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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 cell proliferation.(a) Staining of nuclei with Hoechst (blue) in MCF-7 cells after 3 days in culture. Hypoxic cells remained either untreated (a1), mock-transfected with non-targeting negative control siRNA (a2), transfected with siRNA against CAIX (a3), incubated with the CA inhibitor EZA (a4), or incubated with the MCT1 inhibitor AR-C155858 (a5). (b) Total number of nuclei/mm2 in MCF-7 cell cultures, kept for 0–3 days under the conditions as described in (a). For every data point four dishes of cells were used and five pictures were taken from each dish at random locations, yielding 20 pictures/data point (n = 20/4). (c) Staining of dead MCF-7 cells with propidium iodide (red) after 3 days in culture. Living cells are visualised by phase contrast. Hypoxic cells remained either untreated (c1), mock-transfected with non-targeting negative control siRNA (c2), transfected with siRNA against CAIX (c3), incubated with the CA inhibitor EZA (c4), or incubated with the MCT1 inhibitor AR-C155858 (c5). As positive control, apoptosis was induced by application of staurosporine (c6). (d) Total number of living (grey) and dead (red) cells/mm2, kept for 3 days under the conditions as described in (c). For every data point 4 dishes of cells from two independent batches were used and 15 pictures were taken from each dish at random locations, yielding 60 pictures/data point (n =6 0/4). Data are represented as mean ± SEM.
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f6: Knockdown of CAIX decreases cell proliferation.(a) Staining of nuclei with Hoechst (blue) in MCF-7 cells after 3 days in culture. Hypoxic cells remained either untreated (a1), mock-transfected with non-targeting negative control siRNA (a2), transfected with siRNA against CAIX (a3), incubated with the CA inhibitor EZA (a4), or incubated with the MCT1 inhibitor AR-C155858 (a5). (b) Total number of nuclei/mm2 in MCF-7 cell cultures, kept for 0–3 days under the conditions as described in (a). For every data point four dishes of cells were used and five pictures were taken from each dish at random locations, yielding 20 pictures/data point (n = 20/4). (c) Staining of dead MCF-7 cells with propidium iodide (red) after 3 days in culture. Living cells are visualised by phase contrast. Hypoxic cells remained either untreated (c1), mock-transfected with non-targeting negative control siRNA (c2), transfected with siRNA against CAIX (c3), incubated with the CA inhibitor EZA (c4), or incubated with the MCT1 inhibitor AR-C155858 (c5). As positive control, apoptosis was induced by application of staurosporine (c6). (d) Total number of living (grey) and dead (red) cells/mm2, kept for 3 days under the conditions as described in (c). For every data point 4 dishes of cells from two independent batches were used and 15 pictures were taken from each dish at random locations, yielding 60 pictures/data point (n =6 0/4). Data are represented as mean ± SEM.

Mentions: The increase in glycolytic rate, as observed under hypoxic condition (see Fig. 5), leads to an increase in the production of lactate and protons, which have to be removed from the cell efficiently to suppress intracellular acidification, inhibition of metabolism and ultimately a decrease in cell proliferation. To investigate whether CAIX-mediated augmentation in lactate flux facilitates cancer cell proliferation under hypoxic conditions, we determined the number of MCF-7 cells kept under hypoxia at different conditions for three days (Fig. 6a,b). Indeed, knockdown of CAIX decreased cell proliferation to 27%–43% (as compared to cells transfected with non-targeting negative control siRNA), while transfection with non-targeting negative control siRNA had no significant effects on cell proliferation (102%–114%, as compared to untreated, hypoxic cells). Interestingly, total inhibition of lactate transport with 300 nM AR-C155858 decreased cell proliferation only slightly more than did knockdown of CAIX (24%–30%, as compared to untreated, hypoxic cells). Inhibition of CAIX catalytic activity with 30 μM EZA, however, had no effect on cell proliferation (94%–111% as compared to untreated, hypoxic cells). These results were confirmed on the triple negative breast cancer cell line MDA-MB-231 (Fig. S1g). Like in MCF-7 cells knockdown of CAIX decreased cell proliferation while transfection of the cells with non-targeting negative control siRNA and inhibition of CA catalytic activity with 30 μM EZA had no significant effect on cell proliferation. In contrast to MCF-7 cells inhibition of MCT1/2 transport activity with 300 nm AR-C155858 did not decrease cell proliferation. Since MDA-MB-231 cells primarily express MCT4 instead of MCT1 (Fig. S1d,e; ref. 37,36) a MCT1/2 inhibitor would not fully impair lactate efflux. However, incubation of MDA-MB-231 cells with the isoform-unspecific MCT inhibitor α-cyano-4-hydroxycinnamic acid (cinnamate, 1 mM) resulted in a severe reduction in cell proliferation (Fig. S1h), similar as did AR-C155858 in MCF-7 cells (see Fig. 6b).


