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EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest Hirotoshi Ishigaki a, 1, Toshiyuki Minami a, b, *, 1, Osamu Morimura c, Hidemi Kitai b, Daisuke Horio a, Yuichi Koda a, b, Eriko Fujimoto a, b, Yoshiki Negi a, b, Yasuhiro Nakajima a, Maiko Niki a, b, Shingo Kanemura a, b, Eisuke Shibata a, b, Koji Mikami a, b, Ryo Takahashi a, b, Takashi Yokoi a, b, Kozo Kuribayashi a, b, Takashi Kijima a, b, c a
Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan Department of Thoracic Oncology, Hyogo College of Medicine, Hyogo, Japan c Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2019 Accepted 19 September 2019 Available online xxx
Small-cell lung cancer (SCLC) is characterized by one of neuroendocrine tumors, and is a clinically aggressive cancer due to its rapid growth, early dissemination, and rapid acquisition of multidrug resistance to chemotherapy. Moreover, the standard chemotherapeutic regimen in SCLC has not changed for three decades despite of the dramatic therapeutic improvement in non-SCLC. The development of a novel therapeutic strategy for SCLC has become a pressing issue. We found that expression of Eph receptor A2 (EphA2) is upregulated in three of 13 SCLC cell lines and five of 76 SCLC tumor samples. Genetic inhibition using siRNA of EphA2 significantly suppressed the cellular proliferation via induction of cell cycle arrest in SBC-5 cells. Furthermore, small molecule inhibitors of EphA2 (ALWeIIe41-27 and dasatinib) also exclusively inhibited proliferation of EphA2-positive SCLC cells by the same mechanism. Collectively, EphA2 could be a promising candidate as a therapeutic target for SCLC. © 2019 Elsevier Inc. All rights reserved.
Keywords: EphA2 Cell cycle arrest Small-cell lung cancer Molecular-targeted therapy
1. Introduction Small-cell lung cancer (SCLC) accounts for approximately 15% of primary lung cancers. SCLC exhibits neuroendocrine characteristics and is a clinically aggressive cancer associated with the poorest outcome of all the histological types of lung cancer. The extreme aggressiveness of SCLC is due to its rapid doubling time, widespread metastases, and development of multidrug resistance to chemotherapy [1,2]. Numerous kinds of clinical trials, including those that evaluate antitumor efficacy of molecular-targeted therapy, have been conducted, but eventuated in disappointing results [3]. One of the reasons of the insufficient antitumor effect of moleculartargeted therapy is that definitively actionable molecules with oncogenic driver activity are rarely detected in SCLC [4].
* Corresponding author. Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan. E-mail address: [email protected] (T. Minami). 1 H. Ishigaki and T. Minami contributed equally to this work.
Consequently, the standard therapeutic strategy remains unchanged for more than three decades despite of the dramatic improvement in the treatment of non-SCLC (NSCLC) during the same period of time. Therefore, development of novel therapeutic strategy in SCLC has become a pressing issue [2]. Eph receptors are the largest family of receptor tyrosine kinases (RTKs), and play essential roles in the development of nervous systems [5]. Recent studies have indicated that Eph receptors also affect tumor growth, invasiveness, angiogenesis, and metastasis [6]. Eph receptor family is comprised of the two subclasses, EphA (EphA1-10) and EphB (EphB1-6). Among them, EphA2 has been extensively studied and reported that its overexpression is associated with poor prognosis in various types of cancer such as glioblastoma, esophageal cancer, ovarian cancer, and NSCLC [7,8]. In NSCLC, previous studies demonstrated that EphA2 not only promoted the cellular proliferation but also played key roles in the function of cancer stem cell. Thus, EphA2 can be a promising targetable candidate for the treatment of the patients with NSCLC [9,10]. On the other hand, there has been no report about the biological
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Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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activity of EphA2 in SCLC. In the present study, we found that genetic and pharmacological inhibition of EphA2 could suppress the proliferation of SCLC cells via induction of cell cycle arrest. These results suggested that EphA2-targeted therapy could be a promising strategy in SCLC. 2. Materials and methods 2.1. Cell lines and cell culture The biological properties and the origin of SCLC cell lines, NCIeH69, NCIeH446, NCIeN231, SBC-1, SBC-2, SBC-3, SBC-5, OS-1, OS2RA, OS3R5, Smk, OC-10, and CADO-LC6 were previously described [11]. All the SCLC cells were maintained in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 mg/mL). They were routinely monitored for mycoplasma contamination by Mycoplasma Detection kit (Minerva Biolabs, Berlin, Germany). 2.2. Antibodies and reagents Rabbit monoclonal antibodies (Abs) against EphA2, phosphoAkt (pAkt), Akt, phospho-Erk (pErk), Erk, phospho-Rb (pRb), phospho-p53 (p-p53), p53, p21, and GAPDH were purchased from Cell Signaling Technology (CST, Danvers, MA). Anti-actin goat polyclonal Ab were available from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Counting Kit-8 (CCK-8, a tetrazolium reagent) and Annexin V apoptosis detection kit with propidium iodide (PI) were obtained from Dojindo (Osaka, Japan) and Biolegend (San Diego, CA), respectively. EphA2 inhibitors ALWeIIe41-27 and dasatinib were also purchased from APExBIO (Houston, TX) and Cayman Chemical (Ann Arbor, MI), respectively. 2.3. Immunohistochemistry (IHC) A tissue microarray (TMA) slide containing 80 paraffinembedded clinical SCLC samples was obtained from US Biomax (Rockville, MD). Each five-mm thick section on TMA slide was deparaffinized and incubated in Target Retrieval Solution pH 9.0 (Agilent, Santa Clara, CA) for 40 min at 96 C for antigen retrieval, then endogenous peroxidase activity was blocked with peroxidase blocking solution (Agilent). The sections were allowed to react with diluted anti-EphA2 rabbit monoclonal Ab (1:100) overnight at 4 C. Then, the slides were incubated with a peroxidase-labeled polymer conjugated to secondary anti-rabbit Ab using EnVision FLEX/HRP (Agilent) and developed with 3,30 -deaminobenzidine as the chromogen. 2.4. EphA2 knock down (KD) analysis Cells were grown in 12-well tissue culture-treated plates (Corning, New York, NY) and transfected with either 10 pmol/L of small interfering RNA (siRNA) targeted EPHA2 (siEphA2 #1, Hs_EPHA2_6 FlexiTube siRNA, SI00300188; and siEphA2 #2, Hs_EPHA2_7 FlexiTube siRNA, SI02223508; Qiagen, Venlo, Netherlands) or control siRNA (AllStars Negative Control siRNA, Qiagen) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). After 48 h, cells were harvested and used for the individual experiments.
