ISSN: 2581-5407
Global Journal of Cancer Therapy
Review Article       Open Access      Peer-Reviewed

A prospective for the role of two-pore channels in breast cancer cells

Hengrui Liu*

Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK; Department of Anesthesiology, Health Science Center, Stony Brook University, Stony Brook, NY 11794, USA
*Corresponding author: Hengrui Liu, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK, Department of Anesthesiology, Health Science Center, Stony Brook University, Stony Brook, NY 11794, USA, E-mail: hengrui.liu1@stonybrookmedicine.edu
Received: 02 March, 2020 | Accepted: 28 March, 2020 | Published: 30 March, 2020

Cite this as

Liu H (2020) A prospective for the role of two-pore channels in breast cancer cells. Glob J Cancer Ther 6(1): 001-003. DOI: 10.17352/2581-5407.000026

Breast cancer is one of the most common cancers and the second most frequent cause of cancer death among women worldwide. Recently, potential links between cancer and the calcium mobilizing messenger Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and its intracellular target Two-Pore Channels (TPCs) have been identified. However, their role in breast cancer cells has not been extensively evaluated, and the understanding of underlying pathways is still lacking. Here we proposed a hypothesis that TPC function is crucial for the cell biology and tumorigenesis of the breast cancer cell. Further study can be done in this field, such as screening the expression of TPC1 and TPC2 in different breast cancers, then testing whether altered expression of TPC1 and TPC2 affects breast cancer and breast cancer stem cells. It is also valuable to search for the potential targets of TPCs and NAADP calcium signaling in breast cancer cells. This mini-review aims to propose potential research methods and experimental design for future study and will contribute to the raising and development of the research in this field.

Background

Breast cancer is one of the most common cancers and the second most frequent cause of cancer death among women worldwide [1]. In 2019, more than 10% of all types of cancers are breast cancer, resulting in more than 6% of death in the world [2]. The incidence of breast cancer is predicted to increase by 50% within 25 years [3]. Thus, the study in breast cancer is urging and of utmost importance.

Ion channels are thought to be associated with breast cancer [4]. Recently, potential links between Two-Pore Channels (TPCs) and cancer have been identified. TPCs are eukaryotic intracellular voltage-gated and ligand-gated cation-selective ion channels with one important modulator of such channels being NAADP. There are two known paralogs in the human genome, including TPC1 and TPC2. In humans, TPC1 is sodium selective and TPC2 conducts sodium ions, calcium ions and possibly hydrogen ions [5]. However, their role in breast cancer cells has not been extensively evaluated, and the understanding of underlying pathways is still lacking.

Ong Nam Phuong Nguyen, et al. [6], found that TPC gene knockout or TPC pharmacological inhibitors can decrease the metastasis of 4T1 mouse breast cancer cell lines in an in vivo study, but a whole animal study assessing human cancer cell lines is also needed. Wei Sun, et al. [7], also found that TPCs are overexpressed in 4T1 cells and play a role in cancer cell migration. The expression of TPC1 and TPC2 in some human breast cancer cell lines has been determined by Aisyah H Jahidin, et al. [8] But they did not link the difference of expression level to the subtypes of breast cancer cells. They also found that TPC1/TPC2 knocking out did not affect the cell proliferation in MDA-MB-468 breast cancer cell line, but they failed to reveal the effects of TPCs on other breast cancer cell lines. So far, the role of TPCs in breast cancer cell biology has not been systematically evaluated, hence the study of the role of TPCs in proliferation and migration in breast cancer cells and the exploration of the mechanisms underlying these effects is needed.

Currently, cancer cells are thought to originate from breast Cancer Stem Cells (CSCs) or “stem-like cells”. A recent study showed that CSCs are essential for metastatic tumor growth [9,10]. As the key components of NAADP-regulated calcium channels, TPCs contribute to changes in intracellular Ca2+ which regulate a diverse range of cellular processes including proliferation, differentiation, metabolism, gene transcription and apoptosis [11,12] and TPCs are shown to play a crucial role in cancer cell migration [13-15], which implies that there might be a link between CSCs and TPCs. My hypothesis is that TPC function is crucial for cell biology and tumorigenesis in breast cancer cells.

Potential available methods and strategies

To explore the role of TPC1 and TPC2 in cancer cell biology and tumorigenesis in breast cancer. Here this review lists several prospective study designs.

(1). Although TPCs are expressed in breast cancer cells, their expression profile and function in subtypes of breast cancer cells [16] has not been extensively evaluated. Therefore, we can screen the expression of TPC1 and TPC2 in different subtypes of breast cancers and determine their function in the calcium signaling pathway. We expect to find greater expression of TPCs and altered TPC function in certain types of breast cancer cell lines.

RT-qPCR and Western blot methods will be used to determine the mRNA and protein expression of TPC1 and TPC2 in different subtypes of breast cancer cell lines, MCF-7 (Luminal A), BT474 (Luminal B), MDA-MB-468 (basal-like), MDA-MB-231 (claudin-low), MDA-MB-453 (HER2-positive), etc. Whole-endolysosomal patch-clamp recordings can be used to test the function of the TPCs in breast cancer cell lines and tetrandrine will be used as a negative control. Intracellular NAADP induced calcium signals can be assessed by calcium imaging.

