PMID：30784360.pdf

Page 1 of 28 Thyroid Mary Ann Liebert,Inc.DOI:10.1089/thy.2018.0212 1 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.Ken Shiraiwa1,4,Michiko Matsuse1,Yuka Nakazawa2,Tomoo Ogi2,Keiji Suzuki1,Vladimir Saenko3,Shuhang Xu1,Kazuo Umezawa5,Shunichi Yamashita1,Kazuhiro Tsukamoto4,Norisato Mitsutake1 1Departments of Radiation Medical Sciences,2Genome Repair,3Radiation Molecular Epidemiology,Atomic Bomb Disease Institute,Nagasaki University.4Department of Pharmacotherapeutics,Nagasaki University Graduate School of Biomedical Sciences.5Department of Molecular Target Medicine,Aichi Medical University School of Medicine Ken Shiraiwa,MS Department of Radiation Medical Sciences,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.Department of Pharmacotherapeutics,Nagasaki University Graduate School of Biomedical Sciences.1-7-1 Sakamoto,Nagasaki 852-8501,Japan Michiko Matsuse,PhD Department of Radiation Medical Sciences,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.michikomnagasaki-u.ac.jp Yuka Nakazawa,PhD Department of Genome Repair,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.Current address:Department of Genetics,Research Institute of Environmental Medicine,Nagoya University.Furo-cho,Chikusa-ku,Nagoya 464-8601,Japan yu-nakariem.nagoya-u.ac.jp Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 2 of 28 2 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.Tomoo Ogi,PhD Department of Genome Repair,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.toginagasaki-u.ac.jp Current address:Department of Genetics,Research Institute of Environmental Medicine,Nagoya University.Furo-cho,Chikusa-ku,Nagoya 464-8601,Japan togiriem.nagoya-u.ac.jp Keiji Suzuki,PhD Department of Radiation Medical Sciences,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.kzsuzukinagasaki-u.ac.jp Vladimir Saenko,PhD Department of Radiation Molecular Epidemiology,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.saenkonagasaki-u.ac.jp Shuhang Xu,MD PhD Department of Radiation Medical Sciences,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,J Kazuo Umezawa,PhD Department of Molecular Target Medicine,Aichi Medical University School of Medicine.Nagakute,Aichi 480-1195,Japan.umezawaaichi-med-u.ac.jp Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 3 of 28 3 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.Shunichi Yamashita,MD PhD Department of Radiation Medical Sciences,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.shunnagasaki-u.ac.jp Kazuhiro Tsukamoto,MD PhD Department of Pharmacotherapeutics,Nagasaki University Graduate School of Biomedical Sciences.1-7-1 Sakamoto,Nagasaki 852-8501,Japan ktsukanagasaki-u.ac.jp Norisato Mitsutake,MD PhD Department of Radiation Medical Sciences,Atomic Bomb Disease Institute,Nagasaki University.1-12-4 Sakamoto,Nagasaki 852-8523,Japan.mitsunagasaki-u.ac.jp Running title:JAK3/STAT3/NF-B regulates CSC properties in ATC cells Keywords:JAK,STAT3,NF-B,cancer stem cells,anaplastic thyroid carcinoma Correspondence to:Norisato Mitsutake,MD PhD Department of Radiation Medical Sciences Atomic Bomb Disease Institute Nagasaki University 1-12-4 Sakamoto,Nagasaki 852-8523,Japan Tel:+81-95-819-7116 Fax:+81-95-819-7117 mitsunagasaki-u.ac.jp Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 4 of 28 4 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.Abstract Background:Anaplastic thyroid carcinoma(ATC)is one of the most aggressive and refractory cancers,and a therapy with a new concept needs to be developed.Recently,the research of cancer stem cells(CSCs)has been progressed,and CSCs have been suggested to be responsible for metastasis,recurrence,and therapy resistance.In ATC-CSCs,aldehyde dehydrogenase(ALDH)activity is the most reliable marker to enrich the CSCs;however,it itself is just a marker and is not involved in CSC properties.In the present study,therefore,we aimed to identify key signaling pathways specific for ATC-CSCs.Methods:A siRNA library targeting 719 kinases was used in a sphere formation assay and cell survival assay using ATC cell lines to select target molecules specific for