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Molecular Cancer
Published in: Molecular Cancer 1/2016

Open Access 01-12-2016 | Research

Roles of acid-extruding ion transporters in regulation of breast cancer cell growth in a 3-dimensional microenvironment

Authors: Anne Poder Andersen, Mette Flinck, Eva Kjer Oernbo, Nis Borbye Pedersen, Birgitte Martine Viuff, Stine Falsig Pedersen

Published in: Molecular Cancer | Issue 1/2016

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Abstract

Background

The 3-dimensional (3D) microenvironment of breast carcinomas is characterized by profoundly altered pH homeostasis, reflecting increased metabolic acid production and a confined extracellular space characterized by poor diffusion, yet the relative contributions of specific pH-regulatory transporters to 3D growth are poorly understood. The aim of this work was to determine how 3D spheroid growth of breast cancer cells impacts the expression and spatial organization of major acid extruding proteins, and how these proteins in turn are required for spheroid growth.

Methods

MCF-7 (Luminal-A) and MDA-MB-231 (Triple-negative) human breast cancer cells were grown as ~700-950 μm diameter spheroids, which were subjected to Western blotting for relevant transporters (2- and 3D growth), quantitative immunohistochemical analysis, and spheroid growth assays. Individual transporter contributions were assessed (i) pharmacologically, (ii) by stable shRNA- and transient siRNA-mediated knockdown, and (iii) by CRISPR/Cas9 knockout.

Results

In MCF-7 spheroids, expression of the lactate-H+ cotransporter MCT1 (SLC16A1) increased from the spheroid periphery to its core, the Na+,HCO3 − cotransporter NBCn1 (SLC4A7) was most highly expressed at the periphery, and the Na+/H+ exchanger NHE1 (SLC9A1) and MCT4 (SLC16A3) were evenly distributed. A similar pattern was seen in MDA-MB-231 spheroids, except that these cells do not express MCT1. The relative total expression of NBCn1 and NHE1 was decreased in 3D compared to 2D, while that of MCT1 and MCT4 was unaltered. Inhibition of MCT1 (AR-C155858) attenuated MCF-7 spheroid growth and this was exacerbated by addition of S0859, an inhibitor of Na+,HCO3 − cotransporters and MCTs. The pharmacological data was recapitulated by stable knockdown of MCT1 or NBCn1, whereas knockdown of MCT4 had no effect. CRISPR/Cas9 knockout of NHE1, but neither partial NHE1 knockdown nor the NHE1 inhibitor cariporide, inhibited MCF-7 spheroid growth. In contrast, growth of MDA-MB-231 spheroids was inhibited by stable or transient NHE1 knockdown and by NHE1 knockout, but not by knockdown of NBCn1 or MCT4.

Conclusions

This work demonstrates the distinct expression and localization patterns of four major acid-extruding transporters in 3D spheroids of human breast cancer cells and reveals that 3D growth is dependent on these transporters in a cell type-dependent manner, with potentially important implications for breast cancer therapy.
Appendix
Available only for authorised users
Footnotes
1
Part of these findings have previously been reported in abstract form [27].
 
