Document Type : Original Article(s)

Authors

1 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

2 Department of Medical Laboratory Sciences, Faculty of Paramedicine, Tabriz University of Medical Sciences, Tabriz, Iran

3 Department of Biology, Urmia Branch, Islamic Azad University, Urmia, Iran

4 Department of Microbiology, Faculty of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran

5 Department of Biology, Ahar Branch, Islamic Azad University, Ahar, Iran

6 Department of Biology, Tabriz Branch, Islamic Azad University, Tabriz, Iran

Abstract

Background: This study investigates the relative expression of the Na+, HCO3- cotransport gene NBCn1, and caspase-3 within the tumor microenvironment of human breast cancer, considering the in vivo microenvironment.
Method: In this experimental study, breast cancer MDA-MB-231 cells were cultured under normoxia/hypoxia conditions for 24, 48, and 72 hours with varying glucose concentrations (5.5, 11, and 25 mM). The mRNA expression of NBCn1 and caspase-3 was evaluated using real-time polymerase chain reaction. The stability and binding pocket of NBCn1 were assessed using DispHred and the Computed Atlas of Surface Topography of proteins (CASTp) servers, respectively. The location prediction of the protein was determined using the Transmembrane Helices; Hidden Markov Model (TMHMM) server.
Results: Normoxia led to an increase in NBCn1 expression during all three time periods, displaying heterogeneity. The expression was particularly elevated at glucose concentrations of 25 and 5.5 mM. In hypoxic conditions, gene expression was reduced; however, an increase in glucose concentration enhanced SLC4A7 expression. Specifically, a glucose concentration of 25 mM led to decreased caspase-3 expression under hypoxic conditions. In silico studies revealed that SLC4A7 becomes disordered when the pH falls below 7, with most amino acids in the binding pocket being nonpolar.
Conclusion: The heightened risk of breast cancer metastasis may be linked to the upregulation of SLC4A7 and downregulation of caspase-3 expression, underscoring their fundamental roles in cancer treatment and prevention. SLC4A7 is a transmembrane protein, and its folding is pH-dependent.

Highlights

Rasoul Sharifi (Google Scholar)

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How to cite this article:

Mehra A, Mehrkar R, Fakhri A, Jabbari Fard P, Asgharzadeh MR, Ghaffari Agdam MH, et al. A mimicry of the tumor microenvironment's impact on SLC4A7 (NBCn1) and caspase-3 gene expression in breast cancer, along with in silico traits of NBCn1. Middle East J Cancer. 2024;15(3):176-88. doi:10. 30476/mejc.2023.99391.1942.

