Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Synergism of heat shock protein and histone deacetylase inhibitors in synovial sarcoma Nguyen, Anne 2007

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2007-0536.pdf [ 5.6MB ]
Metadata
JSON: 831-1.0100913.json
JSON-LD: 831-1.0100913-ld.json
RDF/XML (Pretty): 831-1.0100913-rdf.xml
RDF/JSON: 831-1.0100913-rdf.json
Turtle: 831-1.0100913-turtle.txt
N-Triples: 831-1.0100913-rdf-ntriples.txt
Original Record: 831-1.0100913-source.json
Full Text
831-1.0100913-fulltext.txt
Citation
831-1.0100913.ris

Full Text

SYNERGISM OF HEAT SHOCK PROTEIN AND HISTONE DEACETYLASE INHIBITORS IN SYNOVIAL SARCOMA by Anne Nguyen B.Sc, The University of British Columbia, 2005 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE . in THE FACULTY OF GRADUATE STUDIES (Pathology) THE UNIVERSITY OF BRITISH COLUMBIA July 2007 © Anne Nguyen, 2007 A B S T R A C T Synovial sarcoma is a cancer of young adults and is fatal in about half of cases. This malignancy carries an SYT-SSX fusion oncoprotein that is not targeted by existing drugs. Recently, we demonstrated that histone deacetylase inhibitors and the heat shock protein 90 inhibitor 17AAG inhibit synovial sarcoma in preclinical models. In this thesis we tested combinations of 17AAG with the histone deacetylase inhibitor MS-275 for synergism by performing proliferation and apoptosis assays on synovial sarcomas. Synergism was assessed by the median-effect principle of Chou and Talalay. The combination was found to be highly synergistic at multiple time points in both tested cell lines (combination indeces as low as 0.11) suggesting that reduced dose combination therapies may be effective against this disease. We next investigated the mechanism of synergism. While our work has shown that histone deacetylase inhibitors induce synovial sarcoma cell death, others have shown in related systems that these agents activate the N F - K B survival pathway, potentially limiting their therapeutic effect. As 17 AAG can inhibit upstream activators of N F - K B , we proposed that 17AAG may exert its synergistic effect with histone deacetylase inhibitors by abrogating activation of N F - K B . This hypothesis was confirmed in cells exposed to the drugs alone or in combination by quantitating total cellular levels of the N F - K B inhibitor IKB, nuclear levels of the N F - K B subunit RelA, and N F - K B mediated transcription. In each of these assays, adding 17AAG reversed the N F - K B activating effects of MS-275. Additionally we demonstrated that the N F - K B inhibitor BAY-11-7085 also synergizes with MS-275. Overall our data suggests that the N F - K B pathway plays a role in the synergistic activity of 17AAG and MS-275. These findings contribute to the preclinical development of an optimal systemic therapy for synovial sarcoma. ii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ACKNOWLEDGEMENTS ix DEDICATION x SECTION 1 Introduction 1 1.1. Summary 1 1.2. Synovial sarcoma 1 1.2.1. SYT-SSX 1 1.2.2. Treatment of synovial sarcoma 3 1.3. Heat shock proteins 4 1.3.1. Chaperone molecules 4 1.3.2. Hsp90 4 1.3.3. Heat shock protein 90 inhibitors 5 1.4. Acetylation 6 1.4.1. Protein acetylation 6 1.4.2. HDACs 8 1.4.3. HD AC inhibitors 8 1.5. Synergy 11 1.6. Mechanism of synergy: hsp70 11 1.7. Mechanism of synergy: N F - K B 12 1.7.1. N F - K B 12 1.7.2. NF- KB and synovial sarcoma 14 1.7.3. N F - K B , HDAC inhibitors and hsp90 inhibitors 15 SECTION 2 Materials and Methods 17 2.1. Reagents 17 2.2. Monolayer cell culture 17 2.3. Proliferation assays 18 2.4. Annexin V - FITC/Propidium Iodide flow cytometry assay 18 2.5. Synergism analysis 19 2.6. Protein quantification 19 2.7. Total lysate preparation 20 2.8. Nuclear/Cytoplasmic lysate preparations 20 2.9. Immunoblot analysis 21 2.10. Luciferase analysis 22 2.11. Immunoprecipitation 22 2.12. Statistical Analysis 23 iii SECTION 3 Results 24 3.1. 17AAG synergizes with MS-275 to inhibit synovial sarcoma in vitro 24 3.1.1. Synergism in MTT assay 24 3.1.2. Synergism in annexin V FITC/PI apoptosis assay 27 3.2. Mechansim of synergism does not involve MS-275 acetylation of hsp70.... 31 3.3. Mechanism of synergism involves 17AAG abrogation of MS-275 induced N F - K B activation 32 3.3.1. MS-275 suppression oftheNF-KB inhibitory protein IKBOC is opposed by 17AAG 32 3.3.2. MS-275 induction of nuclear levels of the N F - K B subunit RelA are opposed by 17AAG 34 3.3.3. N F - K B transcriptional activity is upregulated by treatment with MS-275 but is downregulated with both 17AAG treatment and combined treatment 35 3.4. MS-275 synergizes with N F - K B inhibitors 36 3.4.1. BAY-11-7085 is a N F - K B inhibitor in synovial sarcoma 36 3.4.2. Synergism between BAY-11-7085 and MS-275 38 SECTION 4 Discussion and Conclusion 41 4.1. Synergy 41 4.2. Mechanism of Synergy 43 4.2.1. Heat shock protein 70 acetylation 44 4.2.2. N F - K B Activation 45 4.2.3. Other Mechanisms 48 4.3. Conclusion 50 REFERENCES 51 iv LIST OF TABLES Table 3.1 Comparison of IC50 Values at 24 and 48 Hour Timepoints for 17AAG and MS-275 as Single Agents and in Combination Using MTT Assay 27 Table 3.2 IC5 0 Values following treatment by MS-275, BAY-11-7085 and the Combination on SYO-1 Cell Proliferation at 24, 48, 72 Hours 40 v LIST OF FIGURES Figure 1.1 N F - K B Activation 13 Figure 3.1 17 AAG Dose Response of SYO-1 cells using.MTT Assay 24 Figure 3.2 MS-275 Dose Response of SYO-1 cells using MTT Assay 25 Figure 3.3 Combination Dose of 17AAG and MS-275 SYO-1 Monolayer Culture Using MTT Assay 26 Figure 3.4 Combination Index and Comparison of Drugs as Single Agents Versus Combination Dose of 17AAG and MS-275 in SYO-1 Using Annexin V/FITC PI Flow Cytometry Assay at 24 Hours 28 Figure 3.5 Combination Index and Comparison of Drugs as Single Agents Versus Combination Dose of 17AAG and MS-275 in SYO-1 Using Annexin V/FITC PI Flow Cytometry Assay at 48 Hours 29 Figure 3.6 Combination Index and Comparison of Drugs as Single Agents Versus Combination Dose of 17AAG and MS-275 in SYO-1 Using Annexin V/FITC PI Flow Cytometry Assay at 72 Hours 30 Figure 3.7 Immunoblots of a-Hsp70 and a-Acetylated Lysine of Immunoprecipitates of Hsp70 31 Figure 3.8 Dose Response of IKBCC to MS-275, 17AAG and Combination Treatment 33 Figure 3.9 Dose Response of RelA to MS-275, 17AAG and Combination Treatment 34 Figure 3.10 Transcriptional Activation of N F - K B Luciferase Reporter Activity Following Treatment : 36 Figure 3.11 BAY-11-7085 Inhibition of N F - K B Luciferase Reporter Activity 37 Figure 3.12 BAY-11-7085 Dose Response of SYO-1 cells Using MTT Assay 38 Figure 3.13 MS-275 + BAY-11-7085 Dose Response and Combination Index Values of SYO-1 Treated Cells Using MTT Assay 39 LIST OF ABBREVIATIONS 17AAG 17-(Allylamino)-17-Demethoxygeldanamycin CI Combination Index CER Cytoplasmic Extraction Reagents EGFR-- Epidermal Growth Factor Receptor FITC Fluorescein Isothiocyanate FBS Fetal Bovine Serum FGFR3 Fibroblast Growth Factor Receptor 3 GNAT Gcn5-related jV-acetyltransferases HAT Histone Acetyltransferase HDAC Histone Deacetylase HER2 Human Epidermal Growth Factor Receptor 2 HSP Heat Shock Protein HRP Horse Radish Peroxidase HSP70 Heat Shock Protein 70 HSP90 Heat Shock Protein 90 IAP Inhibitor-of-poptosis IC50 - Half Maximal Inhibitory Concentration IGF2 Insulin-like Growth Factor 2 IGFBP2 Insulin-like Growth Factor Binding Protein 2 IKK — IKB Kinase IB Immunoblots EP - Immunoprecipitates LAR Luciferase Assay Reagent MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide MYST - MOZ, YBF2/SAS3, SAS2 and TIP60 RA — Retinoic Acid NER Nuclear Extraction Reagents NaBu Sodium Butyrate PBS Phosphate Buffered Saline PI - Propidium Iodide RIP — — Receptor (TNFRSF)-Interacting Serine-Threonine Kinase RIPK4 Receptor-Interacting Serine-Threonine Kinase 4 RPMI Roswell Park Memorial Institute SAHA Suberoyl Anilide Hydroxamic Acid SS Synovial Sarcoma TBS Tris Buffered Saline TSA Trichostatin A TNF Tumour Necrosis Factor TRAP TNFR-associated factor TRAIL— Tumour necrosis (TNF)-related apoptosis-inducing ligand viii ACKNOWLEDGEMENTS I am in debt to and am very grateful for the support of the Nielsen Lab: Dr. Torsten Nielsen Dr. Joanna Lubieniecka Dr. Jefferson Terry Suzanne Liu Dr. Neal Poulin Maggie Cheang I am in debt to and am very grateful for the support of the GPEC/Huntsman Lab: Dr. Dmitry Turbin Samuel Leung Challayne Smith Janine Senz Melinda Miller Much thanks to my Supervisory Committee for their guidance: Susan Porter Wayne Riggs Samuel Aparicio I owe sincere thanks also to the Prostate Center. Much gratitude to Dr. Kazuo Nagashima and Dr. Akira Kawai for providing us with the synovial sarcoma cell lines and to Dr. Michael Underhill for the N F - K B reporter plasmid. Dr. Torsten Nielsen is a scholar of the Michael Smith Foundation for Health Research. This work was supported by a research grant from the Terry Fox Foundation. ix DEDICATION This work is dedicated to my mother Hoang Diep, and my father Son Nguyen for taking good care of me and providing for me lovingly. Also to my sisters Uyen Diep, Dana Nguyen, Kim Nguyen, and my cousins Trung Kien Nguyen and Khai Quang Diep Dinh for their love and support. I would also like to dedicate this to friend Becca Coad for her wonderful companionship. Finally I'd like to dedicate this to my husband Haitham Helal for his unconditional love and support. 1. Introduction 1.1. Summary Clinically-applicable heat shock protein 90 (Hsp90) and histone deacetylase (HDAC) inhibitors have independently been shown to suppress growth and induce apoptosis in synovial sarcoma. The first part of this study tests the hypothesis that such agents may be synergistic against synovial sarcoma, which would permit increased efficacy while reducing dose and minimizing potential side effects. The second part of this study seeks to explain the possible mechanisms of synergism of these agents. This will be done by looking at the effects of both drugs, firstly on Heat Shock Protein 70 (Hsp70) induction and then secondly on the activation of N F - K B . 1.2. Synovial sarcoma Synovial sarcoma is an aggressive malignancy of unknown cellular origin comprising 10% of all sarcomas.1 This disease exhibits an undifferentiated mesenchymal phenotype, which suggests that malignant transformation likely involves dysfunctional cellular differentiation. Histologically, synovial sarcoma displays two major types: biphasic and monophasic.2 The biphasic type consists of epithelial elements arranged in gland-like structures against a spindle cell background. The monophasic type contains only the spindle cell elements. 1.2.1. S Y T - S S X The chromosomal translocation t(X;18)(pl 1.2;ql 1.2), resulting in one of the following fusion oncoproteins, SYT-SSX1, SYT-SSX2 and SYT-SSX4, is demonstrable in over 90% ' Ladanyi M. (2001) "Fusions of the SYT and SSX genes in synovial sarcoma." Oncogene 20: 5755-5762 2 Ibid 1 of these cancers.3'4 Of these fusion oncoproteins, the SYT-SSX1 is the most common and shows significantly reduced metastasis-free survival than SYT-SSX2.5 SYT-SSX4 is extremely rare. The function in oncogenesis of the SYT-SSX fusion oncoprotein is unclear; however there are indications of roles for SYT-SSX in both transcriptional activation and repression. Both the SYT protein and the fusion oncoprotein SYT-SSX bind with SIP/CoAA, a general nuclear transcriptional co-activator and a RNA splicing modulator.6 Together SYT and SIP/CoAA have been found to stimulate estrogen and glucocorticoid receptor-dependent transcriptional activation.7 Furthermore, there is good evidence that this oncoprotein may deregulate epigenetic modulation of the normal proteins. The SYT protein, but not the SSX protein, interacts with both a component of a FED AC complex, mSin3 A 8 and a component of a histone acetyltransferase (HAT) complex, p3009. Gene expression profiling of synovial sarcoma using spotted cDNA microarrays has demonstrated a distinct gene expression pattern as compared to other sarcomas. Within these profiles of synovial sarcoma, high expression of a number of receptor tyrosine kinases and steroid hormone receptors such as retinoic acid (RA) pathway gene products, epidermal ' 3 Ibid 4 Skytting B., Nilsson G., Brodin B., Xie Y., Lundeberg I, Uhlen M. , and Larsson O. (1999) "A novel fusion gene, SYT-SSX4, in synovial sarcoma" J Natl Cancer Inst. 11:974-5 5 Kawai A., Woodruff J., Healey J.H., Brennan M.F., Antonescu C.R., and Ladanyi M. (1998) "SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma" NEngl JMed 338:153-160 6 Perani M. , Antonson P., Hamoudi R , Ingram C.J.E., Cooper C.S., and Garrett. (2005) "The proto-oncoprotein SYT interacts with SYT-interacting protein/co-activator activator (SIP/CoAA), a human nuclear receptor co-activator with similarity to EWS and TLS/FUS family of proteins" J. Biol. Chem. 280: 42863^2876 7 Ibid 8 Ito T., Ouchida M , Ito S., Jitsumori Y., Morimoto Y., Ozaki T., Kawai A., Inoue H., and Shimizu K. (2004) "SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3A, a component of histone deacetylase complex" Lab. Invest. 84: 1484-1490 9 Ogryzko V.V., Schiltz R. L. , Russanova V., Howard B.H., and Nakatani Y. (1996) "The transcriptional coactivators p300 and CBP are histone acetyltransferases" Cell 87: 953-959 2 growth factor receptor (EGFR), receptor-interacting serine-threonine kinase 4 (RTPK4),1U insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor II (IGF2), fibroblast growth factor receptor 3 (FGFR3) and human epidermal growth factor receptor 2 (HER2) have been found.11 1.2.2. Treatment of synovial sarcoma Synovial sarcoma typically affects young adults, with an estimated cumulative 5-year and 10-year survival rates of 68% and 41%, respectively12. Current therapies of synovial sarcoma involve surgical resection followed by either radiotherapy (using megavolt photon or electron beam energies and conventional fractionation) and/or chemotherapy (most commonly using a multiagent regimen containing either cyclophosphamide, or ifosfamide plus doxorubicin, or epirubicin).13 In some cases, actinomycin D, vincristine, diacarbazine or cisplatin is added to the treatment.14 As can be seen by the dismal outcomes of the current treatment, there is a strong need for a more effective therapy for synovial sarcoma. Recently, we15 and others16 have found that both Hsp90 inhibitors 17AAG and the HDAC inhibitor depsipeptide, as single agents, are successful at arresting growth of synovial sarcoma in vitro and at slowing growth in xenografts. Futher investigation of both of these agents can support the development of effective treatment for synovial sarcoma. 1 0 Nielsen TO., West R.B., Linn S.C, Alter O., Knowling M.A., O'Connell J.X., Zhu S., Fero M., Sherlock G., Pollack J.R., Brown P.O., Botstein D., and van de Rijn M. (2002) "Molecular characterisation of soft tissue tumours: a gene expression study" Lancet 359: 1301-1307 11 Allander S.V., Illei P.B., Chen Y., Antonescu C.R., Bittner M., Ladanyi M., Meltzer PS (2002) "Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation" ,4m. J. Pathol. 161: 1587-1595. 1 2 Hasegawa T., Yokoyama R., Matsuno Y., Shimoda T., Hirohashi S..(2001) "Prognostic significance of histologic grade and nuclear expression of beta-catenin in synovial sarcoma" Hum. Pathol. 32: 257-263. 