Literature
1.
Chen JL, Lucas JE, Schroeder T, Mori S, Wu J, Nevins J, et al. The genomic analysis of lactic acidosis and acidosis response in human cancers. PLoS Genet. 2008;4:e1000293.CrossRefPubMedPubMedCentral
2.
Gaedtke L, Thoenes L, Culmsee C, Mayer B, Wagner E. Proteomic analysis reveals differences in protein expression in spheroid versus monolayer cultures of low-passage colon carcinoma cells. J Proteome Res. 2007;6:4111–8.CrossRefPubMed
3.
Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001;294:1708–12.CrossRefPubMed
4.
Pampaloni F, Reynaud EG, Stelzer EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007;8:839–45.CrossRefPubMed
5.
Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA. Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol. 2010;148:3–15.CrossRefPubMed
6.
Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol. 2004;14:198–206.CrossRefPubMed
7.
Andersen AP, Moreira JM, Pedersen SF. Interactions of ion transporters and channels with cancer cell metabolism and the tumour microenvironment. Philos Trans R Soc Lond B Biol Sci. 2014;369:20130098.CrossRefPubMedPubMedCentral
8.
Parks SK, Chiche J, Pouyssegur J. pH control mechanisms of tumor survival and growth. J Cell Physiol. 2011;226:299–308.CrossRefPubMed
9.
Koppenol WH, Bounds PL, Dang CV. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer. 2011;11:325–37.CrossRefPubMed
10.
Webb BA, Chimenti M, Jacobson MP, Barber DL. Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer. 2011;11:671–7.CrossRefPubMed
11.
Schwab A, Fabian A, Hanley PJ, Stock C. Role of ion channels and transporters in cell migration. Physiol Rev. 2012;92:1865–913.CrossRefPubMed
12.
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;316:2538–53.CrossRefPubMed
13.
Le FR, Chiche J, Marchiq I, Naiken T, Ilc K, Murray CM, et al. CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors. Proc Natl Acad Sci U S A. 2011;108:16663–8.CrossRef
14.
Boedtkjer E, Bunch L, Pedersen SF. Physiology, Pharmacology and Pathophysiology of the pH Regulatory Transport Proteins NHE1 and NBCn1: Similarities, Differences, and Implications for Cancer Therapy. Curr Pharm Des. 2012;18:1345–71.CrossRefPubMed
15.
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;132:1288–99.CrossRefPubMed
16.
Dhup S, Dadhich RK, Porporato PE, Sonveaux P. Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. Curr Pharm Des. 2012;18:1319–30.CrossRefPubMed
17.
Cardone RA, Casavola V, Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer. 2005;5:786–95.CrossRefPubMed
18.
Pedersen SF, Stock C. Ion channels and transporters in cancer: pathophysiology, regulation, and clinical potential. Cancer Res. 2013;73:1658–61.CrossRefPubMed
19.
Gorbatenko A, Olesen CW, Morup N, Thiel G, Kallunki T, Valen E, et al. ErbB2 upregulates the Na+, HCO3--cotransporter NBCn1/SLC4A7 in human breast cancer cells via Akt, ERK, Src, and Kruppel-like factor 4. FASEB J. 2014;28:350–63.CrossRefPubMed
20.
Ahmed S, Thomas G, Ghoussaini M, Healey CS, Humphreys MK, Platte R, et al. Newly discovered breast cancer susceptibility loci on 3p24 and 17q23.2. Nat Genet. 2009;41:585–90.CrossRefPubMedPubMedCentral
21.
Chiche J, Le FY, Vilmen C, Frassineti F, Daniel L, Halestrap AP, et al. In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH. Int J Cancer. 2012;130:1511–20.CrossRefPubMed
22.
Lee S, Axelsen TV, Andersen AP, Vahl P, Pedersen SF, Boedtkjer E: Disrupting Na,HCO-cotransporter NBCn1 (Slc4a7) delays murine breast cancer development. Oncogene. 2016;35(16):2112-22. doi: 10.​1038/​onc.​2015.​273.
23.
Busco G, Cardone RA, Greco MR, Bellizzi A, Colella M, Antelmi E, et al. NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space. FASEB J. 2010;24:3903–15.CrossRefPubMed
