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Molecular Biology Reports

, Volume 46, Issue 2, pp 1873–1884 | Cite as

Silencing the OCT4-PG1 pseudogene reduces OCT-4 protein levels and changes characteristics of the multidrug resistance phenotype in chronic myeloid leukemia

  • Aline Portantiolo Lettnin
  • Eduardo Felipe Wagner
  • Michele Carrett-Dias
  • Karina dos Santos Machado
  • Adriano Werhli
  • Andrés Delgado Cañedo
  • Gilma Santos Trindade
  • Ana Paula de Souza VottoEmail author
Original Article
  • 167 Downloads

Abstract

Cancer stem cells show epigenetic plasticity and intrinsic resistance to anti-cancer therapy, rendering capable of initiating cancer relapse and progression. Transcription factor OCT-4 regulates various pathways in stem cells, but its expression can be regulated by pseudogenes. This work evaluated how OCT4-PG1 pseudogene can affect OCT-4 expression and mechanisms related to the multidrug resistance (MDR) phenotype in FEPS cells. Considering that OCT-4 protein is a transcription factor that regulates expression of ABC transporters, level of gene expression, activity of ABC proteins and cell sensitivity to chemotherapy were evaluated after OCT4-PG1 silencing. Besides we set up a STRING network. Results showed that after OCT4-PG1 silencing, cells expressed OCT-4 gene and protein to a lesser extent than mock cells. The gene and protein expression of ABCB1, as well as its activity were reduced. On the other hand, ALOX5 and ABCC1 genes was increased even as the activity of this transporter. Moreover, the silencing cells become sensitive to two chemotherapics tested. The network structure demonstrated that OCT4-PG1 protein interacts directly with OCT-4, SOX2, and NANOG and indirectly with ABC transporters. We conclude that OCT4-PG1 pseudogene plays a key role in the regulation OCT-4 transcription factor, which alters MDR phenotype in the FEPS cell line.

Keywords

ABC transporters Cancer stem cells MRP1 protein P-glycoprotein STRING database 

Abbreviations

CFDA

Carboxy fluorescein diacetate

SC

Stem cells

CSC

Cancer stem cells

DNR

Daunorubicin

hESCs

Human embryonic stem cells

INDO

Indomethacin

MDR

Multidrug resistance

MRP1

Multidrug resistance associated protein

P-gp

P-glycoprotein

Rho 123

Rhodamine 123

VP

Verapamil

Notes

Acknowledgements

The authors thank Dr. Vivian Rumjanek (Tumoral Immunology Laboratory at the Medical Biochemistry Institute of the Federal University of Rio de Janeiro, Brazil) for providing and allowing the use of the FEPS cell line.

