Exosomes released by imatinib‑resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib‑sensitive cells in the presence of imatinib

Hrdinova,Tereza; Hrdinova,Tereza; Toman,Ondrej; Toman,Ondrej; Dresler,Jiri; Dresler,Jiri; Klimentova,Jana; Klimentova,Jana; Salovska,Barbora; Salovska,Barbora; Pajer,Petr; Pajer,Petr; Bartos,Oldrich; Bartos,Oldrich; Polivkova,Vaclava; Polivkova,Vaclava; Linhartova,Jana; Linhartova,Jana; Machova Polakova,Katerina; Machova Polakova,Katerina; Kabickova,Hana; Kabickova,Hana; Brodska,Barbora; Brodska,Barbora; Krijt,Matyas; Krijt,Matyas; Zivny,Jan; Zivny,Jan; Vyoral,Daniel; Vyoral,Daniel; Petrak,Jiri; Petrak,Jiri

doi:10.3892/ijo.2020.5163

Exosomes released by imatinib‑resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib‑sensitive cells in the presence of imatinib

Authors:
- Tereza Hrdinova
- Ondrej Toman
- Jiri Dresler
- Jana Klimentova
- Barbora Salovska
- Petr Pajer
- Oldrich Bartos
- Vaclava Polivkova
- Jana Linhartova
- Katerina Machova Polakova
- Hana Kabickova
- Barbora Brodska
- Matyas Krijt
- Jan Zivny
- Daniel Vyoral
- Jiri Petrak
View Affiliations

Affiliations: Institute of Hematology and Blood Transfusion, 128 20 Prague 2, Czech Republic, Institute of Hematology and Blood Transfusion, 128 20 Prague 2, Czech Republic, Military Health Institute, Military Medical Agency, 160 01 Prague 6, Czech Republic, Faculty of Military Health Sciences, University of Defense in Brno, 500 02 Hradec Kralove, Czech Republic, Department of Genome Integrity, Institute of Molecular Genetics of The Czech Academy of Sciences, 142 20 Prague 4, Czech Republic, Department of Infectious Diseases, First Faculty of Medicine, Charles University and Military University Hospital Prague, 169 02 Prague 6, Czech Republic, Institute of Pathological Physiology, First Faculty of Medicine, Charles University, 128 20 Prague 2, Czech Republic
Published online on: December 23, 2020 https://doi.org/10.3892/ijo.2020.5163
Pages: 238-250

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Abstract

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR‑ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR‑ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib‑resistant K562 (K562IR) cells. The K562IR‑derived exosomes were internalized by imatinib‑sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance‑associated markers using a deep label‑free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon‑induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

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1	Nowell PC and Hungerford DA: A minute chromosome in human chronic granulocytic leukemia. Science. 132:14971960.