  1. Harbeck N, Gnant M. Breast cancer. Lancet. 2017;389(10074):1134-50. doi: 10.1016/S0140-6736(16)31891-8.
  2. Payan N, Presles B, Brunotte F, Coutant C, Desmoulins I, Vrigneaud JM, et al. Biological correlates of tumor perfusion and its heterogeneity in newly diagnosed breast cancer using dynamic first-pass 18F-FDG PET/CT. Eur J Nucl Med Mol Imaging. 2020;47(5):1103-15. doi: 10.1007/s00259-019-04422-4.
  3. Xu X, Sun X, Ma L, Zhang H, Ji W, Xia X, et al. 18F-FDG PET/CT radiomics signature and clinical parameters predict progression-free survival in breast cancer patients: A preliminary study. Front Oncol. 2023;13:1149791. doi: 10.3389/fonc.2023.1149791.
  4. Incio J, Suboj P, Chin SM, Vardam-Kaur T, Liu H, Hato T, et al. Metformin reduces desmoplasia in pancreatic cancer by reprogramming stellate cells and tumor-associated macrophages. PLoS One. 2015;10(12):e0141392.
  5. Zhang C, Fu L, Fu J, Hu L, Yang H, Rong TH, et al. Fibroblast growth factor receptor 2-positive fibroblasts provide a suitable microenvironment for tumor development and progression in esophageal carcinoma. Clin Cancer Res. 2009;15(12):4017-27. doi:10.1158/1078-0432.CCR-08-2824.
  6. Liberti MV, Locasale JW. Correction to: 'The Warburg effect: How does it benefit cancer cells?': [Trends in Biochemical Sciences, 41 (2016) 211]. Trends Biochem Sci. 2016;41(3):287. doi: 10.1016/j.tibs.2016.01.004. Erratum for: Trends Biochem Sci. 2016;41(3):211-8.
  7. Ivashkiv LB. The hypoxia-lactate axis tempers inflammation. Nat Rev Immunol. 2020;20(2):85-6. doi: 10.1038/s41577-019-0259-8.
  8. Corbet C, Feron O. Tumour acidosis: from the passenger to the driver's seat. Nat Rev Cancer. 2017;17(10):577-93. doi: 10.1038/nrc.2017.77.
  9. de la Cruz-López KG, Castro-Muñoz LJ, Reyes-Hernández DO, García-Carrancá A, Manzo-Merino J. Lactate in the regulation of tumor microenvironment and therapeutic approaches. Front Oncol. 2019;9:1143.
  10. Lauritzen G, Stock CM, Lemaire J, Lund SF, Jensen MF, Damsgaard B, et al. The Na+/H+ exchanger NHE1, but not the Na+, HCO3(-) cotransporter NBCn1, regulates motility of MCF7 breast cancer cells expressing constitutively active ErbB2. Cancer Lett. 2012;317(2):172-83.
  11. Hulikova A, Vaughan-Jones RD, Swietach P. Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths. J Biol Chem. 2011;286(16):13815-26. doi: 10.1074/jbc.M111.219899.
  12. Boedtkjer E, Praetorius J, Matchkov VV, Stankevicius E, Mogensen S, Füchtbauer AC, et al. Disruption of Na+,HCO₃⁻ cotransporter NBCn1 (slc4a7) inhibits NO-mediated vasorelaxation, smooth muscle Ca²⁺ sensitivity, and hypertension development in mice. Circulation. 2011;124(17):1819-29. doi: 10.1161/CIRCULATIONAHA.110.015974.
  13. Aghababazadeh M, Dorraki N, Javan FA, Fattahi AS, Gharib M, Pasdar A. Downregulation of Caspase 8 in a group of Iranian breast cancer patients - A pilot study. J Egypt Natl Canc Inst. 2017;29(4):191-5. doi: 10.1016/j.jnci.2017.10.001.
  14. Fonin AV, Stepanenko OV, Sitdikova AK, Antifeeva IA, Kostyleva EI, Polyanichko AM, et al. Folding of poly-amino acids and intrinsically disordered proteins in overcrowded milieu induced by pH change. Int J Biol Macromol. 2019;125:244-55. doi: 10.1016/j.ijbiomac.2018.12.038.
  15. Santos J, Iglesias V, Pintado C, Santos-Suárez J, Ventura S. DispHred: A server to predict pH-dependent order-disorder transitions in intrinsically disordered proteins. Int J Mol Sci. 2020;21(16):5814. doi: 10.3390/ijms21165814.
  16. McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 2013;5(4):a008656. doi: 10.1101/cshperspect.a008656. Erratum in: Cold Spring Harb Perspect Biol. 2015;7(4). pii: a026716. doi: 10.1101/cshperspect.a026716.
  17. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19(9):1639-45. doi: 10.1101/gr.092759.109.
  18. Santos J, Iglesias V, Pintado C, Santos-Suárez J, Ventura S. DispHred: A server to predict pH-dependent order-disorder transitions in intrinsically disordered proteins. Int J Mol Sci. 2020;21(16):5814. doi: 10.3390/ijms21165814.
  19. Zamora WJ, Campanera JM, Luque FJ. Development of a structure-based, pH-dependent lipophilicity scale of amino acids from continuum solvation calculations. J Phys Chem Lett. 2019;10(4):883-9. doi: 10.1021/acs.jpclett.9b00028.
