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Molecular Chaperone HSP90 Is Necessary to Prevent Cellular Senescence Via Lysosomal

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Molecular Chaperone HSP90 Is Necessary to Prevent Cellular Senescence Via Lysosomal Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-0613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Molecular chaperone HSP90 is necessary to prevent cellular senescence via lysosomal 2 degradation of p14ARF 1,7 1,7 2,3 2,4 1 1 3 Su Yeon Han , Aram Ko , Haruhisa Kitano , Chel Hun Choi , Min-Sik Lee , Jinho Seo , 5 6 2 2 1 4 Junya Fukuoka , Soo-Youl Kim , Stephen M. Hewitt , Joon-Yong Chung , Jaewhan Song 5 Affiliations and addresses 1 6 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, 7 Seoul, Korea 2 8 Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, 9 National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 3 10 Department of Thoracic Surgery, Shiga University of Medical Science, Otsu 520-2192, 11 Japan 4 12 Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan 13 University School of Medicine, Seoul 135-710, Republic of Korea 5 14 Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, 15 Nagasaki, 852-8523, Japan 6 16 Cancer Cell and Molecular Biology Branch, Division of Cancer Biology, Research Institute, 17 National Cancer Center, Goyang 410-769, Republic of Korea 7 18 These authors contributed equally to this work. 19 Running title: HSP90-mediated p14ARF degradation in NSCLC 20 Key words: p14ARF, HSP90, NSCLC, Lysosome-dependent degradation, Senescence 21 Funding: This work was supported by grants from the Basic Science Research Program of 22 the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT 23 and Future Planning (2014R1A1A1002589) (S Han, A Ko) and from the National Cancer 1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-0613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 24 Center, Korea (NCC-1420300) (J Song). 25 Correspondence to: Jaewhan Song, PhD, Department of Biochemistry, Yonsei University, 26 Sinchon-dong, Seadaemun-gu, Seoul 120-749, Korea. Tel.: +82 2 2123 7631; Fax: +82 2 362 27 9897; E-mail: [email protected] 28 29 The authors have declared that no conflict of interest exists. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 2 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-0613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 47 Abstract 48 The tumor suppressor function of p14ARF is regulated at a post-translational level via 49 mechanisms yet to be fully understood. Here we report the identification of an 50 unconventional p14ARF degradation pathway induced by the chaperone HSP90 in 51 association with the E3 ubiquitin ligase CHIP. The ternary complex of HSP90, CHIP and 52 p14ARF was required to induce the lysosomal degradation of p14ARF by a ubiquitination- 53 independent but LAMP2A-dependent mechanism. Depletion of HSP90 or CHIP induced 54 p14ARF-dependent senescence in human fibroblasts. Premature senescence observed in cells 55 genetically deficient in CHIP was rescued in cells that were doubly deficient in CHIP and 56 p14ARF. Notably, non-small-cell lung cancer cells (NSCLC) positive for p14ARF were 57 sensitive to treatment with the HSP90 inhibitor geldanamycin. Further, overexpression of 58 HSP90 and CHIP with a concomitant loss of p14ARF correlated with poor prognosis in 59 patients with NSCLC. Our findings identify a relationship between p14ARF and its 60 chaperones that suggest new therapeutic strategies in cancers which overexpress HSP90. 61 62 63 64 65 66 67 68 69 3 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-0613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 70 Introduction 71 Non-small-cell lung cancer (NSCLC) constitutes approximately 85-90% of lung cancers, 72 with a 5-year survival rate of 15.9% (1, 2). NSCLC is a primary subtype of lung cancer that 73 displays relative insensitivity to chemotherapy and radiotherapy compared with small-cell 74 lung cancer (SCLC), which accounts for the majority of other lung cancers (3-7). NSCLC 75 displays genetic and cellular heterogeneity such as mutation and amplification of oncogenes 76 including HER2, MET, FGFR1, FGFR2, ALK, ROS1, NRG1, NTRK1 and RET (2, 8-10). In 77 contrast, a small number of inhibitors with specific targets such as EGRF mutation or EML4– 78 ALK fusion are currently available for clinical NSCLC treatment. Other clinical trials using 79 inhibitors that target driver mutations including FGFR, DDR2, KRAS and PI3K are also 80 currently underway (11). 