Skip to main content
SpringerLink
Log in

Constitutive Expression of NRAS with Q61R Driver Mutation Activates Processes of Epithelial–Mesenchymal Transition and Leads to Substantial Transcriptome Change of Nthy-ori 3–1 Thyroid Epithelial Cells

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

The Q61R mutation of the NRAS gene is one of the most frequent driver mutations of thyroid cancer. Tumors with this mutation are characterized by invasion into blood vessels and formation of distant metastases. To study the role of this mutation in the growth of thyroid cancer, we developed a model system on the basis of thyroid epithelial cell line Nthy-ori 3–1 transduced by a lentiviral vector containing the NRAS gene with the Q61R mutation. It was found that the expression of NRAS(Q61R) in thyroid epithelial cells has a profound influence on groups of genes involved in the formation of intercellular contacts, as well as in processes of epithelial–mesenchymal transition and cell invasion. The alteration in the expression of these genes affects the phenotype of the model cells, which acquire traits of mesenchymal cells and demonstrate increased ability for survival and growth without attachment to the substrate. The key regulators of these processes are transcription factors belonging to families SNAIL, ZEB, and TWIST, and in different types of tumors the contribution of each individual factor can vary greatly. In our model system, phenotype change correlates with an increase in the expression of SNAIL2 and TWIST2 factors, which indicates their possible role in regulating invasive growth of thyroid cancer with the mutation of NRAS(Q61R).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

EMT:

epithelial-mesenchymal transition

TC:

thyroid cancer

References

  1. Goodsell, D. S. (1999) The molecular perspective: the ras oncogene, Oncologist, 4, 263–264.

    CAS  PubMed  Google Scholar 

  2. Fukushima, T., and Takenoshita, S. (2005) Roles of RAS and BRAF mutations in thyroid carcinogenesis, Fukushima J. Med. Sci., 51, 67–75.

    Article  CAS  PubMed  Google Scholar 

  3. Bhaijee, F., and Nikiforov, Y. E. (2011) Molecular analysis of thyroid tumors, Endocr. Pathol., 22, 126–133.

    Article  CAS  PubMed  Google Scholar 

  4. Nikiforov, Y. E., and Nikiforova, M. N. (2011) Molecular genetics and diagnosis of thyroid cancer, Nat. Rev. Endocr., 7, 569–580.

    Article  CAS  Google Scholar 

  5. Jang, E. K., Song, D. E., Sim, S. Y., Kwon, H., Choi, Y. M., Jeon, M. J., Han, J. M., Kim, W. G., Kim, T. Y., Shong, Y. K., and Kim, W. B. (2014) NRAS codon 61 muta–tion is associated with distant metastasis in patients with follicular thyroid carcinoma, Thyroid, 24, 1275–1281.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Melo, M., Gaspar da Rocha, A., Batista, R., Vinagre, J., Martins, M. J., Costa, G., Ribeiro, C., Carrilho, F., Leite, V., Lobo, C., Cameselle–Teijeiro, J. M., Cavadas, B., Pereira, L., Sobrinho–Simoes, M., and Soares, P. (2017) TERT, BRAF, and NRAS in primary thyroid cancer and metastatic disease, J. Clin. Endocr. Metab., 102, 1898–1907.

    Google Scholar 

  7. Sohn, S. Y., Park, W. Y., Shin, H. T., Bae, J. S., Ki, C. S., Oh, Y. L., Kim, S. W., and Chung, J. H. (2016) Highly con–cordant key genetic alterations in primary tumors and matched distant metastases in differentiated thyroid cancer, Thyroid, 26, 672–682.

    Article  CAS  PubMed  Google Scholar 

  8. Gras, B., Jacqueroud, L., Wierinckx, A., Lamblot, C., Fauvet, F., Lachuer, J., Puisieux, A., and Ansieau, S. (2014) Snail family members unequally trigger EMT and thereby differ in their ability to promote the neoplastic transforma–tion of mammary epithelial cells, PLoS One, 9, e92254.

