Poziotinib suppresses ovarian cancer stem cell growth via inhibition of HER4-mediated STAT5 pathway
Heejin Lee a, b, Jun Woo Kim a, b, Dong Kyu Choi a, Ji Hoon Yu a, b, Jae Ho Kim c,
Dong-Seok Lee b, Sang-Hyun Min a, *
a New Drug Development Center, DGMIF, 80 Chumbok-ro, Dong-gu, Daegu, 41061, Republic of Korea
b School of Life Sciences and Biotechnology, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea
c Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Gyeongsangnam-do, Republic of Korea
Abstract
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with an overall 5-year survival rate of only 30%. EOC is associated with drug resistance, frequent recurrence, and poor prog- nosis. A major contributor toward drug resistance might be cancer stem cells (CSCs), which may remain after chemotherapy. Here, we aimed to find therapeutic agents that target ovarian CSCs. We performed a high-throughput screening using the Clinical Compound Library with a sphere culture of A2780 EOCs. Poziotinib, a pan-human epidermal growth factor receptor (HER) inhibitor, decreased sphere formation, viability, and proliferation, and induced G1 cell cycle arrest and apoptosis in ovarian CSCs. In addition, poziotinib suppressed stemness and disrupted downstream signaling of Wnt/b-catenin, Notch, and Hedgehog pathways, which contribute to many characteristics of CSCs. Interestingly, HER4 was over- expressed in ovarian CSCs and Poziotinib reduced the phosphorylation of STAT5, AKT, and ERK, which are regulated by HER4. Our results suggest that HER4 may be a promising therapeutic target for ovarian CSCs, and that poziotinib may be an effective therapeutic option for the prevention of ovarian cancer recurrence.
1. Introduction
Epithelial ovarian cancer (EOC) has the fourth highest mortality rate among gynecological malignancies worldwide, and the sur- vival rate of patients with stage three or higher EOC is less than 50% [1]. Treatment usually involves surgery and chemotherapy, and sometimes radiotherapy. Chemotherapy in ovarian cancer (OC) typically consists of the platinum-based drug, cisplatin, and the taxane-based drug, paclitaxel. Although OC is initially chemo- responsive, the majority of patients experience a relapse with chemoresistance within 5 years [2]. The major cause of OC recur- rence has been known as the cancer stem cell (CSC). CSCs are defined as subpopulations of cells within a tumor that possess the capacity of self-renewal and generate heterogeneous lineages of cancer cells in the tumor [3]. CSCs reside in various solid tumors,where these subpopulations play a critical role in tumor initiation, progression, metastasis, recurrence, and drug resistance [4].
The characteristics of CSCs are regulated by various signaling pathways such as Wnt/beta-catenin, Notch, and Hedgehog (Hh) [5]. Recent studies have shown that the expression of these signaling molecules is higher in CSCs than in non-CSCs. The Wnt/b-catenin canonical signaling pathway appears to be involved in the main- tenance of stemness in both embryonic cells and CSCs [6]. The extracellular Wnt ligand binds to Frizzled cell surface receptors, leading to increased cytoplasmic b-catenin levels and their subse- quent translocation to the nuclei, followed by the induction of key Wnt target genes [6]. The Notch canonical signaling pathway plays an importance role in cell differentiation, proliferation, and apoptosis [7]. Activated Notch signaling increases the transcription of target genes such as HES1 and c-MYC [7]. The Hedgehog pathway regulates embryonic development, cell differentiation, regenera- tion, and stem cell biology [8]. Hedgehog ligands bind to the seven- transmembrane domain receptor Patched (Ptch). Ligand binding to Ptch releases Ptch binding to Smoothened (Smo), a second seven- transmembrane domain receptor protein. This results in a conformational change in Smo and subsequent activation of the downstream signaling pathway, leading to the activation of Gli (Gli1, Gli2, and Gli3) and subsequent transcription of target genes such as Gli1, Ptch1, and hip1. Therefore, to overcome this limitation of CSC-related metastasis, recurrence, and drug resistance, novel treatments are needed to effectively target CSCs.
