cell lines and tissues did not display any mutation that may cause an intrinsic EGFR activity. However, we cannot exclude the presence of micro/ macro deletions or copy number variations, that could explain differences in EGFR expression levels and function. In addition, the differences in responses to EGFR inhibitors could be due to different EGFR-dependent signaling in the employed cell lines. In this respect, our data are in keeping with previous evidence showing that EGF stimulates ERK1/2 phosphorylation [39]. In our model, pERK induction is independent of EGFR expression, since we observed this phenomenon in both cell lines, suggesting that EGF may act also thorough other EGF receptor family members, such as Erbb3 and Erbb4. In addition, our results show that Sunitinib does not affect pERK, on the contrary of what previously reported by Lin et al. [40]. These authors show pERK induction after treatment of NCI-H295 and SW13 cells with Sunitinib concentrations much lower as compared to those we employed, that, in turn, are in keeping with those employed in previous studies demonstrating the antiproliferative effects of Sunitinib [41]. In addition, we found that Erlotinib reduces pERK only in ACC cells expressing high EGFR levels. These data confirm Erlotinib selectivity for EGFR and suggest that ERK activation may be important to tranduce the antiproliferative effects of EGFR blockade. This hypothesis is confirmed by the evidence that an ERK inhibitor completely blocked the proliferative effects of EGF in SW13 cells. However, basal ERK activity does not seem to impact on SW13 cell survival, since a specific ERK inhibitor does not affect basal SW13 cell proliferation. On the contrary, we found that ERK activity is important for NCI-H295 cell survival, since a specific ERK inhibitor hampers cell proliferation, which is not rescued by EGF. The results obtained in the adrenocortical tumor primary culture partially recapitulate what observed in the two cell lines. Indeed, EGF induces cell viability and reduces basal apoptosis of the tumor sample, which expresses higher EGFR levels as compared to the normal counterpart. Both Sunitinib and Erlotinib block these effects, supporting the hypothesis that these actions are mediated by EGFR activation. On the contrary, EGF does not affect cell viability and basal apoptosis in the normal counterpart, where Erlotinib does not exert any antiproliferative or proapoptotic effect. Therefore, these data suggest that EGFR expression levels may drive sensitivity to EGFR inhibitors and may represent a putative predictive marker for medical treatment efficacy in ACC. In addition, these data underline once again the importance of an accurate characterization of the tumor tissue, in order to allow the best drug selection for each patient, in the perspective of a tailored medical therapy. Future trials with Erlotinib enrolling ACC patients whose tumors express high EGFR levels are necessary to validate these results.