polymerase chain reaction (PCR) was performed by using SYBR Green PCR Master Mix (Applied Biosystems) on the Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems) with the following profile: 1 cycle at 94 C for 2 min; 40 cycles at 94 C for 15 s, at 60 C for 1 min, and at 72 C for 1 min. Data analysis was carried out by the ABI sequence detection software using the relative quantification. The threshold cycle (Ct) is expressed as the mean value. The relative expression of each mRNA was calculated by the DCt method. The amount of the EphA2 relative to GAPDH mRNA was expressed as 2-(DCt). Primer sequence are as follows: EphA2 sense: 50 -TTCAGCCACCACAACATCAT-30 , antisense: 50 -TCAGACACCTTGCAGACCAG-30 , GAPDH sense: 50 -GCAAATTCCATGGCACCGT-30 , antisense: 50 -TCGCCCCACTTGATTTTGG-3’. 2.6. Flow cytometry To analyze the phosphorylation status of Rb, cells (2 105) transfected with siRNAs or treated with EphA2 inhibitors for 24 h were incubated with anti-pRb Ab (diluted 1:100) for 45 min at 4 C followed by labeling with Alexa488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA). For the cell cycle and apoptosis analysis, cells were similarly prepared as described above, and were stained with PI alone or Annexin V plus PI using Annexin V apoptosis detection kit with PI (Biolegend) according to the manufacture's instructions. After staining, cells were analyzed by BD LSRFortessa (Becton Dickinson, Franklin Lakes, NJ). 2.7. Cell proliferation assay To assess whether genetic inhibition of EphA2 affect proliferation of EphA2-positive SCLC cells, EphA2 siRNA-transfected cells (5 103/well) were plated onto 96-well tissue culture-treated plates (Corning) and incubate for 72 h. For drug sensitivity analysis, cells were treated with serially diluted EphA2 inhibitor, ALWeIIe41-27 or dasatinib, for 72 h. The relative number of proliferating cells was quantified using CCK-8 following the instruction manual. 2.8. Immunoblotting Cells were transfected with siRNA or treated with EphA2 inhibitors for up to 72 h, then lysed in RIPA lysis buffer (Thermo Fisher Scientific). The whole cell lysates were separated in 5e20% gradient gel (Wako, Osaka, Japan) by SDS-PAGE, thereafter transferred to polyvinylidene difluoride membranes. The membranes were incubated with the proper primary Abs (diluted 1:250e500) overnight at 4 C followed by appropriate horseradish peroxidaseconjugated secondary Abs (diluted 1:1000e4000, Cell Signaling Technology) for 1 h at room temperature. Immunoreactive bands were visualized using a chemiluminescent technique with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific). 2.9. Statistical analysis All the studies for statistical evaluation were performed in triplicate in each experiment and repeated at least thrice. Mean ± SEM values were calculated and differences were evaluated by two-sided Student's t-test. P < 0.05 values were considered statistically significant.
2.5. Quantitative real-time polymerase chain reaction 3. Results Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was synthesized from 100 ng total RNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Waltham, MA). The quantification of real-time
3.1. EphA2 is overexpressed in a subset of SCLC Although EphA2 has been reported to be overexpressed in
Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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various types of cancers including NSCLC, expression of EphA2 in SCLC had remained unknown [7,8]. We first evaluated the expression of EphA2 in SCLC cell lines by immunoblotting. EphA2 turned out to be overexpressed in three of 13 SCLC cell lines (Fig. 1A). To evaluate EphA2 expression in human SCLC clinical samples, we prepared formalin-fixed paraffin-embedded cell blocks of SBC-3, SBC-5 and H69 cells, and performed EphA2 staining by IHC. Consistent with the result of immunoblotting, overexpression of EphA2 was also observed in SBC-3 and SBC-5 cells but not in H69 cells (Fig. 1B). Thus, our IHC method could clearly distinguish between EphA2-positive and -negative SCLC. Then, we performed IHC using human SCLC tissue array obtained from US Biomax. Four of 80 samples were excluded from examination due to having been detached from TMA slide during antigen retrieval process. The remaining 76 SCLC clinical samples were evaluated. As shown in Fig. 1C, overexpression of EphA2 was detected in 5 of 76 samples (6.6%). These results suggested that EphA2 was overexpressed in a
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subset of SCLC and may play some roles in disease progression. 3.2. Genetic and pharmacological inhibition of EphA2 suppressed the proliferation of EphA2-positive SCLC cells To investigate the biological effect of EphA2 on SCLC cells, we knocked down the mRNA expression of EphA2 using siRNA technique in EphA2-positive SBC-5 cells. Despite the highest expression of EphA2 being highest in SBC-5 cells among 13 SCLC cell lines, the expression of EphA2 protein as well as transcript had been successfully suppressed by EphA2 targeting siRNA (siEphA2 #1 or siEphA2 #2) transfection. The inhibitory effect of siEphA2 #2 siRNA on EphA2 expression was particularly durable, lasting up to 5 days. (Supplementary Figs. S1A and B). After the confirmation of successful KD of EphA2 in SBC-5 cells, we performed CCK-8 assay to evaluate whether EphA2 KD affect the proliferative ability in SBC5 cells and found that EphA2 KD significantly suppressed the
Fig. 1. EphA2 is overexpressed in a subset of SCLC. A, Comprehensive analysis of EphA2 expression in SCLC cell lines by immunoblotting. EphA2 is overexpressed in three of 13 SCLC cell lines. All the three EphA2-positive cell lines (SBC-3, SBC-5, and Smk) are of Japanese origin. Similar results were confirmed twice and representative data is shown. B, Establishment of IHC method for the detection of EphA2 expression in paraffin-embedded samples. H69 cells were used as a negative control (left panel). Consistent with the result of immunoblotting, SBC-3 and SBC-5 were stained positive by our IHC system (mid and right panel). Scale Bars, 50 mm. C, Representative histological image of EphA2-negative (left panel) and epositive (right panel) case of human SCLC sample are shown. Scale Bars, 50 mm.
Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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Fig. 2. EphA2 inhibition suppresses the proliferation of EphA2-positive SCLC cells. A, EphA2 KD significantly inhibited the proliferation of EphA2-positive SBC-5 cells. SBC-5 cells were transfected with either control or EphA2 siRNA (siEphA2 #1 and siEphA2 #2). Cells were plated onto 96 well plate 48 h after the transfection, and incubated for further 72 h. The relative number of viable cells was quantified by CCK-8 assay. Results represent mean ± SEM of three independent experiments. B, Representative images of SBC-5 cells transfected with either control (upper) or siEphA2 #1 (bottom, left) or siEphA2 #2 (bottom, right) siRNA. Scale Bars, 250 mm. C, Representative images of SBC-5 cells treated with DMSO (left), 300 nM of ALWeIIe41-27 (middle), and 100 nM of dasatinib for 72 h. Scale Bars, 250 mm. D, SBC-5 cells were transfected with control or siEphA2 #2 siRNA. Thereafter, cells were treated with DMSO or 100 nM of dasatinib for another 72 h. The relative number of viable cells was quantified by CCK-8 assay. Bars, SEM. Experiments were independently repeated thrice in triplicates. *P < 0.05, **P < 0.01, N$S. not significant.
Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
H. Ishigaki et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx Table 1 EphA2 inhibitors selectively inhibited proliferation of EphA2-positive SCLC cells. SCLC cell lines
IC50 of ALWeIIe41-27 (nM)
IC50 of dasatinib (nM)
SBC-3 SBC-5 Smk H69 SBC-2
301.6 ± 8.0 272.9 ± 15.2 36.1 ± 9.7 1266.5 ± 226.9 1604.8 ± 183.3
527.1 ± 73.2 78.1 ± 10.7 48.7 ± 8.1 >10000 >10000
Note: Data are the means ± SEM of at least three independent experiments performed in triplicate.
proliferation of SBC-5 cells. Cell proliferation ratios in siEphA2 #1 siRNA-transfected cells and siEphA2 #2 siRNA-transfected cells were 36.7 ± 1.9% and 29.4 ± 8.3% of control siRNA-transfected cells, respectively. (Fig. 2A and B). We then examined whether pharmacological inhibition of EphA2 could also exert antiproliferative activity against EphA2-positive SCLC cells. ALWeIIe41-27 and dasatinib are known to be able to work as EphA2 inhibitors [10,12]. EphA2-positive SCLC cells (SBC-3, SBC-5, and Smk) are more sensitive to both EphA2 inhibitors compared to EphA2-negative SCLC cells (H69 and SBC-2) (Table 1, Supplementary Figs. S2A and B, and Fig. 2C). We consider that dasatinib has a potential to be applicable to the patients with EphA2-positive SCLC because this drug has been safely used clinically for the treatment of chronic myelogenous leukemia in clinical for 12 years [13]. However, unlike selective EphA2 inhibitor ALW-II-41-27, dasatinib is known to inhibit not only the kinase activity of EphA2 but also of Abl, c-Kit, plateletderived growth factor receptor-b, and Src family [14]. Therefore, we further examined whether dasatinib's antiproliferative effect on the cells is exerted exclusively through EphA2 inhibition. While dasatinib suppressed the proliferation of SBC-5 cells, antiproliferative effect of dasatinib against SBC-5 cells was cancelled when EphA2 was eliminated by siRNA (Fig. 2D). These results indicated that both ALWeIIe41-27 and dasatinib specifically targeted EphA2 and exclusively inhibited the proliferation of EphA2positive SCLC cells. 3.3. EphA2 inhibition induced cell cycle arrest to EphA2-positive SCLC cells Since multiple intracellular signaling pathways including RAS/ MAPK signals have been reported to be linked to EphA2 [5,6], we performed immunoblotting to elucidate the mechanism of the antiproliferative effect of EphA2 inhibition on SCLC cells using SBC5 cells. Unexpectedly, both genetic and pharmacological inhibition of EphA2 did not affect the phosphorylation status of Erk (Supplementary Figs. S3AeC). Moreover, EphA2 inhibition resulted in only a small increase in the number of Annexin V-positive apoptotic cells. (Supplementary Figs. S4A and B). These results implied that the antiproliferative effect of EphA2 inhibition was brought about neither through the inactivation of the proliferation signal nor through inducing remarkable apoptosis. We then examined the effect of EphA2 inhibition on cell cycle of SBC-5 cells by PI staining. Flowcytometric analysis showed that the proportion of G0/G1 phase significantly increased by EphA2 siRNA transfection compared to control siRNA transfection (37.1 ± 2.3% in control siRNA-transfected cells vs. 53.4 ± 3.3% in EphA2 siRNA-transfected cells, P ¼ 0.025) (Fig. 3A and B). Moreover, both EphA2 inhibitors, ALWeIIe41-27 and dasatinib, similarly increased the proportion of G0/G1 phase (34.0 ± 1.0% in DMSO-treated cells vs. 41.5 ± 1.6% in ALW-II-41-27-treated cells, P < 0.01, 34.7 ± 2.8% in DMSO-treated cells vs. 40.6 ± 2.3% in dasatinib-treated cells, P ¼ 0.012) (Fig. 3CeF). These results indicated that EphA2 inhibition induced cell cycle arrest, bringing about its antiproliferative effect on SCLC
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cells. 3.4. EphA2 inhibition induces cell cycle arrest through dephosphorylation of Rb To determine how EphA2 inhibition induced cell cycle arrest in EphA2-positive SCLC cells, we investigated the regulation of cell cycle-associated molecules. Both EphA2 KD and inhibitors caused upregulation of p27 in SBC-5 cells (Fig. 4AeC). Furthermore, flowcytometric analysis demonstrated that genetic and pharmacological EphA2 inhibition remarkably increased the proportion of dephosphorylated Rb in SBC-5 cells (14.7 ± 4.1% in control siRNAtransfected cells vs. 37.0 ± 3.5% in EphA2 siRNA-transfected cells, P ¼ 0.020, 8.7 ± 2.2% in DMSO-treated cells vs. 22.3 ± 3.0% in ALWII-41-27-treated cells, P ¼ 0.040, 5.4 ± 0.4% in DMSO-treated cells vs. 12.9 ± 1.8% in dasatinib-treated cells, P ¼ 0.029) (Fig. 4DeI). Since EphA2 inhibition did not affect either the proliferation signals or cause prominent apoptosis (Supplementary Figs. S3AeC, and Figs. S4A and B), these results demonstrated that EphA2 inhibition induced cell cycle arrest directly through regulating series of cell cycle-associated molecules. 4. Discussion SCLC remains one of the most aggressive and lethal malignancies. Various kinds of genomic alterations have been detected by comprehensive genomic analysis aimed at the development of novel molecular targeted therapy for SCLC. However, most of them do not act as biologically relevant driver oncogenes, and are not yet targetable [4,15]. Moreover, antitumor activity of immunotherapies using immune checkpoint inhibitors, antibody-dependent cellmediated cytotoxicity, and vaccines are still under investigation in SCLC [15,16]. Among them, only anti-program cell death-ligand 1 antibody, atezolizumab, recently showed modest survival benefit in patients with extensive-disease SCLC when added to the standard chemotherapy [17]. Thus, establishment of novel therapeutic strategy is indispensable to bring about a better outcome for the patients with SCLC. The reasons why we focused on EphA2 are as follows. First, we hypothesized that EphA2 plays an important role in the proliferation of SCLC cells because SCLC is one of neuroendocrine tumors and the fundamental role of EphA2 is to develop the nervous system [1,5]. Second, EphA2 is supposed to work as a “hub” among RTKs in SCLC. SCLC cells express various types of RTKs such as c-Kit, c-Met, insulin-like growth factor 1 receptor, ErbB family, and fibroblast growth factor receptor (FGFR) [18,19]. Among these RTKs, EphA2 is reported to be able to interact with ErbB family and FGFR, and transactivate them [20,21]. These previous studies indicated the possibility of EphA2 to be able to work as a “hub” RTK in SCLC. In the present study, we showed that EphA2 was overexpressed in a subset of SCLC. Comprehensive analysis using SCLC cell lines demonstrated that EphA2 was overexpressed in three of 13 SCLC cell lines. Notably, all the three EphA2-positive cell lines were generated from Japanese patients (Fig. 1A). In fact, EphA2-positive ratio (6.6%) in human SCLC clinical samples obtained from Caucasian patients were lower than we expected (Fig. 1C). These results suggest that ethnic differences may exist in EphA2 expression in SCLC, pointing to the need of further evaluations of EphA2 expression by using Japanese SCLC clinical samples. Regarding the possibility of EphA2-targeted therapy, we have demonstrated that both genetic and pharmacological inhibition of EphA2 suppressed the proliferation of these EphA2-positive SCLC cells via inducing cell cycle arrest (Fig. 2AeC, Table 1, and Fig. 3AeF). Most of the patients with SCLC already have widespread metastases at the time of diagnosis [2]. Therefore, taking clinical
Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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Fig. 3. Genetic and pharmacological inhibition of EphA2 induce cell cycle arrest to EphA2-positive SCLC cells. A, Representative cell cycle histogram of SBC-5 cells either transfected with control (left panel) or siEphA2 #2 siRNA (right panel) was analyzed by flowcytometry. B, Quantification of G0/G1 cell cycle phase in SBC-5 cells either transfected with control or siEphA2 #2 siRNA. C, Representative cell cycle histogram of SBC-5 cells treated with DMSO (left panel) or 300 nM of ALWeIIe41-27 (right panel) for 24 h. D, Quantification of G0/ G1 cell cycle phase in SBC-5 cells either treated with DMSO or 300 nM of ALW-II-41-27. E, Representative cell cycle histogram of SBC-5 cells treated with DMSO (left panel) or 100 nM of dasatinib (right panel) for 24 h. F, Quantification of G0/G1 cell cycle phase in SBC-5 cells either treated with DMSO or 100 nM of dasatinib. The experiments were independently repeated at least thrice. *P < 0.05, **P < 0.01.
