Molecular mechanisms and binding partners controlling expression, localization and function of the Na+, HCO3- cotransporter NBCn1 in cancer
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1
University of Copenhagen, Department of Biology, Denmark
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2
University of Copenhagen, Dept. of Veterinary Disease Biology, Denmark
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3
Aarhus University, Dept. of Biomedicine, Denmark
Background and aim. In recent years, dysregulation of acid-base transport in cancer development has received substantial attention given its potential diagnostic and therapeutic relevance. In our previous work, we demonstrated the strong upregulation of the Na+-HCO3- cotransporter NBCn1 (SLC4A7) in MCF-7 breast cancer cells by a truncated, constitutively active ErbB2 (HER2) receptor, ∆NErbB2 (1) and showed that NBCn1 expression is also increased in breast cancer patient tissue (2). The aim of the work presented here was to characterize the molecular mechanisms regulating NBCn1 expression, localization and function in cancer, focusing on breast cancer.
Methods. Methods included promoter mutational analyses, luciferase reporter assays, chromatin immunoprecipitation (ChIP), live imaging of pHi, GST pulldown analysis and mass spec analysis, and validation by co-IP and immunofluorescence analysis. 3D spheroid models and tissue microenvironment-simulating conditions are also employed.
Results. Firstly, we present our recent characterization of the SLC4A7 promoter and identification of its minimal ∆NErbB2-sensitive region, and show that NBCn1 protein expression is regulated by PI3K/Akt1, ERK2, and Src signaling as well as by the two cancer-related transcription factors, Krüppel-like Factor 4 (KLF4) and Specificity protein 1 (Sp1), which oppositely regulate NBCn1 expression. Further, we show that NBCn1 expression is increased by stimulation of full-length ErbB receptors in several cancer cell types. Second, recent data on regulation of NBCn1 mRNA stability and 3’UTR activity by ∆NErbB2 in MCF-7 cells is presented, demonstrating that both mRNA stability and 3’UTR activity of NBCn1 in breast cancer cells are regulated by ∆NErbB2. Third, novel NBCn1 binding partners are presented. The NBCn1 C-terminal tail is known to be important for trafficking of NBCn1 to the plasma membrane. By GST-pulldown and mass spec analysis, we have recently identified a range of novel NBCn1 binding partners. We present here the functional importance of several of these proteins for NBCn1 expression and localization.
Conclusions. NBCn1 is strongly upregulated by ErbB receptor signaling. This involves opposite effects of the highly cancer-relevant transcription factors KLF4 and Sp1 on the NBCn1 promoter, as well as regulation of the 3’UTR of NBCn1. We identify novel NBCn1 binding partners with important roles in control of NBCn1 expression and localization. We suggest that ErbB-mediated NBCn1 upregulation may play important roles in breast cancer development.
References
1. Lauritzen, G., et al. (2010). Exp.Cell Res. 316, 2538-2553
2. Boedtkjer, E., et al. (2013). Int J.Cancer 132, 1288-1299
Keywords:
Bicarbonate transport,
KLF4,
HER2/ERBB2,
SLC4A7,
pH-regulation
Conference:
4th Annual Meeting of the International Society of Proton Dynamics in Cancer, Garching, Germany, 10 Oct - 12 Oct, 2013.
Presentation Type:
Abstract
Topic:
3. pH, cell signalling and growth
Citation:
Pedersen
SF,
Gorbatenko
A,
Olesen
CW,
Andersen
AP,
Lauritzen
G,
Kong
S,
Bianchi
C,
Moreira
JM and
Boedtkjer
E
(2014). Molecular mechanisms and binding partners controlling expression, localization and function of the Na+, HCO3- cotransporter NBCn1 in cancer.
Front. Pharmacol.
Conference Abstract:
4th Annual Meeting of the International Society of Proton Dynamics in Cancer.
doi: 10.3389/conf.fphar.2014.61.00034
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Received:
17 Dec 2013;
Published Online:
07 Feb 2014.
*
Correspondence:
Prof. Stine F Pedersen, University of Copenhagen, Department of Biology, Copenhagen, Denmark, sfpedersen@bio.ku.dk