Funding

This work was supported by the National Program for Academic Cooperation (PROCAD/CAPES) [Grant Numbers 2951/2014], Brazil. Lettnin, A.P. received a graduate fellowship from CAPES. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. 1.
    Villodre ES, Kipper FC, Pereira MB, Lenz G (2016) Roles of OCT4 in tumorigenesis, cancer therapy resistance and prognosis. Cancer Treat Rev N 51:1–9.  https://doi.org/10.1016/j.ctrv.2016.10.003 CrossRefGoogle Scholar
  2. 2.
    Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111CrossRefPubMedGoogle Scholar
  3. 3.
    Peitzsch C, Kurth I, Kunz-Schughart L, Baumann M, Dubrovska A (2013) Discovery of the cancer stem cell related determinants of radioresistance. Radiother Oncol 108:378–387CrossRefPubMedGoogle Scholar
  4. 4.
    Kreso A, Dick JE (2014) Evolution of the cancer stem cell model. Cell Stem Cell 14(3):275–291CrossRefPubMedGoogle Scholar
  5. 5.
    Cojoc M, Mäbert K, Muders MH, Dubrovska A (2015) A role for cancer stem cells in therapy resistance: cellular and molecular mechanisms. Semi Cancer Biol 31:16–27CrossRefGoogle Scholar
  6. 6.
    Simandi Z, Horvath A, Wright LC, Cuaranta-Monroy I, De Luca I, Karolyi K, Sauer S, Deleuze J-F, Gudas LJ, Cowley SM, Nagy L (2016) OCT4 acts as an integrator of pluripotency and signal-induced differentiation. Mol Cell 63:647–661.  https://doi.org/10.1016/j.molcel.2016.06.039 CrossRefPubMedGoogle Scholar
  7. 7.
    Brehm A, Ohbo K, Scholer H (1997) The carboxy-terminal transactivation domain of Oct-4 acquires cell specificity through the POU domain. Mol Cell Biol 17:154–162CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wang X, Dai J (2010) Concise review: isoforms of OCT4 contribute to the confusing diversity in stem cell biology. Stem cells (Dayton Ohio) 28(5):85–93.  https://doi.org/10.1002/stem.419 CrossRefGoogle Scholar
  9. 9.
    Scholer HR, Balling R, Hatzopoulos AK, Suzuki N, Gruss P (1989) Octamer binding proteins confer transcriptional activity in early mouse embryogenesis. EMBO J 8:2551–2557CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW, Staudt LM (1990) A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345(6277):686–692CrossRefPubMedGoogle Scholar
  11. 11.
    Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372–376CrossRefPubMedGoogle Scholar
  12. 12.
    Cavaleri F, Scholer HR (2003) Nanog: a new recruit to the embryonic stem cell ochestra. Cell 113:551–552CrossRefPubMedGoogle Scholar
  13. 13.
    Deyev IE, Polanovsky OL (2004) The oct genes and oct proteins. Mol Biol 38:48–55CrossRefGoogle Scholar
  14. 14.
    Chen Y-C, Hsu H-S, Chen Y-W, Tsai T-H, How C-K, Wang C-Y, Hung S-C, Chang Y-L, Tsai M-L, Lee Y-Y, Ku H-H, Chou S-H (2008) Oct-4 expression maintained cancer stem like properties in lung cancer-derived CD-133 positive cells. PLoS ONE 3 (7):e2637CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Salci KR, Leea JB, Mitchell RR, Orlando L, Fiebig-Comyn A, Shapovalova Z, Bhatia M (2015) Acquisition of pluripotency through continued environmental influence on OCT4-induced plastic human fibroblasts. Stem Cell Res 15:221–230CrossRefPubMedGoogle Scholar
  16. 16.
    Marques DS, Sandrini JZ, Boyle RT, Marins LF, Trindade GF (2010) Relationships between multidrug resistance (MDR) and stem cell markers in human chronic myeloid leukemia cell lines. Leuk Res 34:757–762CrossRefPubMedGoogle Scholar
  17. 17.
    Dean M, Fojo T, Bates S (2005) Tumor stem cells and drug resistance. Nat Rev Cancer 5:275–284.  https://doi.org/10.1038/nrc1590 CrossRefPubMedGoogle Scholar
  18. 18.
    Guo J, Cahill MR, McKenna SL, O’Driscoll CM (2014) Biomimetic nanoparticles for siRNA delivery in the treatment of leukemia. Biotechnol Adv 32(8):1396–1409.  https://doi.org/10.1016/j.biotechadv.2014.08.007 CrossRefPubMedGoogle Scholar