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In conclusion, our results show that VEGF/VEGFR system may not be crucial in modulating ACC proliferation and responsiveness to Sunitinib and that specific EGFR inhibition may represent an alternative medical approach in selected ACC patients. In addition we found that the ERK pathway mediates, at least in part, EGF effects in the ACC cell line that expresses EGFR. Our data suggest that the best therapeutic strategy for ACC patients should be based on an accurate molecular profiling of the patient’s tumor. Ideally, this approach would allow to predict the efficacy of a specific drug on the basis the expression of its molecular target and related pathways, which may represent predictive factors of response. Acknowledgments This work was supported by grants from the Italian Ministry of Education, Research and University (FIRB RBAP11884M, RBAP1153LS, 2010TYCL9B_002), Fondazione Cassa di Risparmio di Ferrara, and Associazione Italiana per la Ricerca sul Cancro (AIRC) in collaboration with Laboratorio in rete del Tecnopolo “Tecnologie delle terapie avanzate” (LTTA) of the University of Ferrara. We thank Pfizer Inc. for providing Sunitinib. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bcp.2015.10.012. References [1] I. Veytsman, L. Nieman, T. Fojo, Management of endocrine manifestations and the use of mitotane as a chemotherapeutic agent for adrenocortical carcinoma, J. Clin. Oncol. 27 (27) (2009) 4619–4629, doi:http://dx.doi.org/10.1200/ JCO.2008.17.2775. [2] M. Terzolo, F. Daffara, A. Ardito, B. Zaggia, V. Basile, L. Ferrari, A. Berruti, Management of adrenal cancer: a 2013 update, J. Endocrinol. Invest. 37 (3) (2014) 207–217. [3] M. Terzolo, A. Angeli, M. Fassnacht, F. Daffara, L. Tauchmanova, P.A. Conton, R. Rossetto, L. Buci, P. Sperone, E. Grossrubatscher, G. Reimondo, E. Bollito, M. Papotti, W. Saeger, S. Hahner, A.C. Koschker, E. Arvat, B. Ambrosi, P. Loli, G. Lombardi, M. Mannelli, P. Bruzzi, F. Mantero, B. Allolio, L. Dogliotti, A. Berruti, Adjuvant mitotane treatment for adrenocortical carcinoma, N. Engl. J. Med. 356 (23) (2007) 2372–2380. [4] M. Fassnacht, M. Terzolo, B. Allolio, FIRM-ACT Study Group, Combination chemotherapy in advanced adrenocortical carcinoma, N. Engl. J. Med. 366 (23) (2012) 2189–2197. [5] M. Fassnacht, M.C. Kreissl, D. Weismann, B. Allolio, New targets and therapeutic approaches for endocrine malignancies, Pharmacol. Ther. 123 (1) (2009) 117–141, doi:http://dx.doi.org/10.1016/j.pharmthera.2009.03.013. [6] M. Kroiss, M. Quinkler, S. Johanssen, E. van, N.P. rp, N. Lankheet, A. Pöllinger, K. Laubner, C.J. Strasburger, S. Hahner, H.H. Müller, B. Allolio, M. Fassnacht, Sunitinib in refractory adrenocortical carcinoma: a phase II, single-arm, openlabel trial, J. Clin. Endocrinol. Metab. 97 (Octoner (10)) (2012) 3495–3503, doi: http://dx.doi.org/10.1210/jc.2012-1419. [7] A.W. Griffioen, L.A. Mans, A.M. de Graaf, P. Nowak-Sliwinska, C.L. de Hoog, T.A. de Jong, F.A. Vyth-Dreese, J.R. van Beijnum, A. Bex, E. Jonasch, Rapid angiogenesis onset after discontinuation of sunitinib treatment of renal cell carcinoma patients, Clin. Cancer Res. 18 (14) (2012) 3961–3971, doi:http://dx. doi.org/10.1158/1078-0432.CCR-12-0002. [8] A. Teulé, O. Casanovas, Relevance of angiogenesis in neuroendocrine tumors, Target. Oncol. 7 (2) (2012) 93–98, doi:http://dx.doi.org/10.1007/s11523-0120217-x. [9] A.F. Okines, A.R. Reynolds, D. Cunningham, Targeting angiogenesis in esophagogastric adenocarcinoma, Oncologist 16 (6) (2011) 844–858, doi: http://dx.doi.org/10.1634/theoncologist.2010-0387. [10] D.J. George, Phase 2 studies of sunitinib and AG013736 in patients with cytokine-refractory renal cell carcinoma, Clin. Cancer Res. 13 (2 Suppl) (2007) 753s–757s. [11] G.S. Omenn, Y. Guan, R. Menon, A new class of protein cancer biomarker candidates: differentially expressed splice variants of ERBB2 (HER2/neu) and ERBB1 (EGFR) in breast cancer cell lines, J. Proteomics 0 (2014) 103–112, doi: http://dx.doi.org/10.1016/j.jprot.2014.04.012 pii: S1874-3919(14) 00179-1. [12] V. Kotoula, E. Sozopoulos, H. Litsiou, G. Fanourakis, T. Koletsa, G. Voutsinas, S. Tseleni-Balafouta, C.S. Mitsiades, A. Wellmann, N. Mitsiades, Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas, Endocr. Relat. Cancer 16 (2) (2009) 565–572, doi:http://dx.doi. org/10.1677/erc-08-0101.