2	Rowley JD: Letter: A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature. 243:290–293. 1973. View Article : Google Scholar : PubMed/NCBI
3	Heisterkamp N, Stephenson JR, Groffen J, Hansen PF, de Klein A, Bartram CR and Grosveld G: Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature. 306:239–242. 1983. View Article : Google Scholar : PubMed/NCBI
4	Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S and Sawyers CL: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 344:1031–1037. 2001. View Article : Google Scholar : PubMed/NCBI
5	Lahaye T, Riehm B, Berger U, Paschka P, Müller MC, Kreil S, Merx K, Schwindel U, Schoch C, Hehlmann R and Hochhaus A: Response and resistance in 300 patients with BCR-ABL-positive leukemias treated with imatinib in a single center: A 4.5-year follow-up. Cancer. 103:1659–1669. 2005. View Article : Google Scholar : PubMed/NCBI
6	Gadzicki D, von Neuhoff N, Steinemann D, Just M, Büsche G, Kreipe H, Wilkens L and Schlegelberger B: BCR-ABL gene amplifi-cation and overexpression in a patient with chronic myeloid leukemia treated with imatinib. Cancer Genet Cytogenet. 159:164–167. 2005. View Article : Google Scholar : PubMed/NCBI
7	Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN and Sawyers CL: Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. 293:876–880. 2001. View Article : Google Scholar : PubMed/NCBI
8	Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J, Goldman JM and Melo JV: Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: Diverse mechanisms of resistance. Blood. 96:1070–1079. 2000. View Article : Google Scholar : PubMed/NCBI
9	Steinbichler TB, Dudas J, Skvortsov S, Ganswindt U, Riechelmann H and Skvortsova II: Therapy resistance mediated by exosomes. Mol Cancer. 18:582019. View Article : Google Scholar : PubMed/NCBI
10	Nehrbas J, Butler JT, Chen DW and Kurre P: Extracellular vesicles and chemotherapy resistance in the AML microenvironment. Front Oncol. 10:902020. View Article : Google Scholar : PubMed/NCBI
11	Johnstone RM, Adam M, Hammond JR, Orr L and Turbide C: Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem. 262:9412–9420. 1987. View Article : Google Scholar : PubMed/NCBI
12	Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ and Lotvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9:654–659. 2007. View Article : Google Scholar : PubMed/NCBI
13	Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, Zheng Y, Hoshino A, Brazier H, Xiang J, et al: Double-stranded DNA in exosomes: A novel biomarker in cancer detection. Cell Res. 24:766–769. 2014. View Article : Google Scholar : PubMed/NCBI
14	Prada I and Meldolesi J: Binding and Fusion of Extracellular vesicles to the plasma membrane of their cell targets. Int J Mol Sci. 17:12962016. View Article : Google Scholar :
15	Maacha S, Bhat AA, Jimenez L, Raza A, Haris M, Uddin S and Grivel JC: Extracellular vesicles-mediated intercellular communication: Roles in the tumor microenvironment and anti-cancer drug resistance. Mol Cancer. 18:552019. View Article : Google Scholar : PubMed/NCBI
16	Raimondo S, Saieva L, Corrado C, Fontana S, Flugy A, Rizzo A, De Leo G and Alessandro R: Chronic myeloid leukemia-derived exosomes promote tumor growth through an autocrine mechanism. Cell Commun Signal. 13:82015. View Article : Google Scholar : PubMed/NCBI
17	Corrado C, Raimondo S, Saieva L, Flugy AM, De Leo G and Alessandro R: Exosome-mediated crosstalk between chronic myelogenous leukemia cells and human bone marrow stromal cells triggers an interleukin 8-dependent survival of leukemia cells. Cancer Lett. 348:71–76. 2014. View Article : Google Scholar : PubMed/NCBI
18	Cai J, Wu G, Tan X, Han Y, Chen C, Li C, Wang N, Zou X, Chen X, Zhou F, et al: Transferred BCR/ABL DNA from K562 extracellular vesicles causes chronic myeloid leukemia in immu-nodeficient mice. PLoS One. 9:e1052002014. View Article : Google Scholar
