Matrix metalloproteinases and heat shock proteins on extracellular vesicles in colorectal cancer patients: association with metabolic status

Introduction. In most patients with colorectal cancer (CRC), the tumor develops against the background of metabolically healthy obesity or metabolic syndrome (more than 60 % of patients), the key pathogenetic moment of which is developing hyperinsulinemia. Metabolic changes are also characteristic of patients with colon polyps (CP), which are currently considered as the most significant precancerous diseases. It has been shown that fractions of small extracellular vesicles (EVs) of adipocyte origin are specifically enriched in extracellular matrix proteins, including matrix metalloproteinases (MMPs), chaperones, and some metabolic enzymes involved in the synthesis of lipids and carbohydrates. This was the reason for choosing exosomal markers in our study. Comparison of protein expression on CD9- and FABP-4 positive vesicles will be useful to explain some clinical issues, such as the effectiveness of thermoradiotherapy or radiotherapy in obese CRC patients; for a more substantiated search for vesicular prognostic markers in obese cancer patients. However, taking into account the lack of data in the literature on the level of MMPs and HSPs expression in the composition of the total pool of EVs and in the composition of FABP4-positive EVs in patients with PTC and CRC patients, the aim of the work was formulated.

Aim. Study of the level of MMPs and heat shock proteins (HSPs) on CD9- and FABP4-positive EVs in patients with CP and CRC in relation to metabolic status.

Materials and methods. The study included 12 patients with CRC (T2-4N0-2M0; mean age 59.6 ± 1.6 years) who were treated at the Department of Abdominal Oncology of the Cancer Research Institute of the Tomsk National Research Medical Center from 2019 to 2021. The comparison group included 10 patients with CP. The level of proteins on the surface of CD9- and FABP4-positive EVs was studied using flow cytometry.

Results. MMP9-positive EVs were detected more often in CRC patients than in CP patients, however, MMP9+MMP2+TIMP-positive EVs were significantly more frequently detected in CP patients. Among the studied heat shock proteins, HSP60 was most often expressed on the surface of EVs, and HSP60-positive EVs were detected on the surface of CD9-positive exosomes in patients with PTC much more often than in CRC. In patients with CRC, compared with patients with СP, among FABP4-positive EVs, the proportion of triple-positive EVs and EVs with the MMP9+MMP2-TIMP1+ phenotype significantly increases, which in general may indicate overexpression of MMP9 and TIMP1 by adipocytes or marcrophages of adipose tissue in patients with CRC. Correlation analysis revealed multiple correlations of individual phenotypes of CD9-positive EVs in patients with CRC with body mass index and serum high density lipoprotein cholesterol levels, while the phenotypes of FABP4-positive EVs were associated mainly with triglyceride levels.

Conclusions. The phenotypes of CD9-positive and FABP4-positive circulating EVs are promising as predictors for clarifying cancer risk in patients with colon polyps, as well as in terms of explaining the effectiveness of the treatment of CRC patients with obesity or metabolic syndrome.

1. Yunusova N.V., Kondakova I.V., Kolomiets L.A. et al. The role of metabolic syndrome variant in the malignant tumors progression. Diabetes Metab Syndr 2018;12(5):807-12. DOI: 10.1016/j.dsx.2018.04.028

2. Yunusova N.V., Kondakova I.V., Kolomiets L.A. et al. Molecular targets for the therapy of cancer associated with metabolic syndrome (transcription and growth factors). Asia Pac J Clin Oncol 2018;14(3):134-40. DOI: 10.1111/ajco.12780

3. Wang J., Wu Y., Guo J. et al. Adipocyte-derived exosomes promote lung cancer metastasis by increasing MMP9 activity via transferring MMP3 to lung cancer cells. Oncotarget 2017;8(47):81880-91. DOI: 10.18632/oncotarget.18737

4. Bouloumie A., Sengenes C., Portolan G. et al. Adipocyte produces matrix metalloproteinases 2 and 9: involvement in adipose differentiation. Diabetes 2001;50(9):2080—6. DOI: 10.2337/diabetes.50.9.2080

5. Weisberg S.P., McCann D., Desai M. et al. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003; 112(12):1796-808. DOI: 10.1172/JCI19246

6. Gao X., Salomon C., Freeman D.J. et al. Extracellular vesicles from adipose tissue — a potential role in obesity and type 2 diabetes? Front Endocrinol (Lausanne) 2017;8:202. DOI: 10.3389/fendo.2017.00202