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 cell proliferation.(a) Staining of nuclei with Hoechst (blue) in MCF-7 cells after 3 days in culture. Hypoxic cells remained either untreated (a1), mock-transfected with non-targeting negative control siRNA (a2), transfected with siRNA against CAIX (a3), incubated with the CA inhibitor EZA (a4), or incubated with the MCT1 inhibitor AR-C155858 (a5). (b) Total number of nuclei/mm2 in MCF-7 cell cultures, kept for 0–3 days under the conditions as described in (a). For every data point four dishes of cells were used and five pictures were taken from each dish at random locations, yielding 20 pictures/data point (n = 20/4). (c) Staining of dead MCF-7 cells with propidium iodide (red) after 3 days in culture. Living cells are visualised by phase contrast. Hypoxic cells remained either untreated (c1), mock-transfected with non-targeting negative control siRNA (c2), transfected with siRNA against CAIX (c3), incubated with the CA inhibitor EZA (c4), or incubated with the MCT1 inhibitor AR-C155858 (c5). As positive control, apoptosis was induced by application of staurosporine (c6). (d) Total number of living (grey) and dead (red) cells/mm2, kept for 3 days under the conditions as described in (c). For every data point 4 dishes of cells from two independent batches were used and 15 pictures were taken from each dish at random locations, yielding 60 pictures/data point (n =6 0/4). Data are represented as mean ± SEM.
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Related In: Results  -  Collection

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f6: Knockdown of CAIX decreases cell proliferation.(a) Staining of nuclei with Hoechst (blue) in MCF-7 cells after 3 days in culture. Hypoxic cells remained either untreated (a1), mock-transfected with non-targeting negative control siRNA (a2), transfected with siRNA against CAIX (a3), incubated with the CA inhibitor EZA (a4), or incubated with the MCT1 inhibitor AR-C155858 (a5). (b) Total number of nuclei/mm2 in MCF-7 cell cultures, kept for 0–3 days under the conditions as described in (a). For every data point four dishes of cells were used and five pictures were taken from each dish at random locations, yielding 20 pictures/data point (n = 20/4). (c) Staining of dead MCF-7 cells with propidium iodide (red) after 3 days in culture. Living cells are visualised by phase contrast. Hypoxic cells remained either untreated (c1), mock-transfected with non-targeting negative control siRNA (c2), transfected with siRNA against CAIX (c3), incubated with the CA inhibitor EZA (c4), or incubated with the MCT1 inhibitor AR-C155858 (c5). As positive control, apoptosis was induced by application of staurosporine (c6). (d) Total number of living (grey) and dead (red) cells/mm2, kept for 3 days under the conditions as described in (c). For every data point 4 dishes of cells from two independent batches were used and 15 pictures were taken from each dish at random locations, yielding 60 pictures/data point (n =6 0/4). Data are represented as mean ± SEM.
Mentions: The increase in glycolytic rate, as observed under hypoxic condition (see Fig. 5), leads to an increase in the production of lactate and protons, which have to be removed from the cell efficiently to suppress intracellular acidification, inhibition of metabolism and ultimately a decrease in cell proliferation. To investigate whether CAIX-mediated augmentation in lactate flux facilitates cancer cell proliferation under hypoxic conditions, we determined the number of MCF-7 cells kept under hypoxia at different conditions for three days (Fig. 6a,b). Indeed, knockdown of CAIX decreased cell proliferation to 27%–43% (as compared to cells transfected with non-targeting negative control siRNA), while transfection with non-targeting negative control siRNA had no significant effects on cell proliferation (102%–114%, as compared to untreated, hypoxic cells). Interestingly, total inhibition of lactate transport with 300 nM AR-C155858 decreased cell proliferation only slightly more than did knockdown of CAIX (24%–30%, as compared to untreated, hypoxic cells). Inhibition of CAIX catalytic activity with 30 μM EZA, however, had no effect on cell proliferation (94%–111% as compared to untreated, hypoxic cells). These results were confirmed on the triple negative breast cancer cell line MDA-MB-231 (Fig. S1g). Like in MCF-7 cells knockdown of CAIX decreased cell proliferation while transfection of the cells with non-targeting negative control siRNA and inhibition of CA catalytic activity with 30 μM EZA had no significant effect on cell proliferation. In contrast to MCF-7 cells inhibition of MCT1/2 transport activity with 300 nm AR-C155858 did not decrease cell proliferation. Since MDA-MB-231 cells primarily express MCT4 instead of MCT1 (Fig. S1d,e; ref. 37,36) a MCT1/2 inhibitor would not fully impair lactate efflux. However, incubation of MDA-MB-231 cells with the isoform-unspecific MCT inhibitor α-cyano-4-hydroxycinnamic acid (cinnamate, 1 mM) resulted in a severe reduction in cell proliferation (Fig. S1h), similar as did AR-C155858 in MCF-7 cells (see Fig. 6b).

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