24.
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;317:172–83.CrossRefPubMed
25.
Stock C, Gassner B, Hauck CR, Arnold H, Mally S, Eble JA, et al. Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange. J Physiol. 2005;567:225–38.CrossRefPubMedPubMedCentral
26.
Hulikova A, Vaughan-Jones RD, Swietach P. Dual role of CO2/HCO3(−) formula buffer in the regulation of intracellular pH of three-dimensional tumor growths. J Biol Chem. 2011;286:13815–26.CrossRefPubMedPubMedCentral
27.
Andersen AP, Ronnov-Jessen L, Hulikova A, Swietach P, Pedersen SF. Development of model systems for analysis of effects of cell-cell and cell-microenvironment interactions on pH regulatory proteins in breast cancer. FASEB J. 27, 471.4. 2013
28.
Pastorek J, Pastorekova S, Callebaut I, Mornon JP, Zelnik V, Opavsky R, et al. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene. 1994;9:2877–88.PubMed
29.
Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A. 1992;89:9064–8.CrossRefPubMedPubMedCentral
30.
Krause S, Maffini MV, Soto AM, Sonnenschein C. The microenvironment determines the breast cancer cells’ phenotype: organization of MCF7 cells in 3D cultures. BMC Cancer. 2010;10:263.CrossRefPubMedPubMedCentral
31.
Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS. The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell. 2002;111:29–40.CrossRefPubMed
32.
Asada K, Miyamoto K, Fukutomi T, Tsuda H, Yagi Y, Wakazono K, et al. Reduced expression of GNA11 and silencing of MCT1 in human breast cancers. Oncology. 2003;64:380–8.CrossRefPubMed
33.
Takahashi E, Abe J, Gallis B, Aebersold R, Spring DJ, Krebs EG, et al. p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1. J Biol Chem. 1999;274:20206–14.CrossRefPubMed
34.
Hulikova A, Harris AL, Vaughan-Jones RD, Swietach P. Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia. J Cell Physiol. 2013;228:743–52.CrossRefPubMed
35.
Kong SC, Gianuzzo A, Novak I, Pedersen SF. Acid–base transport in pancreatic cancer: molecular mechanisms and clinical potential. Biochem Cell Biol. 2014;92:449–59.CrossRefPubMed
36.
Amith SR, Wilkinson JM, Baksh S, Fliegel L. The Na+/H+ exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells. Oncotarget. 2015;6:1262–75.CrossRefPubMed
37.
Weaver VM, Howlett AR, Langton-Webster B, Petersen OW, Bissell MJ. The development of a functionally relevant cell culture model of progressive human breast cancer. Semin Cancer Biol. 1995;6:175–84.CrossRefPubMed
38.
Ghosh S, Spagnoli GC, Martin I, Ploegert S, Demougin P, Heberer M, et al. Three-dimensional culture of melanoma cells profoundly affects gene expression profile: a high density oligonucleotide array study. J Cell Physiol. 2005;204:522–31.CrossRefPubMed
39.
Kolosenko I, Fryknas M, Forsberg S, Johnsson P, Cheon H, Holvey-Bates EG, et al. Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs. Int J Cancer. 2015;136:E51–61.CrossRefPubMed
40.
Kirk P, Wilson MC, Heddle C, Brown MH, Barclay AN, Halestrap AP. CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. EMBO J. 2000;19:3896–904.CrossRefPubMedPubMedCentral
41.
Gallagher SM, Castorino JJ, Philp NJ. Interaction of monocarboxylate transporter 4 with beta1-integrin and its role in cell migration. Am J Physiol Cell Physiol. 2009;296:C414–21.CrossRefPubMed
42.
Grillon E, Farion R, Fablet K, De WM, Tse CM, Donowitz M, et al. The spatial organization of proton and lactate transport in a rat brain tumor. PLoS ONE. 2011;6:e17416.CrossRefPubMedPubMedCentral
43.
Sonveaux P, Vegran F, Schroeder T, Wergin MC, Verrax J, Rabbani ZN, et al. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J Clin Invest. 2008;118:3930–42.PubMedPubMedCentral