Poziotinib is an epithermal growth factor receptor (EGFR/HER1, 2, and 4) inhibitor developed and investigated by Hanmi Pharma- ceutical in phase II clinical trials for the treatment of non-small cell lung and breast cancer. Members of the EGFR tyrosine kinase family, including EGFR (also called HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4), are overexpressed in a variety of tumors [9]. Through homo- or hetero-dimerization, EGFR family members activate several downstream pathways, including STAT, PI3K/AKT, and Ras/Raf/MAPK/ERK signaling pathways that lead to the growth and survival of tumor cells and CSCs [10]. Several pre- clinical and clinical studies have demonstrated the benefits of EGFR inhibition, especially of HER1, 2, and 3 for tumors and CSCs [11]. However, the role of HER4 in CSCs remains unclear. In this study, we aimed to carry out screening of drugs targeting ovarian CSCs using sphere-forming cells (A2780-SP and SKOV3-SP) derived from EOCs (A2780 and SKOV3). Based on our finding that the irreversible pan- HER inhibitor, poziotinib, showed anticancer effects against ovarian CSCs by reducing stemness and inducing apoptosis in CSCs, we further investigated the effects of poziotinib on HER4- overexpressing ovarian CSCs, and demonstrated its potential ben- efits for the treatment of EOC.
2. Materials and methods
2.1. Two-dimensional (2D) and three-dimensional (3D) spheroid cell culture and treatment
EOC cell lines, A2780 cells were cultured in RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Hyclone). SKOV3 cells were pur- chased from American Type Culture Collection (ATCC, USA). SKOV3 cells were cultured in McCoy’s 5A medium (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/strepto- mycin (Hyclone). A2780-SP and SKOV3-SP cells were cultured in CSC medium, i.e., complete medium (CM) containing Neurobasal medium (NBM, Gibco) supplemented with B-27 (Gibco), 10 ng/mL basic fibroblast growth factor (R&D Systems), 20 ng/mL human epidermal growth factor (R&D Systems), Glutamax (Gibco), 4-(2- hydroxyethyl)-1-piperazine ethanesulfonic acid (Gibco), and 2.5 mg/mL amphotericin B (Gibco) in ultra-low attachment (ULA) 100 mm2 plates (Corning). Sphere culture medium (CM) was changed every 2e3 days. Spheres were dissociated into single cells by treatment with Accutase (Gibco). Poziotinib, cisplatin (Med- ChemExpress), and dimethyl sulfoxide (Sigma) were used to treat the cells.
2.2. Cell viability
Ovarian cancer cells (A2780, SKOV3) were seeded in 96-well cell culture plates (Corning) at a density of 1500 cells/well. Ovarian CSCs (A2780-SP, SKOV3-SP) were seeded in ULA round-bottom 96- well plates (Corning) at a density of 1500 cells/well. After centri- fuging the plates at 3000 rpm for 3 min, the plates were incubated for 24 h. The indicated concentrations of compounds were added to the cells and incubated for a total of 6 days. After every 3 days of incubation, the supernatant medium was replaced with fresh me- dium containing compounds. Sphere cells in each well were imaged using EVOS® (Thermo Scientific). We measured the sphere size using ImageJ software (NIH Image). After imaging, sphere cell viability was assessed by CellTiter-Glo® (Promega), and luciferase activity was measured using a Tecan plate reader (Biocompare).