1 3 Ferrari A., Gronchi A., Casanova M., Meazza C, Gandola L., Collini P., Laura Lozza L., Bertulli R., Olmi P., and Casali P.G. (2004) "Synovial sarcoma: A retrospective analysis of 271 patients of all ages treated at a single institution" Cancer 101: 627-634 1 4 Ibid 1 5 Terry J., Lubieniecka J.M., Kwan W., Liu S., and Nielsen T.O. (2005) "Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin prevents synovial sarcoma proliferation via apoptosis in in vitro models" Clin Cancer Res 11:5641 1 6 Ito T, Ouchida M, Morimoto Y, Yoshida A, Jitsumori Y, Ozaki T, Sonobe H, Inoue H, Shimizu K. (2005) "Significant growth suppression of synovial sarcomas by the histone deacetylase inhibitor FK228 in vitro and in vivo" Cancer Lett 224:311 3 1.3. Heat shock proteins 1.3.1. Chaperone molecules Heat shock proteins (Hsp) (Hsp 10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp 110) are required as chaperones of other proteins.17 Protein chaperoning involves both protein holding (the prevention of misfolding and aggregation of proteins) and protein folding (self-association to form large folding chambers for the formation of tertiary structures).18 The function of protein holders (such as Hsp70 and Hsp90) is to bind to unfolded sequences during the mRNA translation of nascent proteins and in response to cellular stress.19 This is essential for cell survival as misfolded and aggregated proteins trigger cells to begin programmed cell death.20 As a consequence of their role as protein holders, Hsp70 and Hsp90 are regulators of cellular metabolism, growth signaling, and cell-cycle progression, and are anti-apoptotic.21 Hsps are implicated as potentiators of cancer as dysregulation of these main functions occur at a multiple stages of malignancy. Indeed, increases in Hsp expression occur both during progression of malignancy as well as during development of 22 treatment resistance. 1.3.2. Hsp90 The most studied and one of the most significant of the heat shock proteins is Hsp90. Hsp90 is a ubiquitous molecular chaperone accounting for 1-2% of total cellular protein.23 Hsp90 assists in folding or complex formation of hundreds of client proteins.24 It is also a 1 7 Calderwood S.K., Khaleque Md.,A., Sawyer D.B., and Ciocca D.R. (2006) "Heat shock proteins in cancer: chaperones of tumorigenesis". Trends Biochem. Sci, 31: 164-172 1 8 Ibid. 1 9 Wegele H., Muller L., and Buchner J. (2004) "Hsp70 and Hsp90-a relay team for protein folding" Rev. Physiol. Biochem. Pharmacol. 151: 1-44. 2 0 Calderwood S.K. et. al. 2006 2 1 Ibid. 2 2 Ibid. 2 3 Pacey S., Banerji U., Judson I., and Workman P. (2006) "Hsp90 inhibitors in the clinic" Handb. Exp. Pharmacol. 172: 331-358 4 constitutive stabilizing component of many large tertiary complexes. Many of its client proteins such as CDK-4, Raf-126, HER227, IKK28, and RIP29 play essential roles in cell growth, division and survival. Importantly, Hsp90 has been shown to assist in the folding of mutant proteins such as mutant p5 330 and Bcr-Abl31, thereby acting as a gatekeeper facilitating oncogenic changes. This unique function makes Hsp90 an attractive target in cancer therapy. 1.3.3. Heat shock protein 90 inhibitor Studies have shown that inhibition of Hsp90 results in degradation of client proteins, inhibition of tumour growth by arresting cells at Gl, differentiation, and activation of apoptosis.32 As a result of the work of several investigators at the preclinical stage, one specific Hsp90 inhibitor, 17-(Allylamino)-17-demthoxygeldanamycin (17AAG) has completed phase I clinical trials33 and is now being tested in phase II trials for a variety of malignancies including metastatic melanoma, breast cancer and ovarian cancer.34 17AAG is 2 5 Calderwood S.K. et. al. 2006 2 6 Schulte T.W., Blagosklonny M.V., Romanova L., Mushinski J.F., Monia B.P., Johnston J.F., Nguyen P.M., Trepel J., and Neckers L.M. (1996) "Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway" Mol Cell Biol 16: 5839-5845 2 7 Xu W., Mimnaugh E, Rosser M.F., Nicchitta C , Marcu M., Yarden Y., Neckers L. (2001) "Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90" J. Biol. Chem. 276: 3702-3708 2 8 Broemer M., Krappmann D., and Scheidereit C. "Requirement of Hsp90 activity for IkappaB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-kappaB activation" (2004) Oncogene 23: 5378-5386 2 9 Lewis J., Devin A., Miller A., Lin Y., Rodriguez Y., Neckers L., and Liu Z., (2000) "Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation" J. Biol. Chem. 275: 10519-10526 3 0 Mtlller P., Ceskova P., and Vojtesek B. (2005) "Hsp90 is essential for restoring cellular functions of temperature-sensitive p53 mutant protein but not for stabilization and activation of wild-type p53: implications for cancer therapy" J. Biol. Chem 280: 6682-6691 3 1 Rahmani M., Reese E., Dai Y., Bauer C , Kramer L.B., Huang M., Jove R., Dent P., and Grant S. (2005) "Cotreatment with Suberanoylanilide Hydroxamic Acid and 17-Allylamino 17-demethoxygeldanamycin Synergistically Induces Apoptisis in Bcr-Abl+ Cells Sensitive and Resistant to STI571 (Imatinib Mesylate) in Association with Down-Regulation of Bcr-Abl, Abrogation of Singal Transducer and Activator of Trancscription 5 Activity, and Bax Conformational Change" Mol. Pharmacol. 67: 1166-1176 3 2 Workman P., (2004) "Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone" Cancer Lett. 206: 149-157 3 3 Neckers L., and Ivy S.P. (2003) "Heat shock protein 90" Curr. Opin. Oncol. 15: 419-124 3 4 Pacey et. al. 2006 5 the first Hsp90 inhibitor to enter clinical trials.35 At a molecular level, 17AAG exerts its inhibitor effect by blocking the binding of ATP to Hsp90.36 Importantly, by impairing Hsp90 function, 17AAG effectively inhibits several tyrosine kinases that play a role in activating growth and the cell cycle, such as EGFR and HER2.37 In addition, Hsp90 inhibitors inhibit the N F - K B pathway' by inhibiting the Hsp90 clients IKB Kinase (EKK)38 and Receptor (TNFRSF)-Interacting Serine-Threonine Kinase (RIP)39. One potentially negative outcome of Hsp90 inhibition is upregulation of Hsp70 in a compensatory fashion. As Hsp70 has pro-growth and survival effects on cells, secondary activation by Hsp90 inhibitor is likely to diminish effectiveness of the anti-tumour properties.40 Importantly, previous work in our lab has shown that in synovial sarcoma, 17-AAG treatment resulted in the degradation of receptor tyrosine kinases such as EGFR, HER2, FGFR3 and c-kit and as well as induction of apoptosis in vitro.41 1.4. Acetylation 1.4.1. Protein acetylation Postranslational modification by acetylation of the lysine residues of histones is widely recognized as a method of transcriptional regulation.42 Early findings suggested that acetylation acts as a transient transcriptional activator by neutralizing the positive charge of Sausville E.A., Tomaszewski IE. , and Ivy P. (2003) "Clinical development of 17-allylamino, 17-demethoxygeldanamycin" Curr. Cancer Drug Targets 3: 377-385 3 6 Prodromou C , Roe S.R., O'Brien R., Ladbury J.E., Piper P.W., and Pearl L.H (1997) "Identification and Structural Characterization of the ATP/ADP-Binding Site in the Hsp90 Molecular Chaperone" Cell 90: 65-75. 3 7 Lang S.A., Klein D., Moser C , Gaumann A., Glockzin G., Dahlke M.H., Dietmaier W., Bolder U., Schlitt H.J., Geissler E.K., and Stoeltzing O. (2007) "Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo" Mol Cancer Then 6:1123-1132. 38Broemer M. et. al. 2004 3 9 Lewis J. et. al. 2000 4 0KiangJ.G., Bowman P.D., WuB.W., Hampton N., Kiang A.G., Zhao B., Juang Y.T., Atkins J.L., and Tsokos G.C. (2004) "Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70" J. Appl. Physiol. 97: 564-569 4 1 Terry J. et. al. 2005 4 2 Marks P.A., Miller T., and Richon V.M. (2003) "Histone deacetylases" Curr. Opin. Pharmacol. 3: 344-351 6 lysine residues on histone proteins causing decondensation of the chromatin. More recent findings suggest that in addition to the regulation of histones, acetylation is essential for the regulation of many other proteins, both inside and outside the nucleus.44 These other proteins include p5 3 4 5 , N F - K B 4 6 , Hsp9047 which have roles in cellular growth, survival and protein folding. This regulation can positively or negatively regulate function. For example Hsp90 is inhibited by acetylation48 whereas both p5 3 4 9 and the RelA subunit of N F - K B 5 0 are activated by acetylation. For these reasons acetylation is thought to have a fundamental role in cellular processes related to malignancy and is another attractive target for cancer therapy51. Acetylation occurs through the action of HAT proteins in a few different families. Some of the HAT families include: the Gcn5-related TY-acetyltransferases (GNAT) superfamily, the MYST group (MOZ, YBF2/SAS3, SAS2 and TIP60), and the p300/CBP family.52 Correspondingly deacetylation is accomplished by HDAC proteins from two families comprising four separate classes.53 The first family, class I, II and IV HDACs, deacetylate the lysine residue by a charge transfer system at the active site onto a zinc2+ ion 4 3 Hess-Stump H . (2005) "Histone deacetylase inhibitors and cancer: from cell biology to the clinic" Eur. J. Cell Biol. 84: 109-121 4 4 Minucci S., and Pelicci P.G. (2006) "Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer" Nat. Rev. Cancer 6: 38-51 4 5 Gu. W. and Roeder R.G. (1997) "Activation of p53 Sequence-Specific D N A Binding by Acetylation of the p53 C -Terminal Domain" Cell 90: 595-606 4 6 Chen L . , Fischle W., Verdin E . , and Greene W.C. (2001) "Duration of Nuclear N F - K B Action Regulated by Reversible Acetylation" Science 293: 1653-1657 4 7 Y u X. , Guo S., Marcu M . G . , Neckers L . , Nguyen D . M . , Chen G.A. , Schrump D.S. (2002) "Modulation of p53, E r b B l , ErbB2, and Raf-1 Expression in Lung Cancer Cells by Depsipeptide FR901228" J. Natl. Cancer Inst. 94: 504-513 4 8 Kovacs J.J., Murphy P.J.M., Gaillard S., Zhao X . , Wu J.T., Nicchitta C . V . , Yoshida M . , Toft D O . , Pratt W.B. , Yao T P . , (2005) "HDAC6 Regulates Hsp90 Acetylation and Chaperone-Dependent Activation of Glucocorticoid Receptor" Mol. Cell 18:601-605 4 9 Gu W. and Roeder R.G. 1997 5 0 ChenL. et.al. 2001 5 1 Rosato R.R., and Grant S. (2005) "Histone deacetylase inhibitors: insights into mechanisms of lethality" Expert Opin. Ther. Targets 9: 809-824 5 2 Hess-Stump H . 2005 5 3 Minucci S., and Pelicci P.G. 2006 7 complexed with the HDAC protein54. Chelation of the HDAC or any other means of inhibition at the active site of the FID AC is the mechanism of action of HDAC inhibitors on 55 2+ this family. The second family, class III HDACs is NAD dependent and is related to yeast homologue Sir2.56 This class of HDAC is not well characterized and is not referred to further in this work. 1.4.2. HDACs Class I HDACs (HDAC1, 2,3 and 8) have been found to be located almost exclusively in the nucleus57 indicating a role in transcriptional regulation. One example of a non-histone target modulated by the Class IHDAC3 is the RelA subunit of N F - K B which.58 Another possible example is Hsp70 which co-immunoprecipitates with HDACs 1,2 and 3.59 The class II HDACs (HDAC4,5,6,7,9 and 10) occur in both the nucleus and cytosol60 and therefore are more likely to have a role in acetylating those proteins whose functions and pathways occur outside the nucleus. HDAC6 which is predominately localized to the cytoplasm is found to be associated with the chaperone protein Hsp90 which it reversibly deacetylates.61 Class IV consists of a single HDAC, HDAC11.62 The existence of class II HDACS outside the nucleus provide further evidence for the importance of acetylation in regulation of the activities of non-histone proteins. 1.4.3. HDAC inhibitors 5 4 D E Ruijter A.J.M., V A N Gennip A.H., Caron H.N., Kemp S., and V A N Kuilenburg A.B.P. "Histone deacetylases (HDACs): characterization of the classical HDAC family " (2003) Biochem. J. 370: 737-749 5 5 Ibid 5 6 Hess-Stump H. 2005 5 7 D E Ruijter A.J.M. et. al. 2003 5 8 Chen et. al. 2001 5 9 Johnson C.A., White D.A., Lavender J.S., O'Neill L.P., Turner B.M. (2001) "Human Class I Histone Deacetylase Complexes Show Enhanced Catalytic Activity in the Presence of ATP and Co-immunoprocipitate with the ATP-dependent Chaperone Protein Hsp70" J. Biol. Chem. 277: 9590-9597 6 A D E Ruijter A.J.M. et. al. 2003 6 1 Kovacs J.J. et al. 2005 6 2 Minucci S., and Pelicci P.G. 2006 8 A variety of histone deacetylase (HDAC) inhibitors have been studied as targeted therapy against many malignancies.63 Histone deacetylase inhibitors (including MS-275) are also currently involved in phase I and II clinical trials of several cancers including leukemias, lymphomas, melanomas and refractory solid tumours,64 and are considered to be promising new therapies for a wide range of cancers. The studies in this thesis focus on the HDAC inhibitor MS-275. Previous research has indicated that MS-275 is an effective inhibitor of HDAC1, 2, and 365 but is a quite poor inhibitor of class II HDACs (HDAC4,6 and 10)66 where much higher doses are required. In contrast, other HDAC inhibitors such as FK228 and trichostatin A (TSA) are considered to be pan HDAC inhibitors. Many explanations for the mechanism of effect of HDAC inhibitors on tumours have been suggested. It is hypothesized that treatment of cells with HDAC inhibitors result in transcriptional changes on certain genes whose net effect preferentially targets malignant cells.67 By this theory HDAC inhibitors would result in net upregulation of tumour-suppressors and other proteins that limit malignant growth and/or secondarily downregulate oncogenes and other proteins that promote malignancy. A second possible mechanism of therapeutic effect includes inhibition of the chaperone function of Hsp90 by acetylation.68 As explained above, Hsp90 is a folding chaperone of many oncogenes and tyrosine kinases whose overexpression and activation have a malignant effect. Additionally, Hsp90 is one of the alternative protein substrates for HDAC669. As a result, HDAC inhibitors can act as 6 3 Hess-Stump H. 2005 6 4 Minucci S., and Pelicci P.G. 2006 6 5 Hu E., Dul E., Sung C M . , Chen Z., Kirkpatrick R., Zhang G.F., Johanson K., Liu R., Lago A., Hofmann G., Macarron R., D E L O S Frailes M. , Perez P., Krawiec J., Winkler J., and Jaye M. (2003) "Identification of Novel Isoform-Selective Inhibitors within Class I Histone Deacetylases" J. Pharmacol. Exp. Ther. 307: 720-728 6 6 Hess-Stumpp H., Bracker T.U., Henderson D., Politz O. (2007) "MS-275, a potent orally available inhibitor of histone deacetylases—The development of an anticancer agent" Int. J. Biochem. Cell Biol, epub ahead of print 6 7 Minucci S., et. al. 2003 6 8 Yu X. et. al. 2002 6 9 Kovacs J.J. et. al.2005 9 anti-tumour agents by inhibiting Hsp90. Thirdly HDAC inhibitors (MS-275, TSA, suberoyl anilide hydroxamic acid (SAHA) and sodium butyrate (NaBu) have been shown to inhibit tumours by increasing the levels of reactive oxygen species (ROS), ' ' ' which induce apoptosis and senescence in cancer cells. Fourthly HDAC inhibitors are able to act through extrinsic receptor-mediated apoptotic pathways, such as death receptors, the Fas pathway and the tumour necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) pathway.74 Contrary to these anti-growth and survival effects, one of the mechanisms of HDAC inhibitors results in upregulation of a survival pathway. By inhibiting HDAC3 and causing net acetylation of the RelA subunit of N F - K B , treatment of cells with HDAC inhibitors allow RelA to remain longer within the nucleus. As N F - K B needs to be located within the nucleus in order to act as a transcription factor, HDAC inhibitors result in a net increase in N F - K B activity.75 Previous work within our lab has shown inhibition of growth and induction of apoptosis by HDAC inhibitors TSA, FK228 and MS-275 on synovial sarcoma cell lines.76'77 This work has been supported by others who have shown that in synovial sarcoma, the HDAC inhibitor FK228 causes histone acetylation, inhibition of growth at nanomolar levels, 7 0 Rosato R.R., Almenara J.A., and Grant S. (2003) "The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIPT/WAFl I" Cancer Res. 63: 3637-3645 1 Moreira J.M.A., Scheipers P., and Serensen P. (2003) "The histone deacetylase inhibitor Trichostatin A modulates CD4+ T cell responses" BMC Cancer 3: 30-47 7 2 Ruefli A.A., Ausserlechner M.J., Bemhard D., Sutton V.R., Tainton K.M., Kofler R., Smyth M.J., and Johnstone R.W. (2001) "The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species" Proc. Natl. Acad. Sci. U.S.A. 98: 10833-10838 7 3 Louis M. , Rosato R R , Brault L., Sandra Osbild S., Battaglia E., Yang X.H., Grant S., and Bagrel D. (2004) "The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress" Int. J. Oncol. 