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Proteomic Analysis of Extracellular Vesicles Derived from MDA-MB-231 Cells in Microgravity

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Abstract

Patients with triple-negative breast cancer (TNBC) have a relatively poor prognosis and cannot benefit from endocrine and/or targeted therapy. Considerable effort has been devoted toward the elucidation of the molecular mechanisms and potential diagnostic/therapeutic targets. However, it is inefficient and often ineffective to study the biological nuances of TNBC in large-scale clinical trials. In contrast, the investigation of the association between molecular alterations induced through controlled variables and relevant physiochemical characteristics of TNBC cells in laboratory settings is simple, definite, and efficient in exploring the molecular mechanisms. In this study, microgravity was selected as the sole variable of study as it can inhibit cancer cell viability, proliferation, metastasis, and chemoresistance. Identifying the key molecules that shift cancer cells toward a less aggressive phenotype may facilitate future TNBC studies. We focused on extracellular vesicles (EV) derived from TNBC MDA-MB-231 cells in microgravity, which mediate intercellular communication by transporting signaling molecules between cells. Our results show that in comparison with cells in full gravity, EV release rate decreased in microgravity while average EV size increased. In addition, we found EVs may be superior to cells in analyzing differentially expressed proteins, especially those that are down-regulated ones and usually unidentified or neglected in analysis of intact cellular contents. Proteomic analysis of both EVs and cells further revealed a significant correlation with GTPases and proliferation of MDA-MB-231 cells in microgravity. Altogether, our findings would further inspire in-depth correlative cancer biological studies and subsequent clinical research.

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Data Availability

Data are available by request from the author for correspondence.

References

  1. Greenup R, Buchanan A, Lorizio W, Rhoads K, Chan S, Leedom T, King R, McLennan J, Crawford B, Marcom PK, Hwang ES (2013) Prevalence of BRCA mutations among women with triple-negative breast cancer (TNBC) in a Genetic Counseling Cohort. Ann Surg Oncol 20(10):3254–3258

    PubMed  Google Scholar 

  2. Siddharth S, Sharma D (2018) Racial disparity and triple-negative breast cancer in African-American women: a multifaceted affair between obesity, biology, and socioeconomic determinants. Cancers 10(12):514

    CAS  PubMed Central  Google Scholar 

  3. Dietze EC, Sistrunk C, Miranda-Carboni G, O’Regan R, Seewaldt VL (2015) Triple-negative breast cancer in African-American women: disparities versus biology. Nat Rev Cancer 15(4):248–254

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13:no. 15 Pt 1, pp. 4429–4434

    PubMed  Google Scholar 

  5. Foulkes WD, Smith IE, Reis-Filho JS (2010) Triple-negative breast cancer. N Engl J Med 363(20):1938–1948

    CAS  PubMed  Google Scholar 

  6. Collignon J, Lousberg L, Schroeder H, Jerusalem G (2016) Triple-negative breast cancer: treatment challenges and solutions. Breast Cancer (Dove Med Press) 8:93–107

    CAS  Google Scholar 

  7. Bizzarri M, Monici M, van Loon JJWA (2015) How microgravity affects the biology of living systems. Biomed Res Int. https://doi.org/10.1155/2015/863075

    Article  PubMed  PubMed Central  Google Scholar 

  8. Shi ZX, Rao W, Wang H, Wang ND, Si JW, Zhao J, Li JC, Wang ZR (2015) Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry. Biochem Biophys Res Commun 457(3):378–384

    CAS  PubMed  Google Scholar 

  9. Lee JM, Mhawech-Fauceglia P, Lee N, Parsanian LC, Lin YG, Gayther SA, Lawrenson K (2013) A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro. Lab Invest 93(5):528–542

    PubMed  Google Scholar 

  10. Takeda M, Magaki T, Okazaki T, Kawahara Y, Manabe T, Yuge L, Kurisu K (2009) Effects of simulated microgravity on proliferation and chemosensitivity in malignant glioma cells. Neurosci Lett 463(1):54–59

    CAS  PubMed  Google Scholar 

  11. Zhao J, Ma H, Wu L, Cao L, Yang Q, Dong H, Wang Z, Ma J, Li Z (2017) The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells. In Vitro Cell Dev Biol Anim 53(8):744–751