2.3. Western blotting
Protein extraction solution (Radioimmunoprecipitation assay buffer containing phosphatase inhibitor and protease inhibitor cocktail) was used to obtain whole cell lysates. Nuclear and cytosol fractions were prepared using a Nuclear and Cytoplasmic Isolation kit (Thermo Scientific). Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blot analysis. After blocking with 5% skimmed milk, the membranes were initially incubated with primary antibodies in blocking buffer overnight at 4 ◦C, followed by HRP-conjugated secondary antibodies for 2 h at room temperature. The primary antibodies used were directed against Vinculin (Santa Cruz Biotechnology, Inc., sc-73614), GAPDH (Santa Cruz Biotechnology, Inc., sc-47724), Lamin B (R&D Systems, MAB8525), ALDH (BD Bio- sciences, #611194), CD133 (Abcam, ab19898), OCT4 (Santa Cruz Biotechnology, Inc., sc-8682), NANOG (Cell Signaling Technology, #3580S), KLF4 (GeneTex, GTX101508), phospho-b-catenin (S552) (Cell Signaling Technology, #9566S), b-catenin (Cell Signaling Technology, #8480S), cleaved PARP (Cell Signaling Technology, #5625S), PARP (Cell Signaling Technology, #9542S), BCL2 (Santa Cruz Biotechnology, Inc., sc-7382), MCL1 (Cell Signaling Technol- ogy, #4572S), Cyclin D1 (Cell Signaling Technology), phospho-AKT (Ser473) (Cell Signaling Technology, #3787S), AKT (Cell Signaling Technology, #9272S), phospho-ERK (Thr202/Tyr204) (Cell Signaling Technology, #9101S), ERK (Cell Signaling Technology, #9102S), phospho-STAT5 (Y694) (Cell Signaling Technology, #9351S), and STAT5 (Cell Signaling Technology, #94205S). The secondary antibodies used were goat anti-mouse IgG-HRP (R&D Systems) and goat anti-rabbit IgG-HRP (R&D Systems). Signals were developed with enhanced chemiluminescence HRP substrate (Bio-Rad) and detected using LAS-3000 mini imager (Fuji film). Signal intensities were calculated with ImageJ software (NIH Image).
2.4. Sphere cell proliferation assay
A2780-SP cells were seeded in ULA flat bottom, 96-well plates (Corning) at a density of 6000 cells/well. After 24 h, indicated concentrations of the compounds were added. After 7 days, we analyzed the spheres using IncuCyte (BioTek).
2.5. Flow cytometry
To isolate cells with ALDH activity, an ALDEFLUOR™ assay kit (STEMCELL Technologies) was used according to the manufacturer’s instructions. Allophycocyanin (APC) mouse anti-human CD117 and CD133 (BD Biosciences) were used to isolate CD117 and CD133 cells. After trypsinization, cells were washed with 1 mL cold phosphate-buffered saline (PBS) with centrifugation at 500g for 5 min. Cells were suspended in PBS and incubated with anti-CD117 and anti-CD133 antibodies. After incubation for 30 min on ice in the dark, cells were washed twice with PBS and resuspended in PBS. The CD117 and CD133 cells were isolated by using a flow cytometry cell sorter (BD FACS AriaIII).
2.6. RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the sample using a TRIzol RNA extraction kit (GeneAll) according to the manufacturer’s in- structions, and 2 mg of the RNA was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit (Applied Biosystems). The synthesized cDNA was amplified by PCR using SYBR Green PCR Master Mix (Applied Biosystems) and a StepOne Real-Time PCR system (Applied Biosystems) with the indicated primers. GAPDH was used as the reference gene. The results are presented relative to control using the ddCt method. The primers used in these experiments were GAPDH (F: 50-GGAGCCAAAAGGGTCATCAT-30, R: 50-GTGATGGCATGGACTGTGGT-30),c-MYC (F: 50-
GGCTCCTGGCAAAAGGTCA-30, R: 50-CTGCGTAGTTGTGCTGATGT-30), GLI-1 (F:50-AGCGTGAGCCTGAATCTGTG-30,R:50-CAGCATG- TACTGGGCTTTGAA-30), HIP-1 (F: 50-ACACGCCAGAACGTGCATA-30, R: 50-CACTGCGTTGCTAGACAGAG-30), PTCH-1 (F: 50-CCAGAAAGTA- TATGCACTGGCA-30, R: 50-GTGCTCGTACATTTGCTTGGG-30), EGFR (F: 50-AGGCACGAGTAACAAGCTCAC-30,R:50-ATGAGGACA- TAACCAGCCACC-30),ERBB2 (F: 50-TGTGACTGCCTGTCCCTACAA-30, R:50-CCAGACCATAGCACACTCGG-30),ERBB4(F:50-GCA- GATGCTACGGACCTTACG-30,R: 50-GACACTGAGTAACACATGCTCC- 30), Pik3r1 (p85a) (F: 50-CTGAAGCTGACACGGAGCAGC-30, R: 50- GACGTGTACGTCGATCATCTC-30),Pik3ca(p110a)(F:50-GTGTGTGGCTGTGACGAATAC-30,R:50-CTATCAATCGGCAGCTGAGAG-30).