25(6): 1701-17011 7 4 Rosato R.R., and Grant S. 2005 7 5 ChenL.et. al. 2001 76Kwan W., Terry J., Liu S., Knowling M. , and Nielsen T. (2005) "Effect of depsipeptide (NSC 630176), a histone deacetylase inhibitor, on human synovial sarcoma in vitro" ASCO annual meeting 7 7 Liu S., Knowling M.A., Clarkson P., Lubieniecka J.M., Cheng H., and Nielsen T.O. (2006) "Clear cell sarcoma and other translocation-associated sarcomas are highly sensitive to histone deacetylase inhibitor MS-275" CTOS 12th annual meeting 10 increased sensitivity of HEK293 cell lines transfected with the SYT-SSX oncogene, and inhibition of growth and invasion in mouse xenografts.78 1.5. Synergy The evidence that both 17AAG and the HDAC inhibitors have shown efficacy in targeting synovial sarcoma in vivo and in vitro has raised the question of whether combining the drugs would produce a synergistic effect. We therefore tested combinations of 17AAG with the histone deacetylase inhibitor MS-275 by MTT proliferation assays and by Annexin V flow cytometry apoptosis assay, using established synovial sarcoma cell line models in vitro. Synergism was assessed by the median-effect principle of Chou and Talalay.79 1.6. Mechanism of Synergy: Opposing effects on the pro survival protein Hsp70 The first mechanism discussed here focuses on acetylation as a method of non-histone protein regulation. Hsp70 is consistently upregulation as a result of treatment of cells with Hsp90 inhibitors.80 As another chaperone folding complex, Hsp70 seems to have a role in malignant progression. Increases in Hsp70 in the lysosomal membranes of tumours have been found.81 When these tumours are depleted of stores of Hsp70, the cells have been found to undergo spontaneous cell death.82 Others.have shown that specific inhibition of Hsp70 on animal models of colon cancer and melanoma results in tumour cell death.83 In addition, increases in the expression of Hsp70 have been correlated with resistance to 7 8 Ito T. et. al. 2005 7 9 Chou TC, and Talalay P. (1984) "Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors'Mcfv. Enzyme Regul. 22:27-55 8 0 Pacey S. et. al 2006 8 1 Nylandsted J., Gyrd-Hansen M., Danielewicz A., Fehrenbacher N., Lademann U., Hoyer-Hansen M., Weber E., Multhoff G., Rohde M. , and Jaatelaa M. (2004) "Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization" J. Exp. Med 200: 425-435 8 2 Ibid. 8 3 Schmitt E, Maingret L, Puig PE, Rerole AL, Ghiringhelli F, Hammann A, Solary E, Kroemer G, Garrido C. (2006) "Heat shock protein 70 neutralization exerts potent antitumor effects in animal models of colon cancer and melanoma" Cancer Res 66:4191-4197 11 chemotherapy.84 The Hsp70 induction in response to Hsp90 inhibition may be a protective mechanism by which tumour cells respond.85 Together these findings suggest that any method of inhibiting Hsp90 and Hsp70 simultaneously would be synergistic. As mentioned above, recent research has shown that Hsp90 is inhibited by acetylation. This acetylation of Hsp90 can be regulated by treatment with HDAC inhibitors.86 Furthermore, Hsp70 has been found to co-imunoprecipitate with HDACs 1,2,3 indicating that similarly to Hsp90, Hsp70 may be inhibited by acetylation mediated by treatment with an HDAC inhibitor. Using the HDAC inhibitor FK228 other researchers have shown that Hsp70 can be hyperacetylated and have given partial evidence that acetylation inhibits Hsp70 efficacy.87 We have sought to test the hypothesis that the HDAC inhibitor MS-275 synergizes with Hsp90 inhibitor by abrogating Hsp90 inhibitor activation of Hsp70. The hypothesis will be tested by looking at acetylation status of the Hsp70 protein following treatment with the HDAC inhibitor MS-275. 1.7. Mechanism of synergy: opposing effects on the pro survival pathway N F - K B 1.7.1. N F - K B Another possible mechanism for synergy involves effects of the HDAC inhibitor and the Hsp90 inhibitor on the survival pathway N F - K B . N F - K B is a transcription factor with a fundamental role in inflammation and immune response.88 It has over two hundred known 8 4 Vargas-Roig L.M., Gago F.E.., Tello O., Aznar J.C. and Ciocca D.R. (1998) "Heat shock protein expression and drug resistance in cancer patients treated with induction chemotherapy" Int. J. Cancer (Pred. Oncol.): 79: 468-475 85Wegele H.,et. al. 2004 8 6 Yu X. et. al. 2002 8 7 Wang Y., Wang S. Y., Zhang X.H., Zhao M., Hou CM., Xu Y.J., Du Z.Y., and Yu X.D. (2007) "FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of HsplO" Biochem. Biophys. Res. Commun. epub ahead of print 8 8 KarinM. (2006) "Nuclear factor-KB in cancer development and progression" Nature 441: 431-436 12 T N F a , H - l , LPS . and others I K K Complex I Inactive NF-kB Active NF-kB imotved in lnn.Ui: immunity. inlhunmalion. . .dl •>urvi\ .il Figure 1.1 N F - K B Activation target genes89 including those involved in cell cycle pathway such as cyclin D 9 0, in signaling such as IL-8, and in survival such as BC1-XL,91 TNFR associated factor (TRAF) 1, TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAPl and c-IAP2,92 and XIAP93. In addition, N F - K B has a role in differentation and development of such cells as osteoclasts, B-lymphocytes,94 and fetal hepatic cells95. As discussed below, a variety of studies have shown N F - K B activation promotes malignancy by protecting cells from apoptosis. The term " N F - K B " actually refers to 5 different subunits RelA (p65), cRel, RelB, p50 ( N F - K B 1), and p52 (NF-KB 1) which homo- or hetero-dimerize in various combinations. 96 8 9 Shishodia S., and Aggarwal B .B . (2004) "Nuclear factor-kB: a friend or a foe in cancer?" Biochem. Pharmacol. 68: 1071-1080 9 0 Hinz M . , Krappmann D. , Eichten A . , Heder A . , Scheidereit C , and Strauss M . (1999) "NF-kappaB function in growth control: regulation of cyclin D l expression and GO/Gl-to-S-phase transition. "Mol Cell Biol. 19; 2690-2698 9 1 Khoshman A . , Tindell C , Laux I., Bae D. , Bennett B. , and Nel A . E . (2000) "The NF-kB Cascade Is Important in Bcl-xL Expression and for the Anti-Apoptotic Effects of the CD28 Receptor in Primary Human CD4 Lymphocytes" J. Immunol. 165:1743-1754 9 2 Wang C , Mayo M . W . , Komeluk R.G. , Goeddel D.V. , Baldwin A.S. Jr. (1998) "NF-KB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAPl and C-IAP2 to suppress caspase-8 activation" Science 281: 1680-1683 9 3 Stehlik C , D E Martin R , Kumabashiri I., Schmid J.A., Binder B.R., and Lipp J. (1998) "Nuclear Factor (NF)-KB regulated X-chromosome-linked iap Gene Expression Protects Endothelial Cells from Tumor Necrosis Factor a-induced Apoptosis" J. Exp. Med. 188: 211-216 9 4 Franzoso G. , Carlson L . , Xing L. , Poljak L . , Shores E.W., Brown K . D . , Antonio Leonardi A . , Tran T., Boyce B.F., and Siebenlist U . (1997) "Requirement for NF-KB in osteoclast and B-cell development" Genes Dev. 11; 3482-3496 9 5 Beg A . A . , Sha W.C. , Bronson R.T., Ghosh S., and Baltimore D. (2002) "Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-KB" Nature 376; 167 -170 9 6 K a r i n M . 2006 13 Inactive N F - K B is bound to an inhibitory protein known as IKB and is localized to the cytoplasm.97 Upstream activation involving many different possible signals such as tumour necrosis factor a (TNF-a)98/RTP (family proteins)99, Ras/PI3Kinase/Akt,100 and IL-1101 result in the activation of IKB Kinase complexes (IKK) (Figure 1.1). IKK then phosphorylates and ubiquitinates the inhibitory I K B 1 0 2 resulting in the release and relocation of N F - K B dimers to the nucleus where they activate transcription of genes103. 1.7.2. N F - K B and synovial sarcoma ReiA A number of studies have indicated a strong role for inflammation and infectious response in the progression of cancer.104 Epidemiological studies have shown that infections diseases such as hepatitis B and C viruses, human papillomaviruses, Epstein-Barr Virus, human T-cell lymphotrophic virus I, human immunodeficiency virus, the bacterium Helicobacter pylori, schistosomes, and liver flukes cause 15.6% of cancers world wide.105 N F - K B has been found to be constitutively activated in a number of tumour cell lines including both solid and hematopoietic cancers.106 Within these cell lines, N F - K B has been 9 7 Alkalay I., Yaron A.,Hatzubai A., Oriani A., Ciechanover A., andBen-Neriah Y. (1995) "Stimulation-dependent M3a phosphorylation marks the NF-KB inhibitor for degradation via the ubiquitin-proteasome pathway" Proc. Natl. Acad. Sci. USA 92: 10599-10603 9 8 Liu Z.G., Hailing Hsu H., Goeddel D.V., and Karin M. (1996) "Dissection of TNF Receptor 1 Effector Functions: JNK Activation Is Not Linked to Apoptosis While.NF-kB Activation Prevents Cell Death" Cell 87: 565-576 9 9 Hsu. H., Huang J., Shu H.B., Baichwal V., and Goeddel D.V. (1996) "TNF-Dependent Recruitment of the Protein Kinase RIP to the TNF Receptor-1 Signaling Complex" Immunity 4; 387-396 100Madrid L.V., Wang C.Y, Guttridge D.C, Schottelius A.J.G., Baldwin A.S., Jr., and Mayo M.W. (2000) "Akt suppresses apoptosis by stimulating the transcriptional activation potential of the RelA/p65 subunit of NF-KB". Mol. Cell. Biol. 20:1626-1638 1 0 1 Moynagh P.N., Williams D.C, O'Neill L.A. (1994) "Activation of NF-kappaB and induction of vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression in human glial cells by BL-1. Modulation by antioxidants." J Immunol 153; 2681-2690 1 0 2 Alkalay et. al. 1995 1 0 3 Ibid. 1 0 4 Karin M. 2006 1 0 5 Pisani P., Parkin D.M., Munoz N., Ferlay J. (1997) "Cancer and infection: estimates of the attributable fraction in 1990" Cancel Epidemiol Biomarkers Prev 6: 387-400 1 0 6 Shishodia S., and Aggarwal B.B. 2004 14 found to assist in resistance to apoptosis and to promote cancer cell survival1U/ but also has a role in promoting angiogenesis and invasion108. Studies of inhibition of N F - K B by the compound BAY-11-7085 have shown that apoptosis is induced by N F - K B inhibition in suspended, re-adhering colon cancer cells.109 BAY-11-7085 treatment has also induced as apoptosis and inhibited cell invasion in cisplatin resistant ovarian cancer cells.110 BAY-11-7085 works by inhibiting the phosphorylation of the inhibitory IKB thereby preventing the release of N F - K B into the nucleus.111 Recent findings within our lab show that the N F - K B pathway may have an important role in the pathogenesis of synovial sarcoma. Our analysis of cDNA microarray gene expression profiling of thousands of genes has demonstrated high levels of upregulation of the protein RIPK4 (data not shown). Importantly RTPK4 is an activator of N F - K B 1 1 2 suggesting there may be constitutive activation of N F - K B within synovial sarcoma. This indicates a possible role of N F - K B in the pathogenesis of synovial sarcoma. 1.7.3. N F - K B , HDAC inhibitor and hsp90 inhibitor In this work I will test the hypothesis that 17AAG and HDAC inhibitor are synergistic against synovial sarcoma and that this synergism may involve opposing effects on the survival pathway N F - K B . While our work has shown that HDAC inhibitors are effective at 1 0 7 Karin M. 2006 1 0 8 Albini A., DelPEva R., Vene R., Ferrari N., Buhler D.R., Noonan D.M., and Fassina G. (2005) "Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kB and Akt as targets" FASEB. 20: 527-529 1 0 9 Scaife C.L., Kuang J., Wills J.C., Trowbridge D.B., Gray P., Manning B.M., Eichwald E.J., Daynes R.A., and Kuwada SK. (2002) "Nuclear Factor B Inhibitors Induce Adhesion-dependent Colon Cancer Apoptosis: Implications for Metastasis" Cancer Res. 62; 6870-6878 "°Mabuchi S., Ohmichi M., Nishio Y., Hayasaka T., Kimura A., Ohta T., Saito M., Kawagoe J., Takahashi K., Yada-Hashimoto N., Sakata M., Motoyama T., Kurachi H., Tasaka K., and Murata Y. (2004) "Inhibition of NFB Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models" 279; 23477-23485 1 1 1 Pierce J.W., Schoenleber R., Jesmok G, Best J., Moore S.A., Collins T., and Gerritsen M.E. (1997) "Novel Inhibitors of Cytokine-induced IkBa Phosphorylation and Endothelial Cell dhesion Molecule Expression Show Anti-inflammatory Effects in Vivo" J. Biol. Chem. Ill; 21096-21103 1 1 2 Moran S.T., Haider K., Ow Y., Milton P., Chen L., and Pillai S. (2003) "Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner" J. Biol. Chem. 278: 21526-21533 15 inducing cell death, HD AC3 has been shown by others to regulate the acetylation of the N F -K B subunit RelA113. Inhibition of HDAC3 with HDAC inhibitor causes increased RelA acetylation resulting in higher levels of activity of this pathway by weakening its binding with the N F - K B inhibitor I K B O . 1 1 4 . Other independent research has confirmed HDAC inhibitor induction of N F - K B activity; an effect which diminished the lethality of these drugs on non-small cell lung cancer cell lines115. This data has prompted researchers to combine HDAC inhibitors with inhibitors of the N F - K B pathway116. Meanwhile 17AAG has been shown to be an effective inhibitor of the N F - K B pathway through its action on IKK 1 1 7 and through the RIP family proteins.118 In this work, we therefore propose that 17AAG may be able to synergize with an HDAC inhibitor by reducing the activation of N F - K B following HDAC inhibitor treatment thereby enhancing the anti-cancer effect of HDAC inhibitor. This hypothesis will be tested on several levels such as by observing protein levels of IKBO. and nuclear RelA using Western blotting techniques, and by investigating N F - K B transcriptional activity using a luciferase assay. Taking this one step further, HDAC inhibitor MS-275 should also synergize with any other inhibitor of N F - K B . T O test this we therefore examined combinations of BAY-11-7085 with the histone deacetylase inhibitor MS-275 by MTT proliferation assays using established synovial sarcoma cell line models in vitro. Synergism was assessed by the median-effect principle of Chou and Talalay. 1 1 3 Chen L. et. al. 2001 1 1 4 Ibid 1 1 5 Mayo M.W., Denlinger C.K, Broad R.M., Yeung F., Reilly E.T., Shi Y., and Jones DR. (2003) "Ineffectiveness of histone deacetylase inhibitors to induce apoptosis involves the transcriptional activation of NF-kappa B through the Akt pathway'' J. Biol. Chem. 278: 18980-18989 1 1 6 Rundall B.K., Denlinger C.E., and Jones D.R., (2004) "Combined histone deacetylase and NF-kappaB inhibition sensitizes non-small cell lung cancer to cell death" Surgery 136 (2): 416-225 "7BroemerM. etal. 2004 1 1 8 Lewis J.,et. al. 2000 16 2. Methods: 2.1. Reagents: 17-AAG was kindly provided under the terms of a Materials Transfer Agreement with the Developmental Therapeutics Branch of the National Cancer Agency (Bethesda, MD) through Kosan Biosciences (Hayward, CA). MS-275 was kindly provided by Schering AG through Berlex Pharmaceuticals (Montville, New Jersey). Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), and trypsin were purchased from Life Technologies (Invitrogen, Mississauga, Ontario, Canada). BAY-11-7085 was purchased from Calbiochem. The pNFKB-Luc plasmid was kindly provided by Dr. T. Michael Underhill of the Department of Cellular and Physiological Sciences, University of British Columbia but was originally from Clontech (Mountain View, CA). This plasmid, pNFKB-Luc is designed for monitoring the activation of N F K B signal transduction pathway. It contains the firefly luciferase (luc) gene from Photinus pyralis. This vector also contains multiple copies of the N F K B consensus sequence fused to a TATA-like promoter (PTAL) region from the Herpes simplex virus thymidine kinase (HSV-TK) promoter. After endogenous N F K B proteins bind to the kappa (K) enhancer element (KB), transcription is induced and the reporter gene is activated. 2.2. Monolayer cell culture. The biphasic synovial sarcoma cell line SYO-1 was kindly provided by Akira Kawai (National Cancer Centre Hospital, Tokyo).1 The presence oft(X;18) in the synovial sarcoma cell lines was confirmed by diagnostic cytogenetic karyotyping, reverse transcription-PCR, and fluorescence in situ hybridization analysis. All monolayer cell 1 Kawai A, Naito N, Yoshida A, Morimoto Y, Ouchida M, Shimizu K, Beppu Y. (2004) "Establishment and characterization of a biphasic synovial sarcoma cell line, SYO-1" Cancer Lett. 204: 105-113. 17 cultures were grown on untreated tissue culture vessels in RPMI 1640 supplemented with 10% FBS under standard incubation conditions (37°C, 95% humidity, 5% C 0 2 ) , 2.3. Proliferation assays Monolayer culture proliferation was assessed by measuring the reduction of 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) from Sigma-Aldrich (St. Louis, MO). Confluent monolayer cultures were trypsinized and replated at 2 x 104 cells per well ("20% confluence), in triplicate, in 48-well plates. These cultures were grown to 50%o confluence, at which time the medium was replaced with media containing the final concentration of vehicle control (0. l%o DMSO), the agent or combination to be tested, and in some cases doxorubicin (for comparative purposes). MTT was dissolved in lx phosphate buffered saline (PBS) to a stock concentration of 5 mg/mL and filter sterilized. MTT was added to a final concentration of 1 mg/mL per well at each time point and incubated under standard conditions for 3 hours, the medium removed and an equal volume of DMSO added. Dissolved MTT formazan for each vessel well was transferred to a 96-well plate in triplicate and the absorbance measured at 570 nm in a PowerWaveX enzyme-linked immunoabsorbent assay plate reader from Bio-Tek Instruments (Winooski, VT). The average reading for each vessel well was used to determine the overall mean for each treatment time point. 2.4. Annexin V - F I T C / P r o p i d i u m Iodide flow cytometry assay Monolayer cultures were plated at 4 xlO5 cells/well on a 6 well plate and treated the following day. At the specified timepoint, trypsinized cells were suspended in lx Binding Buffer and stained with 5pl of Annexin V-fluorescein isothiocyanate (FITC) and 0.5pg of propidium iodide (PI) for 15 minutes at 21°C in the dark. Stained cells were 18 diluted fivefold in lxBinding Buffer. Cells were analyzed on a Coulter® Epics® X-MCL Flow Cytometer from Beckman Coulter (Fullerton, CA) for both FITC and PI signals. 2.5. Synergism analysis: Synergy was quantified according to the protocol published by Chou and Talalay.2 SYO-1 and Fuji cells were treated as single agents in triplicates and the log of the fraction affected/fraction unaffected was plotted as a function of the log of the dose to determine the IC50 from the equation log(fa/fu) = mlog(D)-mlog(IC50). Dose responses of 17AAG and MS-275 in combination were tested at a fixed ratio in triplicates. Combination index (CI) values were obtained from the equation: CIX = Dclx/Dlx + Dc2x/D2x+ DclxDc2x/DlxD2x where Dclx is dose of drug 1 in combination required for achieving X percent of drug efficacy. CI values below 1 are indicative of synergism. 2.6. Protein quantification. Sample protein concentrations were determined by bicinchoninic acid assay as per instruction in the BC A™ Protein Assay Kit (Pierce, Rockford, EL). Briefly, 9 standards were prepared dissolved in vehicle ranging from blank to 2000ug/ml. Samples and unknowns were aliquoted to a 96-well plate in triplicate. Working reagent was prepared as 50 parts reagent A to 1 part reagent B and added to each well of plate at a volume of 200ul. Samples were mixed slightly and incubated for 30 minutes at 37°. Following incubation samples were measured for absorbance at 562 nm in a PowerWaveX enzyme-linked immunoabsorbent assay plate reader from Bio-Tek Instruments (Winooski, VT). 2 Chou T C , and Talalay P. (1984) "Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors" A dv. Enzyme Regul. 22:27-55 19 The average reading for each was used to determine the absorbance for each sample. The protein concentrations of the standards were plotted versus the absorbance reading and a polynomial curve to the fourth power was fitted to the data points. The unknown protein concentrations were calculated according to the equation given by the fitted polynomial curve. 2.7. Total lysate preparation Cellular extracts were prepared in lysis buffer (10 mM Tris (pH 7.5), 1 mM ethylene glycol tetraacetic acid, 150 mM sodium chloride, 1% Triton X-100, 0.5% Nonidet P-40, 1 mMNasVOa, and lmM phenylmethylsulfonyl fluoride from 4 x 105 cells, incubated on ice for 20 minutes, and centrifuged at 10 OOOg to remove cellular debris. 2.8. Nuclear/Cytoplasmic lysate preparations Purified nuclear and cytoplasmic extracts were prepared by using the NE-PER® Nuclear and Cytoplasmic Extraction Reagents (NER/CER) kit from Pierce (Rockford, IL). Briefly, to the CER I and NER reagents, lmM phenylmethylsulfonyl fluoride was added. Samples were resuspended in 1ml of lxPBS and centrifuged at 500g for 3mins. Supernatant was aspirated and cells were resuspended in 200p.l of ice cold CER I by vortexing on high for 15 seconds and incubated on ice for lOmins. CERII was added to samples at a volume of 1 IJJ.1 and mixed by vortexing for 5 seconds twice with a 1 min incubation on ice in-between. Samples were then centrifuged for 5 minutes at 16,000g. Supernatant containing the cytoplasmic extract was then removed and samples were resuspended in 100p.l ice-cold NER by vortexing on high for 15 seconds. Incubation on ice for 10 minutes followed by brief vortexing was repeated 4 times. Samples were then 20 centrifuged at 16,000g for 10 minutes and then supernatant containing nuclear extract was removed. 2.9. Immunoblot analysis. The following primary antibodies: mouse a-acetyl lysine, rabbit ct-p50, rabbit a-p65 were purchased from Abeam (Cambridge, MA), mouse a-IxBa was purchased from Cell Signaling (Beverly, MA), mouse a-Hsp70 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mouse a-Hsp90 was purchased from Stressgen (now Nventa, San Diego, CA), rabbit a-p85 was purchased from Upstate (Millipore, Charlottesville, VA). The following secondary antibodies: goat a-rabbit horse radish peroxidase (HRP) and goat a-mouse HRP were purchased from Pierce Biotechnology (Rockford, IL). Protein samples were loaded onto a 10% polyacrylamide gel from 30% acrylamide/Bis solution from Bio-RAD (Hercules, CA) and run at room temperature for 1.5 hrs at 120V. The proteins on the gel were then transferred to a Trans-Blot® Transfer Medium nitrocellulose membrane from Bio-RAD (Hercules, CA) at room temperature at 400mA and 100-150V for 1 hour. The membrane was then blocked for 1 hour at room temperature in 5% milk tris buffered saline (TBS)-Tween 1%. Primary antibodies were diluted from 1:500 to 1:2000 and secondary antibodies were diluted from 1:10000 in 5% milk TBS-Tween (1%). Membranes were probed with the primary antibody dilution at 4°C overnight. Membranes were washed five times in lx TBS-Tween 1% for 10 minutes at room temperature. Membranes were probed in the secondary antibody dilution at room temperature for 2 hours, and then washed five times in lx TBS-Tween 1% for 10 minutes at room temperature. Membranes were incubated in 1 ml each of SuperSignal® West Femto Luminol/Enhancer Solution and Stable Peroxide Buffer from Pierce 21 (Rockford, IL) at room temperature for 2 mins. Cells were exposed onto photographic film for from lsec to 10 minutes. 2.10. Luciferase analysis SYO-1 cells were plated onto 24 well plates at 4xl04 cells/well. SYO-1 cells were transfected with 0.3p,g of plasmid/well using FuGENE 6 Transfection reagent from Roche Applied Science (Indianapolis, EN) as per instructions from the manufacturer. Briefly, reagent was mixed at ratio of 3 parts reagent to 100 parts serum free RPMI media and incubated at room temperature for 5 minutes. DNA was added directly to solution and incubated for 15 minutes at room temperature. FuGENE mix with DNA was added to cells on 24-well plates at 30ul/well. Cells were treated the following day. After 24 hours treatment, cells were washed with ice cold lxPBS and 100p.l of ice cold lx Passive Lysis Buffer from the Dual-Luciferase® Reporter Assay System kit from Promega (Madison, Wl) and incubated with shaking for 20 minutes at room temperature. Samples were aliquoted 3 times at 20p.l/well to a luciferase plate and twice at 10 p.l/well to a 96 well plate for protein quantification. Samples were injected with 50ul/well of luciferase assay reagent (LAR) II from the kit and read on a EG&G Berthold microplate luminometer 96V (Germany). Samples were normalized with total protein concentration and average values were compared to vehicle control set to 1.00. 2.11. Immunoprecipitat ion Approximately 500 ug of total proteins was incubated with 2 ug of primary antibody at 4 °C for 2 hours, aft er which 20 uL of protein A/G-Plus-Agarose (Santa Cruz Biotechnology) was added to the mixture and incubated overnight at 4 °C. Agarose-antibody-protein complexes were washed three times with lysis buffer. After discarding 22 the supernatant from the final wash, the antibody-protein complexes were resuspended in 25pl lx sample buffer diluted with lysis buffer. Twenty p.1 of each sample was loaded onto 10% polyacrylamide gels, and the immunoprecipitated proteins were separated by gel electrophoresis. 2.12. Statistical Analysis MTT, N F - K B luciferase reporter experiments treated with the drugs as single agents or in combination were done in triplicates and all experiments to determine synergism were repeated at least once. Annexin V-FITC apoptosis assays and western blotting assays were likewise repeated once. Statistical analysis on replicates was performed by calculating the standard deviation, standard error of the mean and 95% confidence intervals. 23 3. Results: 3.1. 17AAG synergizes with MS-275 to inhibit synovial sarcoma in vitro 3.1.1. Synergism in MTT assay Previous work in our laboratory has shown that both Hsp90 inhibitors and histone HDAC inhibitors have anti-cancer activity on synovial sarcoma cell lines. To determine if both agents are able to synergize against synovial sarcoma, an in vitro MTT assay is performed on the synovial sarcoma cell line SYO-1 using the Hsp90 inhibitor 17AAG and the HDAC inhibitor MS-275. SYO-1 cells were grown in monolayer culture and exposed to varying concentrations of each agent alone and in combination at a set ratio in varying doses. IC50 values were determined for the drugs as single agents. Finally, using the Chou and Talaly median dose method, combination index values were determined from the IC50 of the drugs as single agents and the efficacy of the combination treatments; values less than 1 indicate synergism. Both 17AAG and MS-275 are effective at reducing cell proliferation on 17AAG on SYOl Cells 24 hour Doxorubicin 0.5ug/ml 17AAG 0.01 uM| 17AAG 0.05 uM :;; 17AAG 0.1 uM B 17AAG 0.25 uM S: 17AAG 0.5 uM g 17AAG 1 uM 17AAG 2 uM B 17AAG5 uM Dose Response Equation SYOl 17AAG \ v = 0.9104X + 6.1586 y = 0.7036X + 4.46^1 R2 = 0.8932 R z = 0.9041 4-1 TJ ig ti .1 § l x + 2.9602 0.9453 Log Dose Figure 3.1 17AAG Dose Response of SYO-1 cells using MTT Assay 2x l0 4 SYO-1 cel ls a re grown in each well of a 48 wel l plate and t reated with 17AAG. MTT Cel l prol i feration assays are performed at 24, 48 and 72 hours wi th a di lut ion of l m g / m l MTT. 24 synovial sarcoma in a dose and time dependent manner (Figure 3.1, Figure 3.2). The 17-AAGIC50 values at 24 and 48 hours in this assay were 2.1 p M and 0.32 p M respectively (Table 3.1). 17AAG is more effective at inhibiting synovial sarcoma growth than doxorubicin at equimolar concentrations. The MS-275 IC50 values at 24 and 48 hours in this assay were 4.5 p M and 0.56 uM respectively (Table 3.1). MS-275 is also more effective at inhibiting synovial sarcoma growth than doxorubicin at equimolar concentrations. Finally the monolayer cultures were treated in combination at a set ratio of 2 parts 17AAG to 5 parts MS-275 in varying molar concentrations and tested for viable cells. The combination of drugs showed much greater effectiveness for reducing cell numbers of MS275 SYOl D^oxorubicin 0.92 uM gMS-275 0.05uM MS-275 0.25uM ::MS-275 0.5uM gMS-275 luM >;:MS-275 5uM MM S-2 75 10 uM 24 Hour 48 hour 72 hour Dose Response S Y O l 24 Hours MS-274 R 2 = TJ m * c ro -S o c : 33 Z3 ; 769x + 8.396J8 0.9541n . -5 3.4373 26 -1.5 Log Dose Figure 3.2 : M S - 2 7 5 Dose Response of SYO-1 cells using M T T Assay 2xl04 SYO-1 cells are grown in each well of a 48 well plate and treated with MS-275. MTT Cell proliferation assays are performed at 24, 48 and 72 hours with a dilution of lmg/ml MTT. synovial sarcoma in a dose and time dependent manner (Figure 3.3). In the synovial sarcoma cell line SYO-1, a 50% reduction in MTT absorbance at 24 hours was achieved by a combination of 0.25 uM 17AAG with 0.6 LIM MS-275, giving a combination index value of 0.11. This result shows the drug combination is nine times more effective than if the drug effects were simply additive. At 48 hours the IC50 for the drugs in combination was 0.12LLM 25 17AAG and 0.3uM MS-275. Combination index values for 48 hours were as low as 0.34 for a 75% reduction of cell numbers as compared to vehicle control indicating that nearly 3 times 120 100 MTT Assay SYOl 24 Hours MS-275 and 17AAG , o b c o 17AAG 17AAG 17AAG 0.1 0.01 uM + 0.02 uM + uM + MS-MS-275 MS-275 275 0.25 0.025 uM 0.05 uM uM 17AAG0.2 17AAG0.4 UM + MS- uM + MS-275 0.5 275 1 uM uM Combination Index vs Fractional Affect: SYOl 24 Hours, 17AAG + MS-275 •K 1.00 0.50 0.00 1.16 0,40 0.53 ' 0.35 0.21 • 0.11- 0 . l P ' 14 20 40 60 80 100 Fraction of Cells affected MTT Assay SYOl 48 Hours MS-275 and 17AAG 17AAG 17AAG 17AAG 0.1 17AAG 0.2 17AAG 0.4 0.01 uM + 0.02 uM+ uM + MS- uM + MS- uM + MS-MS-275 MS-275 275 0.25 275 0.5 275 1 uM 0.025 uM 0.05 uM uM uM Combination hdexPlots Versus Fractional Aflect: 48 hours SYOlMS-275 and I7AAG 2.00 1.50 % 1.00 0.50 0.00 0 .66&0.68 - •» 0.52 0.34 20 40 60 80 Fraction of Cells affected 100 Figure 3.3 Combinat ion Dose of 17AAG and MS-275 S Y O - 1 Monolayer Culture using M T T Assay . 2 x l 0 4 SYO-1 cells are grown in each wel l o f a 48 wel l plate and treated with MS-275+17AAG. Agents were combined at a set ratio of 2 parts 17AAG to 5 parts MS-275 at vary ing concentrat ions MTT Cell prol i ferat ion assays are performed at 24, 48 and 72 hours wi th a di lut ion of l m g / m l MTT. Combinat ion index values below 1 indicate svnera ism as calculated usina Chou and Ta la lav Median Dose Method. 