19	Jafarzadeh N, Safari Z, Pornour M, Amirizadeh N, Forouzandeh Moghadam M and Sadeghizadeh M: Alteration of cellular and immune-related properties of bone marrow mesenchymal stem cells and macrophages by K562 chronic myeloid leukemia cell derived exosomes. J Cell Physiol. 234:3697–3710. 2019. View Article : Google Scholar
20	Taverna S, Flugy A, Saieva L, Kohn EC, Santoro A, Meraviglia S, De Leo G and Alessandro R: Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis. Int J cancer. 130:2033–2043. 2012. View Article : Google Scholar
21	Mineo M, Garfield SH, Taverna S, Flugy A, De Leo G, Alessandro R and Kohn EC: Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a Src-dependent fashion. Angiogenesis. 15:33–45. 2012. View Article : Google Scholar
22	Min QH, Wang XZ, Zhang J, Chen QG, Li SQ, Liu XQ, Li J, Liu J, Yang WM, Jiang YH, et al: Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR365. Exp Cell Res. 362:386–393. 2018. View Article : Google Scholar
23	Toman O, Kabickova T, Vit O, Fiser R, Polakova KM, Zach J, Linhartova J, Vyoral D and Petrak J: Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target. Oncol Rep. 36:1258–1268. 2016. View Article : Google Scholar : PubMed/NCBI
24	Machova Polakova K, Kulvait V, Benesova A, Linhartova J, Klamova H, Jaruskova M, de Benedittis C, Haferlach T, Baccarani M, Martinelli G, et al: Next-generation deep sequencing improves detection of BCR-ABL1 kinase domain mutations emerging under tyrosine kinase inhibitor treatment of chronic myeloid leukemia patients in chronic phase. J Cancer Res Clin Oncol. 141:887–899. 2015. View Article : Google Scholar
25	Kohlmann A, Grossmann V, Nadarajah N and Haferlach T: Next-generation sequencing-feasibility and practicality in haematology. Br J Haematol. 160:736–753. 2013. View Article : Google Scholar : PubMed/NCBI
26	Linhartova J, Hovorkova L, Soverini S, Benesova A, Jaruskova M, Klamova H, Zuna J and Machova Polakova K: Characterization of 46 patient-specific BCR-ABL1 fusions and detection of SNPs upstream and downstream the breakpoints in chronic myeloid leukemia using next generation sequencing. Mol Cancer. 14:892015. View Article : Google Scholar : PubMed/NCBI
27	Moravcova J, Rulcova J, Polaková KM and Klamova H: Control genes in international standardization of real-time RT-PCR for BCR-ABL. Leuk Res. 33:582–584. 2009. View Article : Google Scholar
28	Rulcova J, Zmekova V, Zemanova Z, Klamova H and Moravcova J: The effect of total-ABL, GUS and B2M control genes on BCR-ABL monitoring by real-time RT-PCR. Leuk Res. 31:483–491. 2007. View Article : Google Scholar
29	Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cavé H, et al: Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program. Leukemia. 17:2318–2357. 2003. View Article : Google Scholar : PubMed/NCBI
30	Müller MC, Cross NCP, Erben P, Schenk P, Hanfstein B, Ernst T, Hehlmann R, Branford S, Saglio G and Hochhaus A: Harmonization of molecular monitoring of CML therapy in Europe. Leukemia. 23:1957–1963. 2009. View Article : Google Scholar : PubMed/NCBI
31	Deprez L, Mazoua S, Corbisier P, Trapmann S, Schimmel H, White H, Cross N and Emons H: The certification of the copy number concentration of solutions of plasmid DNA containing a BCR- ABL b3a2 transcript fragment. Certified reference material: ERM-AD623a, ERM-AD623b, ERM-AD623c, ERM-AD623d, ERM-AD623e ERM-AD623f. Luxembourg: Publications Office of the European Union; 2012. Report number EUR 25248. ISBN: 978-92-79-23343-22012
32	White H, Deprez L, Corbisier P, Hall V, Lin F, Mazoua S, Trapmann S, Aggerholm A, Andrikovics H, Akiki S, et al: A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR. Leukemia. 29:369–376. 2015. View Article : Google Scholar
33	Thery C, Amigorena S, Raposo G and Clayton A: Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter. 3:Unit 3.22. 2006.