7. Furuhashi M. Fatty Acid-binding protein 4 in cardiovascular and metabolic diseases. J Atheroscler Thromb 2019;26(3):216-32. DOI: 10.5551/jat.48710

8. Eguchi A., Lazic M., Armando A.M. et al. Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress. J Mol Med (Berl) 2016;94(11):1241-53. DOI: 10.1007/s00109-016-1446-8

9. DeClercq V., d'Eon B., McLeod R.S. Fatty acids increase adiponectin secretion through both classical and exosome pathways. Biochim Biophys Acta 2015;1851(9):1123—33. DOI: 10.1016/j.bbalip.2015.04.005

10. Kralisch S., Ebert T., Lossner U. et al. Adipocyte fatty acid-binding protein is released from adipocytes by a non-conventional mechanism. Int J Obes (Lond) 2014;38(9):1251-4. DOI: 10.1038/ijo.2013.232

11. Durcin M., Fleury A., Taillebois E. et al. Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles. J Extracell Vesicles 2017;6(1):1305677. DOI: 10.1080/20013078.2017.1305677

12. Tamkovich S.N., Yunusova N.V., Stakheeva M.N. et al. Isolation and characterization of blood plasma in patients with breast cancer and colorectal cancer. Biomedicinskaya Khimiya = Biomedical Chemistry 2017;63(2):165—9. (in Russ.). DOI: 10.18097/PBMC20176302165

13. Tamkovich S.N., Yunusova N.V., Somov A.K. et al. Comparative sub-population analysis of exosomes from blood plasma of cancer patients. Biomedicinskaya Khimiya = Biomedical Chemistry 2018;64(1):110-4. (In Russ.). DOI: 10.18097/PBMC20186401110

14. Yunusova N.V., Zambalova E.A., Patysheva M.R. et al. Exosomal protease cargo as prognostic biomarker in colorectal cancer. Asian Pac J Cancer Prev 2021;22(3):861-9. DOI: 10.31557/APJCP.2021.22.3.861

15. Matthews A.L., Noy P.J., Reyat J.C. Regulation of A disintegrin and metalloproteinase (ADAM) family sheddases ADAM10 and ADAM17: the emerging role of tetraspanins and rhomboids. Platelets 2017;28(4):333-41. DOI: 10.1080/09537104.2016.1184751

16. Gutwein P., Stoeck A., Riedle S. Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells. Clin Cancer Res 2005;11(7):2492-501. DOI: 10.1158/1078-0432.CCR-04-1688

17. Buzas E.I., Tot E.A., Sodar B.V. Molecular interactions at the surface of extracellular vesicles. Semin Immunopathol 2018;40(5):453-64. DOI: 10.1007/s00281-018-0682-0

18. Shimoda M., Khokha R. Metalloproteinases in extracellular vesicles. Biochim Biophys Acta Mol Cell Res 2017;1864(11 Pt. A):1989-2000. DOI: 10.1016/j.bbamcr.2017.05.027

19. Ginestra A., Monea S., Seghezzi G. Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells. J Biol Chem 1997;272(27):17216-22. DOI: 10.1074/jbc.272.27.17216

20. Kaur P., Hurwitz M.D., Krishnan S. et al. Combined hyperthermia and radiotherapy for the treatment of cancer. Cancers (Basel) 2011;3(4):3799-823. DOI: 10.3390/cancers3043799

21. Taha E.A., Ono K., Eguchi T. Roles of extracellular HSPs as biomarkers in immune surveillance and immune evasion. Int J Mol Sci 2019;20(18):4588. DOI: 10.3390/ijms20184588

22. Lv L.H., Wan Y.L., Lin Y. et al. Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro. J Biol Chem 2012;287(19):15874-85. DOI: 10.1074/jbc.M112.340588

23. Bavisotto C.C., Cipolla C., Graceffa G. et al. Immumirphological pattern of molecular chaperones in normal and pathological thyroid tissues and circulating exosomes: potential use in clinics. Int J Mol Sci 2019;20(18):4496. DOI: 10.3390/ijms20184496

24. Hubal M.J., Nadler E.P., Ferrante S.C. et al. Circulating adipocyte-derived exosomal MicroRNAs associated with decreased insulin resistance after gastric bypass. Obesity (Silver Spring) 2017;25(1):102-10. DOI: 10.1002/oby.21709

25. Gustafson C.M., Shepherd A.J., Miller V.M., Jayachandran M. Age-and sex-specific differences in blood-borne microvesicles from apparently healthy humans. Biol Sex Differ 2015;6:10. DOI: 10.1186/s13293-015-0028-8