26 less drugs were required to produce the same effect when combining the drugs as compared to using them single agents The results of these experiments indicate that 17AAG is synergistic with MS-275. Table 3.1 Comparison of IC5o values at 24 and 48 Hour Timepoints for 17AAG and MS-275 as Single Agents and in Combination Using MTT Assay Timepoint (hrs) IC50so Average MS-275 (uM) 17AAG (pM) Combination (pJVI) MS-275 17AAG 24 4.5 2.1 0.6 0.25 48 0.56 0.32 0.3 0.12 3.1.2. Synergism in Annexin V-FITC apoptosis assay To confirm synergistic effect of the agents 17AAG and MS-75 on synovial sarcoma cell lines, another an assay measuring apoptosis was used. SYO-1 cells were grown in monolayer culture, treated with varying concentrations of 17AAG and MS-275 as single agents and in combination at a set ratio and harvested with trypsin. Solubilized cells were then stained with Annexin V-FITC and PI and assayed on a flow cytometer to determine levels of apoptotic/necrotic cells at 24, 48 and 72 hours (Figure 3.4, Figure 3.5, Figure 3.6). Efficacy of 17AAG and MS-275 as single agents on synovial sarcoma was confirmed by these assays at all time points in a time and dose dependent manner. Additionally, by treating the cells in combination at a set ratio of 2 parts 17AAG to 5 parts MS-275, greater apoptosis was observed at lower doses. In the synovial sarcoma cell line SYO-1, a 50% reduction in MTT absorbance at 24 hours was achieved by a combination of 0.7 uM 17AAG with 1.76 u.M MS-275. In comparison, for 50%> efficacy in the flow cytometry assay, 1 U.M 17AAG with 5.6 uM MS-275 was required as single agents. Synergistic effect was similarly 27 observed at 48 and 72 hours. Combination index values were as low as 0.11, 0.07 and 0.2 for 24 hours. 48 hours and 72 hours respectively. 17AAG S Y O l 24 hours Flow Propidium Iodide vs Annexin V FITC 0.79% 6,56% 111 1 J J 0 10 10 DMSO 0.2% 1 2 3 * 10 10 10 10 Doxorubicin 10 u.M 17AAG0.02uM 17AAG 0.2 uM 17AAG0.4uM 17AAG 1 uM 17AAG 2 uM MS-275 S Y O l 24 hours Flow Propidium Iodide vs Annexin V FITC Orrbnation Index Rots vs Fractional Affect: ^ Hxrs, SYOl, 17AAG+MS-275 CD C C o ro C 3 E o U .1.50 M B Q5D Q00 •>0 58 *0.44 VTJ45~ 0.20 676% 879% 15.124 141* 1277% 21.504 3 io* o° io1 io! is1 to" :c' io! io3 IOV IO1 io} io' i iV IO' IO! IO1 10* MS275 0.05 uM MS275 0.5 uM MS275 1 |i M MS275 2 uM MS275 5 u M 17AAG + MS275 S Y O l 24 hours Flow Propidium Iodide vs Annexin V FITC IO ^ io ! 0 50 100 Fraction cf Cells affected m J J J J J1 J J J 10 10 10 10 10 10 10 10 MS275 0.025 MS275 0.1 uM 17AAG 0.01 17AAG 0.04 U.M 0.95% 17« -''JMSB 1 1851 0 io' 13! 10! i i 1.22% Bit MS275 0.25 uM MS275 0.5 uM MS275 1 uM 17AAG0.1uM 17AAG0.2uM 7AAG 0.4 uM Figure 3.4 Combination Index and Comparison of Drugs as Single Agents Versus Combination Dose of 17AAG and MS-275 in SYO-1 using Annexin V/FITC PI Flow Cytometry Assay at 24 Hours 4 x l 0 5 SYO-1 cells are grown in each well of a 6 well plate. Drugs were dosed as single agents and in combination at varying combinations. Combined treatment at a set ratio of 2 parts 17AAG to 5 parts MS-275 at varying concentrations. Cells were stained with propidium iodide and with Annexin V/FITC. Apoptotic cells were calculated from the FACS result and used to calculate combination index values. 28 17MG SYOl 48 hours Flow Propidium Iodide vs Annexin V FITC *1 Ml fa p i ui UK tin tail life 0. u» tn 63::.'-:-:. Sua •. )«;:. 17AAG0.02 MM 1 7 A A G 0 . 2 U M 17AAG0.4nM 17AAG1uM 17AAG20HM MS275 SYQ1 48 hours Flow Propidium Iodide vs Annexin V FITC DMSO 0.2% Doxorubicin 10 \xM Combina t ion Index Plots V e r s u s Fract ional Affect: 48 Hours S Y O l 1 7 A A G + M S 2 7 5 *1 . 77 4 1 . 5 6 i J .0 .79 >0.50 .0.73, » 0 . 7 0 .0 .38 4 0:.07 0 20 40 60 80 100 Fraction of Cells affected £1 a in in* 1 • 1 ci w ia KB-'*: MS275 0.05u MS275 0.5uM MS275 1 uM MS275 2 u M MS275 5uM 17AAG + MS275SYQ1 48 hours Flow Propidium Iodide vs Annexin V U3 11* 12% "1 1 1 "M 1 111111 am w RTC ! 6fcv',/ k01 H3.;:,.. ' - 1 " MS275 0.025nM MS275 0.1 nM + 17AAG0.01uM 17AAG0.O4uM MS275 0.25 uWH 17AAG0.1 uM MS275 0.5uM + 17AAG0.2uM MS275 1 uM + 17AAG0.4nM Figure 3.5 Combination Index and Comparison of Drugs as Single Agents Versus Combination Dose of 17AAG and MS-275 in SYO-1 using Annexin V/FITC PI Flow Cytometry Assay at 48 hours 4xl05 SYO-1 cells are grown in each well of a 6 well plate. Drugs were dosed as single agents and in combination at varying combinations. Combined treatment at a set ratio of 2 parts 17AAG to 5 parts MS-275 at varying concentrations. Cells were stained with propidium iodide and with Annexin V/FITC. Apoptotic cells were calculated from the FACS result and used to calculate combination index values. 29 17AAG SYOl 72 hours How Ropidum Iodide vs Annexin V FITC M m ta I0H Kvl; MB DMSO 0.2% Doxorubicin 10 uM Combination Index Plots Versus Fractional Affect: 72 Hours SYOl 17AAG + MS275 2.00 Si 1-50 j 1.00 'I I u 0.50 0.00 »1.30 •. 1.13 > 1.00 >0.75 4 0.59 4 0.74 rft56 : 0.24.. 0.20 20 40 60 80 Fraction of Cells affected 17AAG0.02uM 17AAG0.2nM 17AAG0.4jiM 17AAG1nM MS275SYQ1 72 haurs Flew Ftopidum lodde vs Annexin V FITC "I 1 "'i 1 1 •< rf •> 17AAG2uM MS275 0.05uM MS275 0.5nM MS275 1nM MS275 2LJVI "mi MS275 5^M " Ujl : in IS un ! &•„• 17AAG0.01nM+ 17AAG0.04nM+ 7AAG0.1 LIM+ 17AAG0.2nM + MS275 0.025LJVI MS275 0.1(1M MS275 0.25uM MS2750.5uM 17AAG0.4^ + MS275 1LJ« Figure 3.6 Combination Index and Comparison of Drugs as Single Agents Versus Combination Dose of 17AAG and MS-275 in SYO-1 using Annexin V/FITC PI Flow Cytometry Assay at 72 hours 4 x l 0 5 S Y O -1 cel ls are g rown in each well of a 6 well plate. Drugs were dosed as single agents and in combinat ion at varying combinat ions. Comb ined t reatment at a set ratio o f 2 parts 17AAG to 5 parts MS-275 at vary ing concentrat ions. Cel ls were sta ined with propidium iodide and with Annex in V / F I T C . Apoptot ic cel ls were calculated f rom the FACS result and used to calculate combinat ion index values. These results confirm that 17AAG and histone deacetylase inhibitors are synergistic against synovial sarcoma in vitro and that the results are not assay specific. It is interesting to note the decrease in cell numbers seen in the MTT assays is demonstrated by the Annexin V/FITC PI flow cytometry assays to be a result of apoptotic cell death. Together both sets of data suggest that low dose combination therapies may be effective against this disease. 30 3.2. Mechanism of synergism does not involve MS-275 acetylation of Hsp70 Treatment of cancer cells with Hsp90 inhibitors has been shown to upregulate the pro-survival protein Hsp70. We have sought to test the hypothesis that the HDAC inhibitor MS-275 is synergistic with Hsp90 inhibitors by abrogating Hsp90 inhibitor-induced activation of Hsp70. We assessed this possibility by looking at the acetylation status of the Hsp70 protein following treatment with the HDAC inhibitor MS-275. If MS-275 is indeed capable of «P cf jf J* jP cf J* J* jf> ,f jf J^IP- a -Hs P 70 + Immunoprecipitate - > 4 — Supernatant ^ 4 Lysate • b ^ J> y J ^ IB: a -Hs P 70 acetylated lysine Hsp70 , < - Hsp70 4- Immunoprecipitate —• 4- Supernatant - • • ^ Lysate • Figure 3.7 Immunoblots (IB) of a - H s p 7 0 and a -Acety la ted Lysine of Immunoprecip i ta tes (IP) of Hsp70 SYO-1 was treated with MS-275 for 24 hours and lysed. 500u.g of lysate was incubated with a-Hsp70 and immunoprecipated. Total volumes of the immunoprecipates were run on a S D S -page gel. Immunoblotting for a -acetylated lysine (a) and a-Hsp70 (b)was then performed. inhibiting Hsp70 via acetylation, then acetylation of Hsp70 proteins should increase following treatment with this agent as a first step. We examined this possibility by precipitating Hsp70 proteins from lysates of SYO-1 cells treated with MS-275 at varying concentrations and then immunoblotting for acetylated lysines (Figure 3.7a), and for Hsp70 31 as a control (Figure 3.7b). In the control experiment where Hsp70 immunoprecipate was immunoblotted with oc-Hsp70, a strong signal for Hsp70 in appeared immunoprecipated lanes with virtually no signal in the supernatant lanes. This indicates successful immunoprecipation of Hsp70 and that total Hsp70 protein levels are not significantly affected by MS-275 treatment. However when Hsp70 immunoprecipate was immunoblotted for a-acetylated lysine, no signal for acetylated proteins was observed at any dose. A band appeared at approximately 70kDa mark in supernantant and total lysate lanes with the a-acetylated lysine lanes but is likely to be coincidental. The results for this experiment indicated that Hsp70 is not acetylated by the HDAC inhibitor MS-275. This suggests that the mechanism for synergism between HDAC inhibitor MS-275 and Hsp90 inhibitor 17AAG does not involve acetylation-mediated inhibition of Hsp70 nor an MS-275-induced decrease in Hsp70 levels. 3.3. Mechanism of synergism involves 17AAG abrogation of MS-275-induced N F -K B activation 3.3.1. MS-275 suppression of the N F - K B inhibitory protein IKBO, is opposed by 17AAG Others have shown that a consequence of HDAC inhibitor treatment of cells is activation of the pro-survival N F - K B pathway. The activation of this particular pathway may diminish cytotoxic effects of HDAC inhibitors. As 17AAG is an inhibitor of the N F - K B pathway, it may abrogate this activation of N F - K B by MS-275. If this is correct, it would provide a mechanism for synergism between 17AAG and MS-275. The IKBCC complex acts as an N F - K B inhibitor by binding to N F - K B dimers in the cytosol, preventing their entry into the nucleus and transporting them back from the nucleus to the cytoplasm. As the degradation of IKBOC is required for N F - K B activation, IKBO. levels are therefore inversely 32 proportional to N F - K B activation. To explore the possibility of N F - K B mediated synergy, protein levels of the N F - K B inhibitory complex IKBO. were measured as a response to 1 7 A A G and MS-275 as single agents and in combination. hcBce levels were obtained by preparing lysates of treated SYO-1 cells, running lOpg of each lysate on a S D S - P A G E gel j# a-p85 a-l K B a-p85 a-l K B a-p85 a-l K B :i.oo 2.50 2.00 , 1.50 1.00 0.50 0.00 I IDACi 01%DMSO 0 25 UMMS-275 1uMMS-275 10uMMS-275 Dose 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Hep90i 0 1% D M S O 0 1 uM 17AAG 0 . 4 u M 1 7 A A G 1 uM 17AAG Dote 3 00 2 50 2 00 0 50 0 00 uumbiiialiun 0 2% DMSO 0 25 uM MS- l u M M S - 2 7 5 * 10uMMS-275 2 75 + 0 1 uM 04 uM 17AAG * 1 u M 17 AAG 1 7AAG Figure 3 . 8 Dose Response of IKBCX to M S - 2 7 5 , 1 7 A A G and Combinat ion Treatment 5 X 1 0 4 SYO-1 cel ls in monolayers are grown in 6 well plates and t reated for 24 hours. Total lysates were prepared and quant i f ied. 10ug of lysate was run on an SDS-page gel . By immunoblot t ing IKBO. and p85 (as a loading control) were detected using a- lKBa and a -p85 ant ibodies. and immunoblotting with a- IKBO. antibody (Figure 3.8). By using these techniques we find that protein levels of the inhibitory complex IKBOC did indeed decrease in synovial sarcoma cells with MS-275 treatment in a dose dependent manner, indicating activation of the N F - K B pathway. However, synovial sarcoma cells treatmented with 1 7 A A G as a single agent or in combination with MS-275, IKB levels are not significantly altered. Thus, treatment with 33 17AAG is successful at reversing MS-275 activation of the pro-survival N F - K B pathway, providing evidence for a mechanism of synergistic cytotoxicity. 3.3.2. M S - 2 7 5 induction of nuclear levels of the N F - K B subunit RelA are opposed by 17AAG N F - K B activation'requires relocation of the N F - K B dimers to the nucleus from the cytoplasm before it can mediate its transcriptional effects on cell survival. To provide further 1 ^ oc-p85 a-RelA 4? & a-p85 a-RelA M r **; | a-p85 W a-RelA 2 3 . 0 0 u 2 . 5 0 o g . 2 . 0 0 | 1.50 O 1.00 ro o) 0 . 5 0 o z 0 0 0 2 . 4 4 2.41 1:33 TIT 0 . 1 % D M S O 0 . 2 5 u M M S - 1 u M M S - 2 7 5 10 u M M S -2 7 5 2 7 5 | 3.00 JE 2.50 o £ 2.00 I 1.50 O 1.00 ro .S> 0.50 o Z 0.00 0.48 0 24 1 -0 . 1 % DMSO 0.1 u M 1 7 A A G 0.4 u M 1 u M 1 7 A A G 1 7 A A G 3.00 2.50 2.00 1.50 1.00 0.50 0.00 1.44 1.00 0.76 0.52 0.2% DMSO 0.25 uM MS- 1 uM MS-275 10 uM MS-275 + 0.1 uM + 0 .4uM 275 + 1 uM 17 AAG 17 AAG 17AAG Figure 3.9 Dose Response of RelA to M S - 2 7 5 , 1 7 A A G and Combinat ion Treatment 5 X 1 0 4 SYO-1 cel ls in monolayers are g rown in 6 well plates and t reated for 24 hours. Nuclear and cytoplasmic extracts were prepared and quant i f ied. 15Lig were run on an SDS-page gel . Using Immunoblot t ing tech in iques, RelA and p85 (as a loading control) were detected using a-RelA and a -p85 ant ibodies. evidence for the hypothesis that 17AAG and MS-275 synergize through effects on N F - K B , nuclear levels of N F - K B subunit RelA are measured. SYO-1 cells are grown on 90mm plates 34 and treated for 24h with MS-275 or 17AAG as single agents and in combination at varying concentrations. Nuclear and cytoplasmic extracts are prepared from treated cells and the nuclear extracts are run on an SDS-page gel. Nuclear RelA levels are observed by immunoblotting with a-RelA antibody (Figure 3.9). The results show that while nuclear levels of RelA increase following treatment with MS-275, nuclear levels of RelA decrease following treatment with 17AAG both as a single agent and in combination with MS-275. In this experiment, treatment with 17 AAG is successful at reversing MS-275 activation of the pro-survival N F - K B pathway, and providing further evidence for this mechanism of synergism. 3 . 3 . 3 . N F - K B transcriptional activity is upregulated by treatment with M S - 2 7 5 but is downregulated with both 1 7 A A G treatment and combined treatment The ultimate experiment to test the hypothesis of synergism between 17AAG and MS-275 through the N F - K B pathway requires observation of the transcriptional potential of N F - K B following treatment with these drugs. In order to detect N F - K B transcriptional activation levels, SYO-1 cells were transfected with a N F - K B luciferase reporter. Cells were then treated with 17AAG and MS-275 as single agents and in combination for 24 hours, and lysed in passive lysis buffer. A part of the samples were aliquoted in triplicate to a luminometer plate and to a 96 well plate. In the luminometer, samples were injected with LARII reagent and luminosity was read. The remaining sample was quantified using copper sulfate/bicinchoninic acid assay as described in methods section. N F - K B reporter activity was normalized to total lysed protein concentration and vehicle control. Vehicle control was set to 1.00 (Fig 3.10). The results show that MS-275 dramatically increases N F - K B transcriptional activity, whereas 17AAG as a single agent decreases 35 transcriptional activity. In the combination, the 17AAG effect predominates as there is a net decrease in N F - K B transcriptional activity. Overall the evidence accumulated from observing levels of IKBCC, nuclear RelA and N F - K B luciferase reporter following drug treatment are all consistent, and support the hypothesis that MS-275 and 17AAG Luciferase Assay MS-275 NF-kB: SYOl Fold Induction NF-kB Activity 24 Hours Treatment Readout Normalized to total Protein Concentration Feb 23, 2007 • IS 0.1% DMSO ^ _ 0.05uM MS-275 \ 0 1 uM MS-275 0 5 uM MS-275 IHOT :¥ 1 uM MS-275 a 5 uM MS-275 Luciferase Assay 17AAG NF-kB: S Y O l Fold Induction NF-kB Activity 24 hrs Treatment Readout Normalized to total Protein Concentration March 8, 2007 8 0.1% DMSO .S 0.1 uM 17 AAG if 0.2 uM 17AAG 0.4 uM 17AAG 1 uM 17AAG 2 uM 17AAG Luciferase Assay MS-275+17AAG NF-kB: SYOl Fold Induction NF-kB Activity 24 hrs Treatment Readout Normalized to total Protein Concentration March 8, 2007 » 0 1% DMSO Figure 3.10 Transcriptional Activation of N F - K B Luciferase Reporter Activity Following Treatment SYO-1 cells were grown as monolayer cultures in 24 well plates and transfected with 0.3ug of N F - K B luciferase reporter plasmid. Cells were treated the following day for 24 hours and lysed with passive lysis buffer. Samples were aliquoted to plates and simultaneously assayed for luminosity by injection with 50ul/well of LARII reagent and protein quantitiy by copper sulfate/bicchionic acide assay. Readings for luminosity were normalized to protein concentration and vehicle control was set to 1 nn synergize by acting through the N F - K B pathway in synovial sarcoma. Whereas M S - 2 7 5 activates this survival pathway, 1 7 A A G deactivates it, abrogating a survival mechanism triggered by M S - 2 7 5 . 