  19. 19.
    Vaidya S, Ghosh K, Vundinti BR (2001) Recent developments in drug resistance mechanism in chronic myeloid leukemia: a review. Eur J Haematol 87:381–393CrossRefGoogle Scholar
  20. 20.
    Haimeur A, Conseil G, Deeley RG, Cole SP (2004) The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation. Curr Drug Metab 5:21–53CrossRefPubMedGoogle Scholar
  21. 21.
    Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2:48–58CrossRefPubMedGoogle Scholar
  22. 22.
    Wu CP, Hsieh CH, Wu YS (2011) The emergence of drug transporter-mediated multidrug resistance to cancer chemotherapy. Mol Pharm 8:1996–2011.  https://doi.org/10.1021/mp200261n CrossRefPubMedGoogle Scholar
  23. 23.
    Leonard GD, Fojo T, Bates SE (2003) The role of ABC transporters in clinical practice. Oncologist 8:411–424CrossRefPubMedGoogle Scholar
  24. 24.
    Fernandes J, Weinlich R, Castilho RO, Kaplan MAC, Amarante-Mendes GP, Gattass CR (2005) Pomolic acid triggers mitochondria-dependent apoptotic cell death in leukemia cell line. Cancer Lett 219:49–55CrossRefPubMedGoogle Scholar
  25. 25.
    Gottesman MM, Ling V (2006) The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research. FEBS Lett 580:998–1009CrossRefPubMedGoogle Scholar
  26. 26.
    Ferguson PJ, Brisson AR, Koropatnick J, Vincent MD (2009) Enhancement of cytotoxicity of natural product drugs against multidrug resistant variant cell lines of human head and neck squamous cell carcinoma and breast carcinoma by tesmilifene. Cancer Lett 274:279–289CrossRefPubMedGoogle Scholar
  27. 27.
    Fatemian T, Othman I, Chowdhury EH (2014) Strategies and validation for siRNA-based therapeutics for the reversal of multi-drug resistance in cancer. Drug Discov 19:71–78Google Scholar
  28. 28.
    Suo G, Han J, Wang X, Zhang J, Zhao Y, Dai J (2005) Oct4 pseudogenes are transcribed in cancers. Biochem Bioph Res Commun 337:1047–1051CrossRefGoogle Scholar
  29. 29.
    Pain D, Chirn G-W, Strassel C, Kemp DM (2005) Multiple retropseudogenes from pluripotent cell-specific gene expression indicates a potential signature for novel gene identification. J Biol Chem 280:6265–6268CrossRefPubMedGoogle Scholar
  30. 30.
    Vanin E (1985) Processed pseudogenes: characteristics and evolution. Ann Rev Genet 19:253–272CrossRefPubMedGoogle Scholar
  31. 31.
    Pink RC, Wicks K, Caley DP, Punch EK, Jacobs L, Carter DRF (2011) Pseudogenes: pseudo-functional or key regulators in health and disease. RNA 17:792–798CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Tutar Y (2012) Pseudogenes. Comp Funct Genom.  https://doi.org/10.1155/2012/424526 CrossRefGoogle Scholar
  33. 33.
    Muro EM, Mah N, Andrade-Navarro MA (2011) Functional evidence of post-transcriptional regulation by pseudogenes. Biochimie 93:1916–1921.  https://doi.org/10.1016/j.biochi.2011.07.024 CrossRefPubMedGoogle Scholar
  34. 34.
    Wang L, Guo Z-Y, Zhang R, Xin B, Chen R, Zhao J, Wang T, Wen W-H, Jia L-T, Yao L-B, Yang A-G (2013) Pseudogene OCT4-pg4 functions as a natural micro RNA sponge to regulate OCT4 expression by competing for miR-145 in hepatocellular carcinoma. Carcinogenesis 34(8):1773–1781.  https://doi.org/10.1093/carcin/bgt139 CrossRefPubMedGoogle Scholar
  35. 35.
    Daflon-Yunes N, Pinto-Silva FE, Vidal RS, Novis BF, Berguetti T, Lopes RRS, Polycarpo C, Rumjanek VM (2013) Characterization of a multidrug-resistant chronic myeloid leukemia cell line presenting multiple resistance mechanisms. Mol Cell Biochem 383:123–135CrossRefPubMedGoogle Scholar
  36. 36.