2.7. Apoptosis (Annexin V (AV)/propidium iodide (PI) staining) and cell cycle (PI staining) analysis
A2780-SP cells were seeded in ULA 6-well plates at a density of 1 × 106 cells/well and grown in CSC medium. After 24 h, the A2780-SP cells were treated with compound(s). Sphere cells were grown for 24 h at 37 ◦C in a humidified atmosphere containing 5% CO2. After 24 h, sphere cells were harvested by centrifugation. The supernatant was decanted, and the cells were gently re-suspended in PBS. The cells were washed once with PBS. The pelleted cells were re-suspended in 0.3 mL of PBS. To fix the cells, 0.7 mL cold ethanol was gently added dropwise to the tube containing 0.3 mL of cell suspension in PBS and left on ice for 1 h. The cells were centrifuged, washed once with cold PBS, and re-centrifuged. For Annexin V (AV)/ propidium iodide (PI) staining, the cell pellet was re-suspended in 0.1 mL of AV binding buffer, followed by the addition of 5 mL of FITC AV and 5 mL PI (BD Biosciences). For PI staining, the cell pellet was re-suspended in 0.1 mL of PBS, followed by the addition of 2 mL of 10 mg/mL RNase A and incubated at 37 ◦C for 1 h. After 1 h, 5 mL of PI solution (BD Biosciences) was added. In both AV/PI and PI staining, the cells were gently vortexed and incubated for 15 min at room temperature in the dark, followed by the addition of 400 mL of AV binding buffer (AV/PI staining) or cold PBS (PI staining) to each tube. Flow cytometry was performed within 1 h.
2.8. Statistical analysis
All data are expressed as mean ± standard deviation (SD) of 2 or 3 independent experiments. Statistically significant differences were determined using t-test or One-way ANOVA with GraphPad Prism 5 (GraphPad, Inc., La Jolla, CA, USA). P < 0.05 was considered to be statistically significant. 3. Results 3.1. Poziotinib selectively inhibited sphere formation and stemness of ovarian CSCs To study selective inhibition of ovarian CSCs, we used epithelial ovarian CSCs (A2780-SP), which have characteristics of CSCs, such as the expression of stem cell markers, high tumorigenic potential, and drug resistance [12]. A2780-SP spheres were generated from A2780 cells when cultured in a low attachment state in CM. For sphere viability measurements, A2780-SP cells were seeded to form spheres and then treated with compounds from the Clinical Compound Library at 10 mM. Sphere viability was measured after 6 days of incubation. This screening revealed that poziotinib reduced sphere viability by more than 90% compared to that in DMSO- treated controls (not shown). The results of the screening were reconfirmed in A2780 and A2780-SP cells by calculating the GI50 values (Fig. 1A). The GI50 value of cisplatin was approximately 3 times higher in A2780-SP cells than in A2780 cells (Fig. 1A), indi- cating greater reduction in viability of A2780 cells by cisplatin compared to that in A2780-SP cells, and hence the resistance of the CSCs to cisplatin, a standard drug for OC. The GI50 value of pozio- tinib was about 12 times lower in A2780-SP cells than in A2780 cells (Fig. 1A). Moreover, poziotinib decreased sphere size and viability in a dose-dependent manner in A2780-SP cells (Fig. 1B). Similar results were observed for SKOV3 cells (Fig. 1C). Poziotinib was more efficient than cisplatin in reducing the viability of SKOV3-SP cells, which are CSC-enriched spheres of SKOV3 cells (Fig. 1C and D). As CSCs with markers, such as ALDH, CD117, and CD133, in A2780 cells have shown similar characteristics as CSCs induced by sphere formation [5], we sorted ALDHþ, CD117þ, and CD133þ cells from the A2780 cell population and treated them with cisplatin and poziotinib