3.4 . M S - 2 7 5 synergizes with N F - K B inhibitors 3 .4 .1 . B A Y - 1 1 - 7 0 8 5 is a N F - K B inhibitor in synovial sarcoma 0.60 0.40 -N 0.20 0.00 i0.05uMMS-275 + O.luM 17MG 0 luM MS-275 + 0.2uM 17AAG ;0.5 uM MS-275 + 0.4uM 17MG 1 uM MS-275 + luM 17AAG 5 uM MS-275 + 2uM 17AAG 36 If 17 AAG is successful at abrogation of MS-275 induced N F - K B activation, and this is indeed one of the mechanisms by which these drugs synergize, then MS-275 should also show synergistic activity with other N F - K B inhibitors. We have sought to confirm this by examining possible synergistic effect between MS-275 and the N F - K B inhibitor BAY-11-7085. The compound BAY-11-7085 was first tested for its abilities to inhibit N F - K B in synovial sarcoma. SYO-1 monolayer cultures in 24 well plates were transfected with the N F - K B luciferase reporter plasmid. Cells were then treated for 24 hours with BAY-11-7085 and lysed. A part of the samples were aliquoted in triplicate to a luminometer plate and to a 96 well plate. In the luminometer cells were injected with LARII reagent and luminosity was read. The other samples were quantified using copper sulfate/bicinchoninic acid assay as Figure 3.11 B A Y - 1 1 - 7 0 8 5 Inhibition of N F - K B Luciferase Reporter Activity SYO-1 cells were grown as monolayer cultures in 24 well plates and transfected with 0.3ug of N F -K B luciferase reporter plasmid. Cells were treated the following day for 24 hours and lysed with passive lysis buffer. Samples were aliquoted to plates and simultaneously assayed for luminosity by injection with 50ul/well of LARII reagent and protein quantitiy by copper sulfate/bicchionic acide assay. Readings for luminosity were normalized to protein concentration and vehicle control was set to 1.00 described in the methods section. N F - K B reporter activity was normalized to total lysed protein concentration and vehicle control. Vehicle control was set to 1.00. As observed from the data (Figure 3.11), BAY-11-7085 is an effective inhibitor of N F - K B luciferase reporter 37 transcriptional activity, indicating a strong repression of N F - K B activity with an IC50 of 4.9 uM for N F - K B luciferase reporter repression. 3.4.2. Synergism between BAY-11-7085 and MS-275 To determine whether MS-275 can synergize with BAY-11-7085, the effect of BAY-11-7085 on cell viability was observed using a 24, 48, and 72 hour MTT assay at varying concentrations on the cell line SYO-1 in vitro (Figure 3.12). From the results of the drugs as BAY-11-7085 MTT SYOl 120 T 100 H— u -Q ro > f: 0.5 uM BAY-11-7085 st 1 uM BAY-11-7085 5 uM BAY-11-7085 10 uM BAY-11-7085 g 20 uM BAY-11-7085 24 Hours 48 Hours 72 Hours Figure 3.12 BAY-11-7085 Dose Response of SYO-1 cells Using MTT Assay 3 x l 0 5 SYO-1 cells are grown in each well of a 24 well plates and treated with 17AAG. MTT Cell proliferation assays are performed at 24, 48 and 72 hours with a dilution of l m g / m l MTT. single agents the IC5 0 values were calculated. Next BAY-11-7085 was combined with MS-257 at a set ratio in varying concentrations and used to treat SYO-1 cells for 24, 48 and 72 hours. These cells were then subjected to an MTT assay. Combination index values as quantifications of synergism were determined using the Chou and Talalay median dose 38 method by taking the data from the IC50 of the drugs as single agents and the efficacy of the drugs in combination (Figure 3.13). The results of these experiments show that BAY-11-MS-275 + BAY-11-7085 MTT SYOl 1 0 3 0 3 i Hours 48 Hours 72 Hours 0.075 LM BAY-11-7085 + 0.025 uM MS 275 0.15 uM BAY-11-7085 + 0.05 uM MS 275 0.75 uM BAY-11-7085 + 0.25 LM MS 275 1.5 l M BAY-11-7085 + 0.5 uMMS 275 5LMBAY-11-7085| + 1UMMS275 2.0 1.5 1.0 0.5 0.0 Combination Index Plots Versus Fractional Affect: 24 Hours S Y O l BAY-11-7085 and MS-275 * u , : " » 0 . 8 6 v0.87 »0 .58 -> 0.48 + 0.26 i0.62 *0.3r>0'36 Fractior?9ffected 100 Cornb2ij=98n Index Plots Versus Fractional Affect: 48 Hours SYOl BAY-11-7085 and MS-275 2.0 1.5 1.0 0.5 0.0 1.67 E0 .73 a 0.74 ° - ^ 5 a.S6 » 0.25 50 Fraction affected 100 Corjprjjrjation Index Plots Versus Fractional Affect: ' 72 hrs S Y O l BAY-11-7085 and MS-275 2.0 x a> <M l l .O . | . 5 o u o.o 0.57 • 0.56 O0.71 *0 .61 * 0 . 3 8 26 50 Fraction affected 100 Figure 3.13: MS-275 + BAY-11 -7085 Dose Response and Combinat ion Index Values of SYO-1 Treated Cells Using MTT Assay 3 x 1 0 s SYO-1 cells are grown in each well of a 24 well plates and treated with MS-275+17AAG. Agents were combined at a set ratio of 3 parts BAY-11-7085 to 1 part MS-275 at varying concentrations. MTT Cell proliferation assays are performed at 24 , 48 and 72 hours with a dilution of lmg/ml MTT. Combination index values below 1 indicate synergism as calculated using Chou and Talalay Median Dose Method. 7085 synergizes with MS-275. As single agents, to reduce cell numbers by 70%, the dose of BAY-11-7085 required is 6.2 uM and for MS275 is 9.5 uM at 24 hours in these assays. In synovial sarcoma cell line SYO-1, 70% reduction in MTT absorbance at 24 hours was achieved by a combination of 1.5 uM BAY-11-7085 and 0.5 uM MS-275. This result is 3 times more effective than if the drugs were simply additive. This shows that the N F - K B activation does diminish synovial sarcoma response to HDAC inhibitors which can be 39 reversed by specifically inhibiting the N F - K B pathway in itself. IC50 values of the drugs as single agents,and in combination are summarized in the table below (Table 3.2) Table 3.2 IC50 Values following Treatment by MS-275, B A Y - 1 1 -7085 and the Combination on SYO-1 Cell Proliferation at 24, 48 and 72 Hours Timepoint (hrs) IC5O50 Average MS-275 (uM) BAY-11-7085 (uM) Combination (uM) MS-275 BAY-11 24 5.2 3.5 0.45 1.4 48 0.56 3.4 0.27 0.8 72 0.36 3.03 0.26 0.77 40 4. Discussion and Conclusion 4.1. Synergy The motivation for this work is to contribute to the development an effective therapy for synovial sarcoma. We combined the two agents 17AAG and MS-275, which have individually demonstrated cytotoxicity on synovial sarcoma, and tested for synergism. 17AAG has shown some success in clinical trials. It has completing several phase I studies for solid tumours such as colorectal, pancreas, liver, ovary, thyroid, renal, and lung carcinomas as well as liposarcoma.l'2 This agent has also progressed to a few phase II trials.3' However, these trials have also demonstrated a number of limitations to this drug. 17AAG caused toxicities in patients such as elevation in circulating liver enzymes (indicative of liver damage), elevation in alkaline phosphatase, elevation in bilirubin, optic neuritis, dyspnea, fatigue, nausea, vomiting, anorexia, diarrhoea, anaemia and low grade fever .4'5'6 Similarily, the HDAC inhibitor MS-275 has completed phase I trials in cancers such as melanoma, sarcoma, leukemia, lymphoma and renal cell, non-small cell lung, breast and colorectal carcinomas, but has shown such toxicities as nausea, vomiting, anorexia, fatigue, hypoalbuminemia and hypocalcemia.7'8 These toxic effects limit the dose that can be 1 Grem J.L., Morrison G., Guo X.D., Agnew E., Takimoto C.H., Thomas R., Szabo E., Grochow L., Grollman F., Hamilton J.M., Neckers L., and Wilson R.H.(2005) "Phase I and pharmacologic study of 17-(Allylamino)-17-demethoxygeldanamycin in adult patients with solid tumors. "J. Clin. Oncol. 23: 1885-1893. 2 Goetz M.P., Toft D., Reid I, Ames M., Stensgard B., Safgren S., Adjei A.A., Sloan I, Atherton P., Vasile V., Salazaar S., Adjei A., Croghan G. and Erlichman C. (2005) "Phase T Trial ofl7-(Allylamino)-17-demethoxygeldanamycin in Patients With Advanced Cancer" J. Clin. Oncol. 23: 1078-1087 3 Pacey S. et. al. 2006 4 Ronnen E.A., Kondagunta G.V., Ishill N., Sweeney S.M., DeLuca J.K., Schwartz L., Bacik J., andMotzer R.J. (2006) "A phase JJ trial of 17-(Allylamino)-17-demethoxygeldanamycin in patients with papillary and clear cell renal cell carcinoma" Invest. New Drugs 24:543-546 5 Grem J.L. et. al. 2005 6 Goetz M.P. et. al. 2005 7 Ryan Q.C., Headlee D., Acharya M., Sparreboom A., Trepel J.B., Ye J., Figg W.D., Hwang K., Chung E.J., Murgo A., Giovanni M.., Elsayed Y., Monga M., Kalnitskiy M., Zwiebel J., and Sausville EA. (2005) "Phase I and Pharmacokinetic Study of MS-275, a Histone Deacetylase Inhibitor, in Patients With Advanced and Refractory Solid Tumors or Lymphoma" J. Clin. Oncol. 23: 3912-3922 41 administered to patients and as a result may limit the efficacy of the drug in combating cancers. Additionally the achievable serum levels of MS-275 are quite low with an average maximum serum concentration of 53.1 ng/ml or 0.14 uM, with a mean half life of 33 hours at the maximum tolerated dose of 8 mg/m2.9 This can be explained by the fact that MS-275 has a high protein binding affinity that limits the unbound (actively available) fraction.10 Should these drugs synergize they would increase the benefit for patients by improving treatment efficacy at a lower dose and concurrently reducing toxic effects. The evidence presented in this thesis demonstrates synergy between 17AAG and MS-275 in synovial sarcoma cells in vitro at a fixed ratio of 2 parts 17AAG to 5 parts MS-275. The fact that MS-275 is mainly a class I HDAC inhibitor and only poorly inhibits the other classes raises some questions about the nature of synergy between these two agents. One such question is whether or not being a specific inhibitor for class I HDAC increases or decreases the efficacy of synergism. Other HDAC inhibitors such as FK228 are effective at inhibiting multiple classes of HDAC. Importantly, acetylation of Hsp90 results in inhibition of this chaperone, and others have shown that by acting to inhibit the class II HDAC 6, FK228 is able to cause Hsp90 acetylation. This suggests that combining Hsp90 inhibitor 17AAG with FK228 or another pan-HDAC inhibitor would result in some redundancy as both target Hsp90. As MS-275 is not effective at targeting HDAC 6, it may be more effective at synergizing with 17AAG. 8 Gojo I., Jiemjit A., Trepel J.B., Sparreboom A., Figg W.D., Rollins S., Tidwell M.L., Greer J., Chung E.J., Lee M. J., Gore S.D., Sausville E.A., Zwiebel J. and Karp J.E. (2007) "Phase I and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias" Blood 109: 2781-2790 9 Ibid. 1 0 Acharya M.R., Sparreboom A., Sausville E.A., Conley B.A., Doroshow J.H., Venitz J., and Figg W.D. (2006) "Interspecies differences in plasma protein binding of MS-275, a novel histone deacetylase inhibitor" Cancer Chemother Pharmacol 57:275-281 42 A few synergy studies using Hsp90 inhibitor and various FID AC inhibitors have shown positive results in hematopoetic malignancies, giving a rationale for organizing clinical trials that combine the two agents. One study has shown synergistic induction of mitochondrial damage through cytochrome c release, caspase-3 and caspase-8 induction; all leading to apoptosis in a variety of human leukemia cells when 17 AAG was combined with the HDAC inhibitors SAHA and NaBu. n ' n These leukemia cells included the fusion protein derived Bcr-Abl+ leukemia resistant to imatinib mesylate. Another study has shown that by combining 17AAG with the HDAC inhibitor cinnamic hydroxamic acid analog (LBH589), synergy was seen in the induction of apoptosis and attenuation of Bcr-Abl, p-AKT, and p-ERKl/2 in human chronic myeloid leukemia blast crisis cells. This combination also induced more apoptosis in imatinib mesylate resistant human chronic myeloid leukemia blast crisis and acute myeloid leukemia cells.13 While these studies are suggestive, the best encouragement for beginning clinical trials in synovial sarcoma with 17AAG and MS-275 in combination, would be future synergy studies of this combination using animal models with synovial sarcoma cancers. 4.2. Mechanism of Synergy This promising result raises further questions about the nature of this disease and the mechanism of action of the inhibitors. Understanding the mechanism of action of these agents to synergize on synovial sarcoma is the focus of the second part of this thesis. Effective and responsible administration of treatment requires in depth understanding of the 1 1 Rahmani M., Yu C, Dai Y., Reese E., Ahmed W., Dent P., and Grant S. (2003) "Coadministration of the Heat Shock Protein 90 Antagonist 17-Allylamino-17-demethoxygeldanamycin with Suberoylanilide Hydroxamic Acid or Sodium Butyrate Synergistically Induces Apoptosis in Human Leukemia Cells" Cancer Res. 63: 8420-8427 1 2 Rahmani M. et. al. 2005 13George P., Bali P., Annavarapu S., Scuto A., Fiskus W., Guo F., Sigua C , Sondarva G., Moscinski L., Atadja P., and Bhalla K. (2005) Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3" Blood 105:1768-1776 43 mechanism of effect of the drug on the tumour in order to predict and understand side effects. In order to understand the specific effects of the drug on each cancer, more molecular studies are required. Increased information of the mechanism of action of this combination may give insight as to which other cancers the combination may be effective in targeting based on the knowledge of activated pathways in those cancers. Additionally suggestions for future drug combinations combining similar drugs or drugs with common targets may result from this study. Most importantly, studying the mechanism gives greater support for the synergy findings based on cell proliferation and apoptosis assays and gives further rationale for clinical studies based on these drugs. As a survival technique in response to treatment with anti-cancer agents, tumours may upregulate a compensatory pathway or trigger cell survival signals. The two hypotheses for mechanism of synergy discussed below consider the effect of one agent to trigger a compensatory pro-survival function following drug treatment and abrogation of the activation of this pathway by the other agent. 