    Delgado-Cañedo A, Dos Santos DG, Chies JAB, Kvitko K, Nardi NB (2006) Optimization of na electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses. Cytotechnology 51:141–148.  https://doi.org/10.1007/s10616-006-9028-1 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–11CrossRefGoogle Scholar
  38. 38.
    Snel B, Lehmann G, Bork P, Huynen MA (2000) STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res 28(18):3442–3444CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, Santos A, Doncheva NT, Roth A, Bork P, Jensen LJ, von Mering C (2017) The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 45:D:362–368CrossRefGoogle Scholar
  40. 40.
    Visvader JE, Lindeman GJ (2012) Cancer stem cells: current status and evolving complexities. Cell Stem Cell 10:717–728CrossRefPubMedGoogle Scholar
  41. 41.
    Giuffrida D, Rogers IM, Nagy A, Calogero AE, Brown TJ, Casper RF (2009) Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth. Cell Prolif 42(6):788–798.  https://doi.org/10.1111/j.1365-2184.2009.00640.x PMID: 19732065CrossRefPubMedGoogle Scholar
  42. 42.
    Zhou S, Abdouh M, Arena V, Arena M, Arena GO (2017) Reprogramming malignant cancer cells by human embryonic stem cell. PLoS ONE 9(1):e0169899.  https://doi.org/10.1371/journal.pone.0169899 CrossRefGoogle Scholar
  43. 43.
    Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, Weinberg RA (2008) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40:499–507CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Hayashi H, Arao T, Togashi Y, Kato H, Fujita Y, De Velasco MA, Kimura H, Matsumoto K, Tanaka K, Okamoto I, Ito A, Yamada Y, Nakagawa K, Nichio K (2013) The OCT4 pseudogene POU5F1B is amplified and promotes an aggressive phenotype in gastric cancer. Oncogene 34:199–208CrossRefPubMedGoogle Scholar
  45. 45.
    Tang Y-A, Chen C-H, Sun SH, Cheng C-P, Tseng VS, Hsu H-S, Su W-C, Lai W-W, Wang Y-C (2015) Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer. Nucleic Acids Res 43(3):1593–1608.  https://doi.org/10.1093/nar/gkv024 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Kobayashi I, Takahashi F, Nurwidya F, Nara T, Hashimoto M, Murakami A, Yagishita S, Tajima K, Hidayat M, Shimada N, Suina K, Yoshioka Y, Sasaki S, Moriyama M, Moriyama H, Takahashi K (2016) Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells. Biochem Biophys Res Commun 473:125–132CrossRefPubMedGoogle Scholar
  47. 47.
    Onichtchouk D (2016) Evolution and functions of Oct4 homologs in non-mammalian vertebrates. Biochim Biophys Acta 1859:770–779.  https://doi.org/10.1016/j.bbagrm.2016.03.013 CrossRefPubMedGoogle Scholar
  48. 48.
    Liedtke S, Enczmann J, Waclawczyk S, Wernet P, Ko¨gler G (2007) Oct4 and its pseudogenes confuse stem cell research. Cell Stem Cell 1:364–366.  https://doi.org/10.1016/j.stem.2007.09.003 CrossRefPubMedGoogle Scholar
  49. 49.
    Jez M, Ambady S, Kashpur O, Grella A, Malcuit C, Vilner L, Rozman P, Dominko T (2014) Expression and differentiation between OCT4A and its Pseudogenes in human ESCs and differentiated adult somatic cells. PLoS ONE 9(7):e104296.  https://doi.org/10.1371/journal.pone.0089546 CrossRefGoogle Scholar
  50. 50.
    Scarola M, Comisso E, Pascolo R, Chiaradia R, Marion RM, Schneider C, Blasco MA, Schoeftner S, Benetti R (2015) Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA. Nat Commun 6:7631.  https://doi.org/10.1038/ncomms8631 CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Carrett-Dias M, Almeida LK, Pereira JL, Almeida DV, Filgueira DMVB, Marins LF, Votto APdeS, Trindade GS (2016) Cell differentiation and the multiple drug resistance phenotype in human erythroleukemic cells. Leuk Res 42:13–20.  https://doi.org/10.1016/j.leukres.2016.01.2018 CrossRefPubMedGoogle Scholar
  52. 52.