44.
Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. J Biol Chem. 2006;281:9030–7.CrossRefPubMed
45.
Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, et al. Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function. Sci Rep. 2015;5:13605.CrossRefPubMedPubMedCentral
46.
Cheng C, Edin NF, Lauritzen KH, Aspmodal I, Christoffersen S, Jian L, et al. Alterations of monocarboxylate transporter densities during hypoxia in brain and breast tumour cells. Cell Oncol (Dordr). 2012;35:217–27.CrossRef
47.
De Saedeleer CJ, Porporato PE, Copetti T, Perez-Escuredo J, Payen VL, Brisson L, et al. Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration. Oncogene. 2014;33:4060–8.CrossRefPubMed
48.
Fredsted J, Pedraz-Cuesta E, Jensen HH, Bornebusch A, Nejsum LN, Kragelund BB et al.: Prolactin signaling stimulates invasion via the Na+/H+ exchanger NHE1 in T47D breast cancer cells. Mol Endocrinol 2015, in revision
49.
De MA, Canese R, Marino ML, Borghi M, Iero M, Villa A, et al. pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity. Int J Cancer. 2010;127:207–19.CrossRef
50.
Wang BY, Zhang J, Wang JL, Sun S, Wang ZH, Wang LP, et al. Intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer. J Exp Clin Cancer Res. 2015;34:85.CrossRefPubMedPubMedCentral
51.
Hulikova A, Aveyard N, Harris AL, Vaughan-Jones RD, Swietach P. Intracellular carbonic anhydrase activity sensitizes cancer cell pH signaling to dynamic changes in CO2 partial pressure. J Biol Chem. 2014;289:25418–30.CrossRefPubMedPubMedCentral
52.
53.
Parks SK, Pouyssegur J. The Na(+)/HCO3(−) Co-Transporter SLC4A4 Plays a Role in Growth and Migration of Colon and Breast Cancer Cells. J Cell Physiol. 2015;230:1954–63.CrossRefPubMed
54.
Heidtmann H, Ruminot I, Becker HM, Deitmer JW. Inhibition of monocarboxylate transporter by N-cyanosulphonamide S0859. Eur J Pharmacol. 2015;762:344–9.CrossRefPubMed
55.
Rotin D, Steele-Norwood D, Grinstein S, Tannock I. Requirement of the Na+/H+ exchanger for tumor growth. Cancer Res. 1989;49:205–11.PubMed
56.
Pouyssegur J, Franchi A, Pages G. pHi, aerobic glycolysis and vascular endothelial growth factor in tumour growth. Novartis Found Symp. 2001;240:186–96.CrossRefPubMed
57.
Zancan P, Sola-Penna M, Furtado CM, Da SD. Differential expression of phosphofructokinase-1 isoforms correlates with the glycolytic efficiency of breast cancer cells. Mol Genet Metab. 2010;100:372–8.CrossRefPubMed
58.
Sugiyama S, Satoh H, Nomura N, Terada H, Watanabe H, Hayashi H. The importance of glycolytically-derived ATP for the Na+/H+ exchange activity in guinea pig ventricular myocytes. Mol Cell Biochem. 2001;217:153–61.CrossRefPubMed
59.
Larsen AM, Krogsgaard-Larsen N, Lauritzen G, Olesen CW, Honore HS, Boedtkjer E, et al. Gram-scale solution-phase synthesis of selective sodium bicarbonate co-transport inhibitor S0859: in vitro efficacy studies in breast cancer cells. ChemMedChem. 2012;7:1808–14.CrossRefPubMed
60.
Sigurethsson HH, Olesen CW, Dybboe R, Lauritzen G, Pedersen SF. Constitutively active ErbB2 regulates cisplatin-induced cell death in breast cancer cells via pro- and antiapoptotic mechanisms. Mol Cancer Res. 2015;13:63–77.CrossRefPubMed
Metadata
Title
Roles of acid-extruding ion transporters in regulation of breast cancer cell growth in a 3-dimensional microenvironment
Authors
Anne Poder Andersen
Mette Flinck
Eva Kjer Oernbo
Nis Borbye Pedersen
Birgitte Martine Viuff
Stine Falsig Pedersen
Publication date
01-12-2016
Publisher
BioMed Central
Published in
Molecular Cancer / Issue 1/2016
Electronic ISSN: 1476-4598
DOI
https://doi.org/10.1186/s12943-016-0528-0

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