34	McDowell EM and Trump BF: Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med. 100:405–414. 1976.PubMed/NCBI
35	Wisniewski JR, Zougman A and Mann M: Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res. 8:5674–5678. 2009. View Article : Google Scholar : PubMed/NCBI
36	Cox J and Mann M: MaxQuant enables high peptide iden-tification rates, individualized p.p.b-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 26:1367–1372. 2008. View Article : Google Scholar : PubMed/NCBI
37	Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV and Mann M: Andromeda: A peptide search engine integrated into the MaxQuant environment. J Proteome Res. 10:1794–1805. 2011. View Article : Google Scholar : PubMed/NCBI
38	Cox J, Hein MY, Luber CA, Paron I, Nagaraj N and Mann M: Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 13:2513–2526. 2014. View Article : Google Scholar : PubMed/NCBI
39	Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M and Cox J: The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 13:731–740. 2016. View Article : Google Scholar : PubMed/NCBI
40	Chawade A, Alexandersson E and Levander F: Normalyzer: A tool for rapid evaluation of normalization methods for omics data sets. J Proteome Res. 13:3114–3120. 2014. View Article : Google Scholar : PubMed/NCBI
41	Waas M, Snarrenberg ST, Littrell J, Jones Lipinski RA, Hansen PA, Corbett JA and Gundry RL: SurfaceGenie: A web-based application for prioritizing cell-type specific marker candidates. Bioinformatics. 36:3447–3456. 2020. View Article : Google Scholar : PubMed/NCBI
42	Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, Dingli F, Loew D, Tkach M and Théry C: Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci USA. 113:E968–E977. 2016. View Article : Google Scholar : PubMed/NCBI
43	Tadokoro H, Umezu T, Ohyashiki K, Hirano T and Ohyashiki JH: Exosomes derived from hypoxic leukemia cells enhance tube formation in endothelial cells. J Biol Chem. 288:34343–34351. 2013. View Article : Google Scholar : PubMed/NCBI
44	Chandran RK, Geetha N, Sakthivel KM, Aswathy CG, Gopinath P, Raj TVA, Priya G, Nair JKKM and Sreedharan H: Genomic amplification of BCR-ABL1 fusion gene and its impact on the disease progression mechanism in patients with chronic myelogenous leukemia. Gene. 686:85–91. 2019. View Article : Google Scholar
45	Hu J, Wang S, Zhao Y, Guo Q, Zhang D, Chen J, Li J, Fei Q and Sun Y: Mechanism and biological significance of the overexpression of IFITM3 in gastric cancer. Oncol Rep. 32:2648–2656. 2014. View Article : Google Scholar : PubMed/NCBI
46	Zhang D, Wang H, He H, Niu H and Li Y: Interferon induced transmembrane protein 3 regulates the growth and invasion of human lung adenocarcinoma. Thorac Cancer. 8:337–343. 2017. View Article : Google Scholar : PubMed/NCBI
47	Yang M, Gao H, Chen P, Jia J and Wu S: Knockdown of interferon-induced transmembrane protein 3 expression suppresses breast cancer cell growth and colony formation and affects the cell cycle. Oncol Rep. 30:171–178. 2013. View Article : Google Scholar : PubMed/NCBI
48	Gan CP, Sam KK, Yee PS, Zainal NS, Lee BKB, Abdul Rahman ZA, Patel V, Tan AC, Zain RB and Cheong SC: IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells. Cell Oncol (Dordr). 42:477–490. 2019. View Article : Google Scholar
49	Liu Y, Lu R, Cui W, Pang Y, Liu C, Cui L, Qian T, Quan L, Dai Y, Jiao Y, et al: High IFITM3 expression predicts adverse prognosis in acute myeloid leukemia. Cancer Gene Ther. 27:38–44. 2019. View Article : Google Scholar : PubMed/NCBI
50	Lehmann JM, Riethmuller G and Johnson JP: MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immuno-globulin superfamily. Proc Natl Acad Sci USA. 86:9891–9895. 1989. View Article : Google Scholar
