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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

Knockdown of CAIX decreases lactate transport in cancer cells.(a) Antibody staining for CAIX (green) in MCF-7 cells, kept under hypoxic conditions. Hypoxic cells either remained untreated (a1), mock-transfected with non-targeting negative control siRNA (a2) or transfected with siRNA against CAIX (a3). Nuclei are stained with Hoechst (blue). (b) Quantification of the fluorescent signal for CAIX as shown in (a). (c) Original recording of the relative change in intracellular lactate concentration in MCF-7 cells kept under hypoxic conditions during application of 1 and 3 mM lactate. Cells were either untreated (black trace), mock-transfected with non-targeting negative control siRNA (green trace) or transfected with siRNA against CAIX (blue trace). (d) Rate of change in lactate level during application of 1 and 3 mM lactate in hypoxic MCF-7 cells, either untreated (gray bars), mock-transfected with non-targeting negative control siRNA (green bars) or transfected with siRNA against CAIX (blue bars). Knock-down of CAIX induced a significant decrease in lactate flux. (e) Original recordings of changes in pHi in hypoxic MCF-7 cells, either untreated (control, gray traces), mock-transfected with non-targeting negative control siRNA (green traces) or transfected with siRNA against CAIX (blue traces). (f,g) Rate of change in pHi, as induced by application (f) and removal (g) of lactate, respectively. (h) Original recordings of the relative change in intracellular lactate concentration in MCF-7 cells kept under normoxic (blue traces) or hypoxic (red traces) conditions during application of lactate in the presence and absence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. (i) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic and hypoxic conditions, respectively, as induced by application of 1 mM lactate in the absence and presence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. Hypoxia induces a significant increase in lactate flux both in the absence and in the presence of CO2/HCO3− and EZA. Knockdown of CAIX induced a significant decrease in the rate of change in pHi, both during addition and removal of lactate. Data are represented as mean ± SEM.
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f3: Knockdown of CAIX decreases lactate transport in cancer cells.(a) Antibody staining for CAIX (green) in MCF-7 cells, kept under hypoxic conditions. Hypoxic cells either remained untreated (a1), mock-transfected with non-targeting negative control siRNA (a2) or transfected with siRNA against CAIX (a3). Nuclei are stained with Hoechst (blue). (b) Quantification of the fluorescent signal for CAIX as shown in (a). (c) Original recording of the relative change in intracellular lactate concentration in MCF-7 cells kept under hypoxic conditions during application of 1 and 3 mM lactate. Cells were either untreated (black trace), mock-transfected with non-targeting negative control siRNA (green trace) or transfected with siRNA against CAIX (blue trace). (d) Rate of change in lactate level during application of 1 and 3 mM lactate in hypoxic MCF-7 cells, either untreated (gray bars), mock-transfected with non-targeting negative control siRNA (green bars) or transfected with siRNA against CAIX (blue bars). Knock-down of CAIX induced a significant decrease in lactate flux. (e) Original recordings of changes in pHi in hypoxic MCF-7 cells, either untreated (control, gray traces), mock-transfected with non-targeting negative control siRNA (green traces) or transfected with siRNA against CAIX (blue traces). (f,g) Rate of change in pHi, as induced by application (f) and removal (g) of lactate, respectively. (h) Original recordings of the relative change in intracellular lactate concentration in MCF-7 cells kept under normoxic (blue traces) or hypoxic (red traces) conditions during application of lactate in the presence and absence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. (i) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic and hypoxic conditions, respectively, as induced by application of 1 mM lactate in the absence and presence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. Hypoxia induces a significant increase in lactate flux both in the absence and in the presence of CO2/HCO3− and EZA. Knockdown of CAIX induced a significant decrease in the rate of change in pHi, both during addition and removal of lactate. Data are represented as mean ± SEM.

Mentions: To determine whether lactate transport is augmented by CAIX, we knocked down CAIX in hypoxic MCF-7 cells (Fig. 3). Transfection of cells with siRNA against CAIX reduced expression of CAIX to 46%, while transfection of cells with non-targeting negative control siRNA had no effect on CAIX expression level (Fig. 3a,b). The rate of lactate flux, as measured with Laconic, decreased to ~50% when CAIX was knocked down in hypoxic MCF-7 cells (Fig. 3c,d). These data fit well to the doubling in lactate transport in hypoxic cells as compared to normoxic cells (see Fig. 1b). Like lactate flux, the rate of change in pHi (ΔpHi/Δt), as induced by application and removal of lactate, also decreased after knockdown of CAIX under hypoxic conditions in MCF-7 cells, while transfection with control siRNA had no significant effect on ΔpHi/Δt (Fig. 3e–g).


