1 Hyperactive mTOR Induces Neuroendocrine Differentiation in Prostate Cancer Cell 2 with Concurrent Up-regulation of IRF1 3 4 Authors: Mayuko Kanayama1, 2, Toshiya Hayano3, Michinori Koebis2, Tatsuya Maeda4, Yoko 5 Tabe5, Shigeo Horie1, and Atsu Aiba2 6 7 Affiliations: 1Department of Urology, Juntendo University Graduate School of Medicine, 8 Tokyo, Japan, 2Laboratory of Animal Resources, Center for Disease Biology and Integrated 9 Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 10 3Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 11 Shiga, Japan, 4Institute of Molecular and Cellular Biosciences, The University of Tokyo, 12 Tokyo, Japan, 5Department of Clinical Laboratory Medicine, Juntendo University Graduate 13 School of Medicine, Tokyo, Japan. 14 Correspondence: Atsu Aiba, Laboratory of Animal Resources, Center for Disease Biology 15 and Integrated Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 16 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: +81-3-5841-3638, Fax: +81-3-5841-3679, 17 E-mail: [email protected] 18 The running title: The role of IRF1 in active mTOR-induced NED 19 This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative 20 Areas (Comprehensive Brain Science Network), Grant Numbers 221S0003 (to A.A.), and 21 Grant-in-Aid for Scientific Research (B), JSPS KAKENHI Grant Numbers 25291042 and 22 17H03802 (to T.M.) from the Ministry of Education, Science, Sports and Culture of Japan. 23 Disclosure of Potential Conflicts of Interest: The authors have no conflict of interest. 1 1 Abstract 2 BACKGROUND 3 Neuroendocrine-differentiated prostate cancer (NEPCa) is refractory to androgen deprivation 4 therapy and shows a poor prognosis. The underlying mechanisms responsible for 5 neuroendocrine differentiation (NED) are yet to be clarified. In this study, we investigated the 6 role of mammalian target of rapamycin (mTOR) in NEPCa. 7 METHODS 8 We utilized a gain-of-function analysis by establishing a human PCa LNCaP stable line that 9 expresses hyperactive mTOR (LNCaP-mTOR). Then, we employed a comprehensive mass 10 spectrometric analysis to identify a key transcription factor in LNCaP-mTOR, followed by a 11 loss-of-function analysis using CRISPR/Cas system. 12 RESULTS 13 The activation of mTOR induced NED. We observed significant cell growth arrest in NED of 14 LNCaP-mTOR, which accompanied increased expression of p21WAF1/CIP1. A comprehensive 15 mass spectrometric analysis identified interferon regulatory factor 1 (IRF1) as a key 16 transcription factor in growth arrest of LNCaP-mTOR. The disruption of IRF1 gene in 17 LNCaP-mTOR reversed cell growth arrest along with the suppression of its target p21WAF1/CIP1. 18 These results indicate that the growth arrest in NED is at least in part dependent on IRF1 19 through the induction of p21WAF1/CIP1. 20 CONCLUSIONS 21 We identified active mTOR as a novel inducer of NED, and elucidated a mechanism 22 underlying the malignant transformation of NEPCa by recapitulating NED in vitro. 23 Keywords: Hyperactive mTOR, Interferon Regulatory Factor 1, LNCaP, NED 2 1 1 INTRODUCTION 2 To date, several reports have suggested that neuroendocrine (NE) differentiation (NED) is 3 responsible for progression of prostate cancer (PCa) to hormone-refractory state.1-3 4 NE-differentiated PCa (NEPCa) is thought to accelerate PCa progression through several 5 mechanisms.2,4,5 Nevertheless, the underlying mechanisms responsible for NED development 6 are yet to be fully clarified. 7 Meanwhile, the activation of the PI3K/Akt/mTOR pathway is a frequent event in many types 8 of cancers including PCa, such as PTEN deletion and activating mutations in PIK3CA.6 9 Interestingly, the activation of mTOR pathway has been reported in NE tumors of other 10 tissues.7,8 Thus, we hypothesized that activation of mTOR may induce NED in PCa as well. 11 To prove this hypothesis and investigate the molecular mechanisms of NEPCa progression, 12 we performed a gain-of-function analysis by establishing a PCa stable line that expresses 13 hyperactive mTOR. 14 Here, we demonstrate that hyperactive mTOR induces NED in LNCaP with matching 15 phenotypes reported earlier including the growth arrest,9-16and this growth arrest is at least in 16 part dependent on interferon regulator factor 1 (IRF1) through the induction of p21WAF1/CIP1. 17 In LNCaP stably expressing hyperactive mTOR (LNCaP-mTOR), we focused on IRF1 18 based on the results of a comprehensive proteomic analysis. Furthermore, IRF1 knockout in 19 LNCaP-mTOR by CRISPR/Cas system resulted in a partial recovery from growth arrest. 20 Together, we elucidated the mechanism underlying the malignant transformation of NED by 21 generating two types of NED models in vitro depending on IRF1 gene status. 