Breast cancer tissues with complete clinical-pathological data (including cancer subtype) will be collected from patients and made into paraffin-embedded tissue microarrays. mRNA and protein expression of TPC1 and TPC2 in patients’ tumors will be determined by in situ RNA hybridization and immunohistochemistry methods.

(2). TPCs play a role in cancer cells, but their role in breast cancer cell biology has not been systematically evaluated, so I will test whether the expression of TPC1 and TPC2 affects breast cancer cells. I expect to find the knocking out of TPCs can decrease breast cancer cell migration and metastasis.

Cell viability, migration, invasion, and adhesion viability in Tpcn1/2+/+, Tpcn1/2−/−, Tpcn1−/−, and Tpcn2−/− breast cancer cell lines will be determined using MTT, Cell Cytotoxicity Assay Kit, apoptosis ELISA Kit [17], Flow Cytometry, wound healing, transwell, matrices assay, colony-forming assay, trypsin detaching assay, etc. Intracellular NAADP induced calcium signals will be assessed by calcium imaging. The breast cancer subtypes that have more TPC expression will be compared with those that have less expression.

NSG mice and Tpcn1/2+/+, Tpcn1/2−/−, Tpcn1−/−, and Tpcn2−/− luciferase MDA-MB-231 (human breast cancer cell lines) will be used in this study [18]. Cells will be injected into the breast pads. Bioluminescence signals will be measured by luciferase imaging. The primary tumor will be excised and collected after four weeks and the primary recurrent and metastasis will be evaluated.

(3). As Cancer Stem Cells (CSCs) have been shown to be essential for metastatic tumor growth and TPCs is shown to play a crucial role in cancer cell migration, I expect to find that knocking out of TPCs will decrease the population of CSCs and that the CSCs have higher levels of TPC expression.

Self-renewal ability of Tpcn1/2+/+, Tpcn1/2−/−, Tpcn1−/−, and Tpcn2−/− breast cancer cell lines will be determined using serial replating assays [19]. The level of BCSCs will be determined by CSC marker [20,21], (CD44+ CD24−/low and ALDH+) stain followed by flow cytometry and immunohistochemistry methods. The number of breast CSCs and cancer cells in tumor and metastatic tissue in animal models will be determined by cell marker staining using the immunohistochemistry method.

Immuno-magnetic cell sorting of stem cell subpopulation will be used [22]. Breast cancer cell lines will be exposed to FITC-conjugated anti-CD44, anti-ALDH, and anti-LGR5 antibody, and further labeled with dextran-coated magnetic nanoparticles using bispecific tetrameric antibody complexes (TAC). These cells will be subjected to immuno-magnetic cell separation and CD44+, ALDH+, and LGR5+ cells will be identified as CSCs. Sphere culture [23,24] , will also be used as another method to extract cancer stem-like cells. Cells will be cultured in ultralow attachment plates and the sphere passage cells will be identified as CSCs. The expression of TPCs in CSCs will be tested.

(4). To have a better understanding of TPCs in breast cancer biology, the precise mechanisms of TPCs action, and their links with NAADP calcium signaling, I will search for potential targets of TPCs and NAADP calcium signaling in breast cancer cells. I expect to find some potential targets that can explain the effect of TPCs on breast cancer cells.

Total protein and total RNA of Tpcn1/2+/+, Tpcn1/2−/−, Tpcn1−/−, and Tpcn2−/− breast cancer cells will be extracted, and proteomic analysis and gene array methods will be used to screen altered protein and mRNA expression. QPCR and western blotting methods can be used to confirm the results, the method was described previously [25,26]. The gene knockout method will be used to test the potential targets of TPCs and NAADP induced calcium signals. Sorted CSCs will also be studied to search for potential targets underlying the effect of TPCs on breast CSCs.

Conclusion

This review hopes to propose a prospective study for the role of two-pore channels in breast cancer cells and will contribute to the raising and development of the research in this field.

The author thanks for the support of Zongxiong Liu, Weifen Chen, and Yaqi Yang.