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)

Knockdown of CAIX decreases lactate transport in cancer cells.(a) Antibody staining for CAIX (green) in MCF-7 cells, kept under hypoxic conditions. Hypoxic cells either remained untreated (a1), mock-transfected with non-targeting negative control siRNA (a2) or transfected with siRNA against CAIX (a3). Nuclei are stained with Hoechst (blue). (b) Quantification of the fluorescent signal for CAIX as shown in (a). (c) Original recording of the relative change in intracellular lactate concentration in MCF-7 cells kept under hypoxic conditions during application of 1 and 3 mM lactate. Cells were either untreated (black trace), mock-transfected with non-targeting negative control siRNA (green trace) or transfected with siRNA against CAIX (blue trace). (d) Rate of change in lactate level during application of 1 and 3 mM lactate in hypoxic MCF-7 cells, either untreated (gray bars), mock-transfected with non-targeting negative control siRNA (green bars) or transfected with siRNA against CAIX (blue bars). Knock-down of CAIX induced a significant decrease in lactate flux. (e) Original recordings of changes in pHi in hypoxic MCF-7 cells, either untreated (control, gray traces), mock-transfected with non-targeting negative control siRNA (green traces) or transfected with siRNA against CAIX (blue traces). (f,g) Rate of change in pHi, as induced by application (f) and removal (g) of lactate, respectively. (h) Original recordings of the relative change in intracellular lactate concentration in MCF-7 cells kept under normoxic (blue traces) or hypoxic (red traces) conditions during application of lactate in the presence and absence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. (i) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic and hypoxic conditions, respectively, as induced by application of 1 mM lactate in the absence and presence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. Hypoxia induces a significant increase in lactate flux both in the absence and in the presence of CO2/HCO3− and EZA. Knockdown of CAIX induced a significant decrease in the rate of change in pHi, both during addition and removal of lactate. Data are represented as mean ± SEM.
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Related In: Results  -  Collection

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f3: Knockdown of CAIX decreases lactate transport in cancer cells.(a) Antibody staining for CAIX (green) in MCF-7 cells, kept under hypoxic conditions. Hypoxic cells either remained untreated (a1), mock-transfected with non-targeting negative control siRNA (a2) or transfected with siRNA against CAIX (a3). Nuclei are stained with Hoechst (blue). (b) Quantification of the fluorescent signal for CAIX as shown in (a). (c) Original recording of the relative change in intracellular lactate concentration in MCF-7 cells kept under hypoxic conditions during application of 1 and 3 mM lactate. Cells were either untreated (black trace), mock-transfected with non-targeting negative control siRNA (green trace) or transfected with siRNA against CAIX (blue trace). (d) Rate of change in lactate level during application of 1 and 3 mM lactate in hypoxic MCF-7 cells, either untreated (gray bars), mock-transfected with non-targeting negative control siRNA (green bars) or transfected with siRNA against CAIX (blue bars). Knock-down of CAIX induced a significant decrease in lactate flux. (e) Original recordings of changes in pHi in hypoxic MCF-7 cells, either untreated (control, gray traces), mock-transfected with non-targeting negative control siRNA (green traces) or transfected with siRNA against CAIX (blue traces). (f,g) Rate of change in pHi, as induced by application (f) and removal (g) of lactate, respectively. (h) Original recordings of the relative change in intracellular lactate concentration in MCF-7 cells kept under normoxic (blue traces) or hypoxic (red traces) conditions during application of lactate in the presence and absence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. (i) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic and hypoxic conditions, respectively, as induced by application of 1 mM lactate in the absence and presence of 5% CO2/15 mM HCO3− and 30 μM EZA, respectively. Hypoxia induces a significant increase in lactate flux both in the absence and in the presence of CO2/HCO3− and EZA. Knockdown of CAIX induced a significant decrease in the rate of change in pHi, both during addition and removal of lactate. Data are represented as mean ± SEM.
Mentions: To determine whether lactate transport is augmented by CAIX, we knocked down CAIX in hypoxic MCF-7 cells (Fig. 3). Transfection of cells with siRNA against CAIX reduced expression of CAIX to 46%, while transfection of cells with non-targeting negative control siRNA had no effect on CAIX expression level (Fig. 3a,b). The rate of lactate flux, as measured with Laconic, decreased to ~50% when CAIX was knocked down in hypoxic MCF-7 cells (Fig. 3c,d). These data fit well to the doubling in lactate transport in hypoxic cells as compared to normoxic cells (see Fig. 1b). Like lactate flux, the rate of change in pHi (ΔpHi/Δt), as induced by application and removal of lactate, also decreased after knockdown of CAIX under hypoxic conditions in MCF-7 cells, while transfection with control siRNA had no significant effect on ΔpHi/Δt (Fig. 3e–g).

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