    Google Scholar 

  9. Cifone, M. A., and Fidler, I. J. (1980) Correlation of pat–terns of anchorage–independent growth with in vivo behav–ior of cells from a murine fibrosarcoma, Proc. Natl. Acad. Sci. USA, 77, 1039–1043.

    Article  CAS  PubMed  Google Scholar 

  10. Brabletz, T., Kalluri, R., Nieto, M. A., and Weinberg, R. A. (2018) EMT in cancer, Nat. Rev. Cancer, 18, 128–134.

    Article  CAS  PubMed  Google Scholar 

  11. Puisieux, A., Brabletz, T., and Caramel, J. (2014) Oncogenic roles of EMT–inducing transcription factors, Nat. Cell. Biol., 16, 488–494.

    Article  CAS  PubMed  Google Scholar 

  12. Cano, A., Perez–Moreno, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., del Barrio, M. G., Portillo, F., and Nieto, M. A. (2000) The transcription factor snail controls epithe–lial–mesenchymal transitions by repressing E–cadherin expression, Nat. Cell. Biol., 2, 76–83.

    Article  CAS  PubMed  Google Scholar 

  13. Vu, T., and Datta, P. K. (2017) Regulation of EMT in colo–rectal cancer: a culprit in metastasis, Cancers (Basel), 9, E171.

    Google Scholar 

  14. Vasko, V., Espinosa, A. V., Scouten, W., He, H., Auer, H., Liyanarachchi, S., Larin, A., Savchenko, V., Francis, G. L., de la Chapelle, A., Saji, M., and Ringel, M. D. (2007) Gene expression and functional evidence of epithelial–to–mesenchymal transition in papillary thyroid carcinoma invasion, Proc. Natl. Acad. Sci. USA, 104, 2803–2808.

    Article  CAS  PubMed  Google Scholar 

  15. Lemoine, N. R., Mayall, E. S., Jones, T., Sheer, D., McDermid, S., Kendall–Taylor, P., and Wynford–Thomas, D. (1989) Characterization of human thyroid epithelial cells immortalized in vitro by simian virus 40 DNA trans–fection, Br. J. Cancer, 60, 897–903.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Khosravi–Far, R., White, M. A., Westwick, J. K., Solski, P. A., Chrzanowska–Wodnicka, M. (1996) Oncogenic Ras activation of Raf/mitogen–activated protein kinase–independent path–ways is sufficient to cause tumorigenic transformation, Mol. Cell. Biol., 16, 3923–3933.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Prokofjeva, M. M., Proshkina, G. M., Lebedev, T. D., Shulgin, A. A., Spirin, P. V., Prassolov, V. S., and Deyev, S. M. (2017) Lentiviral gene delivery to plasmolipin–express–ing cells using Mus caroli endogenous retrovirus envelope protein, Biochimie, 142, 226–233.

    Article  CAS  PubMed  Google Scholar 

  18. Schwartz, A. M., Putlyaeva, L. V., Covich, M., Klepikova, A. V., Akulich, K. A., Vorontsov, I. E., Korneev, K. V., Dmitriev, S. E., Polanovsky, O. L., Sidorenko, S. P., Kulakovskiy, I. V., and Kuprash, D. V. (2016) Early B–cell factor 1 (EBF1) is critical for transcriptional control of SLAMF1 gene in human B cells, Biochim. Biophys. Acta, 1859, 1259–1268.

    Article  CAS  PubMed  Google Scholar 

  19. Afanasyeva, M. A., Britanova, L. V., Korneev, K. V., Mitkin, N. A., Kuchmiy, A. A., and Kuprash, D. V. (2014) Clusterin is a potential lymphotoxin beta receptor target that is upregulated and accumulates in germinal centers of mouse spleen during immune response, PLoS One, 9, e98349.