  1. DeSantis CE, Ma J, Gaudet MM, Newman LA, Miller KD, et al. (2019) Breast cancer statistics, 2019. CA: a cancer journal for clinicians 69: 438-451. Link: https://bit.ly/33SkGAG
  2. Observatory TGC (2018) GLOBOCAN. Breast Cancer. Link: https://bit.ly/3buzvMe
  3. Grunfeld E, Coyle D, Whelan T (2004) Family caregiver burden: Results of a longitudinal study of breast cancer patients and their principal caregivers. CMSJ 2004. 170: 1795-1801. Link: https://bit.ly/3auKoxv
  4. Liu H, Dilger JP, Lin J (2020) The Role of Transient Receptor Potential Melastatin 7 (TRPM7) in Cell Viability: A Potential Target to Suppress Breast Cancer Cell Cycle. Cancers 12: 131. Link: https://bit.ly/2w1E9SY
  5. Kintzer AF, Stroud RM (2016) Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana. Nature 531: 258-262. Link: https://bit.ly/2wPDpkf
  6. Nguyen ONP, Grimm C, Schneider LS, Chao YK, Atzberger C, et al. (2017) Two-pore channel function is crucial for migration of invasive cancer cells. Cancer Res 77: 1427-1438. Link: https://bit.ly/3bBcN56
  7. Sun W, Yue J (2018) TPC2 mediates autophagy progression and extracellular vesicle secretion in cancer cells. Experimental cell research 370: 478-489. Link: https://bit.ly/2wKtD2E
  8. Jahidin AH, Stewart TA, Thompson EW, Roberts-Thomson SJ,  Monteith GR (2016) Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells. Biochem Biophys Res Commun 477: 731-736. Link: https://bit.ly/3aomfZv
  9. E Melo FDS, Kurtova AV, Harnoss JM, Kljavin N, Hoeck JD, et al. (2017) A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 543: 676-680. Link: https://bit.ly/2wOrbYZ
  10. Liu H (2020) A Prospective for the Potential Effect of Local Anesthetics on Stem-Like Cells in Colon Cancer. Global J Med Res. Link: https://bit.ly/2UKkZJJ
  11. Favia A, Desideri M, Gambara G, D’Alessio A, Ruas M, et al. (2014) VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2–dependent Ca2+ signaling. Proceedings of the National Academy of Sciences 111: E4706-E4715. Link: https://bit.ly/39tfOTR
  12. Barrige MJ, Bootman MD, Roderick HL (2003) Calcium signaling: Dynamics, Homeostasis, and Remodeling. Nat Rev Mol Cell Biol. 4: 517-529. Link: https://bit.ly/2yj4E7a
  13. Paul NR, Jacquemet G, Caswell PT (2015) Endocytic trafficking of integrins in cell migration. Current Biology 25: R1092-R1105. Link: https://bit.ly/3dDMCwC
  14. Huttenlocher A, Horwitz AR (2011) Integrins in cell migration. Cold Spring Harbor perspectives in biology 3: a005074. Link: https://bit.ly/39pJEZ5
  15. Hämälistö S, Jäättelä M (2016) Lysosomes in cancer-living on the edge (of the cell). Curr Opin Cell Biol 39: 69-76. Link: https://bit.ly/3dE2e3b
  16. Prat A, Perou CM (2011) Deconstructing the molecular portraits of breast cancer. Mol Oncol 5: 5-23. Link: https://bit.ly/2WQznm6
  17. Liu H, Xiong Y, Zhu X, Gao H, Yin S, et al. (2017) Icariin improves osteoporosis, inhibits the expression of PPARγ, C/EBPα, FABP4 mRNA, N1ICD and jagged1 proteins, and increases Notch2 mRNA in ovariectomized rats. Exp Ther Med 13: 1360-1368. Link: https://bit.ly/3dCkXMs
  18. Kocatürk B, Versteeg HH (2015) Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. J Vis Exp 96. Link: https://bit.ly/3aoYPDg
  19. Jin Y, Zhou J, Xu F, Jin B, Cui L, et al. (2016) Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia. J Clin Invest 126: 3961-3980. Link: https://bit.ly/2Jq5P76
  20. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences 100: 3983-3988. Link: https://bit.ly/2QXiORX
  21. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, et al. (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell stem cell 1: 555-567. Link: https://bit.ly/2JqzzAA
  22. Patel S, Waghela B, Shah K, Vaidya F, Mirza S, et al. (2018) Silibinin, A Natural Blend In Polytherapy Formulation For Targeting Cd44v6 Expressing Colon Cancer Stem Cells. Scientific reports 8: 16985. Link: https://go.nature.com/2JnYJjl
  23. Cao L, Zhou Y, Zhai B, Liao J, Xu W, et al. (2011) Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines. BMC gastroenterology 11: 71. Link: https://bit.ly/2UMas0r
  24. Weiswald LB, Bellet D, Dangles-Marie V (2015) Spherical cancer models in tumor biology. Neoplasia 17: 1-15. Link: https://bit.ly/2wAS1nz
  25. Liu H, Xiong Y, Zhu X, Gao H, Yin S, et al. (2017) Icariin improves osteoporosis, inhibits the expression of PPARγ, C/EBPα, FABP4 mRNA, N1ICD and jagged1 proteins, and increases Notch2 mRNA in ovariectomized rats. Exp Ther Me 13: 1360-1368. Link: https://bit.ly/3dCkXMs
  26. Liu H, Xiong Y, Wang H, Yang L, Wang C, et al. (2018) Effects of water extract from epimedium on neuropeptide signaling in an ovariectomized osteoporosis rat model. J Ethnopharmacol 221: 126-136. Link: https://bit.ly/39rNtgB
© 2020 Liu H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.