application of EphA2-targeted therapy into consideration, systemic therapy with small molecule EphA2 inhibitor would be the most favorable treatment. While both EphA2 inhibitors ALWeIIe41-27 and dasatinib exclusively inhibited the proliferation of EphA2positive SCLC cells (Table 1, Supplementary Figs. S2A and B, and Fig. 2C), only dasatinib is currently applicable in clinical settings because of its safe use in the treatment of chronic myeloid leukemia (CML) for more than 12 years [13]. According to the
pharmacokinetic analysis of the previous phase III clinical trial against CML, the maximum serum concentration (Cmax) of dasatinib reached 54.6 ng/mL (111.9 nM) when it was orally administered at 100 mg once daily [22]. Our experimental results showed that the half maximal inhibitory concentration (IC50) of dasatinib was achievable under Cmax in two of three EphA2-positive SCLC cells (Table 1). A phase II clinical study to evaluate the antitumor efficacy of
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Fig. 4. EphA2 inhibition regulates cell cycle-associated molecules and brings about dephosphorylation of Rb in EphA2-positive SCLC cells. A, Expression of cell cycle-associated protein p27 in SBC-5 cells transfected with either control or siEphA2 #2 siRNA. B, Phosphorylation status of EphA2 and p27 expression in SBC-5 cells treated with either DMSO or 300 nM of ALWeIIe41-27 for 8 h. C, Phosphorylation status of EphA2 and p27 expression in SBC-5 cells treated with either DMSO or 100 nM of dasatinib for 8 h. D, Flowcytometric analysis of the phosphorylation status of Rb in SBC-5 cells transfected with either control (left) or siEphA2 #2 siRNA (right). E, Quantification of relative number of phospho-Rb-negative SBC-5 cells transfected with either control or siEphA2 #2 siRNA. F, Phosphorylation status of Rb in SBC-5 cells treated with DMSO or 300 nM of ALWeIIe41-27 for 24 h. G, Quantification of relative number of phospho-Rb-negative SBC-5 cells treated with either DMSO or ALW-II-41-27. H, Phosphorylation status of Rb in SBC-5 cells treated with DMSO or 100 nM of dasatinib for 24 h. I, Quantification of relative number of phospho-Rb-negative SBC-5 cells treated with either DMSO or dasatinib. Anti-phosophorylated Rb specific antibody was used in these flowcytometric analysis. The experiments were repeated at least thrice. *P < 0.05, **P < 0.01.
dasatinib was performed in patients with chemosensitive-relapsed SCLC. In that study, dasatinib did not show satisfactory antitumor effect with a disease control rate of 16% [23]. One possible explanation of this insufficient outcome of dasatinib is that activating
driver mutations have not been identified in c-Kit and c-SRC, the two molecules regarded as target molecules of dasatinib in SCLC [24]. However, we presume that dasatinib or other EphA2 inhibitor will exert antitumor effect against EphA2-positive SCLC through
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preventing EphA2 from transactivating various kinds of other RTKs. Although the incidence of EphA2-positive SCLC is relatively rare (Fig. 1AeC), EphA2-targeted therapy would bring about a clinical benefit in patients with SCLC when applied to the appropriate candidates selected by using EphA2 expression as a biomarker. In conclusion, EphA2-targeted therapy is promising for a subset of patients with SCLC. It would be more practical to use EphA2targeted therapy in combination with cytotoxic drugs because EphA2 inhibition can cause cell cycle arrest but cannot induce critical apoptosis to SCLC cells. Author contributions H$I., T.M., O.M., and T.K., conceived the study and designed experiments; H$I., T.M., O.M., D.H., and H.K. carried out experiments; H$I., T.M., O.M., H$K., Y$N., M.N., Y$K., E.F., Y$N., S$K., and E.S. analyzed the data; H$I., T.M., K.M., R.T., T.Y., K$K., and T.K., wrote the manuscript, which was reviewed and edited by the other coauthors. Disclosure of potential conflict of interests No potential conflicts of interest were disclosed. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (18K15962 to T.M., 18K15963 to S$K., 18K08161 to T.K.). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.076. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.076 References [1] J.K. Sabari, B.H. Lok, J.H. Laird, et al., Unravelling the biology of SCLC: implications for therapy, Nat. Rev. Clin. Oncol. 14 (2017) 549e561. [2] L.A. Byers, C.M. Rudin, Small cell lung cancer: where do we go from here? Cancer 121 (2015) 664e672. [3] H. Mamdani, R. Induru, S.I. Jalal, Novel therapies in small cell lung cancer,
Transl. Lung Cancer Res. 4 (2015) 533e544. [4] J. George, J.S. Lim, S.J. Jang, et al., Comprehensive genomic profiles of small cell lung cancer, Nature 524 (2015) 47e53. [5] E.B. Pasquale, Eph-ephrin bidirectional signaling in physiology and disease, Cell 133 (2008) 38e52. [6] E.B. Pasquale, Eph receptors and ephrins in cancer: bidirectional signalling and beyond, Nat. Rev. Cancer 10 (2010) 165e180. [7] J. Wykosky, W. Debinski, The EphA2 receptor and ephrinA1 ligand in solid tumors: function and therapeutic targeting, Mol. Cancer Res. 6 (2008) 1795e1806. [8] L. Faoro, P.A. Singleton, G.M. Cervantes, et al., EphA2 mutation in lung squamous cell carcinoma promotes increased cell survival, cell invasion, focal adhesions, and mammalian target of rapamycin activation, J. Biol. Chem. 285 (2010) 18575e18585. [9] W. Song, Y. Ma, J. Wang, et al., JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer, Cancer Res. 74 (2014) 2444e2454. [10] K.R. Amato, S. Wang, A.K. Hastings, et al., Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC, J. Clin. Investig. 124 (2014) 2037e2049. [11] T. Minami, T. Kijima, Y. Otani, et al., HER2 as therapeutic target for overcoming ATP-binding cassette transporter-mediated chemoresistance in small cell lung cancer, Mol. Cancer Ther. 11 (2012) 830e841. [12] Q. Chang, C. Jorgensen, T. Pawson, et al., Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer, Br. J. Canc. 99 (2008) 1074e1082. [13] M. Talpaz, G. Saglio, E. Atallah, et al., Dasatinib dose management for the treatment of chronic myeloid leukemia, Cancer 124 (2018) 1660e1672. [14] L.J. Lombardo, F.Y. Lee, P. Chen, et al., Discovery of N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays, J. Med. Chem. 47 (2004) 6658e6661. [15] N. Tsoukalas, E. Aravantinou-Fatorou, P. Baxevanos, et al., Advanced small cell lung cancer (SCLC): new challenges and new expectations, Ann. Transl. Med. 6 (2018) 145. [16] T. Minami, Y. Kinehara, O. Morimura, et al., Challenges for the development of immunotherapy in small-cell lung cancer, Med Res Arch 6 (2018). [17] L. Horn, A.S. Mansfield, A. Szcze˛ sna, et al., First-line atezolizumab plus chemotherapy in extensive-stage small-cell lung cancer, N. Engl. J. Med. 379 (2018) 2220e2229. [18] F. Koinis, A. Kotsakis, V. Georgoulias, Small cell lung cancer (SCLC): no treatment advances in recent years, Transl. Lung Cancer Res. 5 (2016) 39e50. [19] A.M. Schultheis, M. Bos, K. Schmitz, et al., Fibroblast growth factor receptor 1 (FGFR1) amplification is a potential therapeutic target in small-cell lung cancer, Mod. Pathol. 27 (2014) 214e221. [20] J. Chen, W. Song, K. Amato, Eph receptor tyrosine kinases in cancer stem cells, Cytokine Growth Factor Rev. 26 (2015) 1e6. [21] T. Sawada, D. Arai, X. Jing, et al., Trans-Activation between EphA and FGFR regulates self-renewal and differentiation of mouse embryonic neural stem/ progenitor cells via differential activation of FRS2a, PLoS One 10 (2015) e0128826. [22] X. Wang, A. Roy, A. Hochhaus, et al., Differential effects of dosing regimen on the safety and efficacy of dasatinib: retrospective exposure-response analysis of a Phase III study, Clin. Pharmacol. 5 (2013) 85e97. [23] A.A. Miller, H. Pang, L. Hodgson, et al., A phase II study of dasatinib in patients with chemosensitive relapsed small cell lung cancer (Cancer and Leukemia Group B 30602), J. Thorac. Oncol. 5 (2010) 380e384. [24] P. Bordi, M. Tiseo, F. Barbieri, et al., Gene mutations in small-cell lung cancer (SCLC): results of a panel of 6 genes in a cohort of Italian patients, Lung Cancer 86 (2014) 324e328.
