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Etoposide and Topoisomerase II Inhibition for Aggressive Prostate Cancer: Data from a Translational Study

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Etoposide and Topoisomerase II Inhibition for Aggressive Prostate Cancer: Data from a Translational Study Etoposide and topoisomerase II inhibition for aggressive prostate cancer: data from a translational study. Carlo Cattrini ( [email protected] ) Centro Nacional de Investigaciones Oncológicas https://orcid.org/0000-0003-4785-9480 Matteo Capaia Department of Internal Medicine and Medical Specialties (DIMI), School of Medicine, University of Genoa, Genoa, Italy Francesco Boccardo Department of Internal Medicine and Medical Specialties (DIMI), School of Medicine, University of Genoa, Genoa, Italy. Academic Unit of Medical Oncology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy Paola Barboro Academic Unit of Medical Oncology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy Research Keywords: prostate cancer, mCRPC; VP-16; etoposide; TOP2A; NEPC; AVPC; translational study Posted Date: July 17th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-42290/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on October 19th, 2020. See the published version at https://doi.org/10.1016/j.ctarc.2020.100221. Page 1/19 Abstract Background. Etoposide phosphate (VP-16) is a topoisomerase 2 (TOP2) inhibitor that demonstrated limited activity in patients with metastatic castration-resistant prostate cancer (mCRPC). In our study, we investigated the sensitivity of prostate cancer (PCa) cells (LNCaP, 22Rv1, PC3, DU145, PDB and MDB) to VP-16 and the possible relationship between VP-16 activity and TOP2 expression. We also compared the activity of VP-16 with that of docetaxel, enzalutamide and olaparib. In the datasets analysis, we explored the prevalence and clinical signicance of TOP2 genetic and transcriptomic alterations in mCRPC. Methods. Cell cultures and crystal violet cell proliferation assays were performed. Specic antibodies were used in western blots analyses of cell protein extracts. Large scale datasets were analyzed in cBioportal and STRING was used for the functional enrichment analysis. Results. VP-16 was active in all PCa cell lines analyzed and this drug demonstrated increased activity in the aggressive PC3 and DU145 cells. VP-16 was more cytotoxic compared to the other treatments, except for LNCaP and 22Rv1, which were more sensitive to docetaxel. Of note, the maintainment of antiandrogen treatment in bicalutamide-resistant MDB and PDB increased sensitivity to VP-16, docetaxel and enzalutamide. Compared to LNCaP, TOP2A was found to be overexpressed in 22Rv1, DU145 and PC3, whereas TOP2B was overexpressed in 22Rv1 and PDB. In the mCRPC datasets analysis, TOP2A mRNA overexpression was associated with worse patients’ prognosis, with the molecular features of neuroendocrine prostate cancer (NEPC) and with lower androgen receptor (AR) score. Patients overexpressing TOP2A mRNA were more likely to harbor RB1 loss. Conclusions. Specic subpopulations of patients with aggressive variant prostate cancer (AVPC) could benet from VP-16 treatment. TOP2A overexpression, rather than TOP2B, might be a good biomarker to predict response to VP-16. Further investigations are warranted to determine the role of TOP2A in mCRPC and to assess the ecacy of VP-16, alone or in combination with other agents, for the treatment of patients with mCRPC. Key points: · Etoposide is a chemotherapeutic agent that shows signicant activity in preclinical models of prostate cancer and increased activity is observed in the most aggressive cells. · Overexpression of topoisomerase II alfa and decreased functionality of the androgen receptor signaling seem to be associated with response to etoposide. · Specic patients with castration-resistant prostate cancer could benet from etoposide treatment, alone or in combination with other agents. 1. Introduction Page 2/19 Topoisomerase II (TOP2) inhibitors have a long history in the treatment of prostate cancer (PCa). Mitoxatrone was one of the rst chemotherapeutic agents to be approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) in 1996. Tannock and colleagues published a phase 3 study that demonstrated its benet on pain palliation[1]. However, this drug has never shown a survival advantage in patients with PCa and its use is currently reserved to selected patients without any further treatment option[2]. Same fate has occurred to etoposide phosphate, commonly known as VP-16. This TOP2 inhibitor in combination with estramustine phosphate was included in the National Comprehensive Cancer Network (NCCN) guidelines for the treatment of mCRPC since 1997[3]. However, given the inconvenient risk-benet ratio, this combination has been progressively abandoned by the majority of the centers. The use of