3 1 2 MATERIALS AND METHODS 2 2.1 Generation of a stable line expressing active mTOR with the Tet-On 3G system 3 The tetracycline-inducible (Tet-on) 3G bidirectional expression system was purchased from 4 Clontech (Montain View, CA, USA). A stable line that expresses EGFP and active mTOR 5 upon doxycycline (Dox, Sigma-Aldrich, St. Louis, MO, USA) administration 6 (LNCaP-mTOR) was generated in accordance with the manufacturer’s protocol. 7 2.2 Cell lines and cell culture 8 The human PCa cell line LNCaP was purchased from RIKEN BRC (Tsukuba, Japan) cell 9 bank. LNCaP was maintained in the RPMI 1640 medium supplemented with 10% FBS and 10 1×Penicillin-Streptomycin-Glutamine (Gibco, Walthman, MA, USA). When antibiotics were 11 used for selection or induction, they were added to the medium at the following 12 concentrations: G418 at 500 μg/mL for selection and at 100 μg/mL for maintenance, 13 puromycin at 0.25 μg/mL for both selection and maintenance, hygromycin at 100~125 μg/mL 14 for both selection and maintenance, and Dox at 1 μg/mL for induction of the Tet-On system. 15 For the inhibition of mTOR pathway, cells were incubated with an mTOR inhibitor, 16 rapamycin (Cell Signaling Technology (CST), Danvers, MA, USA), for 7 days at 17 concentrations of 100 nM, 10 nM, 1 nM, or 0.1 nM. For cell counting, 4.6×105 cells of 18 LNCaP were seeded into one dish with or without Dox. The number of cells was counted 19 manually with a hemocytometer. 20 2.3 Xenotransplantation of LNCaP-mTOR and immunohistochemical analysis 21 Experimental procedures were approved by the Institutional Animal Care and Use Committee 22 of the University of Tokyo (Permit Number: M-P14-011). Eight NOD/SCID mice at 7 weeks 23 of age were subcutaneously implanted with 1.0×107 LNCaP-mTOR suspended in Matrigel 24 HC (Corning, New York, USA) at one site of each flank. Mice were given either 1 mg/mL 25 Dox or pure water. After 4~6 months, when tumors became palpable (100 mg~1 g in wet 4 1 weight), animals were euthanized and tumors were excised. Rapamycin was administrated at 2 a dose of 2 mg/kg every other day by intraperitoneal injection for 1 month after tumors 3 became palpable, during which oral Dox administration was continued. For a histological 4 analysis, excised tumors were fixed with 4% formaldehyde, then, paraffin sections were made 5 in accordance with standard protocols. Antibodies used for immunohistochemistry were 6 anti-phospho-S6 ribosomal protein Ser235/236 (1:200; CST 2211), anti-NSE (1:200; a gift 7 from Dr. Sakimura at Niigata University) and anti-chromogranin A (1:100; ab15160, from 8 abcam, Cambridge, UK). The staining was developed with DAB substrate. 9 2.4 Analysis of cellular morphology 10 Phase-contrast images of cells were acquired with a light microscope (BZ 8000 KEYENCE, 11 Osaka, Japan). For quantification of the length of cellular processes and the number of 12 processes, phase-contrast images of LNCaP-mTOR cultured with or without Dox were taken on 13 Day 7. For randomly chosen 20 cells, the sum of length of processes and branches stemming 14 from one cell was calculated, and the number of processes per cell was counted, using image-J 15 software. The results were compared by two-tail paired t-test. Also, in order to investigate the 16 effects of Dox withdrawal from Dox-treated cells, LNCaP-mTOR was cultured with Dox for 17 one week, followed by Dox withdrawal from the medium. GFP emission and cellular 18 morphology were serially observed with a microscope. 19 For electron microscopic images, cells were fixed with 2.5% glutaraldehyde in 0.1 M PB for 1 20 h at 4℃, followed by incubation with 1% osmium in 0.1 M PB for 1 h at 4℃. After dehydration 21 with ethanol, embedding in Epon (Epok 812, Okenshoji, Tokyo, Japan) was done in accordance 22 with routine protocols. Thin sections (80 nm) were cut with glass or diamond knife and picked 23 up on grid mesh. Sections were stained with uranyl acetate for 30 min at room temperature, 24 followed by staining with lead citrate for 3 min. Images were captured with a scanning electron 25 microscope (Hitachi HT 7700, Tokyo, Japan). 5 1 2.5 RNA extraction and RT-PCR analysis 2 Total RNA was isolated from cells with TRIzol reagent (Invitrogen, Walthman, MA, USA). 3 Extracted RNA was converted to cDNA using RNA PCR Kit AMV Ver.3.0 (TaKaRa, Shiga, 4 Japan). The equal amount of cDNA was PCR-amplified with TaKaRa Ex Taq. The primer sets 5 used were as follows: the primer pair for IRF1: 5’-AATTCCAACCAAATCCCGGGG-3’ and 6 5’-AGGCATCCTTGTTGATGTCCCAG-3’, IRF6: 5’-GTGCCCATGAACCCAGTGAAG-3’ 7 and 5’-CTGATCCAGCTCATCTTCCTCATC-3’, interferon (IFN)-β: 5’-AGCACTGG 8 CTGGAATGAGACTATTG-3’ and 5’-ACTGCTCATGAGTTTTCCCCTGG-3’, GAPDH: 9 5’-AGCACCAGGTGGTCTCCTC-3’ and 5’-CCCTGTTGCTGTAGCCAAATTC-3’.