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Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

Granja S, Marchiq I, Le Floch R, Moura CS, Baltazar F, Pouysségur J - Oncotarget (2015)

Bottom Line: The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells.Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines.Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

ABSTRACT
Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG- cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG- cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

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Effect of BSG disruption in lung carcinoma cell line H1975A: Immunoblot analysis for MCT1, MCT4 and BSG in wt and BSG−/− H1975 cell lines maintained in normoxia or hypoxia (1% O2) for 48h. ARD1 was used as a loading control; B: Total biomass of wt and BSG−/− H1975 cells was evaluated over time using the sulphorhodamine B assay; C: Time-course of the intracellular lactate concentration in response to glucose (25mM) added in the presence of either DMSO or iMCT1/2 (300nM).
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Figure 4: Effect of BSG disruption in lung carcinoma cell line H1975A: Immunoblot analysis for MCT1, MCT4 and BSG in wt and BSG−/− H1975 cell lines maintained in normoxia or hypoxia (1% O2) for 48h. ARD1 was used as a loading control; B: Total biomass of wt and BSG−/− H1975 cells was evaluated over time using the sulphorhodamine B assay; C: Time-course of the intracellular lactate concentration in response to glucose (25mM) added in the presence of either DMSO or iMCT1/2 (300nM).

Mentions: First, we disrupted the BASIGIN (BSG) gene in three different non-small lung cancer cell lines (A549, H1975 and H292) by using Zinc Finger Nucleases. Knocked-out candidates lacking expression of BSG (Figs. 3A, 4A) were identified and selected for further characterization. The site that was cut was amplified by PCR and the sequence and mutational independent status of the clones was confirmed by sequencing. Next, we analysed the expression of the MCT1 and MCT4 isoforms in normoxia and hypoxia 1% O2 in these cells Immunoblotting showed that the MCT1 protein was not detectable in BSG−/− A549 cells compared to wild-type (wt) cells, while MCT4 expression was highly reduced. We also observed that the non-detectable MCT4 expression in normoxia in BSG−/− cells remained inducible in hypoxia (Fig. 3A). Similar results were obtained for the H1975 (Fig. 4A) and H292 cell lines (data not shown). Knockout (KO) of the BSG gene in these cells induced a huge decrease in the protein level of MCT1 and MCT4 in both normoxia and hypoxia.


Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

Granja S, Marchiq I, Le Floch R, Moura CS, Baltazar F, Pouysségur J - Oncotarget (2015)

Effect of BSG disruption in lung carcinoma cell line H1975A: Immunoblot analysis for MCT1, MCT4 and BSG in wt and BSG−/− H1975 cell lines maintained in normoxia or hypoxia (1% O2) for 48h. ARD1 was used as a loading control; B: Total biomass of wt and BSG−/− H1975 cells was evaluated over time using the sulphorhodamine B assay; C: Time-course of the intracellular lactate concentration in response to glucose (25mM) added in the presence of either DMSO or iMCT1/2 (300nM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466644&req=5

Figure 4: Effect of BSG disruption in lung carcinoma cell line H1975A: Immunoblot analysis for MCT1, MCT4 and BSG in wt and BSG−/− H1975 cell lines maintained in normoxia or hypoxia (1% O2) for 48h. ARD1 was used as a loading control; B: Total biomass of wt and BSG−/− H1975 cells was evaluated over time using the sulphorhodamine B assay; C: Time-course of the intracellular lactate concentration in response to glucose (25mM) added in the presence of either DMSO or iMCT1/2 (300nM).
Mentions: First, we disrupted the BASIGIN (BSG) gene in three different non-small lung cancer cell lines (A549, H1975 and H292) by using Zinc Finger Nucleases. Knocked-out candidates lacking expression of BSG (Figs. 3A, 4A) were identified and selected for further characterization. The site that was cut was amplified by PCR and the sequence and mutational independent status of the clones was confirmed by sequencing. Next, we analysed the expression of the MCT1 and MCT4 isoforms in normoxia and hypoxia 1% O2 in these cells Immunoblotting showed that the MCT1 protein was not detectable in BSG−/− A549 cells compared to wild-type (wt) cells, while MCT4 expression was highly reduced. We also observed that the non-detectable MCT4 expression in normoxia in BSG−/− cells remained inducible in hypoxia (Fig. 3A). Similar results were obtained for the H1975 (Fig. 4A) and H292 cell lines (data not shown). Knockout (KO) of the BSG gene in these cells induced a huge decrease in the protein level of MCT1 and MCT4 in both normoxia and hypoxia.

Bottom Line: The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells.Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines.Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

ABSTRACT
Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG- cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG- cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

Show MeSH
Related in: MedlinePlus