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EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest Hirotoshi Ishigaki a, 1, Toshiyuki Minami a, b, *, 1, Osamu Morimura c, Hidemi Kitai b, Daisuke Horio a, Yuichi Koda a, b, Eriko Fujimoto a, b, Yoshiki Negi a, b, Yasuhiro Nakajima a, Maiko Niki a, b, Shingo Kanemura a, b, Eisuke Shibata a, b, Koji Mikami a, b, Ryo Takahashi a, b, Takashi Yokoi a, b, Kozo Kuribayashi a, b, Takashi Kijima a, b, c a
Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan Department of Thoracic Oncology, Hyogo College of Medicine, Hyogo, Japan c Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2019 Accepted 19 September 2019 Available online xxx
Small-cell lung cancer (SCLC) is characterized by one of neuroendocrine tumors, and is a clinically aggressive cancer due to its rapid growth, early dissemination, and rapid acquisition of multidrug resistance to chemotherapy. Moreover, the standard chemotherapeutic regimen in SCLC has not changed for three decades despite of the dramatic therapeutic improvement in non-SCLC. The development of a novel therapeutic strategy for SCLC has become a pressing issue. We found that expression of Eph receptor A2 (EphA2) is upregulated in three of 13 SCLC cell lines and five of 76 SCLC tumor samples. Genetic inhibition using siRNA of EphA2 significantly suppressed the cellular proliferation via induction of cell cycle arrest in SBC-5 cells. Furthermore, small molecule inhibitors of EphA2 (ALWeIIe41-27 and dasatinib) also exclusively inhibited proliferation of EphA2-positive SCLC cells by the same mechanism. Collectively, EphA2 could be a promising candidate as a therapeutic target for SCLC. © 2019 Elsevier Inc. All rights reserved.
Keywords: EphA2 Cell cycle arrest Small-cell lung cancer Molecular-targeted therapy
1. Introduction Small-cell lung cancer (SCLC) accounts for approximately 15% of primary lung cancers. SCLC exhibits neuroendocrine characteristics and is a clinically aggressive cancer associated with the poorest outcome of all the histological types of lung cancer. The extreme aggressiveness of SCLC is due to its rapid doubling time, widespread metastases, and development of multidrug resistance to chemotherapy [1,2]. Numerous kinds of clinical trials, including those that evaluate antitumor efficacy of molecular-targeted therapy, have been conducted, but eventuated in disappointing results [3]. One of the reasons of the insufficient antitumor effect of moleculartargeted therapy is that definitively actionable molecules with oncogenic driver activity are rarely detected in SCLC [4].
* Corresponding author. Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan. E-mail address: [email protected] (T. Minami). 1 H. Ishigaki and T. Minami contributed equally to this work.
Consequently, the standard therapeutic strategy remains unchanged for more than three decades despite of the dramatic improvement in the treatment of non-SCLC (NSCLC) during the same period of time. Therefore, development of novel therapeutic strategy in SCLC has become a pressing issue [2]. Eph receptors are the largest family of receptor tyrosine kinases (RTKs), and play essential roles in the development of nervous systems [5]. Recent studies have indicated that Eph receptors also affect tumor growth, invasiveness, angiogenesis, and metastasis [6]. Eph receptor family is comprised of the two subclasses, EphA (EphA1-10) and EphB (EphB1-6). Among them, EphA2 has been extensively studied and reported that its overexpression is associated with poor prognosis in various types of cancer such as glioblastoma, esophageal cancer, ovarian cancer, and NSCLC [7,8]. In NSCLC, previous studies demonstrated that EphA2 not only promoted the cellular proliferation but also played key roles in the function of cancer stem cell. Thus, EphA2 can be a promising targetable candidate for the treatment of the patients with NSCLC [9,10]. On the other hand, there has been no report about the biological
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Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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activity of EphA2 in SCLC. In the present study, we found that genetic and pharmacological inhibition of EphA2 could suppress the proliferation of SCLC cells via induction of cell cycle arrest. These results suggested that EphA2-targeted therapy could be a promising strategy in SCLC. 2. Materials and methods 2.1. Cell lines and cell culture The biological properties and the origin of SCLC cell lines, NCIeH69, NCIeH446, NCIeN231, SBC-1, SBC-2, SBC-3, SBC-5, OS-1, OS2RA, OS3R5, Smk, OC-10, and CADO-LC6 were previously described [11]. All the SCLC cells were maintained in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 mg/mL). They were routinely monitored for mycoplasma contamination by Mycoplasma Detection kit (Minerva Biolabs, Berlin, Germany). 2.2. Antibodies and reagents Rabbit monoclonal antibodies (Abs) against EphA2, phosphoAkt (pAkt), Akt, phospho-Erk (pErk), Erk, phospho-Rb (pRb), phospho-p53 (p-p53), p53, p21, and GAPDH were purchased from Cell Signaling Technology (CST, Danvers, MA). Anti-actin goat polyclonal Ab were available from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Counting Kit-8 (CCK-8, a tetrazolium reagent) and Annexin V apoptosis detection kit with propidium iodide (PI) were obtained from Dojindo (Osaka, Japan) and Biolegend (San Diego, CA), respectively. EphA2 inhibitors ALWeIIe41-27 and dasatinib were also purchased from APExBIO (Houston, TX) and Cayman Chemical (Ann Arbor, MI), respectively. 2.3. Immunohistochemistry (IHC) A tissue microarray (TMA) slide containing 80 paraffinembedded clinical SCLC samples was obtained from US Biomax (Rockville, MD). Each five-mm thick section on TMA slide was deparaffinized and incubated in Target Retrieval Solution pH 9.0 (Agilent, Santa Clara, CA) for 40 min at 96 C for antigen retrieval, then endogenous peroxidase activity was blocked with peroxidase blocking solution (Agilent). The sections were allowed to react with diluted anti-EphA2 rabbit monoclonal Ab (1:100) overnight at 4 C. Then, the slides were incubated with a peroxidase-labeled polymer conjugated to secondary anti-rabbit Ab using EnVision FLEX/HRP (Agilent) and developed with 3,30 -deaminobenzidine as the chromogen. 2.4. EphA2 knock down (KD) analysis Cells were grown in 12-well tissue culture-treated plates (Corning, New York, NY) and transfected with either 10 pmol/L of small interfering RNA (siRNA) targeted EPHA2 (siEphA2 #1, Hs_EPHA2_6 FlexiTube siRNA, SI00300188; and siEphA2 #2, Hs_EPHA2_7 FlexiTube siRNA, SI02223508; Qiagen, Venlo, Netherlands) or control siRNA (AllStars Negative Control siRNA, Qiagen) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). After 48 h, cells were harvested and used for the individual experiments.
