As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2-7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

Precise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pHi, pHe) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na+/H+ exchanger NHE1 and Na+, HCO3− transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pHe to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pHi changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pHi regulation by the two transporters.

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2, and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+/H+ exchanger NHE1, the Na+‐ cotransporter NBCn1, and the lactate‐H+ cotransporters MCT1 and −4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA‐mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA‐MB‐231 triple‐negative breast cancer (TNBC) cell line significantly reduced steady‐state intracellular pH (pHi) and capacity for pHi recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA‐MB‐231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor‐free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.

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.

NHE1 Ser703 phosphorylation in spheroids and 2D culture and during spheroid growth. MCF-7 and MDA-MB-231 spheroids (3D) and 2D cultures were grown 4 and 9 (MDA-MB-231 spheroids only) days in parallel, followed by lysis and Western blotting with antibodies directed against pSer703-NHE1 and total NHE1. Left and top panels in A and B, respectively, show representative Western blots, while right and lower panels, respectively, show quantifications of band intensities normalized to that of corresponding 2D or 3D culture on day 4. A: MCF-7. Note that the level of pSer703-NHE1 was normalized to the mean total NHE1 level from five other experiments (the total NHE1 data shown in Fig. 3a). Data is shown as mean + SD. 2n. B: MDA-MB-231. Data is shown as mean + SEM. 3n. A two-tailed, paired Student’s t-test was used to test for statistically significant difference in means between two groups. * indicates p

Increased metabolism and insufficient blood supply cause acidic waste product accumulation in solid cancers. During carcinogenesis, cellular acid extrusion is upregulated but the underlying molecular mechanisms and their consequences for cancer growth and progression have not been established. Genome-wide association studies have indicated a possible link between the Na+,HCO3–-cotransporter NBCn1 (SLC4A7) and breast cancer. We tested the functional consequences of NBCn1 knockout (KO) for breast cancer development. NBCn1 protein expression increased 2.5-fold during breast carcinogenesis and was responsible for the increased net acid extrusion and alkaline intracellular pH of breast cancer compared with normal breast tissue. Genetic disruption of NBCn1 delayed breast cancer development: tumor latency was ~50% increased while tumor growth rate was ~65% reduced in NBCn1 KO compared with wild-type (WT) mice. Breast cancer histopathology in NBCn1 KO mice differed from that in WT mice and included less aggressive tumor types. The extracellular tumor microenvironment in NBCn1 KO mice contained higher concentrations of glucose and lower concentrations of lactate than that in WT mice. Independently of NBCn1 genotype, the cleaved fraction of poly(ADP-ribose) polymerase (PARP)-1 and expression of monocarboxylate transporter (MCT)1 increased while phosphorylation of Akt and ERK1 decreased as functions of tumor volume. Cell proliferation, evaluated from Ki-67 and phospho-histone H3 staining, was ~60% lower in breast cancer of NBCn1 KO than that of WT mice when corrected for variations in tumor size. We conclude that NBCn1 facilitates acid extrusion from breast cancer tissue, maintains the alkaline intracellular environment and promotes aggressive cancer development and growth.

Event Abstract Back to Event Molecular mechanisms and binding partners controlling expression, localization and function of the Na+, HCO3- cotransporter NBCn1 in cancer Stine F. Pedersen1*, Andrej Gorbatenko1, Christina W. Olesen1, Anne P. Andersen1, Gitte Lauritzen1, Su Chii Kong1, Carolina Bianchi1, Jose M. Moreira2 and Ebbe Boedtkjer3 1 University of Copenhagen, Department of Biology, Denmark 2 University of Copenhagen, Dept. of Veterinary Disease Biology, Denmark 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 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. 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 Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Stine F Pedersen Andrej Gorbatenko Christina W Olesen Anne P Andersen Gitte Lauritzen Su Chii Kong Carolina Bianchi Jose M Moreira Ebbe Boedtkjer Google Stine F Pedersen Andrej Gorbatenko Christina W Olesen Anne P Andersen Gitte Lauritzen Su Chii Kong Carolina Bianchi Jose M Moreira Ebbe Boedtkjer Google Scholar Stine F Pedersen Andrej Gorbatenko Christina W Olesen Anne P Andersen Gitte Lauritzen Su Chii Kong Carolina Bianchi Jose M Moreira Ebbe Boedtkjer PubMed Stine F Pedersen Andrej Gorbatenko Christina W Olesen Anne P Andersen Gitte Lauritzen Su Chii Kong Carolina Bianchi Jose M Moreira Ebbe Boedtkjer Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Major changes in intra- and extracellular pH homoeostasis are shared features of most solid tumours. These changes stem in large part from the metabolic shift of most cancer cells towards glycolytic metabolism and other processes associated with net acid production. In combination with oncogenic signalling and impact from factors in the tumour microenvironment, this upregulates acid-extruding plasma membrane transport proteins which maintain intracellular pH normal or even more alkaline compared with that of normal cells, while in turn acidifying the external microenvironment. Mounting evidence strongly indicates that this contributes significantly to cancer development by favouring e.g. cancer cell migration, invasion and chemotherapy resistance. Finally, while still under-explored, it seems likely that non-cancer cells in the tumour microenvironment also exhibit altered pH regulation and that this may contribute to their malignant properties. Thus, the physical tumour microenvironment and the cancer and stromal cells within it undergo important reciprocal interactions which modulate the tumour pH profile, in turn severely impacting on the course of cancer progression. Here, we summarize recent knowledge of tumour metabolism and the tumour microenvironment, placing it in the context of tumour pH regulation, and discuss how interfering with these properties may be exploited clinically.

