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The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy.

Lukey MJ, Greene KS, Erickson JW, Wilson KF, Cerione RA - Nat Commun (2016)

Bottom Line: We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression.Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity.These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

No MeSH data available.


Related in: MedlinePlus

c-Jun increases GLS expression and BPTES sensitivity in breast cancer cells.(a) Western blot analysis of GLS and c-Jun levels in whole-cell lysates of MDA-MB-468 cells, stably carrying either pCDNA3.1 or the JUN-V5 expression vector pCDNA3.1-JUN. Relative band intensities are indicated for GLS. (b) Glutaminase activity assay using mitochondria isolated from the derivative MDA-MB-468 cell lines. Activity is expressed per mg of total cellular protein, and data presented are the mean±s.d. of triplicate assays. (c) RT–PCR analysis of the derivative MDA-MB-468 cell lines, showing relative abundance of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Western blot analysis showing c-Myc levels in the derivative cell lines. (e) Western blot analysis showing that treatment of vector-control, or JUN-overexpressing, MDA-MB-468 cells with the c-Myc inhibitor 10058-F4 at concentrations up to 60 μM for 48 h has little effect on GLS levels. Relative band intensities are indicated for GLS. (f) Western blot analysis showing that treatment with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h leads to a dose-dependent decrease in GLS levels. The effect is especially pronounced in JUN-overexpressing cells. (g) Cell proliferation assays for MDA-MB-468 cells stably carrying pCDNA3.1 or the JUN expression vector. Cells were seeded in 12-well dishes at a density of 1 × 104 cells per well, cultured for 6 days in the absence or presence of 9 μM (left panel), 12 μM (middle panel) or 15 μM (right panel) BPTES±1 mM dimethyl α-KG. Inhibition of proliferation by BPTES treatment is plotted as a percentage. The data presented are the mean±s.d. of triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.
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f6: c-Jun increases GLS expression and BPTES sensitivity in breast cancer cells.(a) Western blot analysis of GLS and c-Jun levels in whole-cell lysates of MDA-MB-468 cells, stably carrying either pCDNA3.1 or the JUN-V5 expression vector pCDNA3.1-JUN. Relative band intensities are indicated for GLS. (b) Glutaminase activity assay using mitochondria isolated from the derivative MDA-MB-468 cell lines. Activity is expressed per mg of total cellular protein, and data presented are the mean±s.d. of triplicate assays. (c) RT–PCR analysis of the derivative MDA-MB-468 cell lines, showing relative abundance of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Western blot analysis showing c-Myc levels in the derivative cell lines. (e) Western blot analysis showing that treatment of vector-control, or JUN-overexpressing, MDA-MB-468 cells with the c-Myc inhibitor 10058-F4 at concentrations up to 60 μM for 48 h has little effect on GLS levels. Relative band intensities are indicated for GLS. (f) Western blot analysis showing that treatment with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h leads to a dose-dependent decrease in GLS levels. The effect is especially pronounced in JUN-overexpressing cells. (g) Cell proliferation assays for MDA-MB-468 cells stably carrying pCDNA3.1 or the JUN expression vector. Cells were seeded in 12-well dishes at a density of 1 × 104 cells per well, cultured for 6 days in the absence or presence of 9 μM (left panel), 12 μM (middle panel) or 15 μM (right panel) BPTES±1 mM dimethyl α-KG. Inhibition of proliferation by BPTES treatment is plotted as a percentage. The data presented are the mean±s.d. of triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.

Mentions: Using the MDA-MB-468 breast cancer cell line, which exhibits relatively low endogenous levels of c-Jun and moderate levels of GLS (Fig. 4a), we generated derivative cell lines that stably overexpress JUN-V5 or carry the empty plasmid vector. We then collected vector-control cells and JUN-overexpressing cells at ∼60% confluency from RPMI medium (10% FBS, 2 mM glutamine), and probed whole-cell lysates for GLS, c-Jun and V5-tag by western blot (Fig. 6a). This showed that GLS levels are elevated in the JUN-overexpressing cells relative to the vector-control cells. Mitochondria isolated from the two cell lines were assayed for glutaminase activity, which was markedly higher in the JUN-overexpressing cells (Fig. 6b). Moreover, RT–PCR analysis confirmed that GLS transcript levels were upregulated in JUN-overexpressing cells relative to vector-control cells (Fig. 6c). We made equivalent derivative cell lines of the MCF7 parental cell line, which has very low endogenous levels of both c-Jun and GLS (Fig. 4a), and similarly found that GLS expression was upregulated in JUN-overexpressing cells (Supplementary Fig. 10a).