  20. Iglesias V, Pintado-Grima C, Santos J, Fornt M, Ventura S. Prediction of the effect of pH on the aggregation and conditional folding of intrinsically disordered proteins with SolupHred and DispHred. Methods Mol Biol. 2022;2449:197-211. doi: 10.1007/978-1-0716-2095-3_8.
  21. Tian W, Chen C, Lei X, Zhao J, Liang J. CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res. 2018;46(W1):W363-W367. doi: 10.1093/nar/gky473.
  22. Rasoulpoor S, Asgharzadeh MR, Shabani S. A mimic of the tumor microenvironment on GPR30 gene expression in breast cancer. Multidiscip Cancer Invest. 2022;6(2):1-8.
  23. Zhang Y, Peng Q, Zheng J, Yang Y, Zhang X, Ma A, et al. The function and mechanism of lactate and lactylation in tumor metabolism and microenvironment. Genes Dis. 2022;10(5):2029-37. doi: 10.1016/j.gendis.2022.10.006.
  24. Lauritzen G, Jensen MB, Boedtkjer E, Dybboe R, Aalkjaer C, Nylandsted J, et al. NBCn1 and NHE1 expression and activity in DeltaNErbB2 receptor-expressing MCF-7 breast cancer cells: contributions to pHi regulation and chemotherapy resistance. Exp Cell Res. 2010;316(15):2538-53.
  25. Toft NJ, Axelsen TV, Pedersen HL, Mele M, Burton M, Balling E, et al. Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival. Elife. 2021;10:e68447.
  26. Lee S, Toft NJ, Axelsen TV, Espejo MS, Pedersen TM, Mele M, et al. Carbonic anhydrases reduce the acidity of the tumor microenvironment, promote immune infiltration, decelerate tumor growth, and improve survival in ErbB2/HER2-enriched breast cancer. Breast Cancer Res. 2023;25(1):46. doi: 10.1186/s13058-023-01644-1.
  27. Wong P, Kleemann HW, Tannock IF. Cytostatic potential of novel agents that inhibit the regulation of intracellular pH. Br J Cancer. 2002;87(2):238-45. doi: 10.1038/sj.bjc.6600424.
  28. McDonald PC, Chafe SC, Supuran CT, Dedhar S. Cancer therapeutic targeting of hypoxia induced carbonic anhydrase IX: from bench to bedside. Cancers (Basel). 2022;14(14):3297. doi: 10.3390/cancers14143297.
  29. Cardone RA, Casavola V, Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer. 2005;5(10):786-95.
  30. Boedtkjer E, Moreira JM, Mele M, Vahl P, Wielenga VT, Christiansen PM, et al. Contribution of Na+,HCO3(-)-cotransport to cellular pH control in human breast cancer: a role for the breast cancer susceptibility locus NBCn1 (SLC4A7). Int J Cancer. 2013;132(6):1288-99. doi: 10.1002/ijc.27782.
  31. Chen Y, Choong LY, Lin Q, Philp R, Wong CH, Ang BK, et al. Differential expression of novel tyrosine kinase substrates during breast cancer development. Mol Cell Proteomics. 2007;6(12):2072-87. doi: 10.1074/mcp.M700395-MCP200.
  32. Hagemann T, Wilson J, Burke F, Kulbe H, Li NF, Plüddemann A, et al. Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006;176(8):5023-32.
  33. Mandal R, Raab M, Rödel F, Krämer A, Kostova I, Peña-Llopis S, et al. The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer. Cell Mol Life Sci. 2022;79(12):597. doi: 10.1007/s00018-022-04598-3. Erratum in: Cell Mol Life Sci. 2023;80(3):64.
  34. Hernandez L, Kim MK, Noonan AM, Sagher E, Kohlhammer H, Wright G, et al. A dual role for Caspase8 and NF-κB interactions in regulating apoptosis and necroptosis of ovarian cancer, with correlation to patient survival. Cell Death Discov. 2015;1:15053. doi: 10.1038/cddiscovery.2015.53.
  35. Nowak M, Klink M. The role of tumor-associated macrophages in the progression and chemoresistance of ovarian cancer. Cells. 2020;9(5):1299. doi: 10.3390/cells9051299.
  36. Maelfait J, Beyaert R. Non-apoptotic functions of caspase-8. Biochem Pharmacol. 2008;76(11):1365-73. doi: 10.1016/j.bcp.2008.07.034.
  37. Afzaljavan F, Vahednia E, Barati Bagherabad M, Vakili F, Moezzi A, Hosseini A, et al. Genetic contribution of caspase-8 variants and haplotypes to breast cancer risk and prognosis: a case-control study in Iran. BMC Med Genomics. 2023;16(1):72. doi: 10.1186/s12920-023-01484-0.
  38. Moelbert S, Emberly E, Tang C. Correlation between sequence hydrophobicity and surface-exposure pattern of database proteins. Protein Sci. 2004;13(3):752-62.
  39. Rapaport DC. Configurational properties of polymers in a good solvent. J Phys A Math Gen. 1976;9(9):1521-37. doi:10.1088/0305-4470/9/9/013.