4.2.1. Heat Shock Protein 70 Acetylation The first hypothesis for mechanism of synergy between 17 AAG and MS-275 focused on the effects of both drugs on Hsp70. Hsp70 may be important for tumour progression and development, and specific targeting of this protein has been shown to result in apoptosis in some cancers. Importantly Hsp70 is upregulated in response to Hsp90 inhibition. As Hsp70 has a similar role in the cell it may serve to compensate for Hsp90 inhibition and thereby limit the effects of 17AAG and thus prolonging cell survival. This makes any drug effective at targeting Hsp70 attractive for combining with 17AAG. As Hsp70 has been found to be 44 acetylated by the HDAC inhibitor FK228 , and as Hsp70 has been found to be associated with HDAC 1,2 and 3 from class I15, it is worthwhile to investigate the possibility of MS-275 as being an inhibitor for Hsp70. The work presented here has shown that MS-275 is not able to acetylate Hsp70 suggesting that MS-275 has no effect on this protein. From this it can be concluded that effects on Hsp70 inhibition by acetylation are not a likely mechanism of synergy between 17AAG and MS-275. However it remains worthwhile to study the effects of other HDAC inhibitors on Hsp70 acetylation. Specifically because of the results from a recent paper showing that Hsp70 can be acetylated by the HDAC inhibitor FK228,16 investigating Hsp70 activation by treatment with 17 AAG and the corresponding abrogation of activation by FK228 would be potentially useful for clinical studies. Additionally, a quantified synergy study between inhibitors that specifically target Hsp90 and Hsp70 on any cancer responsive to Hsp90 alone (including synovial sarcoma)17 would be worthwhile. 4.2.2. N F - K B Activation The second hypothesis for synergy between 17AAG and MS-275 suggests mediation by the pro-survival N F - K B pathway. While HDAC inhibitors have been demonstrated to activate N F - K B , 17AAG inhibits this pathway. Given that HDAC inhibitor-induced activation of a pro-survival pathway may limit its effectiveness as an anti-cancer agent, 17AAG may have an important role in combination treatment through abrogating N F - K B activation. By treating synovial sarcoma cells in vitro with either 17 AAG, MS-275 or the combination, and observing for effects on N F - K B activation at three different stages we are 1 4 Wang Y., et. al. (2007) 1 5 Johnson C.A. et. al. (2001) 1 6 Wang et. al. 2007 1 7 Terry et. al. 2005 45 able to support this hypothesis To elaborate, i K B a protein (which inhibits the N F - K B dimer and whose protein levels are inversely proportional to N F - K B activation) decreases with increasing doses of MS-275, whereas 17 AAG and the combination treatment both maintain or increase levels of IKBCC. Similarly the RelA subunit of the N F - K B dimer, which is only able to activate transcription if it is within the nucleus, is present in greater quantity within the nucleus following MS-275 treatment but is present in lower quantities within the nucleus following both 17AAG and the combination treatment. Finally the transcriptional activity of N F - K B itself, quantified using the readings from an N F - K B luciferase reporter plasmid system, is increased with MS-275 treatment and decreased with 17AAG or combined treatments. All of this evidence demonstrates 17AAG abrogation of MS-275-mediated N F -K B activation when the combined treatment is given. Proceeding one step further, the N F - K B inhibitor BAY-11-7085 has been tested in combination with MS-275 to determine synergistic effect on synovial sarcoma. Firstly the ability of BAY-11-7085 to inhibit N F - K B has been demonstrated by a decrease in the transcriptional activity of N F - K B as seen using the N F - K B luciferase reporter. Next the efficacy of BAY-11-7085 as a single agent to reduce cell viability of SYO-1 in vitro using MTT assays has been demonstrated. Finally the combination of BAY-11-7085 and MS-275 on SYO-1 in MTT assays were analyzed using the Chou and Talalay median dose method and synergy between the two agents was determined. This provides further evidence that it is the inhibition of N F - K B , an effect common between 17 AAG and BAY-11-7085, that underlies the synergistic enhancement of apoptosis in HDAC inhibitor-treated synovial sarcoma cells. 46 These findings are nevertheless limited by the incomplete knowledge of the role in N F - K B in synovial sarcoma and in cancers in general. The N F - K B pathway can be activated in many different ways, suggesting it plays a key role in both normal and oncogenic signaling. Furthermore, in certain cellular contexts N F - K B plays a role in activating cell death rather than resisting apoptosis. Before inhibition of the pathway in synovial sarcoma patients can be strongly advocated, it would be interesting to examine further the effects of genes regulated by N F - K B in synovial sarcoma itself. Evidence from cDNA tissue microarray expression profiling has demonstrated that the N F - K B activator RIPK4 is highly upregulated in synovial sarcoma. In addition the work in this thesis gives evidence that N F - K B subunit RelA can be found in the nucleus of control treated synovial sarcoma cells, suggesting constitutive activation of N F - K B . However, more work is needed to show that N F - K B plays a role in synovial sarcoma oncogenesis that is enhanced as a side effect of HDAC inhibitor treatment. By demonstrating that the N F - K B pathway is constitutively active in synovial sarcoma and that this activation upregulates genes that cause the pathogenesis of synovial sarcoma, the importance of inhibiting the N F -K B pathway and abrogating HDAC inhibitor-induced N F - K B activation would be emphasized. Additionally, other N F - K B inhibitors need to be investigated for efficacy in synovial sarcoma. The effects shown in the results section of BAY-11-7085 are promising but there exists many other drugs which also inhibit N F - K B that may be effective alone or in combination with HDAC inhibitors. BAY-11-7085, as an agent to be used in synovial sarcoma clinical trials, has three shortcomings. Firstly it is currently not tested in any clinical trials. Secondly we found that this drug is toxic against fibroblast and human mesenchymal 47 cells (data not shown). Thirdly other researchers have shown that it does not specifically inhibit N F - K B but can also act via p38 kinase18. This also raises the possibility that the mechanism of synergy of MS-275 and BAY-11-7085 did not only occur through N F - K B but through additional synergistic effects on other pathways. Based on these limitations useful future directions to take involve exposing synovial sarcoma cell lines to various levels of specific N F - K B inhibition, such as (1) use of siRNA against upstream activators or against the N F - K B subunit itself, (2) other drugs that have been shown to inhibit the N F - K B and/or (3) introducing mutant forms of the subunits by transfection of a plasmid then observing for efficacy at reducing cell proliferation and inducing apoptosis. It would also be interesting to study whether or not MS-275 can synergize with these various methods of N F - K B inhibition. Other important points of investigation include looking at a the effect of 17AAG, MS-275 and the combination on number downstream targets of N F - K B , focusing on those with known roles in cancer pathogenesis such as Bcl-219, VEGF, IL-820, and MMP.921. The expression of these proteins following treatment can be studied using quantitative RT-PCR and protein levels using Western blot techniques 4.2.3. Other Mechanisms An important characteristic of both 17AAG and the HDAC inhibitor MS-275 drugs is that each of them regulate several functions within the cell, leading to diverse effects. Hsp90 1 8 Hu X.Janssen W.E., Moscinski L.C., Bryington M , Dangsupa A., Rezai-Zadeh N., Babbin B.A., Zuckerman K.S. (2001) "An IkappaBalpha inhibitor causes leukemia cell death through a p38 MAP kinase-dependent, NF-kappaB-independent mechanism" Cancer Res. 6:6290-6296. 1 9 Catz S.D., and Johnson J.F. (2001) "Transcriptional regulation of bcl-2 by nuclear factor KB and its significance in prostate cancer" Oncogene 20: 7342-7351 2 0 MukaidaN., Mahe Y., and Matsushima K. (1990) "Cooperative Interaction of Nuclear Factor-KB- and ds-Regulatory Enhancer Binding Protein-like Factor Binding Elements in Activating the Interleukin-8 Gene by Pro-inflammatory Cytokines" J. Biol. Chem. 265: 21128-21133 2 1 Yokoo T., and Kitamura M. (1996) "Dual regulation of EL-1 beta mediated matrix metalloproteinase-9 expression in mesangial cells by NF-KB and AP-1" Am. J. Physiol. 270: F123-30 48 has hundreds of putative clients with different functions, and acetylation not only regulates the transcription of an unknown number of genes, but also regulates non-histone proteins directly. This information leads us to suggest that the mechanism of synergy between the two agents may not be confined to a single pathway. Other worthwhile pathways to investigate for synergistic effects are those which are fundamentally involved in cancer progression. One particular example involves cell cycle progression. Hsp90 has been found to play an important role in the cell cycle by inducing GO/1 cell cycle arrest and cell death in a dose- and time-dependent manner in Mantle-cell lymphoma cell lines.22 This effect was associated with the downregulation of cyclin DI, cdk4 and Akt, depletion of Bid, and activation of the intrinsic/mitochondrial caspase pathway.23 Similarly others have found HDAC inhibitors to be responsible for the induction of p21/WAF-l24'25 which functions as a well-characterized cyclin-dependent kinase (cdk) inhibitor.26 Furthermore specific absence of HDAC 1 in MCF7 cells resulted in either arrest at the Gl phase of the cell cycle, or at the G2/M transition, resulting in loss of mitotic cells, cell growth inhibition and an increase in the percentage of apoptotic cells.27 As both drugs have an effect on cell cycle inhibition yet seem to arrest growth at different phases, it is possible that the mechanism of synergism involves the action of the drugs to stop growth at multiple points. 2 2 Georgakis G.V., Li Y., and Younes A. (2006) "The heat shock protein 90 inhibitor 17-AAG induces cell cycle arrest and apoptosis in mantle cell lymphoma cell lines by depleting cyclin DI, Akt, Bid and activating caspase 9" BrJ. Haematol. 135:68-71 2 3 Ibid. 2 4 Okamoto H., Fujioka Y., Takahashi A., Takahashi T., Taniguchi T., Ishikawa Y., and Yokoyama M. (2006) "Trichostatin A, an Inhibitor of Histone Deacetylase, Inhibits Smooth Muscle Cell Proliferation via Induction of p21WAFl" J. Atheroscler. Thromb. 13: 183-191 2 5 Archer S. Y. Johnson J., Kim H.J., Ma O., Mou H., Daesety V., Meng S., and Hodin R.A "The histone deacetylase inhibitor butyrate downregulates cyclin BI gene expression via a p21AVAF-l-dependent mechanism in human colon cancer cells" Am J Physiol Gastrointest Liver Physiol 289: G696-G703 2 6 Gartel A.L., and Radhakrishnan S.K. (2005) "Lost in Transcription: p21 Repression, Mechanisms, and Consequences" Cancer Res. 65: 3980-3985 2 7 Senese S., Zaragoza K., Minardi S., Muradore I., Ronzoni S., Passafaro A., Bernard L., Draetta G.F., Alcalay M., Seiser C, and Chiocca S. (2007) "A role for histone deacetylase 1 in human tumor cell proliferation" Mol Cell Biol. [Epub ahead of print] 49 4.3. Conclusion 17AAG and MS-275 have a synergistic anti-proliferative and pro-apoptotic effect on synovial sarcoma. As single agents, the IC50 for 17AAG is 2.1 u.M and for MS275 is 4.5 uM at 24 hours in these assays. In the synovial sarcoma cell line SYO-1, a 50% reduction in MTT absorbance at 24 hours was achieved by a combination of 0.24 uM 17AAG with 0.64 pJVl MS-275. This result is nine times more effective than if the drugs were simply additive. Synergistic effects were still present at later assay time points and using the annexin V-FITC PI assay to measure apoptosis. Using western blot analysis of whole cell lysates we find that 17AAG and MS-275 in combination reverse the activation of N F - K B seen with MS-275 alone, as measured by levels of the N F - K B inhibitory complex IKBOC complex. Net effects of these drugs on nuclear levels of active N F - K B subunit RelA and on N F - K B luciferase reporter transcription are consistent with these findings. In addition the N F - K B inhibitor BAY-11-7085 is also synergistic with MS-2754. Overall our data shows that N F - K B pathway plays an important role in the synergistic activity of 17AAG and MS-275. These results open the door for future studies by putting forward a promising combination treatment that should be further developed and examined. The mechanistic study of the two drugs gives insight on the agents and suggests ways for which the interaction of the drugs can be further examined. Most importantly this research opens the door for future development in clinical trials based on 17AAG and MS-275, or related agents. 50 References 1 Acharya M.R., Sparreboom A., Sausville E.A., Conley B.A., Doroshow J.H., Venitz J., and Figg W.D. (2006) "Interspecies differences in plasma protein binding of MS-275, a novel histone deacetylase inhibitor" Cancer Chemother Pharmacol 57:275-281 2 Albini A, Dell'Eva R., Vene R., Ferrari N., Buhler D.R., Noonan D.M., and Fassina G. (2005) "Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kB and Akt as targets" FASEB. 20: 527-529 3 Alkalay I, Yaron A., Hatzubai A., Oriani A., Ciechanover A., and Ben-Neriah Y. (1995) "Stimulation-dependent hcBa phosphorylation marks the N F - K B inhibitor for degradation via the ubiquitin-proteasome pathway" Proc. Natl. Acad. Sci. USA 92: 10599-10603 4 Allander S.V., Illei P.B., Chen Y., Antonescu C.R., Bittner M., Ladanyi M., Meltzer PS (2002) "Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation" Am. J. Pathol. 161: 1587-1595 5 Archer S.Y. Johnson J., Kim H.J., Ma O., Mou H., Daesety V., Meng S., and Hodin RA "The histone deacetylase inhibitor butyrate downregulates cyclin BI gene expression via a p21AVAF-l-dependent mechanism in human colon cancer cells" Am J Physiol Gastrointest Liver Physiol 289: G696-G703 6 Beg A. A , Sha W.C., Bronson R.T., Ghosh S., and Baltimore D. (2002) "Embryonic lethality and liver degeneration in mice lacking the RelA component of N F - K B " Nature 376; 167- 170 7 Broemer M., Krappmann D., and Scheidereit C. "Requirement of Hsp90 activity for IkappaB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-kappaB activation" (2004) Oncogene 23: 5378-5386 8 Calderwood S.K., Khaleque Md.,A., Sawyer D.B., and Ciocca DR. (2006) Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem. Sci, 31: 164-172 9 Catz S.D., and Johnson J.F. (2001) "Transcriptional regulation of bcl-2 by nuclear factor K B and its significance in prostate cancer" Oncogene 20: 7342-7351 10 Chen L., Fischle W., Verdin E., and Greene W.C. (2001) "Duration of Nuclear N F - K B Action Regulated by Reversible Acetylation" Science 293: 1653-1657 11 Ferrari A, Gronchi A., Casanova M., Meazza C, Gandola L., Collini P., Laura Lozza L., Bertulli R., Olmi P., and Casali P.G. (2004) "Synovial sarcoma: A retrospective analysis of 271 patients of all ages treated at a single institution" Cancer 101: 627-634 51 12 Franzoso G., Carlson L., Xing L., Poljak L., Shores E.W., Brown K.D., Antonio Leonardi A., Tran T., Boyce B.F., and Siebenlist U. (1997) "Requirement for NF-kappa B in osteoclast and B-cell development" Genes Dev. 11; 3482-3496 13 Gartel A.L., and Radhakrishnan SK. (2005) "Lost in Transcription: p21 Repression, Mechanisms, and Consequences" Cancer Res. 65: 3980-3985 14 Georgakis G.V., Li Y., and Younes A. (2006) "The heat shock protein 90 inhibitor 17-AAG induces cell cycle arrest and apoptosis in mantle cell lymphoma cell lines by depleting cyclin Dl, Akt, Bid and activating caspase 9" Br J. Haematol. 135: 68-71 15 George P., Bali P., Annavarapu S., Scuto A., Fiskus W., Guo F., Sigua C, Sondarva G., Moscinski L., Atadja P., and Bhalla K. (2005) Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3" Blood 105:1768-1776 16 Goetz M.P., Toft D., Reid J., Ames M., Stensgard B., Safgren S., Adjei A.A., Sloan J., Atherton P., Vasile V., Salazaar S., Adjei A., Croghan G. and Erlichman C. (2005) "Phase I Trial ofl7-(Allylamino)-17-demethoxygeldanamycin in Patients With Advanced Cancer" J. Clin. Oncol. 