81 HSP90 is a major factor that maintains the stability of a variety of proteins. Thus, 82 perturbation of the status of HSP90 would disturb cellular homeostasis, leading to cancer cell 83 death. HSP90 is involved in various cellular mechanisms such as protein folding and 84 activation, cell cycle control, and transcriptional regulation (12-14). In addition to enabling 85 cancer cells to cope with drastic changes in protein homeostasis, HSP90 displays tumorigenic 86 properties via stabilization of the oncogenic proteins such as HER2, AKT, TERT, Raf1, 87 mutant p53, etc (14-27). Because inhibiting HSP90 leads to destabilization of its client 88 oncoproteins, several HSP90 inhibitors were developed as cancer drugs and underwent 89 clinical trials in various human cancers (28). Treatment with geldanamycin (GA) inhibits 90 HSP90 ATPase activity, which is essential for the degradation of HSP90 client oncoproteins 91 and stimulates cell cycle arrest and apoptosis (29). Recent reports have also suggested that 92 GA treatment induces cellular senescence by an unknown mechanism (30). Although HSP90 4 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-0613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 93 may play a role in cellular senescence through TERT stabilization, the accurate mechanism of 94 HSP90 inhibition-induced cellular senescence remains to be identified (26, 31). C-terminus 95 of HSP70-interacting protein (CHIP) is one of the major co-chaperones of HSP90 and can 96 regulate the ubiquitylation and degradation of HSP90 target proteins. CHIP contains the N- 97 terminal TPR domain and C-terminal U-Box domain, which are required for the interaction 98 with chaperones and for the recruitment of the E2 enzyme and ubiquitylation, respectively 99 (32, 33). In association with HSP70, CHIP typically induces the degradation of HSP90 client 100 proteins when HSP90 fails to induce their structural maturation into their native forms 101 possibly through a hostile environment or HSP90 inhibition by drugs (33). 102 p14ARF is an alternative reading frame product of the INK4a/ARF locus and functions as a 103 tumor suppressor through p53-dependent and p53-independent pathways (34). p14ARF is 104 transcriptionally induced by oncogenic signaling such as c-myc and causes oncogene-induced 105 senescence, which results in tumor suppression (35). While p14ARF has been reported to be 106 regulated by post-translational modifications (10, 11, 36), the roles of molecular chaperones 107 in directly modulating p14ARF levels and the related cellular senescence have not been 108 identified. Here, we demonstrate that HSP90 plays a powerful oncogenic role by inducing 109 p14ARF degradation, which prevents cellular senescence. The ternary complex of HSP90, 110 CHIP and p14ARF was required to induce the ubiquitylation-independent and LAMP2A- 111 dependent lysosomal degradation of p14ARF. Furthermore, HSP90 and CHIP overexpression 112 together with a loss of p14ARF expression correlated with poor prognosis in advanced 113 human NSCLC and served as an important prognostic marker. This study is the first to 114 identify molecular chaperone-mediated degradation of the tumor suppressor p14ARF and to 115 demonstrate its clinical importance in NSCLC. 5 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-0613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 116 Materials and methods 117 Cell culture and transfection 118 An embryonic kidney cell line (293T), a cervical cancer cell line (HeLa), a human NSCLC 119 cell line (H1299), the IMR90 fibroblast line, the HFF line and human lung cancer cell lines 120 including A549, NCI-H460, NCI-H522, NCI-H23 and NCI-H226 were provided by 121 American Type Culture Collection (ATCC, Manassas, VA). The human lung cancer cell lines 122 including NCI-H322M, HOP62 and EKVX were provided from the U.S. National Cancer 123 Institute (NCI; MTA no. 2702-09). PC-9 cells were purchased in 2015 from Sigma-Aldrich 124 Corporation (St. Louis, MO). 293T, HeLa, IMR90 and HFF were obtained in 2014. H1299 125 was purchased in 2012. Rest of cell lines were obtained in 2009. 126 All lung cancer cell lines except PC-9 and H1299 were tested and authenticated annually by 127 STR test in National Cancer Center Core Facility, Korea. STR profile was analyzed using 128 Gene Mapper v 5.0 software. All the provided cell lines were aliquoted and frozen within 129 passage 10, and experiments were performed using thawed cell lines from passage 7 to 18.
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