647

[13] M. Nakamura, Y. Miki, J. Akahira, R. Morimoto, F. Satoh, S. Ishidoya, Y. Arai, T. Suzuki, Y. Hayashi, H. Sasano, An analysis of potential surrogate markers of target-specific therapy in archival materials of adrenocortical carcinoma, Endocr. Pathol. 20 (1) (2009) 17–23, doi:http://dx.doi.org/10.1007/s12022009-9058-2. [14] T. Gagliano, E. Gentilin, K. Benfini, C. Di Pasquale, M. Tassinari, S. Falletta, C. Feo, F. Tagliati, E. degli Uberti, M.C. Zatelli, Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells, Endocrine 47 (3) (2014) 943–951, doi:http://dx.doi.org/10.1007/s12020-0140374-z. [15] M.C. Zatelli, R. Rossi, E.C. degli Uberti, Androgen influences transforming growth factor-beta1 gene expression in human adrenocortical cells, J. Clin. Endocrinol. Metab. 85 (2) (2000) 847–852. [16] M.C. Zatelli, D. Piccin, F. Tagliati, A. Bottoni, A. Luchin, C. Vignali, A. Margutti, M. Bondanelli, G.C. Pansini, M.R. Pelizzo, M.D. Culler, E.C. degli Uberti, Selective activation of somatostatin receptor subtypes differentially modulates secretion and viability in human medullary thyroid carcinoma primary cultures: potential clinical perspectives, J. Clin. Endocrinol. Metab. 91 (6) (2006) 2218–2224. [17] M.C. Zatelli, M. Minoia, C. Martini, F. Tagliati, M.R. Ambrosio, M. Schiavon, M. Buratto, F. Calabrese, E. Gentilin, G. Cavallesco, L. Berdondini, F. Rea, E.C. degli Uberti, Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids, Endocr. Relat. Cancer 17 (3) (2010) 719–729, doi: http://dx.doi.org/10.1677/ERC-10-0097. [18] T. Gagliano, M. Bellio, E. Gentilin, D. Molè, F. Tagliati, M. Schiavon, N.G. Cavallesco, L.G. Andriolo, M.R. Ambrosio, F. Rea, E. degli Uberti, M.C. Zatelli, mTOR, p70S6K, AKT, and ERK1/2 levels predict sensitivity to mTOR and PI3K/ mTOR inhibitors in human bronchial carcinoids, Endocr. Relat. Cancer 20 (4) (2013) 463–475, doi:http://dx.doi.org/10.1530/ERC-13-0042. [19] M.C. Zatelli, E. Gentilin, F. Daffara, F. Tagliati, G. Reimondo, G. Carandina, M.R. Ambrosio, M. Terzolo, E.C. degli Uberti, Therapeutic concentrations of mitotane (o,p0 -DDD) inhibit thyrotroph cell viability and TSH expression and secretion in a mouse cell line model, Endocrinology 151 (6) (2010) 2453–2461, doi:http://dx.doi.org/10.1210/en.2009-1404. [20] D. Molè, E. Gentilin, A. Ibañez-Costa, T. Gagliano, M.D. Gahete, F. Tagliati, R. Rossi, M.R. Pelizzo, G. Pansini, R.M. Luque, J.P. Castaño, E. degli Uberti, M.C. Zatelli, The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells, Endocrine (2015) 1–11 [Epub ahead of print]. [21] F. Tagliati, M.C. Zatelli, A. Bottoni, D. Piccin, A. Luchin, M.D. Culler, E.C. degli Uberti, Role of complex cyclin d1/cdk4 in somatostatin subtype 2 receptormediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro, Endocrinology 147 (7) (2006) 3530–3538. [22] D. Molè, T. Gagliano, E. Gentilin, F. Tagliati, C. Pasquali, M.R. Ambrosio, G. Pansini, E.C. degli