at 10 mM for 6 days. Results similar to those obtained with A2780-SP cells were observed in sorted CSCs. Poziotinib effectively reduced the viability of sorted CSCs compared to that of A2780 cells (Fig. 1E). We determined the effects of poziotinib on the proliferation of A2780-SP cells using real-time live imaging. Poziotinib reduced not only viability, but also prolif- eration in a dose-dependent manner (Fig. 1F). Next, we tested whether poziotinib affected the protein levels of stemness markers in CSCs. A2780-SP cells treated with CSC medium showed increased expression of stemness markers, including ALDH, CD133, OCT4, NANOG, and KLF4, compared with those treated with NBM (non- CSCs) (Fig. 1G). Poziotinib dose-dependently reduced the protein levels of ALDH, CD133, OCT4, NANOG, and KLF4 (Fig.1G). These data suggest that poziotinib significantly decreased the stem cell prop- erties of A2780-SP cells. Fig. 1. Poziotinib decreased growth and stemness of ovarian CSCs.(A), (C) The GI50 graph of each drug from the ATP-based cell viability assay in A2780, A2780-SP, SKOV3, and SKOV3-SP cells. Cells were treated with cisplatin and poziotinib for 6 days. (B), (D) A2780-SP and SKOV3-SP cells were treated with cisplatin or poziotinib at the indicated concentration. (B) Representative image of A2780-SP cells after 6 days of treatment (upper panel), and the sphere size and viability were quantified (bottom panel). (D) The sphere size and viability were quantified in SKOV3-SP cells after 6 days of treatment. (E) ATP-based cell viability assay of A2780, A2780-SP, A2780 cells sorted by stem cell markers (ALDHþ, CD117þ, and CD133þ). Cells were treated with cisplatin or poziotinib (10 mM) for 6 days. (F) Proliferation rates of A2780-SP cells treated with poziotinib were analyzed using IncuCyte for 7 days. (G) Expression levels of stemness markers were determined by western blotting. A2780-SP cells were treated with poziotinib at the indicated concentrations for 24 h. 3.2. Poziotinib suppressed b-catenin nuclear translocation and downregulated Notch and Hh target genes in ovarian CSCs Wnt/b-catenin, Notch, and Hedgehog (Hh) signaling pathways play important roles in maintaining the properties of CSCs [5]. Therefore, we tested the downstream components of these signaling pathways after treatment with poziotinib. The trans- location of b-catenin to the nucleus was increased when A2780-SP cells were grown in CM, but significantly reduced when treated with poziotinib in a dose-dependent manner (Fig. 2A). Moreover, a concomitant increase in phosphorylation of b-catenin on Ser 552, which is associated with enhanced transcription, was also detected in the A2780-SP cells grown in CM; poziotinib dramatically reduced the phosphorylation of b-catenin (Fig. 2B). We also investigated the mRNA levels of a Notch target gene (c-MYC), and Hh target genes (GLI-1, HIP-1, and PTCH-1) in A2780-SP cells (Fig. 2C). All four target genes were highly expressed in the A2780-SP cells grown in CM compared with those grown in NBM, and these mRNA levels were significantly decreased with poziotinib treatment (Fig. 2C). Thus, poziotinib inhibited Wnt/b-catenin, Notch, and Hh signaling in ovarian CSCs. 3.3. Poziotinib induced G1 cell cycle arrest and apoptosis in ovarian CSCs Poziotinib induces G1 arrest and apoptosis in cancer cells [9], however, its role for cell cycle arrest and apoptosis in ovarian CSCs has not been investigated. Our results also showed that poziotinib increased the population of A2780-SP cells in the G1 phase and decreased the population of these cells in the S and G2-M phases in a dose-dependent manner (Fig. 3A). Furthermore, poziotinib