    Book  Google Scholar 

  20. Kim, B. A., Jee, H. G., Yi, J. W., Kim, S. J., Chai, Y. J., Choi, J. Y., and Lee, K. E. (2017) Expression profiling of a human thyroid cell line stably expressing the BRAFV600E mutation, Cancer Genomics Proteomics, 14, 53–67.

    Article  CAS  PubMed  Google Scholar 

  21. Roskoski, R., Jr. (2012) ERK1/2 MAP kinases: structure, function, and regulation, Pharmacol. Res., 66, 105–143.

    Article  CAS  PubMed  Google Scholar 

  22. Pauta, M., Rotllan, N., Fernandez–Hernando, A., Langhi, C., Ribera, J., Lu, M., Boix, L., Bruix, J., Jimenez, W., Suarez, Y., Ford, D. A., Baldan, A., Birnbaum, M. J., Morales–Ruiz, M., and Fernandez–Hernando, C. (2016) Akt–mediated foxo1 inhibition is required for liver regener–ation, Hepatology, 63, 1660–1674.

    Article  CAS  PubMed  Google Scholar 

  23. Giordano, T. J., Kuick, R., Thomas, D. G., Misek, D. E., Vinco, M., Sanders, D., Zhu, Z., Ciampi, R., Roh, M., Shedden, K., Gauger, P., Doherty, G., Thompson, N. W., Hanash, S., Koenig, R. J., and Nikiforov, Y. E. (2005) Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation–specific gene expression profiles discovered by DNA microarray analysis, Oncogene, 24, 6646–6656.

    CAS  PubMed  Google Scholar 

  24. Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources, Nat. Protoc., 4, 44–57.

    Article  CAS  PubMed  Google Scholar 

  25. Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009) Bioinformatics enrichment tools: paths toward the compre–hensive functional analysis of large gene lists, Nucleic Acids Res., 37, 1–13.

    Article  CAS  PubMed  Google Scholar 

  26. Sponziello, M., Rosignolo, F., Celano, M., Maggisano, V., Pecce, V., De Rose, R. F., Lombardo, G. E., Durante, C., Filetti, S., Damante, G., Russo, D., and Bulotta, S. (2016) Fibronectin–1 expression is increased in aggressive thyroid cancer and favors the migration and invasion of cancer cells, Mol. Cell. Endocrinol., 431, 123–132.

    Article  CAS  PubMed  Google Scholar 

  27. Da, C., Wu, K., Yue, C., Bai, P., Wang, R., Wang, G., Zhao, M., Lv, Y., and Hou, P. (2017) N–cadherin promotes thyroid tumorigenesis through modulating major signaling pathways, Oncotarget, 8, 8131–8142.

    Article  PubMed  Google Scholar 

  28. Peng, X. G., Chen, Z. F., Zhang, K. J., Wang, P. G., Liu, Z. M., Chen, Z. J., Hou, G. Y., and Niu, M. (2015) VEGF Trapon inhibits tumor growth in papillary thyroid carcino–ma, Eur. Rev. Med. Pharmacol. Sci., 19, 235–240.

    PubMed  Google Scholar 

  29. Kawakami, T., Tokunaga, T., Hatanaka, H., Kijima, H., Yamazaki, H., Abe, Y., Osamura, Y., Inoue, H., Ueyama, Y., and Nakamura, M. (2002) Neuropilin 1 and neuropilin 2 co–expression is significantly correlated with increased vascularity and poor prognosis in non–small cell lung carci–noma, Cancer, 95, 2196–2201.

    Article  CAS  PubMed  Google Scholar 

  30. Dowling, C. M., Hayes, S. L., Phelan, J. J., Cathcart, M. C., Finn, S. P., Mehigan, B., McCormick, P., Coffey, J. C., O’Sullivan, J., and Kiely, P. A. (2017) Expression of protein kinase C gamma promotes cell migration in colon cancer, Oncotarget, 8, 72096–72107.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Martin, T. A., Lane, J., Harrison, G. M., and Jiang, W. G. (2013) The expression of the Nectin complex in human breast cancer and the role of Nectin–3 in the control of tight junctions during metastasis, PLoS One, 8, e82696.

