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Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, Becker HM - Sci Rep (2015)

Bottom Line: Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction.Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1.Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Zoology/Membrane Transport, FB Biologie, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.

ABSTRACT
The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

No MeSH data available.


Related in: MedlinePlus

Expression of CAIX but not of MCT1 and MCT4 is upregulated under hypoxic conditions.(a) Determination of the Km value for lactate in MCF-7 cells under normoxic (21% O2, grey) and hypoxic (1% O2, blue) conditions, respectively, as determined by the rate of change in pHi during application of 0.3, 1, 3, 10 and 30 mM lactate. Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for MCT1 (b), MCT2 (c) and MCT4 (d), respectively. For positive control of MCT2, lysate from MCT2-expressing oocytes was used. Actin was used as loading control. (e) Relative change in the RNA level of MCT1 and MCT4 in MCF-7 cells after three days under hypoxic conditions. (f) Relative change in the RNA level of NHE1 and NBCn1 in MCF-7 cells after three days under hypoxic conditions. (g) Relative change in the RNA level of CAII and CAIX in MCF-7 cells after three days under hypoxic conditions. Expression level of CAIX is strongly upregulated, while the expression levels of MCT1, MCT4, NHE1 and NBCe1 show no significant changes. (h) Western blot of MCF-7 cell lysate, labelled for CAIX and actin as loading control. (i) Quantification of CAIX protein level by western blot analysis in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively. (j) Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for NHE1 and actin. Data are represented as mean ± SEM.
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f2: Expression of CAIX but not of MCT1 and MCT4 is upregulated under hypoxic conditions.(a) Determination of the Km value for lactate in MCF-7 cells under normoxic (21% O2, grey) and hypoxic (1% O2, blue) conditions, respectively, as determined by the rate of change in pHi during application of 0.3, 1, 3, 10 and 30 mM lactate. Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for MCT1 (b), MCT2 (c) and MCT4 (d), respectively. For positive control of MCT2, lysate from MCT2-expressing oocytes was used. Actin was used as loading control. (e) Relative change in the RNA level of MCT1 and MCT4 in MCF-7 cells after three days under hypoxic conditions. (f) Relative change in the RNA level of NHE1 and NBCn1 in MCF-7 cells after three days under hypoxic conditions. (g) Relative change in the RNA level of CAII and CAIX in MCF-7 cells after three days under hypoxic conditions. Expression level of CAIX is strongly upregulated, while the expression levels of MCT1, MCT4, NHE1 and NBCe1 show no significant changes. (h) Western blot of MCF-7 cell lysate, labelled for CAIX and actin as loading control. (i) Quantification of CAIX protein level by western blot analysis in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively. (j) Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for NHE1 and actin. Data are represented as mean ± SEM.

Mentions: To analyse which MCT isoforms mediate lactate/H+ flux under normoxic and hypoxic conditions, we determined the Km value for lactate in MCF-7 cells under both conditions by measuring the rate of change in pHi during application of different lactate concentrations (Fig. 2a). Both, under normoxia and hypoxia, the Km value was ~5 mM. For MCT1 a Km value between 3–8 mM had been determined in various cell types282930. For MCT2 the Km value was found to be 0.7431, while for MCT4 Km values between 17–35 mM have been reported32. Together with the total inhibition of lactate-induced acidification with AR-C155858, these results lead to the conclusion that lactate flux in MCF-7 cells is exclusively mediated by MCT1. This conclusion is further supported by western blot analysis for MCT1, MCT2 and MCT4 (Fig. 2b–d). Only for MCT1 sharp bands could be observed for MCF-7 cells both under normoxic and hypoxic conditions (Fig. 2b). For MCT2 no bands could be observed in MCF-7 cells under either condition, while cell lysate from MCT2-expressing Xenopus oocytes, used as positive control, produced a single sharp band at ~40 kDa (Fig. 2c). For MCT4 only faint bands where visible in MCF-7 cells under normoxia or hypoxia (Fig. 1d), while the same antibody produced robust bands in MDA-MB-231 cells (Fig. S1d).


Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, Becker HM - Sci Rep (2015)

Expression of CAIX but not of MCT1 and MCT4 is upregulated under hypoxic conditions.(a) Determination of the Km value for lactate in MCF-7 cells under normoxic (21% O2, grey) and hypoxic (1% O2, blue) conditions, respectively, as determined by the rate of change in pHi during application of 0.3, 1, 3, 10 and 30 mM lactate. Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for MCT1 (b), MCT2 (c) and MCT4 (d), respectively. For positive control of MCT2, lysate from MCT2-expressing oocytes was used. Actin was used as loading control. (e) Relative change in the RNA level of MCT1 and MCT4 in MCF-7 cells after three days under hypoxic conditions. (f) Relative change in the RNA level of NHE1 and NBCn1 in MCF-7 cells after three days under hypoxic conditions. (g) Relative change in the RNA level of CAII and CAIX in MCF-7 cells after three days under hypoxic conditions. Expression level of CAIX is strongly upregulated, while the expression levels of MCT1, MCT4, NHE1 and NBCe1 show no significant changes. (h) Western blot of MCF-7 cell lysate, labelled for CAIX and actin as loading control. (i) Quantification of CAIX protein level by western blot analysis in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively. (j) Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for NHE1 and actin. Data are represented as mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4559800&req=5

f2: Expression of CAIX but not of MCT1 and MCT4 is upregulated under hypoxic conditions.(a) Determination of the Km value for lactate in MCF-7 cells under normoxic (21% O2, grey) and hypoxic (1% O2, blue) conditions, respectively, as determined by the rate of change in pHi during application of 0.3, 1, 3, 10 and 30 mM lactate. Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for MCT1 (b), MCT2 (c) and MCT4 (d), respectively. For positive control of MCT2, lysate from MCT2-expressing oocytes was used. Actin was used as loading control. (e) Relative change in the RNA level of MCT1 and MCT4 in MCF-7 cells after three days under hypoxic conditions. (f) Relative change in the RNA level of NHE1 and NBCn1 in MCF-7 cells after three days under hypoxic conditions. (g) Relative change in the RNA level of CAII and CAIX in MCF-7 cells after three days under hypoxic conditions. Expression level of CAIX is strongly upregulated, while the expression levels of MCT1, MCT4, NHE1 and NBCe1 show no significant changes. (h) Western blot of MCF-7 cell lysate, labelled for CAIX and actin as loading control. (i) Quantification of CAIX protein level by western blot analysis in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively. (j) Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O2) and hypoxic (1% O2) conditions, labelled for NHE1 and actin. Data are represented as mean ± SEM.
Mentions: To analyse which MCT isoforms mediate lactate/H+ flux under normoxic and hypoxic conditions, we determined the Km value for lactate in MCF-7 cells under both conditions by measuring the rate of change in pHi during application of different lactate concentrations (Fig. 2a). Both, under normoxia and hypoxia, the Km value was ~5 mM. For MCT1 a Km value between 3–8 mM had been determined in various cell types282930. For MCT2 the Km value was found to be 0.7431, while for MCT4 Km values between 17–35 mM have been reported32. Together with the total inhibition of lactate-induced acidification with AR-C155858, these results lead to the conclusion that lactate flux in MCF-7 cells is exclusively mediated by MCT1. This conclusion is further supported by western blot analysis for MCT1, MCT2 and MCT4 (Fig. 2b–d). Only for MCT1 sharp bands could be observed for MCF-7 cells both under normoxic and hypoxic conditions (Fig. 2b). For MCT2 no bands could be observed in MCF-7 cells under either condition, while cell lysate from MCT2-expressing Xenopus oocytes, used as positive control, produced a single sharp band at ~40 kDa (Fig. 2c). For MCT4 only faint bands where visible in MCF-7 cells under normoxia or hypoxia (Fig. 1d), while the same antibody produced robust bands in MDA-MB-231 cells (Fig. S1d).

Bottom Line: Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction.Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1.Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Zoology/Membrane Transport, FB Biologie, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.

ABSTRACT
The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

No MeSH data available.


Related in: MedlinePlus