polymerase chain reaction (PCR) was performed by using SYBR Green PCR Master Mix (Applied Biosystems) on the Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems) with the following profile: 1 cycle at 94 C for 2 min; 40 cycles at 94 C for 15 s, at 60 C for 1 min, and at 72 C for 1 min. Data analysis was carried out by the ABI sequence detection software using the relative quantification. The threshold cycle (Ct) is expressed as the mean value. The relative expression of each mRNA was calculated by the DCt method. The amount of the EphA2 relative to GAPDH mRNA was expressed as 2-(DCt). Primer sequence are as follows: EphA2 sense: 50 -TTCAGCCACCACAACATCAT-30 , antisense: 50 -TCAGACACCTTGCAGACCAG-30 , GAPDH sense: 50 -GCAAATTCCATGGCACCGT-30 , antisense: 50 -TCGCCCCACTTGATTTTGG-3’. 2.6. Flow cytometry To analyze the phosphorylation status of Rb, cells (2 105) transfected with siRNAs or treated with EphA2 inhibitors for 24 h were incubated with anti-pRb Ab (diluted 1:100) for 45 min at 4 C followed by labeling with Alexa488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA). For the cell cycle and apoptosis analysis, cells were similarly prepared as described above, and were stained with PI alone or Annexin V plus PI using Annexin V apoptosis detection kit with PI (Biolegend) according to the manufacture's instructions. After staining, cells were analyzed by BD LSRFortessa (Becton Dickinson, Franklin Lakes, NJ). 2.7. Cell proliferation assay To assess whether genetic inhibition of EphA2 affect proliferation of EphA2-positive SCLC cells, EphA2 siRNA-transfected cells (5 103/well) were plated onto 96-well tissue culture-treated plates (Corning) and incubate for 72 h. For drug sensitivity analysis, cells were treated with serially diluted EphA2 inhibitor, ALWeIIe41-27 or dasatinib, for 72 h. The relative number of proliferating cells was quantified using CCK-8 following the instruction manual. 2.8. Immunoblotting Cells were transfected with siRNA or treated with EphA2 inhibitors for up to 72 h, then lysed in RIPA lysis buffer (Thermo Fisher Scientific). The whole cell lysates were separated in 5e20% gradient gel (Wako, Osaka, Japan) by SDS-PAGE, thereafter transferred to polyvinylidene difluoride membranes. The membranes were incubated with the proper primary Abs (diluted 1:250e500) overnight at 4 C followed by appropriate horseradish peroxidaseconjugated secondary Abs (diluted 1:1000e4000, Cell Signaling Technology) for 1 h at room temperature. Immunoreactive bands were visualized using a chemiluminescent technique with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific). 2.9. Statistical analysis All the studies for statistical evaluation were performed in triplicate in each experiment and repeated at least thrice. Mean ± SEM values were calculated and differences were evaluated by two-sided Student's t-test. P < 0.05 values were considered statistically significant.
2.5. Quantitative real-time polymerase chain reaction 3. Results Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was synthesized from 100 ng total RNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Waltham, MA). The quantification of real-time
3.1. EphA2 is overexpressed in a subset of SCLC Although EphA2 has been reported to be overexpressed in
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various types of cancers including NSCLC, expression of EphA2 in SCLC had remained unknown [7,8]. We first evaluated the expression of EphA2 in SCLC cell lines by immunoblotting. EphA2 turned out to be overexpressed in three of 13 SCLC cell lines (Fig. 1A). To evaluate EphA2 expression in human SCLC clinical samples, we prepared formalin-fixed paraffin-embedded cell blocks of SBC-3, SBC-5 and H69 cells, and performed EphA2 staining by IHC. Consistent with the result of immunoblotting, overexpression of EphA2 was also observed in SBC-3 and SBC-5 cells but not in H69 cells (Fig. 1B). Thus, our IHC method could clearly distinguish between EphA2-positive and -negative SCLC. Then, we performed IHC using human SCLC tissue array obtained from US Biomax. Four of 80 samples were excluded from examination due to having been detached from TMA slide during antigen retrieval process. The remaining 76 SCLC clinical samples were evaluated. As shown in Fig. 1C, overexpression of EphA2 was detected in 5 of 76 samples (6.6%). These results suggested that EphA2 was overexpressed in a
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subset of SCLC and may play some roles in disease progression. 3.2. Genetic and pharmacological inhibition of EphA2 suppressed the proliferation of EphA2-positive SCLC cells To investigate the biological effect of EphA2 on SCLC cells, we knocked down the mRNA expression of EphA2 using siRNA technique in EphA2-positive SBC-5 cells. Despite the highest expression of EphA2 being highest in SBC-5 cells among 13 SCLC cell lines, the expression of EphA2 protein as well as transcript had been successfully suppressed by EphA2 targeting siRNA (siEphA2 #1 or siEphA2 #2) transfection. The inhibitory effect of siEphA2 #2 siRNA on EphA2 expression was particularly durable, lasting up to 5 days. (Supplementary Figs. S1A and B). After the confirmation of successful KD of EphA2 in SBC-5 cells, we performed CCK-8 assay to evaluate whether EphA2 KD affect the proliferative ability in SBC5 cells and found that EphA2 KD significantly suppressed the
Fig. 1. EphA2 is overexpressed in a subset of SCLC. A, Comprehensive analysis of EphA2 expression in SCLC cell lines by immunoblotting. EphA2 is overexpressed in three of 13 SCLC cell lines. All the three EphA2-positive cell lines (SBC-3, SBC-5, and Smk) are of Japanese origin. Similar results were confirmed twice and representative data is shown. B, Establishment of IHC method for the detection of EphA2 expression in paraffin-embedded samples. H69 cells were used as a negative control (left panel). Consistent with the result of immunoblotting, SBC-3 and SBC-5 were stained positive by our IHC system (mid and right panel). Scale Bars, 50 mm. C, Representative histological image of EphA2-negative (left panel) and epositive (right panel) case of human SCLC sample are shown. Scale Bars, 50 mm.