CSC properties.The functions of the selected candidates were confirmed by sphere formation,cell survival,soft-agar,and nude mice xenograft assays using small compound inhibitors.Results:We focused on PDGFR,JAK,and PIM,whose siRNAs had a higher inhibitory effect on sphere formation and also a lower or no effect on regular cell growth in both FRO and KTC3 cells.Next,we used inhibitors of PDGFR,JAK,STAT3,PIM and NF-B,and all of them successfully suppressed sphere formation in a dose-dependent manner but not regular cell growth,conforming the screening results.Inhibition of the JAK/STAT3 and NF-B pathways also reduced anchorage-independent growth in soft agar and tumor growth in nude mice.Conclusions:These results suggest that JAK/STAT3 and NF-B signals play important roles in ATC-CSCs.Targeting these signaling pathways may be a promising approach to treat ATC.Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 5 of 28 5 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.Introduction Thyroid carcinoma is the most common endocrine malignancy and its incidence is growing worldwide.More than 90%of thyroid carcinomas are differentiated types consisting of papillary thyroid carcinoma(PTC)and follicular thyroid carcinoma(FTC),and their overall prognosis is favorable.However,anaplastic thyroid carcinoma(ATC),which is an undifferentiated type accounting for 12%of all thyroid cancer cases,is one of the deadliest human neoplasms,and its mean survival is less than one year even with multimodal treatments(1,2).To overcome this situation,a therapy with a new concept needs to be developed.In recent years,the cancer stem cell(CSC)theory has emerged as an attractive model to explain many aspects of carcinogenesis including metastasis,recurrence,and therapy resistance(3,4).CSCs are a small subpopulation in the cancer tissue and either self-renew or give rise to non-CSCs to produce heterogeneous tumors.Conventional chemo-and radio-therapy have been developed to target non-CSCs,but CSCs are highly resistant to these treatments.Thus,targeting CSCs is a reasonable approach to treat refractory cancers such as ATC.In ATC,previous studies have identified several biomarkers to enrich CSCs.Among these markers,aldehyde dehydrogenase(ALDH)activity is the most reliable and widely used(5,6).Todaro et al.have identified CSCs as a small subpopulation with high ALDH activity;the CSCs were highly tumorigenic in immunocompromised mice while non-CSCs were not(5).However,the ALDH activity itself is just a marker and does not have a functional role in CSC properties(7).To target CSCs,it is necessary to identify functional molecules that are important for survival and self-renewal of CSCs,rather than a marker.In the present study,we focused on kinases as targets because they are an important component of cell signaling pathways and can be blocked by small compounds.If inhibiting different molecules on a same signaling pathway is effective to suppress CSC properties,it is convincing that the pathway is important,which increases the possibility of developing clinical applications.Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 6 of 28 6 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.In this study,we used a siRNA library targeting 719 kinases to screen for important molecules for CSC properties.As a result,the JAKSTAT3NF-B signaling cascade emerged.Several inhibitors of this pathway successfully suppressed some CSCs abilities but not growth of regular cancer cells,suggesting that this pathway is important for CSC functions and may be an attractive target to treat ATC.Materials and methods Cell cultures FRO,KTC3,and THJ16T were established from human ATCs.FRO was obtained from Dr.James Fagin(currently Memorial Sloan-Kettering Cancer Center,NY,USA).KTC3 was kindly provided by Dr.Junichi Kurebayashi(Kawasaki Medical School,Okayama,Japan)(8).THJ16T was obtained from Dr.John Copland(Mayo Clinic,FL,USA).ACT1 was obtained from Dr.Naoyoshi Onoda(Osaka City University;originally established by Dr.Seiji Ohata of Tokushima University(9).8505C was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT,Japan.All