    CAS  PubMed  Google Scholar 

  12. Kowal J, Tkach M, Théry C (2014) Biogenesis and secretion of exosomes. Curr Opin Cell Biol 29:116–125

    CAS  PubMed  Google Scholar 

  13. Tkach M, Thery C (2016) Communication by extracellular vesicles: where we are and where we need to go. Cell 164(6):1226–1232

    CAS  PubMed  Google Scholar 

  14. Osaki M, Okada F (2019) Exosomes and their role in cancer progression. Yonago Acta Med 62(2):182–190

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Maacha S, Bhat AA, Jimenez L, Raza A, Haris M, Uddin S, Grivel JC (2019) Extracellular vesicles-mediated intercellular communication: roles in the tumor microenvironment and anti-cancer drug resistance. Mol Cancer 18(1):55

    PubMed  PubMed Central  Google Scholar 

  16. Nawaz M, Shah N, Zanetti BR, Maugeri M, Silvestre RN, Fatima F, Neder L, Valadi H (2018) Extracellular vesicles and matrix remodeling enzymes: the emerging roles in extracellular matrix remodeling, progression of diseases and tissue repair. Cells 7(10):167

    CAS  PubMed Central  Google Scholar 

  17. Ricklefs FL, Alayo Q, Krenzlin H, Mahmoud AB, Speranza MC, Nakashima H, Hayes JL, Lee K, Balaj L, Passaro C, Rooj AK, Krasemann S, Carter BS, Chen CC, Steed T, Treiber J, Rodig S, Yang K, Nakano I, Lee H, Weissleder R, Breakefield XO, Godlewski J, Westphal M, Lamszus K, Freeman GJ, Bronisz A, Lawler SE, Chiocca EA (2018) Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Sci Adv 4(3):eaar2766

    PubMed  PubMed Central  Google Scholar 

  18. Whiteside TL (2016) Tumor-derived exosomes and their role in cancer progression. Adv Clin Chem 74:103–141

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Torrano V, Royo F, Peinado H, Loizaga-Iriarte A, Unda M, Falcon-Perez JM, Carracedo A (2016) Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer. Curr Opin Pharmacol 29:47–53

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Lawrence RT, Perez EM, Hernandez D, Miller CP, Haas KM, Irie HY, Lee SI, Blau CA, Villen J (2015) The proteomic landscape of triple-negative breast cancer. Cell Rep 11(6):990

    CAS  PubMed  Google Scholar 

  21. Subedi P, Schneider M, Philipp J, Azimzadeh O, Metzger F, Moertl S, Atkinson MJ, Tapio S (2019) Comparison of methods to isolate proteins from extracellular vesicles for mass spectrometry-based proteomic analyses. Anal Biochem 584:113390

    CAS  PubMed  Google Scholar 

  22. Wen Y, Chen Y, Wang G, Abhange K, Xue F, Quinn Z, Mao W, Wan Y (2020) Factors influencing measurement of the secretion rate of extracellular vesicles. Analyst 145:5870–5877. https://doi.org/10.1039/D0AN01199A

    Article  CAS  PubMed  Google Scholar 

  23. Mao W, Wen Y, Lei H, Lu R, Wang S, Wang Y, Chen R, Gu Y, Zhu L, Abhange KK, Quinn ZJ, Chen Y, Xue F, Zheng M, Wan Y (2019) Isolation and retrieval of extracellular vesicles for liquid biopsy of malignant ground-glass opacity. Anal Chem 91(21):13729–13736

    CAS  PubMed  Google Scholar 

  24. Wang L, Abhange KK, Wen Y, Chen Y, Xue F, Wang G, Tong J, Zhu C, He X, Wan Y (2019) Preparation of engineered extracellular vesicles derived from human umbilical cord mesenchymal stem cells with ultrasonication for skin rejuvenation. ACS Omega 4(27):22638–22645

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Wan Y, Liu B, Lei H, Zhang B, Wang Y, Huang H, Chen S, Feng Y, Zhu L, Gu Y, Zhang Q, Ma H, Zheng SY (2018) Nanoscale extracellular vesicle-derived DNA is superior to circulating cell-free DNA for mutation detection in early-stage non-small-cell lung cancer. Ann Oncol 29(12):2379–2383