etoposide in PCa is currently limited to combination with platinum agents to treat the aggressive forms of neuroendocrine PCa (NEPC)[4]. Both isoforms of TOP2, TOP2A and TOP2B, are the targets of mitoxantrone and etoposide. TOP2A is tightly related to cell proliferation, whereas TOP2B is also expressed in non-dividing cells[5]. TOP2A is repressed by RB1 and TP53/DREAM pathway and it is upregulated in presence of RB1 or TP53 inactivation[6, 7]. This isoenzyme enhances androgen-receptor (AR) signalling and it has been associated with PCa aggressiveness, metastatic progression and reduced survival[8-12]. Haffner and colleagues have also revealed the strict relationship between AR and TOP2B, and this isoenzyme has been involved in the production of TMPRSS2-ERG fusions, which are frequently observed in patients with PCa[13]. Etoposide acts as a TOP2 poison through the trapping and stabilization of TOP2-DNA complexes, generating DNA breaks[5]. Mitoxantrone, similarly to doxorubicin, stabilizes these TOP2-DNA complexes less eciently than etoposide and acts as a TOP2 catalytic inhibitor at high concentrations, rather than a TOP2 poison[14]. Given the dose-dependent stabilization of TOP2-DNA complexes induced by etoposide, this drug appears to be the best candidate to exert cytotoxic activity in PCa that overexpress TOP2. In addition, etoposide has been shown to potently inhibit AR ligand binding, further supporting its use in the context of PCa[15]. In the present study, we analyzed the effect of etoposide in different in vitro models of PCa compared to other standard treatments and the possible relationship between the expression of TOP2 isoforms and etoposide activity. We also explored the association between TOP2 expression and clinical data from patients with mCRPC, in order to provide useful information for the selection of patients potentially candidates to etoposide treatment. 2. Materials And Methods 2.1. Cell cultures and chemicals In vitro experiments were conducted using cell models that harbor distinct molecular alterations and mimic different phases of PCa progression (Table S1): LNCaP (hormone-sensitive), 22Rv1 (AR-V7 Page 3/19 variant), PC3 and DU145 (mCRPC). PDB and MDB cells were obtained from LNCaP following the selective pressure of the antiandrogen bicalutamide, as previously described[16]. The human prostate cancer cell lines LNCaP, 22Rv1, DU145 and PC3 were purchased from American Type Culture Collection (CRL1740, CRL2505, HTB81, CRL1435) and cultured in RPMI 1640 medium added with 10% FBS, 2mM glutamine and 1% penicillin/streptomicin solution (all from Celbio, Milano, Italy). The medium for LNCaP and 22Rv1 was also supplemented with 0.25% glucose, 10 mM sodium pyruvate and 10 mM HEPES. Bicalutamide- resistant cell lines PDB and MDB were cultured in phenol red-free RPMI-1640 containing 10% charcoal stripped fetal bovine serum, 1% penicillin, 1% streptomycin, 1% glutamine, 10 µM of bicalutamide (Sigma), with or without 0.1 nM 5-α-dihydrotestosterone (DHT, Sigma) for PDB and MDB respectively. The mycoplasma testing in all cell lines was checked routinely. The STR analysis for authenticating cell lines was carried out in the Biological Bank of our institute. Etoposide (341205) was from Merck-Millipore while enzalutamide (MDV3100), docetaxel (S1148) and olaparib (AZD2281) were from Sellechem. For each chemical, concentrate solution in DMSO was prepared, maintained at -20 °C and freshly diluted in appropriate medium in each experiment. 2.2. Cell proliferation assays The crystal violet assay was performed to evaluate the treatment effects of chemotherapeutic agents on cell proliferation. Cells were seeded at different concentrations depending on their doubling time (Table S1) in 96-well plates and treated for 120h with various concentrations of etoposide (0.1-10 µM), docetaxel (0.1-10 nM), enzalutamide (0.1-20 µM) and olaparib (1-20 µM). In the middle of treatment fresh medium with drug was added. Data of cell proliferation were calculated as percentages of the absorbance value read for untreated cell (0.1% DMSO). At least three independent experiments, each performed 10 times, were done. Using ED50plus v1.0 software, we evaluate the drug concentration required for 50% growth inhibition of the cells (IC50). To compare etoposide treatment responses among PCa in vitro models, we evaluated for each cell line the drug concentration required for 50% viability inhibition (IC50) and its ratio to IC50 of LNCaP used as reference (Rln= IC50 cell line evaluated / IC50 LNCaP). If Rln<1, etoposide treatment was more effective than in LNCaP cell line, whereas if Rln>1 etoposide was less effective than in LNCaP. To test the effectiveness of etoposide treatment in comparison with other antineoplastic agents, we evaluate the ratio of cell growth inhibition of each drug to etoposide
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