Ukr.Biochem.J. 2024; Volume 96, Issue 6, Nov-Dec, pp. 29-35

doi: https://doi.org/10.15407/ubj96.06.029

Generation of the MCF-7 cell sublines with CRISPR/Cas9 mediated disruption of estrogen receptor alfa (ESR1) expression

04.12.2024   Uncategorized   No comments

L. O. Savinska1, S. A. Kvitchenko1,2, S. S. Palchevskyi1,
I. V. Kroupskaya1, A. V. Mazov1, O. M. Garifulin1, V. V. Filonenko1*

1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
2ESC “Institute of Biology and Medicine”, Taras Shevchenko National Univercity of Kyiv, Ukraine;
*e-mail: filonenko@imbg.org.ua

Received: 28 October 2024; Revised: 05 November 2024;
Accepted: 21 November 2024; Available on-line: 17 December 2024

Supported by the literature, our initial hypothesis was that Estrogen Receptor alfa (ESR1) may function as a master regulator by influencing the expression of epithelial-to-mesenchymal transition (EMT)-related genes in cancer cells. To explore this further, we used the CRISPR/Cas9 gene editing system to create MCF-7 sublines with down-regulated ESR1 expression and analyzed its impact on EMT initiation. By applying two distinct types of gRNA for gene editing, we established six MCF-7 cell sublines with either nearly complete or partial down-regulation of the ESR1 isoforms. Unexpectedly, the data obtained revealed no discernible impact of ESR1 down-regulation on EMT manifestation as Western blot and Real-Time qPCR analysis of selected clones revealed no changes in EMT markers expression. We suggested that those of the ESR1 isoforms, the expression of which was not affected by gene editing, could be crucial for the initiation of EMT. The obtained cell models will be used further to evaluate the activity of ESR1 isoforms.

Keywords: , , ,

References:

  1. Saxton RA, Sabatini DM. mTOR Signaling in growth, metabolism, and disease. Cell. 2017;169(2):361-371. PubMed, CrossRef
  2. Filonenko VV, Tytarenko R, Azatjan SK, Savinska LO, Gaydar YuA, Gout I, Usenko VS, Lyzogubov VV. Immunohistochemical analysis of S6K1 and S6K2 localization in human breast tumors. Exp Oncol. 2004;26(4):294-299.
  3. Lyzogubov V, Khozhaenko Y, Usenko V, Antonjuk S, Ovcharenko G, Tykhonkova I, Filonenko V. Immunohistochemical analysis of Ki-67, PCNA and S6K1/2 expression in human breast cancer. Exp Oncol. 2005;27(2):141-144.
  4. van der Hage JA, van den Broek LJ, Legrand C, Clahsen PC, Bosch CJ, Robanus-Maandag EC, van de Velde CJ, van de Vijver MJ. Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in node-negative premenopausal early breast cancer patients. Br J Cancer. 2004;90(8):1543-1550. PubMed, PubMedCentral, CrossRef
  5. Chen B, Yang L, Zhang R, Gan Y, Zhang W, Liu D, Chen H, Tang H. Hyperphosphorylation of RPS6KB1, rather than overexpression, predicts worse prognosis in non-small cell lung cancer patients. PLoS One. 2017 Aug 9;12(8):e0182891. PubMed, PubMedCentral, CrossRef
  6. Lyzogubov VV, Usenko VS, Khojaenko YuS, Lytvyn DI, Soldatkina MA , Rodnin NV, Filonenko V, Pogribniy PV. Immunohistochemical analysis of p70S6 kinase α in human thyroid tissue upon pathology. Eksperim Onkol. 2003;25(4):304-306.
  7. Ismail HM. Overexpression of s6 kinase 1 in brain tumours is associated with induction of hypoxia-responsive genes and predicts patients’ survival. J Oncol. 2012;2012:416927. PubMed, PubMedCentral, CrossRef
  8. Fan W, Wang W, Mao X, Chu S, Feng J, Xiao D, Zhou J, Fan S. Elevated levels of p-Mnk1, p-eIF4E and p-p70S6K proteins are associated with tumor recurrence and poor prognosis in astrocytomas. J Neurooncol. 2017;131(3):485-493. PubMed, CrossRef
  9. Li SH, Chen CH, Lu HI, Huang WT, Tien WY, Lan YC, Lee CC, Chen YH, Huang HY, Chang AY, Lin WC. Phosphorylated p70S6K expression is an independent prognosticator for patients with esophageal squamous cell carcinoma. Surgery. 2015;157(3):570-580. PubMed, CrossRef
  10. Sridhar J, Komati R, Kumar S. Targeting RPS6K1 for Refractory Breast Cancer Therapy. In: Mayrovitz HN, editor. Breast Cancer. Brisbane (AU): Exon Publications. Online first 21 Feb 2022. CrossRef
  11. Grove JR, Banerjee P, Balasubramanyam A, Coffer PJ, Price DJ, Avruch J, Woodgett JR. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. Mol Cell Biol. 1991;11(11):5541-5550. PubMed, PubMedCentral, CrossRef
  12. Tavares MR, Pavan IC, Amaral CL, Meneguello L, Luchessi AD, Simabuco FM. The S6K protein family in health and disease. Life Sci. 2015;131:1-10. PubMed, CrossRef
  13. Zaiets IV, Sivchenko AS, Khoruzhenko AI, Savinska LO, Filonenko VV. The p60-S6K1 isoform of ribosomal protein S6 kinase 1 is a product of alternative mRNA translation. Ukr Biochem J. 2018;90(4):25-35. CrossRef
  14. Zaiets IV, Holiar VV, Sivchenko AS, Smialkova VV, Filonenko VV. p60-S6K1 represents a novel kinase active isoform with the mode of regulation distinct from p70/p85-S6K1 isoforms. Ukr Biochem J. 2019;91(4):17-25. CrossRef
  15. Garifulin OM, Zaiets IV, Kosach VR, Horak IR, Khoruzhenko AI, Gotsulyak Nya, Savinska LO, Kroupskaya IV, Martsynyuk MYe, Drobot LB, Filonenko VV. Alterations in S6K1 isoforms expression induce Epithelial to Mesenchymal Transition and Estrogen Receptor 1 Silencing in human breast adenocarcinoma MCF-7 cells. Biopolym Cell. 2023;39(3):189-200. CrossRef
  16. Kosach VR, Hotsuliak NYa, Zaiets IV, Skorokhod OM, Savinska LO, Khoruzhenko AI, Filonenko VV. Alterations in expression of S6K1 isoforms in MCF7 cells have a strong impact on the locomotor activity as well as on S6K1 and Akt signaling. Biopolym Cell. 2020;36(2):110-121. CrossRef
  17. Gibson DA, Saunders PT. Estrogen dependent signaling in reproductive tissues – a role for estrogen receptors and estrogen related receptors. Mol Cell Endocrinol. 2012;348(2):361-372. PubMed, CrossRef
  18. Faltas CL, LeBron KA, Holz MK. Unconventional Estrogen Signaling in Health and Disease. Endocrinology. 2020;161(4):bqaa030. PubMed, PubMedCentral, CrossRef
  19. Maruani DM, Spiegel TN, Harris EN, Shachter AS, Unger HA, Herrero-González S, Holz MK. Estrogenic regulation of S6K1 expression creates a positive regulatory loop in control of breast cancer cell proliferation. Oncogene. 2012;31(49):5073-5080. PubMed, PubMedCentral, CrossRef
  20. 20. Yamnik RL, Digilova A, Davis DC, Brodt ZN, Murphy CJ, Holz MK. S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation. J Biol Chem. 2009;284(10):6361-6369. PubMed, CrossRef
  21. Pon YL, Zhou HY, Cheung AN, Ngan HY, Wong AS. p70 S6 kinase promotes epithelial to mesenchymal transition through snail induction in ovarian cancer cells. Cancer Res. 2008;68(16):6524-6532. PubMed, CrossRef
  22. Ip CK, Cheung AN, Ngan HY, Wong AS. p70 S6 kinase in the control of actin cytoskeleton dynamics and directed migration of ovarian cancer cells. Oncogene. 2011;30(21):2420-2432. PubMed, CrossRef
  23. Artemenko M, Zhong SSW, To SKY, Wong AST. p70 S6 kinase as a therapeutic target in cancers: More than just an mTOR effector. Cancer Lett. 2022;535:215593. PubMed, CrossRef
  24. Al Saleh S, Al Mulla F, Luqmani YA. Estrogen receptor silencing induces epithelial to mesenchymal transition in human breast cancer cells. PLoS One. 2011;6(6):e20610. PubMed, PubMedCentral, CrossRef
  25. Bouris P, Skandalis SS, Piperigkou Z, Afratis N, Karamanou K, Aletras AJ, Moustakas A, Theocharis AD, Karamanos NK. Estrogen receptor alpha mediates epithelial to mesenchymal transition, expression of specific matrix effectors and functional properties of breast cancer cells. Matrix Biol. 2015;43:42-60. PubMed, CrossRef
Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.