51	Lei X, Guan CW, Song Y and Wang H: The multifaceted role of CD146/MCAM in the promotion of melanoma progression. Cancer Cell Int. 15:32015. View Article : Google Scholar : PubMed/NCBI
52	Wu GJ and Dickerson EB: Frequent and increased expression of human METCAM/MUC18 in cancer tissues and metastatic lesions is associated with the clinical progression of human ovarian carcinoma. Taiwan J Obstet Gynecol. 53:509–517. 2014. View Article : Google Scholar : PubMed/NCBI
53	Watson-Hurst K and Becker D: The role of N-cadherin, MCAM and beta3 integrin in melanoma progression, proliferation, migration and invasion. Cancer Biol Ther. 5:1375–1382. 2006. View Article : Google Scholar : PubMed/NCBI
54	Tripathi SC, Fahrmann JF, Celiktas M, Aguilar M, Marini KD, Jolly MK, Katayama H, Wang H, Murage EN, Dennison JB, et al: MCAM Mediates Chemoresistance in Small-Cell Lung Cancer via the PI3K/AKT/SOX2 signaling pathway. Cancer Res. 77:4414–4425. 2017. View Article : Google Scholar : PubMed/NCBI
55	Liang YK, Zeng D, Xiao YS, Wu Y, Ouyang YX, Chen M, Li YC, Lin HY, Wei XL, Zhang YQ, et al: MCAM/CD146 promotes tamoxifen resistance in breast cancer cells through induction of epithelial-mesenchymal transition, decreased ERα expression and AKT activation. Cancer Lett. 386:65–76. 2017. View Article : Google Scholar
56	Ye H, Adane B, Khan N, Sullivan T, Minhajuddin M, Gasparetto M, Stevens B, Pei S, Balys M, Ashton JM, et al: Leukemic stem cells evade chemotherapy by metabolic adaptation to an adipose tissue niche. Cell Stem Cell. 19:23–37. 2016. View Article : Google Scholar : PubMed/NCBI
57	Landberg N, von Palffy S, Askmyr M, Lilljebjörn H, Sandén C, Rissler M, Mustjoki S, Hjorth-Hansen H, Richter J, Ågerstam H, et al: CD36 defines primitive chronic myeloid leukemia cells less responsive to imatinib but vulnerable to anti-body-based therapeutic targeting. Haematologica. 103:447–455. 2018. View Article : Google Scholar :
58	Bailey CC, Zhong G, Huang IC and Farzan M: IFITM-Family Proteins: The Cell's First Line of Antiviral Defense. Annu Rev Virol. 1:261–283. 2014. View Article : Google Scholar
59	Wrensch F, Winkler M and Pöhlmann S: IFITM proteins inhibit entry driven by the MERS-coronavirus spike protein: Evidence for cholesterol-independent mechanisms. Viruses. 6:3683–3698. 2014. View Article : Google Scholar : PubMed/NCBI
60	Nollet M, Stalin J, Moyon A, Traboulsi W, Essaadi A, Robert S, Malissen N, Bachelier R, Daniel L, Foucault-Bertaud A, et al: A novel anti-CD146 antibody specifically targets cancer cells by internalizing the molecule. Oncotarget. 8:112283–112296. 2017. View Article : Google Scholar
61	Stalin J, Nollet M, Dignat-George F, Bardin N and Blot-Chabaud M: Therapeutic and Diagnostic Antibodies to CD146: Thirty Years of Research on Its Potential for Detection and Treatment of Tumors. Antibodies (Basel). 6:172017. View Article : Google Scholar
62	Wang H, Tang F, Bian E, Zhang Y, Ji X, Yang Z and Zhao B: IFITM3/STAT3 axis promotes glioma cells invasion and is modulated by TGF-β. Mol Biol Rep. 47:433–441. 2020. View Article : Google Scholar
63	Johnston PA and Grandis JR: STAT3 signaling: Anticancer strategies and challenges. Mol Interv. 11:18–26. 2011. View Article : Google Scholar : PubMed/NCBI
64	Hochhaus A, Baccarani M, Silver RT, Schiffer C, Apperley JF, Cervantes F, Clark RE, Cortes JE, Deininger MW, Guilhot F, et al: European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia. 34:966–984. 2020. View Article : Google Scholar : PubMed/NCBI
65	Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, Inuganti A, Griss J, Mayer G, Eisenacher M, et al: The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res. 47:D442–D450. 2019. View Article : Google Scholar :