23: 1078-1087 17 Gojo I., Jiemjit A., Trepel J.B., Sparreboom A., Figg W.D., Rollins S., Tidwell M.L., Greer I, Chung E.J., Lee M.J., Gore S.D., Sausville E.A., Zwiebel J. and Karp J.E. (2007) "Phase I and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias" Blood 109: 2781-2790 18 Grem J.L., Morrison G., Guo X.D., Agnew E., Takimoto C.H., Thomas R., Szabo E., Grochow L., Grollman F., Hamilton J.M., Neckers L., and Wilson R.H.(2005) "Phase I and pharmacologic study of 17-(Allylamino)-17-demethoxygeldanamycin in adult patients with solid tumors. " J. Clin. Oncol. 23: 1885-1893. 19 Gu. W. and Roeder R.G. (1997) "Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain" Cell 90: 595-606 20 Hasegawa T., Yokoyama R., Matsuno Y., Shimoda T., Hirohashi S..(2001) "Prognostic significance of histologic grade and nuclear expression of beta-catenin in synovial sarcoma" Hum. Pathol. 32: 257-263. 21 Hinz M., Krappmann D., Eichten A., Heder A., Scheidereit C , and Strauss M. (1999) "NF-kappaB function in growth control: regulation of cyclin Dl expression and G0/G1-to-S-phase transition." Mol Cell Biol. 19; 2690-2698 22 Hess-Stump H. (2005) "Histone deacetylase inhibitors and cancer: from cell biology to the clinic" Eur. J. Cell Biol. 84: 109-121 52 23 Hess-Stumpp H., Bracker T.U., Henderson D., Politz O. (2007) "MS-275, a potent orally available inhibitor of histone deacetylases—The development of an anticancer agent" Int. J. Biochem. Cell Biol, epub ahead of print 24 Hsu. H., Huang I, Shu H.B., Baichwal V., and Goeddel D.V. (1996) "TNF-Dependent Recruitment of the Protein Kinase RIP to the TNF Receptor-1 Signaling Complex" Immunity 4; 387-396 25 Hu E., Dul E., Sung CM., Chen Z., Kirkpatrick R., Zhang G.F., Johanson K., Liu R., Lago A., Hofmann G., Macarron R., DE LOS Frailes M., Perez P., Krawiec J., Winkler J., and Jaye M. (2003) "Identification of Novel Isoform-Selective Inhibitors within Class I Histone Deacetylases" J. Pharmacol. Exp. Ther. 307: 720-728 26 Hu X.Janssen W.E., Moscinski L.C., Bryington M., Dangsupa A., Rezai-Zadeh N., Babbin B. A., Zuckerman K.S. (2001) "An IkappaBalpha inhibitor causes leukemia cell death through a p38 MAP kinase-dependent, NF-kappaB-independent mechanism" Cancer Res. 6:6290-6296 27 Ito T., Ouchida M., Ito S., Jitsumori Y., Morimoto Y., Ozaki T., Kawai A., Inoue H., and Shimizu K. (2004) "SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3A, a component of histone deacetylase complex" Lab. Invest. 84: 1484-1490 28 Ito T, Ouchida M, Morimoto Y, Yoshida A, Jitsumori Y, Ozaki T, Sonobe H, Inoue H, Shimizu K. (2005) "Significant growth suppression of synovial sarcomas by the histone deacetylase inhibitor FK228 in vitro and in vivo" Cancer Lett 224:311 29 Johnson C.A., White D.A., Lavender J.S., O'Neill L.P., Turner B.M. (2001) "Human Class I Histone Deacetylase Complexes Show Enhanced Catalytic Activity in the Presence of ATP and Co-immunoprocipitate with the ATP-dependent Chaperone Protein Hsp70" J. Biol. Chem. 277: 9590-9597 30 Karin M. (2006) "Nuclear factor-KB in cancer development and progression" Nature 441: 431-436 Karin M., Yamamoto Y., and Wang QM. (2004) "The IKK N F - K B system: a treasure trove for drug development" Nat Rev Drug Discov. 3: 17-26 32 Kawai A., Woodruff J., Healey J.H., Brennan M.F., Antonescu C.R., and Ladanyi M. (1998) "SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma" N Engl J Med 338:153-160 33 Khoshman A., Tindell C , Laux I., Bae D., Bennett B., and Nel AE. (2000) "The NF-kB Cascade Is Important in Bcl-xL Expression and for the Anti-Apoptotic Effects of the CD28 Receptor in Primary Human CD4 Lymphocytes" J. Immunol. 165:1743-1754 53 34 Kiang J.G., BowmanP.D., Wu B.W., HamptonN., Kiang AG., Zhao B., Juang Y.T., Atkins J.L., and Tsokos G.C. (2004) "Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70" J. Appl. Physiol. 97: 564-569 35 Kovacs J.J., Murphy P.J.M., Gaillard S., Zhao X., Wu J.T., Nicchitta C.V., Yoshida M., Toft D.O., Pratt W.B., Yao T.P., (2005) "HDAC6 Regulates Hsp90 Acetylation and Chaperone-Dependent Activation of Glucocorticoid Receptor" Mol. Cell 18: 601-605 36 Kwan W., Terry J., Liu S., Knowling M. , and Nielsen T. (2005) "Effect of depsipeptide (NSC 630176), a histone deacetylase inhibitor, on human synovial sarcoma in vitro" ASCO annual meeting 37 Ladanyi M. (2001) "Fusions of the SYT and SSX genes in synovial sarcoma." Oncogene 20: 5755-5762 38 Lang S.A., Klein D., Moser C, Gaumann A., Glockzin G., Dahlke M.H., Dietmaier W., Bolder U., Schlitt H.J., Geissler E.K., and Stoeltzing O. (2007) "Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo" Mol Cancer Ther. 6:1123-1132. 39 Lewis J., Devin A., Miller A., Lin Y., Rodriguez Y., Neckers L., and Liu Z., (2000) "Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation" J. Biol. Chem. 275: 10519-10526 40 Liu S, Knowling M. A , Clarkson P., Lubieniecka J.M., Cheng H. , and Nielsen T.O. (2006) "Clear cell sarcoma and other translocation-associated sarcomas are highly sensitive to histone deacetylase inhibitor MS-275" CTOS 12th annual meeting 41 Liu Z.G., Hailing Hsu H., Goeddel D.V., and Karin M. (1996) "Dissection of TNF Receptor 1 Effector Functions: JNK Activation Is Not Linked to Apoptosis While NF-kB Activation Prevents Cell Death" Cell 87: 565-576 42 Louis M., Rosato R.R., Brault L., Sandra Osbild S., Battaglia E., Yang X.H., Grant S., and Bagrel D. (2004) "The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress"/^ . J. Oncol. 25(6): 1701-17011 43 Madrid L.V., Wang C.Y., Guttridge D C , Schottelius A.J.G., Baldwin AS., Jr., and Mayo M.W. (2000) "Akt suppresses apoptosis by stimulating the transcriptional activation potential of the RelA/p65 subunit ofNF-KB". Mol. Cell. Biol. 20:1626-1638 44 Marks P.A., Miller T., and Richon V.M. (2003) "Histone deacetylases" Curr. Opin. Pharmacol. 3: 344-351 45 Mabuchi S., Ohmichi M., Nishio Y., Hayasaka T., Kimura A., Ohta T., Saito M., Kawagoe J., Takahashi K., Yada-Hashimoto N., Sakata M., Motoyama T., Kurachi H , Tasaka K., and Murata Y. (2004) "Inhibition of NFB Increases the Efficacy of Cisplatin 54 in in Vitro and in Vivo Ovarian Cancer Models" 279; 23477-23485 46 Mayo M.W., Denlinger C.E., Broad R.M., Yeung F., Reilly E.T., Shi Y., and Jones DR. (2003) "Ineffectiveness of histone deacetylase inhibitors to induce apoptosis involves the transcriptional activation of NF-kappa B through the Akt pathway" J. Biol. Chem. 278: 18980-18989 47 Minucci S., and Pelicci P.G. (2006) "Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer" Nat. Rev. Cancer 6: 38-51 48 Moreira J.M. A., Scheipers P., and S0rensen P. (2003) "The histone deacetylase inhibitor Trichostatin A modulates CD4+ T cell responses" BMC Cancer 3: 30-47 49 Moran S.T., Haider K., Ow Y., Milton P., Chen L., and Pillai S. (2003) "Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner"/. Biol. Chem. 278: 21526-21533 50 Moynagh P.N., Williams D.C, O'Neill LA. (1994) "Activation of NF-kappaB and induction of vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression in human glial cells by IL-1. Modulation by antioxidants." J Immunol 153; 2681-2690 51 Miiller P., Ceskova P., and Vojtesek B. (2005) "Hsp90 is essential for restoring cellular functions of temperature-sensitive p53 mutant protein but not for stabilization and activation of wild-type p53: implications for cancer therapy" J. Biol. Chem 280: 6682-6691 52 Mukaida N., Mahe Y., and Matsushima K. (1990) "Cooperative Interaction of Nuclear Factor-KB- and cw-Regulatory Enhancer Binding Protein-like Factor Binding Elements in Activating the Interleukin-8 Gene by Pro-inflammatory Cytokines" J. Biol. Chem. 265: 21128-21133 53 Neckers L., and Ivy S.P. (2003) "Heat shock protein 90" Curr. Opin. Oncol. 15: 419-424 54 Nielsen TO., West R.B., Linn S.C, Alter O., Knowling M.A, O'Connell J.X., Zhu S., Fero M., Sherlock G., Pollack J.R., Brown P.O., Botstein D., and van de Rijn M. (2002) "Molecular characterisation of soft tissue tumours: a gene expression study" Lancet 359: 1301-1307 55 Nylandsted J., Gyrd-Hansen M., Danielewicz A., Fehrenbacher N., Lademann U., Hoyer-Hansen M., Weber E., Multhoff G., Rohde M., and Jaatelaa M. (2004) "Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization" J. Exp. Med. 200: 425-435 56 Okamoto H., Fujioka Y., Takahashi A, Takahashi T., Taniguchi T., Ishikawa Y., and Yokoyama M. (2006) "Trichostatin A, an Inhibitor of Histone Deacetylase, Inhibits 55 Smooth Muscle Cell Proliferation via Induction of p21WAFl" J. Atheroscler. Thromb. 13: 183-191 57 Ogryzko V.V., Schiltz R. L., Russanova V., Howard B.H., and Nakatani Y. (1996) "The transcriptional coactivators p300 and CBP are histone acetyltransferases" Cell 87: 953-959 58 Pacey S., Banerji U., Judson I., and Workman P. (2006) "Hsp90 inhibitors in the clinic" Handb. Exp. Pharmacol. 172: 331-358 59 Pierce J.W., Schoenleber R., Jesmok G., Best J., Moore S.A., Collins T., and Gerritsen M.E. (1997) "Novel Inhibitors of Cytokine-induced IkBa Phosphorylation and Endothelial Cell dhesion Molecule Expression Show Anti-inflammatory Effects in Vivo" J. Biol. Chem. 272; 21096-21103 60 Pisani P., Parkin D.M., Munoz N., Ferlay J. (1997) "Cancer and infection: estimates of the attributable fraction in 1990" Cancel Epidemiol Biomarkers Prev 6: 387-400 61 Perani M., Antonson P., Hamoudi R., Ingram C.J.E., Cooper C.S., and Garrett. (2005) "The proto-oncoprotein SYT interacts with SYT-interacting protein/co-activator activator (SIP/CoAA), a human nuclear receptor co-activator with similarity to EWS and TLS/FUS family of proteins" J. Biol. Chem. 280: 42863-42876 62 Prodromou C, Roe S.R., O'Brien R., Ladbury J.E., Piper P.W., and Pearl L.H (1997) "Identification and Structural Characterization of the ATP/ADP-Binding Site in the Hsp90 Molecular Chaperone" Cell 90: 65-75. 63 Rahmani M., Reese E., Dai Y., Bauer C, Kramer L.B., Huang M., Jove R., Dent P., and Grant S. (2005) "Cotreatment with Suberanoylanilide Hydroxamic Acid and 17-Allylamino 17-demethoxygeldanamycin Synergistically Induces Apoptisis inBcr-Abl+ Cells Sensitive and Resistant to STI571 (Imatinib Mesylate) in Association with Down-Regulation of Bcr-Abl, Abrogation of Singal Transducer and Activator of Trancscription 5 Activity, and Bax Conformational Change" Mol. Pharmacol. 67: 1166-1176 64 Ronnen E.A., Kondagunta G.V., Ishill N., Sweeney S.M., DeLuca J.K., Schwartz L., Bacik J., and Motzer R.J. (2006) "A phase II trial of 17-(Allylamino)-17-demethoxygeldanamycin in patients with papillary and clear cell renal cell carcinoma" Invest. New Drugs 24:543-546 65 Rosato R.R., Almenara J. A., and Grant S. (2003) "The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIPl/WAFl 1" Cancer Res. 63: 3637-3645 66 Rosato R.R., and Grant S. (2005) "Histone deacetylase inhibitors: insights into mechanisms of lethality" Expert Opin. Ther. Targets 9: 809-824 56 ) 67 Ruefli A.A., Ausserlechner M.J., Bernhard D., Sutton V.R., Tainton KM., Kofler R., Smyth M.J., and Johnstone R.W. (2001) "The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species" Proc. Natl. Acad. Sci. U.S.A. 98: 10833-10838 68 Rundall B.K., Denlinger C.E., and Jones D.R., (2004) "Combined histone deacetylase and NF-kappaB inhibition sensitizes non-small cell lung cancer to cell death" Surgery 136 (2): 416-225 69 DE Ruijter A.J.M., VAN Gennip A.H., Caron H.N., Kemp S., and VAN Kuilenburg A.B.P. "Histone deacetylases (HDACs): characterization of the classical HDAC family " (2003) Biochem. J. 370: 737-749 70 Ryan Q.C., Headlee D., Acharya M , Sparreboom A., Trepel J.B., Ye J., Figg W.D., Hwang K., Chung E.J., Murgo A., Giovanni M.., Elsayed Y., Monga M., Kalnitskiy M., Zwiebel J., and Sausville EA. (2005) "Phase I and Pharmacokinetic Study of MS-275, a Histone Deacetylase Inhibitor, in Patients With Advanced and Refractory Solid Tumors or Lymphoma" J. Clin. Oncol. 23: 3912-3922 71 Sausville E.A., Tomaszewski J.E., and Ivy P. (2003) "Clinical development of 17-allylamino, 17-demethoxygeldanamycin" Curr. Cancer Drug Targets 3: 377-385 72 Scaife C.L., Kuang J., Wills J.C., Trowbridge D.B., Gray P., Manning B.M., Eichwald E.J., Daynes R.A., and Kuwada S.K. (2002) "Nuclear Factor B Inhibitors Induce Adhesion-dependent Colon Cancer Apoptosis: Implications for Metastasis" Cancer Res. 62; 6870-6878 73 Schmitt E, Maingret L, Puig PE, Rerole AL, Ghiringhelli F, Hammann A, Solary E, Kroemer G, Garrido C. (2006) "Heat shock protein 70 neutralization exerts potent antitumor effects in animal models of colon cancer and melanoma" Cancer Res. 66:4191-4197 74 Schulte T.W., Blagosklonny M.V., Romanova L., Mushinski J.F., Monia B.P., Johnston J.F., Nguyen P.M., Trepel J., and Neckers L.M. (1996) "Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1 -MEK-mitogen-activated protein kinase signalling pathway" Mol Cell Biol 16: 5839-5845 75 Senese S., Zaragoza K., Minardi S., Muradore I., Ronzoni S., Passafaro A., Bernard L., Draetta G.F., Alcalay M., Seiser C, and Chiocca S. (2007) "A role for histone deacetylase 1 in human tumor cell proliferation" Mol Cell Biol. [Epub ahead of print] 76 Shishodia S., and Aggarwal B.B. (2004) "Nuclear factor-kB: a friend or a foe in cancer?" Biochem. Pharmacol. 68: 1071-1080 57 77 Skytting B., Nilsson G., Brodin B., Xie Y., Lundeberg J., Uhlen M., and Larsson O. (1999) "A novel fusion gene, SYT-SSX4, in synovial sarcoma" JNatl Cancer Inst. 11:974-5 78 Stehlik C, DE Martin R., Kumabashiri I., Schmid J.A., Binder B.R., and Lipp J. (1998) "Nuclear Factor (NF)-KB regulated X-chromosome-linked tap Gene Expression Protects Endothelial Cells from Tumor Necrosis Factor a-induced Apoptosis" J. Exp. Med. 188: 211-216 79 Terry J., Lubieniecka J.M., Kwan W., Liu S., and Nielsen T.O. (2005) "Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin prevents synovial sarcoma proliferation via apoptosis in in vitro models" Clin Cancer Res 11:5641 80 Vargas-Roig L.M., Gago F.E., Tello O., Aznar J.C. and Ciocca DR. (1998) "Heat shock protein expression and drug resistance in cancer patients treated with induction chemotherapy" Int. J. Cancer (Pred. Oncol.) 79: 468-475 81 Wang C, Mayo M.W., Korneluk R.G., Goeddel D.V., Baldwin AS. Jr. (1998) " N F - K B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAPl and C-IAP2 to suppress caspase-8 activation" Science 281: 1680-1683 82 Wang Y., Wang S.Y., Zhang X.H., Zhao M., Hou CM., Xu Y.J., Du Z.Y., and Yu X.D. (2007) "FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70" Biochem. Biophys. Res. Commun. epub ahead of print 83 Wegele H., Muller L., Buchner J. (2004) "Hsp70 and Hsp90-a relay team for protein folding" Rev. Physiol. Biochem. Pharmacol. 151: 1-4 84 Workman P., (2004) "Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone" Cancer Lett. 206: 149-157 85 Xu W., Mimnaugh E, Rosser M.F., Nicchitta C, Marcu M., Yarden Y., Neckers L. (2001) "Sensitivity of mature Erbb2 togeldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90" J. Biol. Chem. 276: 3702-3708 86 Yokoo T., and Kitamura M. (1996) "Dual regulation of IL-1 beta mediated matrix metalloproteinase-9 expression in mesangial cells by N F - K B and AP-1" Am. J. Physiol. 270: F123-30 87 Yu X., Guo S., Marcu M.G., Neckers L , Nguyen D.M., Chen G.A., Schrump D.S. (2002) "Modulation of p53, ErbBl, ErbB2, and Raf-1 Expression in Lung Cancer Cells by DepsipeptideFR901228V. Natl. Cancer Inst. 94: 504-513 58 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0100913/manifest

Comment

Related Items