    Wang XK, He JH, Xu JH, Ye S, Wang F, Zhang H, Huang ZC, To KK, Fu LW (2014) Afatinib enhances the efficacy of conventional chemotherapeutic agents by eradicating cancer stem-like cells. Cancer Res 74(16):4431–4445.  https://doi.org/10.1158/0008-5472.CAN-13-3553 CrossRefPubMedGoogle Scholar
  53. 53.
    Chen Y, Hu Y, Zhang H, Peng C, Li S (2009) Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nat Genet 41:783–792CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Loe DW, Deeley RG, Cole SP (1998) Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res 58:5130–5136PubMedGoogle Scholar
  55. 55.
    Bergman PJ (2003) Mechanisms of anticancer drug resistance. Vet Clin Small Anim 33(3):651–667CrossRefGoogle Scholar
  56. 56.
    Lee CH (2010) Reversing agents for ATP-binding cassette drug transporters. Methods Mol Bio 596:325–340.  https://doi.org/10.1007/978-1-60761-416-6_14 CrossRefGoogle Scholar
  57. 57.
    Li Y, Atkinson K, Zhang T (2017) Combination of chemotherapy and cancer stem cell targeting agents: Preclinical and clinical studies. Cancer Lett 396:103–109.  https://doi.org/10.1016/j.canlet.2017.03.008 CrossRefPubMedGoogle Scholar
  58. 58.
    Fletcher JI, Williams RT, Henderson MJ, Norris MD, Haber M (2016) ABC transporters as mediators of drug resistance and contributors tocancer cell biology. Drug Resist Upat 26:1–9.  https://doi.org/10.1016/j.drup.2016.03.001 CrossRefGoogle Scholar
  59. 59.
    De Groot DJA, Van der Deen M, Le TKP, Regeling A, De Jong S, De Vries EGE (2007) Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1. Br J Cancer 97:1077–1083CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Nicholls FA, Ahokas JT (1984) Inhibition of purified glutathione Stransferases by indomethacin. Biochem Biophys Res Commun 119:1034–1038CrossRefPubMedGoogle Scholar
  61. 61.
    Takeuchi K, Tanaka A, Kato S, Amagase K, Satoh H (2010) Roles of COX inhibition in pathogenesis of NSAID-induced small intestinal damage. Clin Chim Acta 411:459–466CrossRefPubMedGoogle Scholar
  62. 62.
    Guo Y-C, Chang C-M, Hsu W-L, Chiu S-J, Tsai Y-T, Chou Y-H, Hou M-F, Wang J-Y, Lee M-H, Tsai K-L, Chang W-C (2013) Indomethacin inhibits cancer cell migration via attenuation of cellular calcium mobilization. Molecules 18(6):6584–6596.  https://doi.org/10.3390/molecules18066584 CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Moreira MAM, Bagni C, de Pinho MB, Mac-Cormick TM, dos Santos Mota M, Pinto-Silva FE, Daflon-Yunes N, Rumjanek VM (2014) Changes in gene expression profile in two multidrug resistant cell lines derived from a same drug sensitive cell line. Leuk Res 38(8):983–987.  https://doi.org/10.1016/j.leukres.2014.06.001 CrossRefPubMedGoogle Scholar
  64. 64.
    Chambers I, Tomlinson SR (2009) The transcriptional foundation of pluripotency. Development 136:2311–2322CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Aline Portantiolo Lettnin
    • 1
    • 2
  • Eduardo Felipe Wagner
    • 2
  • Michele Carrett-Dias
    • 1
  • Karina dos Santos Machado
    • 3
  • Adriano Werhli
    • 3
  • Andrés Delgado Cañedo
    • 4
  • Gilma Santos Trindade
    • 1
  • Ana Paula de Souza Votto
    • 1
    • 2
    • 5
    Email author
  1. 1.Post-Graduate Program in Physiological Sciences - PPGCFFederal University of Rio Grande -FURGRio GrandeBrazil
  2. 2.Laboratory of Cell Culture, Institute of Biological Sciences - ICBFederal University of Rio Grande -FURGRio GrandeBrazil
  3. 3.Center of Computational Sciences - C3Federal University of Rio Grande -FURGRio GrandeBrazil
  4. 4.Federal University of Pampa - UNIPAMPASão GabrielBrazil
  5. 5.Instituto de Ciências BiológicasUniversidade Federal do Rio Grande – FURGRio GrandeBrazil

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