Uberti, M.C. Zatelli, Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors, Endocr. Relat. Cancer 18 (4) (2011) 439–450, doi:http://dx. doi.org/10.1530/ERC-11-0055. [23] M.C. Zatelli, G. Trasforini, S. Leoni, G. Frigato, M. Buratto, F. Tagliati, R. Rossi, L. Cavazzini, E. Roti, E.C. degli Uberti, BRAF V600E mutation analysis increasesdiagnostic accuracy for papillary thyroid carcinoma in fine-needle aspiration biopsies, Eur. J. Endocrinol. 161 (3) (2009) 467–473, doi:http://dx. doi.org/10.1530/EJE-09-0353. [24] M. Rossi, M. Buratto, F. Tagliati, R. Rossi, S. Lupo, G. Trasforini, G. Lanza, P. Franceschetti, S. Bruni, E. degli Uberti, M.C. Zatelli, Relevance of BRAF(V600E) mutation testing versus RAS point mutations and RET/PTC rearrangements evaluation in the diagnosis of thyroid cancer, Thyroid 25 (2) (2015) 221–228, doi:http://dx.doi.org/10.1089/thy.2014.0338. [25] H.J. Cao, L.Z. Zheng, N. Wang, L.Y. Wang, Y. Li, D. Li, Y.X. Lai, X.L. Wang, L. Qin, Src blockage by siRNA inhibits VEGF-induced vascular hyperpemeability and osteoclast activity—an in vitro mechanism study for preventing destructive repair of osteonecrosis, Bone 74 (2015) 58–68, doi:http://dx.doi.org/10.1016/j. bone.2014.12.060. [26] N. Kozer, D. Barua, S. Orchard, E.C. Nice, A.W. Burgess, W.S. Hlavacek, A.H. Clayton, Exploring higher-order EGFR oligomerisation and phosphorylation—a combined experimental and theoretical approach, Mol. Biosyst. 9 (7) (2013) 1849–1863, doi:http://dx.doi.org/10.1039/c3mb70073a. [27] E.J. Morris, S. Jha, C.R. Restaino, P. Dayananth, H. Zhu, A. Cooper, D. Carr, Y. Deng, W. Jin, S. Black, B. Long, J. Liu, E. Dinunzio, W. Windsor, R. Zhang, S. Zhao, M.H. Angagaw, E.M. Pinheiro, J. Desai, L. Xiao, G. Shipps, A. Hruza, J. Wang, J. Kelly, S. Paliwal, X. Gao, B.S. Babu, L. Zhu, P. Daublain, L. Zhang, B.A. Lutterbach, M.R. Pelletier, U. Philippar, P. Siliphaivanh, D. Witter, P. Kirschmeier, W.R. Bishop, D. Hicklin, D.G. Gilliland, L. Jayaraman, L. Zawel, S. Fawell, A.A. Samatar, Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors, Cancer Discov. 3 (7) (2013) 742–750, doi:http://dx.doi.org/ 10.1158/2159-8290.CD-13-0070. [28] A.W. Griffioen, L.A. Mans, A.M. de Graaf, P. Nowak-Sliwinska, C.L. de Hoog, T.A. de Jong, F.A. Vyth-Dreese, J.R. van Beijnum, A. Bex, E. Jonasch, Rapid angiogenesis onset after discontinuation of sunitinib treatment of renal cell carcinoma patients, Clin. Cancer Res. 18 (14) (2012) 3961–3971, doi:http://dx. doi.org/10.1158/1078-0432.CCR-12-0002. [29] A.F. Okines, A.R. Reynolds, D. Cunningham, Targeting angiogenesis in esophagogastric adenocarcinoma, Oncologist 16 (6) (2011) 844–858, doi: http://dx.doi.org/10.1634/theoncologist.2010-0387. [30] A. Fiorio Pla, A. Brossa, M. Bernardini, T. Genova, G. Grolez, A. Villers, X. Leroy, N. Prevarskaya, D. Gkika, B. Bussolati, Differential sensitivity of prostate tumor