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Fig. 2. EphA2 inhibition suppresses the proliferation of EphA2-positive SCLC cells. A, EphA2 KD significantly inhibited the proliferation of EphA2-positive SBC-5 cells. SBC-5 cells were transfected with either control or EphA2 siRNA (siEphA2 #1 and siEphA2 #2). Cells were plated onto 96 well plate 48 h after the transfection, and incubated for further 72 h. The relative number of viable cells was quantified by CCK-8 assay. Results represent mean ± SEM of three independent experiments. B, Representative images of SBC-5 cells transfected with either control (upper) or siEphA2 #1 (bottom, left) or siEphA2 #2 (bottom, right) siRNA. Scale Bars, 250 mm. C, Representative images of SBC-5 cells treated with DMSO (left), 300 nM of ALWeIIe41-27 (middle), and 100 nM of dasatinib for 72 h. Scale Bars, 250 mm. D, SBC-5 cells were transfected with control or siEphA2 #2 siRNA. Thereafter, cells were treated with DMSO or 100 nM of dasatinib for another 72 h. The relative number of viable cells was quantified by CCK-8 assay. Bars, SEM. Experiments were independently repeated thrice in triplicates. *P < 0.05, **P < 0.01, N$S. not significant.
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H. Ishigaki et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx Table 1 EphA2 inhibitors selectively inhibited proliferation of EphA2-positive SCLC cells. SCLC cell lines
IC50 of ALWeIIe41-27 (nM)
IC50 of dasatinib (nM)
SBC-3 SBC-5 Smk H69 SBC-2
301.6 ± 8.0 272.9 ± 15.2 36.1 ± 9.7 1266.5 ± 226.9 1604.8 ± 183.3
527.1 ± 73.2 78.1 ± 10.7 48.7 ± 8.1 >10000 >10000
Note: Data are the means ± SEM of at least three independent experiments performed in triplicate.
proliferation of SBC-5 cells. Cell proliferation ratios in siEphA2 #1 siRNA-transfected cells and siEphA2 #2 siRNA-transfected cells were 36.7 ± 1.9% and 29.4 ± 8.3% of control siRNA-transfected cells, respectively. (Fig. 2A and B). We then examined whether pharmacological inhibition of EphA2 could also exert antiproliferative activity against EphA2-positive SCLC cells. ALWeIIe41-27 and dasatinib are known to be able to work as EphA2 inhibitors [10,12]. EphA2-positive SCLC cells (SBC-3, SBC-5, and Smk) are more sensitive to both EphA2 inhibitors compared to EphA2-negative SCLC cells (H69 and SBC-2) (Table 1, Supplementary Figs. S2A and B, and Fig. 2C). We consider that dasatinib has a potential to be applicable to the patients with EphA2-positive SCLC because this drug has been safely used clinically for the treatment of chronic myelogenous leukemia in clinical for 12 years [13]. However, unlike selective EphA2 inhibitor ALW-II-41-27, dasatinib is known to inhibit not only the kinase activity of EphA2 but also of Abl, c-Kit, plateletderived growth factor receptor-b, and Src family [14]. Therefore, we further examined whether dasatinib's antiproliferative effect on the cells is exerted exclusively through EphA2 inhibition. While dasatinib suppressed the proliferation of SBC-5 cells, antiproliferative effect of dasatinib against SBC-5 cells was cancelled when EphA2 was eliminated by siRNA (Fig. 2D). These results indicated that both ALWeIIe41-27 and dasatinib specifically targeted EphA2 and exclusively inhibited the proliferation of EphA2positive SCLC cells. 3.3. EphA2 inhibition induced cell cycle arrest to EphA2-positive SCLC cells Since multiple intracellular signaling pathways including RAS/ MAPK signals have been reported to be linked to EphA2 [5,6], we performed immunoblotting to elucidate the mechanism of the antiproliferative effect of EphA2 inhibition on SCLC cells using SBC5 cells. Unexpectedly, both genetic and pharmacological inhibition of EphA2 did not affect the phosphorylation status of Erk (Supplementary Figs. S3AeC). Moreover, EphA2 inhibition resulted in only a small increase in the number of Annexin V-positive apoptotic cells. (Supplementary Figs. S4A and B). These results implied that the antiproliferative effect of EphA2 inhibition was brought about neither through the inactivation of the proliferation signal nor through inducing remarkable apoptosis. We then examined the effect of EphA2 inhibition on cell cycle of SBC-5 cells by PI staining. Flowcytometric analysis showed that the proportion of G0/G1 phase significantly increased by EphA2 siRNA transfection compared to control siRNA transfection (37.1 ± 2.3% in control siRNA-transfected cells vs. 53.4 ± 3.3% in EphA2 siRNA-transfected cells, P ¼ 0.025) (Fig. 3A and B). Moreover, both EphA2 inhibitors, ALWeIIe41-27 and dasatinib, similarly increased the proportion of G0/G1 phase (34.0 ± 1.0% in DMSO-treated cells vs. 41.5 ± 1.6% in ALW-II-41-27-treated cells, P < 0.01, 34.7 ± 2.8% in DMSO-treated cells vs. 40.6 ± 2.3% in dasatinib-treated cells, P ¼ 0.012) (Fig. 3CeF). These results indicated that EphA2 inhibition induced cell cycle arrest, bringing about its antiproliferative effect on SCLC
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cells. 3.4. EphA2 inhibition induces cell cycle arrest through dephosphorylation of Rb To determine how EphA2 inhibition induced cell cycle arrest in EphA2-positive SCLC cells, we investigated the regulation of cell cycle-associated molecules. Both EphA2 KD and inhibitors caused upregulation of p27 in SBC-5 cells (Fig. 4AeC). Furthermore, flowcytometric analysis demonstrated that genetic and pharmacological EphA2 inhibition remarkably increased the proportion of dephosphorylated Rb in SBC-5 cells (14.7 ± 4.1% in control siRNAtransfected cells vs. 37.0 ± 3.5% in EphA2 siRNA-transfected cells, P ¼ 0.020, 8.7 ± 2.2% in DMSO-treated cells vs. 22.3 ± 3.0% in ALWII-41-27-treated cells, P ¼ 0.040, 5.4 ± 0.4% in DMSO-treated cells vs. 12.9 ± 1.8% in dasatinib-treated cells, P ¼ 0.029) (Fig. 4DeI). Since EphA2 inhibition did not affect either the proliferation signals or cause prominent apoptosis (Supplementary Figs. S3AeC, and Figs. S4A and B), these results demonstrated that EphA2 inhibition induced cell cycle arrest directly through regulating series of cell cycle-associated molecules. 4. Discussion SCLC remains one of the most aggressive and lethal malignancies. Various kinds of genomic alterations have been detected by comprehensive genomic analysis aimed at the development of novel molecular targeted therapy for SCLC. However, most of them do not act as biologically relevant driver oncogenes, and are not yet targetable [4,15]. Moreover, antitumor activity of immunotherapies using immune checkpoint inhibitors, antibody-dependent cellmediated cytotoxicity, and vaccines are still under investigation in SCLC [15,16]. Among them, only anti-program cell death-ligand 1 antibody, atezolizumab, recently showed modest survival benefit in patients with extensive-disease SCLC when added to the standard chemotherapy [17]. Thus, establishment of novel therapeutic strategy is indispensable to bring about a better outcome for the patients with SCLC. The reasons why we focused on EphA2 are as follows. First, we hypothesized that EphA2 plays an important role in the proliferation of SCLC cells because SCLC is one of neuroendocrine tumors and the fundamental role of EphA2 is to develop the nervous system [1,5]. Second, EphA2 is supposed to work as a “hub” among RTKs in SCLC. SCLC