    Book  Google Scholar 

  32. Kremenevskaja, N., von Wasielewski, R., Rao, A. S., Schofl, C., Andersson, T., and Brabant, G. (2005) Wnt–5a has tumor suppressor activity in thyroid carcinoma, Oncogene, 24, 2144–2154.

    Article  CAS  PubMed  Google Scholar 

  33. Kaur, S., Kroczynska, B., Sharma, B., Sassano, A., Arslan, A. D., Majchrzak–Kita, B., Stein, B. L., McMahon, B., Altman, J. K., Su, B., Calogero, R. A., Fish, E. N., and Platanias, L. C. (2014) Critical roles for Rictor/Sin1 com–plexes in interferon–dependent gene transcription and gen–eration of antiproliferative responses, J. Biol. Chem., 289, 6581–6591.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kaur, S., Sassano, A., Majchrzak–Kita, B., Baker, D. P., Su, B., Fish, E. N., and Platanias, L. C. (2012) Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses, Proc. Natl. Acad. Sci. USA, 109, 7723–7728.

    Article  PubMed  Google Scholar 

  35. Cooney, R. N. (2002) Suppressors of cytokine signaling (SOCS): inhibitors of the JAK/STAT pathway, Shock, 17, 83–90.

    Article  PubMed  Google Scholar 

  36. Cancer Genome Atlas Research Network (2014) Integrated genomic characterization of papillary thyroid carcinoma, Cell, 159, 676–690.

    Article  CAS  Google Scholar 

  37. Thiery, J. P., and Sleeman, J. P. (2006) Complex networks orchestrate epithelial–mesenchymal transitions, Nat. Rev. Mol. Cell. Biol., 7, 131–142.

    Article  CAS  PubMed  Google Scholar 

  38. Mueller, N., Wicklein, D., Eisenwort, G., Jawhar, M., Berger, D., Stefanzl, G., Greiner, G., Boehm, A., Kornauth, C., Muellauer, L., Sehner, S., Hoermann, G., Sperr, W. R., Staber, P. B., Jaeger, U., Zuber, J., Arock, M., Schumacher, U., Reiter, A., and Valent, P. (2018) CD44 is a RAS/STAT5–regulated invasion receptor that triggers dis–ease expansion in advanced mastocytosis, Blood, 132, 1936–1950.

    Article  CAS  PubMed  Google Scholar 

  39. Jia, L., Liu, W., Guan, L., Lu, M., and Wang, K. (2015) Inhibition of calcium–activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer, PLoS One, 10, e0136584.

    Book  Google Scholar 

  40. Drak Alsibai, K., and Meseure, D. (2018) Tumor microen–vironment and noncoding RNAs as co–drivers of epithelial–mesenchymal transition and cancer metastasis, Dev. Dyn., 247, 405–431.

    Article  PubMed  Google Scholar 

  41. Yoh, K. E., Regunath, K., Guzman, A., Lee, S. M., Pfister, N. T., Akanni, O., Kaufman, L. J., Prives, C., and Prywes, R. (2016) Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells, Proc. Natl. Acad. Sci. USA, 113, 6107–6116.

    Article  CAS  Google Scholar 

  42. Kim, H., Choi, J. A., and Kim, J. H. (2014) Ras promotes transforming growth factor–beta (TGF–beta)–induced epithelial–mesenchymal transition via a leukotriene B4 receptor–2–linked cascade in mammary epithelial cells, J. Biol. Chem., 289, 22151–22160.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wu, D., Zhao, B., Qi, X., Peng, F., Fu, H., Chi, X., Miao, Q. R., and Shao, S. (2018) Nogo–B receptor promotes epithelial–mesenchymal transition in non–small cell lung cancer cells through the Ras/ERK/Snail1 pathway, Cancer Lett., 418, 135–146.