When studying cancer development it is essential to employ models that recapitulate the tumor microenvironment (TME). We recently showed that truncated ErbB2 (ΔNErbB2) increases acid extrusion via the Na+/H+ exchanger NHE1 and the Na+,HCO3− cotransporter NBCn1 and increases NBCn1 expression in MCF‐7 breast cancer cells. Further, NHE1 inhibition strongly sensitized ΔNErbB2‐expressing MCF‐7 cells to chemotherapy‐induced death. Here, we explore the impact of specific aspects of the TME on pH‐regulatory and survival/motility signaling. Employing MCF‐7 cells +/− inducible ΔNErbB2 expression we show that: (i) TME simulation (pHe 6.5, 1% O2, 7.5 mM lactate) strongly increases Akt‐ and ERK1/2 phosphorylation/activity; TME mimicking conditions has no additional effect on NBCn1 expression but further increases ERK1/2 activity in ΔNErbB2+ cells (ii) in 2D coculture with primary human breast fibroblasts, MCF‐7 cells upregulate the lactate‐H+ cotransporter MCT1 and the epithelial‐mesenchymal transition (EMT) marker α‐smooth muscle actin; (iii) in MCF‐7 spheroids NHE1 expression and ERM‐ and Akt (S473) phosphorylation are greatly reduced compared to 2D monoculture, and pERM and apparently also NBCn1 localize to the spheroid periphery. In conclusion, the TME profoundly impacts on pH regulatory ion transporters and survival/motility signaling in MCF‐7 breast cancer cell models.Funding: Danish Cancer Society, Danish Research Council

To study cancer development it is essential to employ models that recapitulate the tumor microenvironment. We previously showed that the Na+/H+ exchanger NHE1 and the Na+, HCO3− cotransporter NBCn1 are essential for pH regulation in MCF‐7 breast cancer cells and their activity increased by ΔNErbB2 expression. Furthermore, NHE1 inhibition strongly sensitized ΔNErbB2‐ expressing MCF‐7 cells to chemotherapy‐induced death. Here, we explore the impact of specific aspects of the tumor microenvironment on pH‐regulatory and survival/motility signaling programs. Simulation of the tumor microenvironment (pHe 6.5, 1% O2, 7.5 mM lactate) elicited marked upregulation of Akt activity (Akt S473 phosphorylation) in MCF‐7 cells ± ΔNErbB2. In 2D co‐culture with primary human breast fibroblasts, MCF‐7 cells appeared to upregulate the lactate‐H+ cotransporter MCT1 and EMT markers such as α‐smooth muscle actin. Finally, in MCF‐7 spheroids (3D multicellular growths), the expression patterns of NHE1, NBCn1 and pAkt/Akt were altered compared to 2D mono‐culture controls. In conclusion, the tumor microenvironment profoundly impacts on pH regulatory ion transport proteins and survival signaling in breast cancer cell models. Funding: Novo Foundation.

Stability of transient siRNA-mediated knockdown of NHE1 in MDA-MB-231 cells. A. MDA-MB-231 cells were transfected with 100 nM siNHE1. 48 h after transfection, cells were reseeded and the stability of the knockdown in 2D culture was monitored for 120 h. Left panel show Western blots and right panel show quantifications of band intensities normalized to corresponding Mock control. B: Western blots showing CRISPR/Cas9-mediated knockout (KO) of NHE1 in MCF-7 and MDA-MB-231 cells. WT: wild-type. (PDF 135 kb)