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wan Y, Maurer M, He HZ, Xia YQ, Hao SJ, Zhang WL, Yee NS, Zheng SY (2019) Enrichment of extracellular vesicles with lipid nanoprobe functionalized nanostructured silica. Lab Chip 19(14):2346–2355

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Wan Y, Wang L, Zhu C, Zheng Q, Wang G, Tong J, Fang Y, Xia Y, Cheng G, He X, Zheng SY (2018) Aptamer-conjugated extracellular nanovesicles for targeted drug delivery. Cancer Res 78(3):798–808

    CAS  PubMed  Google Scholar 

  28. Jhala DV, Kale RK, Singh RP (2014) Microgravity alters cancer growth and progression. Curr Cancer Drug Targets 14(4):394–406

    CAS  PubMed  Google Scholar 

  29. Moreno-Villanueva M, Wong M, Lu T, Zhang Y, Wu H (2017) Interplay of space radiation and microgravity in DNA damage and DNA damage response. NPJ Microgravity 3:14

    PubMed  PubMed Central  Google Scholar 

  30. Michaletti A, Gioia M, Tarantino U, Zolla L (2017) “Effects of microgravity on osteoblast mitochondria: a proteomic and metabolomics profile. Sci Rep 7(1):15376

    PubMed  PubMed Central  Google Scholar 

  31. Pisanu ME, Noto A, De Vitis C, Masiello MG, Coluccia P, Proietti S, Giovagnoli MR, Ricci A, Giarnieri E, Cucina A, Ciliberto G, Bizzarri M, Mancini R (2014) Lung cancer stem cell lose their stemness default state after exposure to microgravity. Biomed Res Int. https://doi.org/10.1155/2014/470253

    Article  PubMed  PubMed Central  Google Scholar 

  32. Zhang J, Espinoza LA, Kinders RJ, Lawrence SM, Pfister TD, Zhou M, Veenstra TD, Thorgeirsson SS, Jessup JM (2013) NANOG modulates stemness in human colorectal cancer. Oncogene 32(37):4397–4405

    CAS  PubMed  Google Scholar 

  33. Lu Y, Zhu H, Shan H, Lu J, Chang X, Li X, Lu J, Fan X, Zhu S, Wang Y, Guo Q, Wang L, Huang Y, Zhu M, Wang Z (2013) Knockdown of Oct4 and Nanog expression inhibits the stemness of pancreatic cancer cells. Cancer Lett 340(1):113–123

    CAS  PubMed  Google Scholar 

  34. Arun RP, Sivanesan D, Vidyasekar P, Verma RS (2017) PTEN/FOXO3/AKT pathway regulates cell death and mediates morphogenetic differentiation of colorectal cancer cells under simulated microgravity. Sci Rep 7:1–15

    CAS  Google Scholar 

  35. Wang H, Tang H-Y, Tan GC, Speicher DW (2011) Data analysis strategy for maximizing high-confidence protein identifications in complex proteomes such as human tumor secretomes and human serum. J Proteome Res 10(11):4993–5005

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Laulagnier K, Grand D, Dujardin A, Hamdi S, Vincent-Schneider H, Lankar D, Salles JP, Bonnerot C, Perret B, Record M (2004) PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes. Febs Lett 572:1–3

    Google Scholar 

  37. Wan Y, Cheng G, Liu X, Hao SJ, Nisic M, Zhu CD, Xia YQ, Li WQ, Wang ZG, Zhang WL, Rice SJ, Sebastian A, Albert I, Belani CP, Zheng SY (2017) Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes. Nat Biomed Eng 1:1–11

    Google Scholar 

  38. Shields JM, Pruitt K, McFall A, Shaub A, Der CJ (2000) Understanding Ras: ‘it ain’t over ‘til it’s over.’ Trends Cell Biol 10(4):147–154

    CAS  PubMed  Google Scholar 

  39. Lim KH, O’Hayer K, Adam SJ, Kendall SD, Campbell PM, Der CJ, Counter CM (2006) Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr Biol 16(24):2385–2394