cells were cultured in a growth medium(GM)consisting of RPMI1640,10%fetal bovine serum,and penicillin/streptomycin at 37C in a humidified atmosphere with 5%CO2.Cell growth was measured using a Cell Counting Kit-8(Dojindo).The following inhibitors were used:Imatinib(Novartis),JAK Inhibitor I(Calbiochem),STA-21(Santa Cruz),AZD1208(Selleckchem),and DHMEQ(synthesized by KU).Sphere formation assay The cells were incubated in serum-free DMED/F-12(1:1)supplemented with 20 ng/ml EGF,20 ng/ml bFGF and B27 without vitamin A(Thermo Fisher Scientific)in a HydroCell plate(CellSeed).Spheres with a diameter of 100 m or more were counted.Images were captured using a phase contrast microscope(Olympus).Combination drug effects on sphere formation were evaluated using CompuSyn software(ComboSyn).Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 7 of 28 7 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.siRNA screening A MISSION siRNA Human Kinase Panel(Sigma-Aldrich)was used.This panel includes siRNAs for 719 human kinase genes.For sphere formation,cells were seeded in a 96-well HydroCell plate,and each siRNA was trasnsfected at 10 nM using X-treme GENE siRNA transfection reagent(Roche).For each gene,three different siRNAs were mixed and used in the same well.After incubating for 96 hours,the cells were stained with Hoechst 33342(Sigma-Aldrich),and 100 m spheres were counted using an ArrayScan VTI(Thermo Fisher Scientific).For regular cell growth,cells were seeded in a regular 96-well plate,and transfection was performed as described above.After incubation for 96 hours,cell viability was determined using a Cell Counting Kit-8.For control,cells were transfected with Cy3-labeled scrambled RNA.Transfection efficiency was determined by a fluorescent microscope,and it was almost 100%.Soft agar colony formation assay Cells were mixed with 0.33%agar/GM and plated on a solidified 0.5%agar/GM.The agar layers were further overlaid with the GM containing appropriate concentrations of the inhibitors that were replaced every 23 days.After incubation for 10(FRO cells)or 20(THJ16T cells)days,images were captured using a digital camera,and the number of colonies was counted using Fiji software(10).In vivo xenograft experiments All procedures were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals of Nagasaki University with approval of the institutional animal care and use committee.FRO cells(1106)resuspended in the growth medium were injected s.c.into both flanks of 6-week-old male BALB/c nu/nu mice(CLEA Japan).Then they were randomly assigned into three groups.Tumor volumes were calculated according to the formula:a2b0.4,where a is the smallest tumor diameter and b is the diameter perpendicular to a.STA-21 or DHMEQ solution in DMSO/PBS(ratio 1:1)was injected i.p.daily for one week,beginning from day 1 after tumor cell implantation.Control group mice received vehicle injections only.Downloaded by MIDWESTERN UNIVERSITY MULTISITE from at 02/21/19.For personal use only.Page 8 of 28 8 Thyroid The JAK/STAT3 and NF-B signaling pathways regulate cancer stem cell properties in anaplastic thyroid cancer cells.(DOI:10.1089/thy.2018.0212)This paper has been peer-reviewed and accepted for publication,but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.ALDEFLUOR assay To measure the ALDH activity,cells were labeled using an ALDEFLUOR assay kit(StemCell Technologies)following the manufacturers protocol.The cells were then analyzed using a FACSJazz cell sorter(BD Biosciences).The data were further processed with FlowJo software(FlowJo).Statistical Analysis Differences between groups were examined for statistical significance with one-way ANOVA followed by Tukeys post test.A p-value not exceeding 0.05 was considered statistically significant.Data were analyzed with PRISM 6 software(GraphPad Software).Results siRNA screening to identify important cell signaling for CSC properties.We and others have demonstrated that sphere formation assay is valuable to evaluate CSC properties(3,4,6).In our previous report using eight thyroid cancer cell lines,the ability