The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy.

Lukey MJ, Greene KS, Erickson JW, Wilson KF, Cerione RA - Nat Commun (2016)

c-Jun increases GLS expression and BPTES sensitivity in breast cancer cells.(a) Western blot analysis of GLS and c-Jun levels in whole-cell lysates of MDA-MB-468 cells, stably carrying either pCDNA3.1 or the JUN-V5 expression vector pCDNA3.1-JUN. Relative band intensities are indicated for GLS. (b) Glutaminase activity assay using mitochondria isolated from the derivative MDA-MB-468 cell lines. Activity is expressed per mg of total cellular protein, and data presented are the mean±s.d. of triplicate assays. (c) RT–PCR analysis of the derivative MDA-MB-468 cell lines, showing relative abundance of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Western blot analysis showing c-Myc levels in the derivative cell lines. (e) Western blot analysis showing that treatment of vector-control, or JUN-overexpressing, MDA-MB-468 cells with the c-Myc inhibitor 10058-F4 at concentrations up to 60 μM for 48 h has little effect on GLS levels. Relative band intensities are indicated for GLS. (f) Western blot analysis showing that treatment with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h leads to a dose-dependent decrease in GLS levels. The effect is especially pronounced in JUN-overexpressing cells. (g) Cell proliferation assays for MDA-MB-468 cells stably carrying pCDNA3.1 or the JUN expression vector. Cells were seeded in 12-well dishes at a density of 1 × 104 cells per well, cultured for 6 days in the absence or presence of 9 μM (left panel), 12 μM (middle panel) or 15 μM (right panel) BPTES±1 mM dimethyl α-KG. Inhibition of proliferation by BPTES treatment is plotted as a percentage. The data presented are the mean±s.d. of triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.
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f6: c-Jun increases GLS expression and BPTES sensitivity in breast cancer cells.(a) Western blot analysis of GLS and c-Jun levels in whole-cell lysates of MDA-MB-468 cells, stably carrying either pCDNA3.1 or the JUN-V5 expression vector pCDNA3.1-JUN. Relative band intensities are indicated for GLS. (b) Glutaminase activity assay using mitochondria isolated from the derivative MDA-MB-468 cell lines. Activity is expressed per mg of total cellular protein, and data presented are the mean±s.d. of triplicate assays. (c) RT–PCR analysis of the derivative MDA-MB-468 cell lines, showing relative abundance of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Western blot analysis showing c-Myc levels in the derivative cell lines. (e) Western blot analysis showing that treatment of vector-control, or JUN-overexpressing, MDA-MB-468 cells with the c-Myc inhibitor 10058-F4 at concentrations up to 60 μM for 48 h has little effect on GLS levels. Relative band intensities are indicated for GLS. (f) Western blot analysis showing that treatment with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h leads to a dose-dependent decrease in GLS levels. The effect is especially pronounced in JUN-overexpressing cells. (g) Cell proliferation assays for MDA-MB-468 cells stably carrying pCDNA3.1 or the JUN expression vector. Cells were seeded in 12-well dishes at a density of 1 × 104 cells per well, cultured for 6 days in the absence or presence of 9 μM (left panel), 12 μM (middle panel) or 15 μM (right panel) BPTES±1 mM dimethyl α-KG. Inhibition of proliferation by BPTES treatment is plotted as a percentage. The data presented are the mean±s.d. of triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.
Mentions: Using the MDA-MB-468 breast cancer cell line, which exhibits relatively low endogenous levels of c-Jun and moderate levels of GLS (Fig. 4a), we generated derivative cell lines that stably overexpress JUN-V5 or carry the empty plasmid vector. We then collected vector-control cells and JUN-overexpressing cells at ∼60% confluency from RPMI medium (10% FBS, 2 mM glutamine), and probed whole-cell lysates for GLS, c-Jun and V5-tag by western blot (Fig. 6a). This showed that GLS levels are elevated in the JUN-overexpressing cells relative to the vector-control cells. Mitochondria isolated from the two cell lines were assayed for glutaminase activity, which was markedly higher in the JUN-overexpressing cells (Fig. 6b). Moreover, RT–PCR analysis confirmed that GLS transcript levels were upregulated in JUN-overexpressing cells relative to vector-control cells (Fig. 6c). We made equivalent derivative cell lines of the MCF7 parental cell line, which has very low endogenous levels of both c-Jun and GLS (Fig. 4a), and similarly found that GLS expression was upregulated in JUN-overexpressing cells (Supplementary Fig. 10a).

Bottom Line: We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression.Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity.These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

No MeSH data available.


Related in: MedlinePlus