cells express various types of RTKs such as c-Kit, c-Met, insulin-like growth factor 1 receptor, ErbB family, and fibroblast growth factor receptor (FGFR) [18,19]. Among these RTKs, EphA2 is reported to be able to interact with ErbB family and FGFR, and transactivate them [20,21]. These previous studies indicated the possibility of EphA2 to be able to work as a “hub” RTK in SCLC. In the present study, we showed that EphA2 was overexpressed in a subset of SCLC. Comprehensive analysis using SCLC cell lines demonstrated that EphA2 was overexpressed in three of 13 SCLC cell lines. Notably, all the three EphA2-positive cell lines were generated from Japanese patients (Fig. 1A). In fact, EphA2-positive ratio (6.6%) in human SCLC clinical samples obtained from Caucasian patients were lower than we expected (Fig. 1C). These results suggest that ethnic differences may exist in EphA2 expression in SCLC, pointing to the need of further evaluations of EphA2 expression by using Japanese SCLC clinical samples. Regarding the possibility of EphA2-targeted therapy, we have demonstrated that both genetic and pharmacological inhibition of EphA2 suppressed the proliferation of these EphA2-positive SCLC cells via inducing cell cycle arrest (Fig. 2AeC, Table 1, and Fig. 3AeF). Most of the patients with SCLC already have widespread metastases at the time of diagnosis [2]. Therefore, taking clinical
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Fig. 3. Genetic and pharmacological inhibition of EphA2 induce cell cycle arrest to EphA2-positive SCLC cells. A, Representative cell cycle histogram of SBC-5 cells either transfected with control (left panel) or siEphA2 #2 siRNA (right panel) was analyzed by flowcytometry. B, Quantification of G0/G1 cell cycle phase in SBC-5 cells either transfected with control or siEphA2 #2 siRNA. C, Representative cell cycle histogram of SBC-5 cells treated with DMSO (left panel) or 300 nM of ALWeIIe41-27 (right panel) for 24 h. D, Quantification of G0/ G1 cell cycle phase in SBC-5 cells either treated with DMSO or 300 nM of ALW-II-41-27. E, Representative cell cycle histogram of SBC-5 cells treated with DMSO (left panel) or 100 nM of dasatinib (right panel) for 24 h. F, Quantification of G0/G1 cell cycle phase in SBC-5 cells either treated with DMSO or 100 nM of dasatinib. The experiments were independently repeated at least thrice. *P < 0.05, **P < 0.01.
application of EphA2-targeted therapy into consideration, systemic therapy with small molecule EphA2 inhibitor would be the most favorable treatment. While both EphA2 inhibitors ALWeIIe41-27 and dasatinib exclusively inhibited the proliferation of EphA2positive SCLC cells (Table 1, Supplementary Figs. S2A and B, and Fig. 2C), only dasatinib is currently applicable in clinical settings because of its safe use in the treatment of chronic myeloid leukemia (CML) for more than 12 years [13]. According to the
pharmacokinetic analysis of the previous phase III clinical trial against CML, the maximum serum concentration (Cmax) of dasatinib reached 54.6 ng/mL (111.9 nM) when it was orally administered at 100 mg once daily [22]. Our experimental results showed that the half maximal inhibitory concentration (IC50) of dasatinib was achievable under Cmax in two of three EphA2-positive SCLC cells (Table 1). A phase II clinical study to evaluate the antitumor efficacy of
Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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Fig. 4. EphA2 inhibition regulates cell cycle-associated molecules and brings about dephosphorylation of Rb in EphA2-positive SCLC cells. A, Expression of cell cycle-associated protein p27 in SBC-5 cells transfected with either control or siEphA2 #2 siRNA. B, Phosphorylation status of EphA2 and p27 expression in SBC-5 cells treated with either DMSO or 300 nM of ALWeIIe41-27 for 8 h. C, Phosphorylation status of EphA2 and p27 expression in SBC-5 cells treated with either DMSO or 100 nM of dasatinib for 8 h. D, Flowcytometric analysis of the phosphorylation status of Rb in SBC-5 cells transfected with either control (left) or siEphA2 #2 siRNA (right). E, Quantification of relative number of phospho-Rb-negative SBC-5 cells transfected with either control or siEphA2 #2 siRNA. F, Phosphorylation status of Rb in SBC-5 cells treated with DMSO or 300 nM of ALWeIIe41-27 for 24 h. G, Quantification of relative number of phospho-Rb-negative SBC-5 cells treated with either DMSO or ALW-II-41-27. H, Phosphorylation status of Rb in SBC-5 cells treated with DMSO or 100 nM of dasatinib for 24 h. I, Quantification of relative number of phospho-Rb-negative SBC-5 cells treated with either DMSO or dasatinib. Anti-phosophorylated Rb specific antibody was used in these flowcytometric analysis. The experiments were repeated at least thrice. *P < 0.05, **P < 0.01.
dasatinib was performed in patients with chemosensitive-relapsed SCLC. In that study, dasatinib did not show satisfactory antitumor effect with a disease control rate of 16% [23]. One possible explanation of this insufficient outcome of dasatinib is that activating
driver mutations have not been identified in c-Kit and c-SRC, the two molecules regarded as target molecules of dasatinib in SCLC [24]. However, we presume that dasatinib or other EphA2 inhibitor will exert antitumor effect against EphA2-positive SCLC through
Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076
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preventing EphA2 from transactivating various kinds of other RTKs. Although the incidence of EphA2-positive SCLC is relatively rare (Fig. 1AeC), EphA2-targeted therapy would bring about a clinical benefit in patients with SCLC when applied to the appropriate candidates selected by using EphA2 expression as a biomarker. In conclusion, EphA2-targeted therapy is promising for a subset of patients with SCLC. It would be more practical to use EphA2targeted therapy in combination with cytotoxic drugs because EphA2 inhibition can cause cell cycle arrest but cannot induce critical apoptosis to SCLC cells. Author contributions H$I., T.M., O.M., and T.K., conceived the study and designed experiments; H$I., T.M., O.M., D.H., and H.K. carried out experiments; H$I., T.M., O.M., H$K., Y$N., M.N., Y$K., E.F., Y$N., S$K., and E.S. analyzed the data; H$I., T.M., K.M., R.T., T.Y., K$K., and T.K., wrote the manuscript, which was reviewed and edited by the other coauthors. Disclosure of potential conflict of interests No potential conflicts of interest were disclosed. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (18K15962 to T.M., 18K15963 to S$K., 18K08161 to T.K.). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.076. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.076 References [1] J.K. Sabari, B.H. Lok, J.H. Laird, et al., Unravelling the biology of SCLC: implications for therapy, Nat. Rev. Clin. Oncol. 14 (2017) 549e561. [2] L.A. Byers, C.M. Rudin, Small cell lung cancer: where do we go from here? Cancer 121 (2015) 664e672. [3] H. Mamdani, R. Induru, S.I. Jalal, Novel therapies in small cell lung cancer,
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Please cite this article as: H. Ishigaki et al., EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.076