648

[31]

[32]

[33]

[34]

[35]

[36]

T. Gagliano et al. / Biochemical Pharmacology 98 (2015) 639–648 derived endothelial cells to sorafenib and sunitinib, BMC Cancer 14 (2014) 939, doi:http://dx.doi.org/10.1186/1471-2407-14-93. P. Ghatalia, C.J. Morgan, T.K. Choueiri, P. Rocha, G. Naik, G. Sonpavde, Pancreatitis with vascular endothelial growth factor receptor tyrosine kinase inhibitors, Crit. Rev. Oncol. Hematol. 94 (1) (2015) 136–145, doi:http://dx.doi. org/10.1016/j.critrevonc.2014.11.008. P.R. Massey, J.S. Okman, J. Wilkerson, E.W. Cowen, Tyrosine kinase inhibitors directed against the vascular endothelial growth factor receptor (VEGFR) have distinct cutaneous toxicity profiles: a meta-analysis and review of the literature, Support. Care Cancer 23 (6) (2015) 1827–1835, doi:http://dx.doi. org/10.1007/s00520-014-2520-9. C. O’Sullivan, M. Edgerly, M. Velarde, J. Wilkerson, A.M. Venkatesan, S. Pittaluga, S.X. Yang, D. Nguyen, S. Balasubramaniam, T. Fojo, The VEGF inhibitor axitinib has limited effectiveness as a therapy for adrenocortical cancer, J. Clin. Endocrinol. Metab. 99 (4) (2014) 1291–1297, doi:http://dx.doi. org/10.1210/jc.2013-2298. S. Wortmann, M. Quinkler, C. Ritter, M. Kroiss, S. Johanssen, S. Hahner, B. Allolio, M. Fassnacht, Bevacizumab plus capecitabine as a salvage therapy in advanced adrenocortical carcinoma, Eur. J. Endocrinol. 162 (2) (2010) 349–356, doi:http://dx.doi.org/10.1530/EJE-09-0804. A. Berruti, P. Sperone, A. Ferrero, et al., Phase II study of weekly paclitaxel and sorafenib as second/third-line therapy in patients with adrenocortical carcinoma, Eur. J. Endocrinol. 166 (3) (2012) 451–458, doi:http://dx.doi.org/ 10.1530/EJE-11-0918. N.A. Lankheet, L.M. Knapen, J.H. Schellens, J.H. Beijnen, N. Steeghs, A.D. Huitema, Plasma concentrations of tyrosine kinase inhibitors imatinib,

[37]

[38]

[39]

[40]

[41]

erlotinib, and sunitinib in routine clinical outpatient cancer care, Ther. Drug Monit. 36 (3) (2014) 326–334, doi:http://dx.doi.org/10.1097/ FTD.0000000000000004. K. Motoshima, Y. Nakamura, K. Sano, Y. Ikegami, T. Ikeda, K. Mizoguchi, S. Takemoto, M. Fukuda, S. Nagashima, T. Iida, K. Tsukamoto, S. Kohno, I.I. Phase, trial of erlotinib in patients with advanced non-small-cell lung cancer harboring epidermal growth factor receptor mutations: additive analysis of pharmacokinetics, Cancer Chemother. Pharmacol. 72 (6) (2013) 1299–1304. M. Quinkler, S. Hahner, S. Wortmann, S. Johanssen, P. Adam, C. Ritter, C. Strasburger, B. Allolio, M. Fassnacht, Treatment of advanced adrenocortical carcinoma with erlotinib plus gemcitabine, J. Clin. Endocrinol. Metab. 93 (6) (2008) 2057–2062, doi:http://dx.doi.org/10.1210/jc.2007-2564. K. Takahashi-Niki, I. Kato-Ose, H. Murata, H. Maita, S.M. Iguchi-Ariga, H. Ariga, Epidermal growth factor-dependent activation of the ERK pathway by DJ-1 through its direct binding to c-Raf, J. Biol. Chem. 290 (29) (2015) 17838–17847, doi:http://dx.doi.org/10.1074/jbc.M115.666271 pii: jbc.M115.666271. [Epub ahead of print]. C.I. Lin, E.E. Whang, J. Moalem, D.T. Ruan, Strategic combination therapy overcomes tyrosine kinase coactivation in adrenocortical carcinoma, Surgery 152 (6) (2012) 1045–1050, doi:http://dx.doi.org/10.1016/j.surg.2012.08.035. M. Kroiss, M. Reuss, D. Kühner, S. Johanssen, M. Beyer, M. Zink, M.F. Hartmann, V. Dhir, S.A. Wudy, W. Arlt, S. Sbiera, B. Allolio, M. Fassnacht, Sunitinib inhibits cell proliferation and alters steroidogenesis by down-regulation of HSD3B2 in adrenocortical carcinoma cells, Front. Endocrinol. (Lausanne) 2 (27) (2011) , doi:http://dx.doi.org/10.3389/fendo.2011.00027.