    CAS  PubMed  Google Scholar 

  40. Camonis JH, White MA (2005) Ral GTPases: corrupting the exocyst in cancer cells. Trends Cell Biol 15(6):327–332

    CAS  PubMed  Google Scholar 

  41. Vial E, Sahai E, Marshall CJ (2003) ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. Cancer Cell 4(1):67–79

    CAS  PubMed  Google Scholar 

  42. Alkasalias T, Alexeyenko A, Hennig K, Danielsson F, Lebbink RJ, Fielden M, Turunen SP, Lehti K, Kashuba V, Madapura HS, Bozoky B, Lundberg E, Balland M, Guven H, Klein G, Gad AK, Pavlova T (2017) RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo. Proc Natl Acad Sci USA 114(8):E1413–E1421

    CAS  PubMed  Google Scholar 

  43. Gulhati P, Bowen KA, Liu J, Stevens PD, Rychahou PG, Chen M, Lee EY, Weiss HL, O’Connor KL, Gao T, Evers BM (2011) mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res 71(9):3246–3256

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Yoshioka K, Nakamori S, Itoh K (1999) “Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells. Cancer Res 59(8):2004–2010

    CAS  PubMed  Google Scholar 

  45. Liu K, Sun MM, Zhao ZH, Wei N, Jiang GZ, Wang ZY, Zhang L, Zhu XY, Dai LP, Yang HM, Wang T, Chen KS (2019) Effect of RhoC silencing on multiple myeloma xenografts and angiogenesis in nude mice. J Biol Regul Homeost Agents 33(5):1387–1394

    CAS  PubMed  Google Scholar 

  46. Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G, Wakeham A, Khokha R, Mak TW (2005) RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev 19(17):1974–1979

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Ioannou MS, McPherson PS (2016) Regulation of cancer cell behavior by the small GTPase Rab13. J Biol Chem 291(19):9929–9937

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Qadir MI, Parveen A, Ali M (2015) Cdc42: role in cancer management. Chem Biol Drug Des 86(4):432–439

    CAS  PubMed  Google Scholar 

  49. Weber G (1983) Enzymes of purine metabolism in cancer. Clin Biochem 16(1):57–63

    CAS  PubMed  Google Scholar 

  50. Barfeld SJ, Fazli L, Persson M, Marjavaara L, Urbanucci A, Kaukoniemi KM, Rennie PS, Ceder Y, Chabes A, Visakorpi T, Mills IG (2015) Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer. Oncotarget 6(14):12587–12602

    PubMed  PubMed Central  Google Scholar 

  51. Yin J, Ren W, Huang X, Deng J, Li T, Yin Y (2018) Potential mechanisms connecting purine metabolism cancer therapy. Front Immunol 9:1697

    PubMed  PubMed Central  Google Scholar 

  52. Yang L, Achreja A, Yeung TL, Mangala LS, Jiang D, Han C, Baddour J, Marini JC, Ni J, Nakahara R, Wahlig S, Chiba L, Kim SH, Morse J, Pradeep S, Nagaraja AS, Haemmerle M, Kyunghee N, Derichsweiler M, Plackemeier T, Mercado-Uribe I, Lopez-Berestein G, Moss T, Ram PT, Liu J, Lu X, Mok SC, Sood AK, Nagrath D (2016) Targeting stromal glutamine synthetase in tumors disrupts tumor microenvironment-regulated cancer cell growth. Cell Metab 24(5):685–700

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was partially supported by Binghamton University Faculty Startup Fund 910252-35, Binghamton University S3IP award ADLG195, and NIH subward 1R01CA230339-01.

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Authors and Affiliations

  1. The Pq Laboratory of Micro/Nano BiomeDx, Department of Biomedical Engineering, Binghamton University-SUNY, Binghamton, New York, 13902, USA

    Yundi Chen, Fei Xue, Andrea Russo & Yuan Wan

  2. Biotechnology Building, Room 2625, 65 Murray Hill Road, Vestal, New York, 13850, USA

    Yuan Wan

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  1. Yundi Chen
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  2. Fei Xue
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  4. Yuan Wan
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Y.C., F.X., and Y. W. participated in the conceptualization and protocols; Y.C., and F.X. produced the data; all authors analyzed and interpreted the data. All authors prepared the manuscript and approved the final version of the manuscript.

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Correspondence to Yuan Wan.

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Chen, Y., Xue, F., Russo, A. et al. Proteomic Analysis of Extracellular Vesicles Derived from MDA-MB-231 Cells in Microgravity. Protein J 40, 108–118 (2021). https://doi.org/10.1007/s10930-020-09949-2

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