significantly increased the population of AV /PIe and AV / PI cells (Fig. 3B). Poziotinib also increased cleaved PARP protein levels, but reduced the levels of anti-apoptotic proteins, including BCL2 and MCL1, in A2780-SP cells in a dose dependent manner (Fig. 3C). In addition, poziotinib reduced the levels of Cyclin D1 protein, which regulates the G1 phase of the cell cycle in A2780-SP cells (Fig. 3C). These data suggest that poziotinib induced G1 cell cycle arrest and apoptosis in ovarian CSCs. 3.4. Inhibition of HER4 led to a reduction in pSTAT5, pAKT, and pERK in HER4-overexpressing ovarian CSCs Poziotinib is a pan-HER inhibitor [9], but the expression of HER family genes in ovarian CSCs have not been reported yet. Therefore, we checked the mRNA levels of HER family genes (EGFR, ERBB2, ERBB4) in A2780 and A2780-SP cells (Fig. 4A). Among them, ERBB4 (HER4) was highly expressed in A2780-SP cells compared to that in A2780 cells (Fig. 4A). HER4 regulates STAT5, AKT, and ERK signaling pathways [13]. Therefore, we measured protein phosphorylation levels of STAT5, AKT, and ERK in ovarian CSCs after treatment with poziotinib. Poziotinib significantly decreased the phosphorylation of STAT5, AKT, and ERK in a dose-dependent manner (Fig. 4B). STAT5 activates AKT through the expression of p85 regulatory (PIK3R1) and p110 catalytic (PIK3CA) subunits of PI3K [14]. Hence, we checked the mRNA levels of p85a and p110a in A2780-SP cells (Fig. 4C). The expression of p85a and p110a was higher in A2780-SP cells than in A2780 cells, but was decreased after poziotinib treatment (Fig. 4C). Taken together, these data suggest (Fig. 4D) that HER4 showed higher expression in ovarian CSCs than in OC cells, resulting in an increase in the phosphorylation of STAT5, AKT, and ERK, and the transcription of p85a and p110a in HER4-expressing ovarian CSCs. Thus, the signaling pathways of STAT5, AKT, and ERK activated by HER4 might improve the survival and prolifera- tion of ovarian CSCs. Signaling via these pathways was dramatically reduced by poziotinib via inhibition of HER4. Fig. 2. Downstream inhibition of the Wnt, Notch, and Hh pathways. (A) Protein levels of cytosol and nuclear b-catenin were detected in poziotinib-treated A2780-SP cells by immunoblotting, and the results are shown in a graph (right panel). (B) Levels of phospho-b-catenin (S552) and b-catenin were detected in poziotinib-treated A2780-SP cells by immunoblotting, and the results are shown in a graph (right panel). (C) Quantitative RT-PCR for cMYC, GLI-1, HIP-1, and PTCH1 mRNA levels in poziotinib-treated A2780-SP cells. Data are expressed as mean ± standard deviation (SD) of 2 or 3 inde- pendent experiments, *P < 0.05, **P < 0.01, and ***P < 0.001. Fig. 3. Poziotinib induced G1 arrest and apoptosis. (A) Cell cycle was analyzed by propidium iodide (PI) staining in A2780-SP cells 24 h after poziotinib treatment (1, 10 mM). The percentage of cells in the G0, G1, S, and G2-M phases in independent duplicate cultures were plotted (right panel). Bars, ± SD of duplicate cultures. (B) Apoptosis was analyzed by Annexin V (AV)/PI staining in A2780-SP cells 24 h after poziotinib treatment (1, 10 mM). The percentage of AVþ/PIe and AVþ/PI þ cells is expressed in a graph (right panel). (C) Expression of pro- and anti-apoptotic proteins was measured in poziotinib-treated A2780-SP cells (1, 10 mM) by immunoblotting. Fig. 4. Poziotinib inhibited downstream of HER4 signaling in ovarian CSCs. (A) Quantitative RT-PCR for EGFR, ERBB2 (HER2), and ERBB4 (HER4) mRNA levels in A2780 and A2780-SP cells. Data are expressed as mean ± standard deviation (SD) of 3 in- dependent