Insights into the Proteome of Gastrointestinal Stromal Tumors-Derived Exosomes Reveals New Potential Diagnostic Biomarkers
2017; Elsevier BV; Volume: 17; Issue: 3 Linguagem: Inglês
10.1074/mcp.ra117.000267
ISSN1535-9484
AutoresSafinur Atay, Daniel W. Wilkey, Mohammed Milhem, Michael L. Merchant, Andrew K. Godwin,
Tópico(s)Vascular Malformations and Hemangiomas
ResumoDeveloping tumors continuously release nano-sized vesicles that represent circulating "fingerprints" of the tumor's identity. In gastrointestinal stromal tumor (GIST), we have previously reported that these tumors release "oncosomes" carrying the constitutively activated tyrosine kinase (TK) receptor KIT. Despite the clinical utility of TK inhibitors, such as imatinib mesylate (IM), recurrence and metastasis are clinical problems that urge the need to identify new tumor-derived molecules. To this aim, we performed the first high quality proteomic study of GIST-derived exosomes (GDEs) and identified 1,060 proteins composing the core GDE proteome (cGDEp). The cGDEp was enriched in diagnostic markers (e.g. KIT, CD34, ANO1, PROM1, PRKCQ, and ENG), as well as proteins encoded by genes previously reported expressed in GIST (e.g. DPP4, FHL1, CDH11, and KCTD12). Many of these proteins were validated using cell lines, patient-derived KIT+ exosomes, and GIST tissues. We further show that in vitro and in vivo-derived GDE, carry proteins associated with IM response, such as Sprouty homolog 4 (SPRY4), surfeit 4 (SURF4), ALIX, and the cGMP-dependent 3′,5′-cyclic phosphodiesterase 2A (PDE2A). Additionally, we report that the total exosome levels and exosome-associated KIT and SPRY4 protein levels have therapeutic values. In fact, molecular characterization of in vivo-derived KIT+ exosomes indicate significant sorting of p-KITTyr719, total KIT, and SPRY4 after IM-treatment of metastatic patients as compared with the pre-IM levels. Our data suggest that analysis of circulating exosomes levels and molecular markers of IM response in GIST patients with primary and metastatic disease is suitable to develop liquid based biopsies for the diagnosis, prognosis, and monitoring of response to treatment of these tumors. In summary, these findings provide the first insight into the proteome of GIST-derived oncosomes and offers a unique opportunity to further understand their oncogenic elements which contribute to tumorigenesis and drug resistance. Data are available via ProteomeXchange with identifier PXD007997. Developing tumors continuously release nano-sized vesicles that represent circulating "fingerprints" of the tumor's identity. In gastrointestinal stromal tumor (GIST), we have previously reported that these tumors release "oncosomes" carrying the constitutively activated tyrosine kinase (TK) receptor KIT. Despite the clinical utility of TK inhibitors, such as imatinib mesylate (IM), recurrence and metastasis are clinical problems that urge the need to identify new tumor-derived molecules. To this aim, we performed the first high quality proteomic study of GIST-derived exosomes (GDEs) and identified 1,060 proteins composing the core GDE proteome (cGDEp). The cGDEp was enriched in diagnostic markers (e.g. KIT, CD34, ANO1, PROM1, PRKCQ, and ENG), as well as proteins encoded by genes previously reported expressed in GIST (e.g. DPP4, FHL1, CDH11, and KCTD12). Many of these proteins were validated using cell lines, patient-derived KIT+ exosomes, and GIST tissues. We further show that in vitro and in vivo-derived GDE, carry proteins associated with IM response, such as Sprouty homolog 4 (SPRY4), surfeit 4 (SURF4), ALIX, and the cGMP-dependent 3′,5′-cyclic phosphodiesterase 2A (PDE2A). Additionally, we report that the total exosome levels and exosome-associated KIT and SPRY4 protein levels have therapeutic values. In fact, molecular characterization of in vivo-derived KIT+ exosomes indicate significant sorting of p-KITTyr719, total KIT, and SPRY4 after IM-treatment of metastatic patients as compared with the pre-IM levels. Our data suggest that analysis of circulating exosomes levels and molecular markers of IM response in GIST patients with primary and metastatic disease is suitable to develop liquid based biopsies for the diagnosis, prognosis, and monitoring of response to treatment of these tumors. In summary, these findings provide the first insight into the proteome of GIST-derived oncosomes and offers a unique opportunity to further understand their oncogenic elements which contribute to tumorigenesis and drug resistance. Data are available via ProteomeXchange with identifier PXD007997. Tumor development and progression is highly dependent on the productive and bi-directional exchange of information between tumor cells and host stromal cells (1.Wang M. Zhao J. Zhang L. Wei F. Lian Y. Wu Y. Gong Z. Zhang S. Zhou J. Cao K. Li X. Xiong W. Li G. Zeng Z. Guo C. Role of tumor microenvironment in tumorigenesis.J. Cancer. 