    Article  CAS  PubMed  Google Scholar 

  44. Maiques, O., Barcelo, C., Panosa, A., Pijuan, J., Orgaz, J. L., Rodriguez–Hernandez, I., Matas–Nadal, C., Tell, G., Vilella, R., Fabra, A., Puig, S., Sanz–Moreno, V., Matias–Guiu, X., Canti, C., Herreros, J., Marti, R. M., and Macia, A. (2018) T–type calcium channels drive migration/inva–sion in BRAFV600E melanoma cells through Snail1, Pigment Cell. Melanoma Res., 31, 484–495.

    Article  CAS  PubMed  Google Scholar 

  45. Mittal, D., Gubin, M. M., Schreiber, R. D., and Smyth, M. J. (2014) New insights into cancer immunoediting and its three component phases–elimination, equilibrium and escape, Curr. Opin. Immunol., 27, 16–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, R., Ma, Q., Ji, L., Yao, Y., Ma, M., and Wen, Q. (2018) miR–622 suppresses tumor formation by directly targeting VEGFA in papillary thyroid carcinoma, Onco. Targets Ther., 11, 1501–1509.

    PubMed  Google Scholar 

  47. Sheng, L., Zhang, S., and Xu, H. (2017) Effect of slug–mediated down–regulation of E–cadherin on invasiveness and metastasis of anaplastic thyroid cancer cells, Med. Sci. Monit., 23, 138–143.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Borrello, M. G., Alberti, L., Fischer, A., Degl’innocenti, D., Ferrario, C., Gariboldi, M., Marchesi, F., Allavena, P., Greco, A., Collini, P., Pilotti, S., Cassinelli, G., Bressan, P., Fugazzola, L., Mantovani, A., and Pierotti, M. A. (2005) Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene, Proc. Natl. Acad. Sci. USA, 102, 14825–14830.

    Article  CAS  PubMed  Google Scholar 

  49. Meng, X., Kong, D. H., Li, N., Zong, Z. H., Liu, B. Q., Du, Z. X., Guan, Y., Cao, L., and Wang, H. Q. (2014) Knockdown of BAG3 induces epithelial–mesenchymal transition in thyroid cancer cells through ZEB1 activation, Cell. Death. Dis., 5, e1092.

    Book  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991, Moscow, Russia

    D. E. Demin, M. A. Afanasyeva, A. N. Uvarova, M. M. Prokofjeva, A. M. Gorbachova, A. S. Ustiugova, L. V. Putlyaeva, K. A. Tatosyan, P. V. Belousov & A. M. Schwartz

  2. Moscow Institute of Physics and Technology, 141701, Moscow, Russia

    D. E. Demin & A. M. Schwartz

  3. Institute for Information Transmission Problems, Russian Academy of Sciences, 127051, Moscow, Russia

    A. V. Klepikova

  4. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    A. V. Klepikova

Authors
  1. D. E. Demin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. A. Afanasyeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. N. Uvarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. M. Prokofjeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. M. Gorbachova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. S. Ustiugova
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. V. Klepikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. L. V. Putlyaeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. K. A. Tatosyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. P. V. Belousov
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. A. M. Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. M. Schwartz.

Additional information

Russian Text © D. E. Demin, M. A. Afanasyeva, A. N. Uvarova, M. M. Prokofjeva, A. M. Gorbachova, A. S. Ustiugova, A. V. Klepikova, L. V. Putlyaeva, K. A. Tatosyan, P. V. Belousov, A. M. Schwartz, 2019, published in Biokhimiya, 2019, Vol. 84, No. 4, pp. 560–570.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM18–285, February 4, 2019.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Demin, D.E., Afanasyeva, M.A., Uvarova, A.N. et al. Constitutive Expression of NRAS with Q61R Driver Mutation Activates Processes of Epithelial–Mesenchymal Transition and Leads to Substantial Transcriptome Change of Nthy-ori 3–1 Thyroid Epithelial Cells. Biochemistry Moscow 84, 416–425 (2019). https://doi.org/10.1134/S0006297919040096

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297919040096

Keywords

Search

Navigation