experiments, *P < 0.05. (B) Levels of HER4 and of related phosphorylated protein were detected in poziotinib-treated A2780-SP cells by immunoblotting. (C) Quantitative RT-PCR for p85a and p110a mRNA levels in poziotinib-treated A2780-SP cells. Data are expressed as mean ± standard deviation (SD) of 3 independent experiments, *P < 0.05,**P < 0.01, and ***P < 0.001. (D) A proposed regulatory mechanism of the HER4-mediated STAT5 axis pathway in ovarian CSCs. 4. Discussion Cancer stem cells (CSCs) are a small population of cells in tumor tissue that are thought to induce tumor initiation and differentia- tion [4]. CSCs have the ability to initiate metastasis and are resistant to chemotherapy and radiotherapy [2]. These characteristics are considered as the cause of cancer recurrence and emphasize the need for inhibitors that block CSC activities. We screened com- pounds from the Clinical Compound Library for inhibition of sphere formation and viability in ovarian CSCs. Repositioning of bioactive compounds used in clinical trials is advantageous in terms of reduction in development time and costs. In this study, poziotinib, a pan-HER inhibitor, effectively inhibited sphere formation and viability of ovarian CSCs derived from various EOC cells, such as A2780 and SKOV3, and of CSCs sorted on the basis of their expression of ovarian CSC markers. Our results demonstrate the crucial role played by the EGFR family in maintaining ovarian CSCs. For the first time, we reveal here that HER4 is highly expressed in ovarian CSCs compared to that in OC cells and probably triggers the phosphorylation of AKT, STAT5, and ERK in the CSCs. Although the mechanisms regulating CSC properties are not yet clear, it has recently been shown that the activation of HER4 increases phosphorylation of STAT5, which increases the transcription of p85a and p110a. Thus, HER4 activates the PI3K/AKT pathway, and signals from phosphorylated AKT, STAT, and ERK might increase the survival and proliferation of ovarian CSCs. EGFR or HER2 may modulate the STAT3 signaling cascade to regulate stemness, pro- liferation, and survival of cancer and CSCs [15]. However, the role of HER4-mediated STAT5 in ovarian CSCs and the effects of poziotinib on HER4-overexpressing ovarian CSC growth have not yet been reported. Thus, different members of the EGFR family may affect the maintenance of various CSCs depending on the type of the cancer and the expression levels of these EGFR family members in the CSCs. Wnt/b-catenin, Notch, and Hh signaling pathways regulate the characteristics of CSCs, particularly, the maintenance of stemness [5]. Treatment of ovarian CSCs with poziotinib reduced b-catenin translocation to the nucleus and increased phosphorylation of b- catenin on Ser 552. In addition, poziotinib treatment of ovarian CSCs inhibited the expression of target genes of Notch and Hedgehog signaling pathways, such as c-MYC, GLI-1, HIP-1, and PTCH-1. Thus, poziotinib suppressed b-catenin nuclear trans- location and downregulated Notch and Hh target genes in ovarian CSCs. Poziotinib also induced G1 cell cycle arrest and early apoptosis in ovarian CSCs. In conclusion, this study demonstrates that poziotinib could be used to prevent the recurrence of OC by reducing the stemness and inducing apoptosis of ovarian CSCs through disruption of HER4-mediated STAT5, AKT, and ERK signaling.
Declaration of competing interest
The authors declare no potential conflicts of interest.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) grants (NRF-2015M3A9C7030181 and NRF- 2016M3A9E4947797) funded by the Korean Government.
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