2017; 8: 761-773Crossref PubMed Scopus (433) Google Scholar). 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Although the exact mechanism(s) responsible for the formation and release of exosomes is still in its preliminary stages of investigation, these vesicles are known to arise from the limiting membrane of multivesicular bodies (MVBs) 1The abbreviations used are: MBVs, multivesicular bodies;GIST, Gastrointestinal stromal tumors;TK, tyrosine kinase;IM, imatinib mesylate;GDE, GIST-derived exosomes;cGDEp, core GDE proteome;ILVs, intraluminal vesicles;TDEs, tumor-derived exosomes;RTK, receptor tyrosine kinase;TEX, GIST-T1 cell derived exosomes;882 Exo, GIST-882 cell derived exosomes;NTA, nanoparticle tracking analysis;TRPS, tunable resistive pulse sensing;FDR, false discovery rate;iBAQ, intensity based absolute quantification;PCA, principle component analysis;TEM, transmission electron microscopy;1° SMC, primary smooth muscle cells;1° SMC Exo, primary smooth muscle cells derived exosomes;CM, conditioned media;OGR, overexpression gene rank;IPA, ingenuity pathway analysis;HPE, healthy donor-derived exosomes;GPE, GIST patient-derived exsosomes;Unf, unfractionated total exosomes;beads-KIT, anti-KIT antibody coated dynabeads;beads-IC, control IgG coated beads;PR, partial response;NOD, no evidence of disease;PD, progressive disease;NR, nonreducing;R, reducing;FDRBH, false discovery rate with Bonferroni Hochberg correction;LMS, leiomyosarcoma;FS, fibrosarcoma;SFT, solid fibrous tumor;MFH, malignant fibrous histiocytoma;CP, canonical pathway;Fx, biological function;FC, fold change;TL, tumor lysate;NAGT, normal adjacent gastric tissue;DGA, diffuse gastric adenocarcinoma;GA, gastric adenocarcinoma;GITA, gastric interstitial type adenocarcinoma;GMA, gastric mixed adenocarcinoma;ANOVA, analysis of variance;GO, gene ontology;SMT, smooth muscle tissue. 1The abbreviations used are: MBVs, multivesicular bodies;GIST, Gastrointestinal stromal tumors;TK, tyrosine kinase;IM, imatinib mesylate;GDE, GIST-derived exosomes;cGDEp, core GDE proteome;ILVs, intraluminal vesicles;TDEs, tumor-derived exosomes;RTK, receptor tyrosine kinase;TEX, GIST-T1 cell derived exosomes;882 Exo, GIST-882 cell derived exosomes;NTA, nanoparticle tracking analysis;TRPS, tunable resistive pulse sensing;FDR, false discovery rate;iBAQ, intensity based absolute quantification;PCA, principle component analysis;TEM, transmission electron microscopy;1° SMC, primary smooth muscle cells;1° SMC Exo, primary smooth muscle cells derived exosomes;CM, conditioned media;OGR, overexpression gene rank;IPA, ingenuity pathway analysis;HPE, healthy donor-derived exosomes;GPE, GIST patient-derived exsosomes;Unf, unfractionated total exosomes;beads-KIT, anti-KIT antibody coated dynabeads;beads-IC, control IgG coated beads;PR, partial response;NOD, no evidence of disease;PD, progressive disease;NR, nonreducing;R, reducing;FDRBH, false discovery rate with Bonferroni Hochberg correction;LMS, leiomyosarcoma;FS, fibrosarcoma;SFT, solid fibrous tumor;MFH, malignant fibrous histiocytoma;CP, canonical pathway;Fx, biological function;FC, fold change;TL, tumor lysate;NAGT, normal adjacent gastric tissue;DGA, diffuse gastric adenocarcinoma;GA, gastric adenocarcinoma;GITA, gastric interstitial type adenocarcinoma;GMA, gastric mixed adenocarcinoma;ANOVA, analysis of variance;GO, gene ontology;SMT, smooth muscle tissue. by a mechanism of inward budding, leading to the formation of intraluminal vesicles (ILVs) (9.Johnstone R.M. 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With the rapid advances in understanding of the genomic aberration driving GIST and with the successful clinical use of tyrosine kinase inhibitors (TKI), like imatinib mesylate (IM; Gleevec®) and sunitinib malate (Sutent) for the treatment of patients with inoperable or metastatic GIST; GIST has become the quintessential model for molecular targeted therapy (36.Joensuu H. Kindblom L.G. Gastrointestinal stromal tumors–a review.Acta Orthopaedica Scand. Supplement. 2004; 75: 62-71Crossref PubMed Google Scholar, 43.Godwin A.K. Bench to bedside and back again: personalizing treatment for patients with GIST.Mol. Cancer Ther. 2011; 10: 2026-2027Crossref PubMed Scopus (5) Google Scholar). However, despite the initial success of IM in the advanced setting with a clinical benefit of 80%, disease progression remains a perplexing problem, as the vast majority of patients eventually developing resistance. Importantly, once a GIST becomes metastatic, the median disease-specific survival of patients is only ∼19 months with second- and third-line therapies (44.Patel S. Managing progressive disease in patients with GIST: factors to consider besides acquired secondary tyrosine kinase inhibitor resistance.Cancer Treatment Rev. 2012; 38: 467-472Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 45.Jayanthi N.V. Emergence of secondary resistance to imatinib in recurrent gastric GIST.J. Gastrointestinal. 2010; 14 (author reply 2020): 2018-2019Crossref PubMed Scopus (1) Google Scholar). Therefore, there is an urgent need for additional therapeutic strategies and/or discovery of therapeutic markers that will significantly enhance follow-up and predict response to therapy for these tumors. One possible source of biomarkers, as reported by us ad others, is the presence in the systemic circulation of GIST patients of modulatory KIT positive nanovesicles (31.Atay S. Tetik A. Bozok Cetintas V. Yakar Tuluce S. Tuluce K. Kayikcioglu M. Eroglu Z. Beta-myosin heavy-chain mutations R403QLW, V606M, K615N and R663H in patients with hypertrophic cardiomyopathy.Anadolu Kardiyol Derg. 2014; 14: 244-250PubMed Google Scholar). To date, most proteomic studies of exosomes have primarily focused on exosomes derived from carcinomas (16.Simpson R.J. Jensen S.S. Lim J.W. Proteomic profiling of exosomes: current perspectives.Proteomics. 2008; 8: 4083-4099Crossref PubMed Scopus (569) Google Scholar). The purpose of this study was to perform a comprehensive analysis and characterization of the vesicular proteome of highly purified GIST-derived exosomes (GDEs) and further establish the first prototypical proteome signature of these vesicles. Using quantitative proteomic profiling, we report insights into the selective oncogenic cargo of GDEs, demonstrating expected enrichment of common exosome-related markers, as well as proteins involved in tumor progression, angiogenesis, kinase signaling pathways, and immune regulatory components. Importantly, our findings also provide clues to understand the roles of these vesicles in tumorigenesis, drug response, and provide a comprehensive protein signature of GIST that can be used as a resource for the discovery of new diagnostic biomarkers and therapeutic targets. Myometrial samples were obtained from premenopausal women undergoing hysterectomy (3.Quail D.F. Joyce J.A. Microenvironmental regulation of tumor progression and metastasis.Nature Med. 2013; 19: 1423-1437Crossref PubMed Scopus (0) Google Scholar). The human GIST-T1 cells were previously established from a patient with metastatic imatinib-naive GIST and display an imatinib-sensitive KIT mutation in exon 11 (V560-Y579del) (46.Taguchi T. Sonobe H. Toyonaga S. Yamasaki I. Shuin T. Takano A. Araki K. Akimaru K. Yuri K. Conventional and molecular cytogenetic characterization of a new human cell line, GIST-T1, established from gastrointestinal stromal tumor.Lab. Invest. 2002; 82: 663-665Crossref PubMed Scopus (84) Google Scholar). The imatinib-sensitive GIST882 cells were derived from a primary, imatinib-naive GIST, and exhibit a homozygous missense mutation in KIT exon 13 (K642E) (47.Gupta A. Roy S. Lazar A.J. Wang W.L. McAuliffe J.C. Reynoso D. McMahon J. Taguchi T. Floris G. Debiec-Rychter M. Schoffski P. Trent J.A. Debnath J. Rubin B.P. Autophagy inhibition and antimalarials promote cell death in gastrointestinal stromal tumor (GIST).Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 14333-14338Crossref PubMed Scopus (0) Google Scholar). The KIT mutation status of the cell lines was confirmed using the TruSeq Amplicon - Cancer Panel (TSACP) from Illumina. These cells have been extensively characterized previously and are representative of the behavior, genotype, and phenotype of GIST (31.Atay S. Tetik A. Bozok Cetintas V. Yakar Tuluce S. Tuluce K. Kayikci
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