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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

Inhibition of c-Jun suppresses GLS expression and BPTES sensitivity in human breast cancer cell lines.(a) ChIP analysis showing that c-Jun binds to the GLS promoter. Complexes containing c-Jun were immunoprecipitated from cross-linked, digested, chromatin isolated from MDA-MB-231 cells. A parallel immunoprecipitation using rabbit IgG was carried out as a negative control. Following reversal of cross-links and purification of DNA, RT–PCR was run using primers designed to amplify a 196-bp fragment centred on the putative c-Jun binding site at position −188 bp relative to the TSS. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (b) Western blot analysis showing that treatment of MDA-MB-231 or TSE cells with the JNK inhibitor SP600125 (15 μM) leads to decreased phosphorylation of c-Jun, and decreased GLS levels. (c) Treatment of MDA-MB-231 cells with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h results in a dose-dependent decrease in GLS. (d) Western blot analysis showing that transient transfection of MDA-MB-231 cells with a constitutively activated JNK fusion construct results in increased c-Jun phosphorylation and upregulated GLS levels. Cells were collected 48 h after transfection. (e) Western blot analysis showing that in the drug-resistant breast cancer cell line BT-549, treatment with 15 μM SP600125 has little effect on c-Jun phosphorylation and does not lead to decreased GLS levels (left panels). However, knockdown of JUN expression using siRNAs leads to decreased GLS levels. Relative band intensities are indicated. (f) Representative BPTES dose curves showing the effect of BPTES on the proliferation of breast cancer cell lines over 6 days. Curves were fitted using SigmaPlot, with data from triplicate assays. (g) Sensitivity of breast cancer cell lines to GLS inhibition, as indicated by inhibition of proliferation over 6 days by 2 μM BPTES. Of the high-c-Jun lines, only the drug-resistant BT-549 cells were not highly sensitive to BPTES. None of the low-c-Jun lines were highly sensitive. Data presented are the mean±s.d. of triplicate assays. (h) BPTES dose curves for MDA-MB-231 cells±15 μM SP600125, showing that inhibition of JNK desensitizes cells to GLS inhibition (the IC50 for BPTES shifts from 1.8 to 12 μM). Curves were fitted using SigmaPlot, with data from triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots.
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f5: Inhibition of c-Jun suppresses GLS expression and BPTES sensitivity in human breast cancer cell lines.(a) ChIP analysis showing that c-Jun binds to the GLS promoter. Complexes containing c-Jun were immunoprecipitated from cross-linked, digested, chromatin isolated from MDA-MB-231 cells. A parallel immunoprecipitation using rabbit IgG was carried out as a negative control. Following reversal of cross-links and purification of DNA, RT–PCR was run using primers designed to amplify a 196-bp fragment centred on the putative c-Jun binding site at position −188 bp relative to the TSS. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (b) Western blot analysis showing that treatment of MDA-MB-231 or TSE cells with the JNK inhibitor SP600125 (15 μM) leads to decreased phosphorylation of c-Jun, and decreased GLS levels. (c) Treatment of MDA-MB-231 cells with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h results in a dose-dependent decrease in GLS. (d) Western blot analysis showing that transient transfection of MDA-MB-231 cells with a constitutively activated JNK fusion construct results in increased c-Jun phosphorylation and upregulated GLS levels. Cells were collected 48 h after transfection. (e) Western blot analysis showing that in the drug-resistant breast cancer cell line BT-549, treatment with 15 μM SP600125 has little effect on c-Jun phosphorylation and does not lead to decreased GLS levels (left panels). However, knockdown of JUN expression using siRNAs leads to decreased GLS levels. Relative band intensities are indicated. (f) Representative BPTES dose curves showing the effect of BPTES on the proliferation of breast cancer cell lines over 6 days. Curves were fitted using SigmaPlot, with data from triplicate assays. (g) Sensitivity of breast cancer cell lines to GLS inhibition, as indicated by inhibition of proliferation over 6 days by 2 μM BPTES. Of the high-c-Jun lines, only the drug-resistant BT-549 cells were not highly sensitive to BPTES. None of the low-c-Jun lines were highly sensitive. Data presented are the mean±s.d. of triplicate assays. (h) BPTES dose curves for MDA-MB-231 cells±15 μM SP600125, showing that inhibition of JNK desensitizes cells to GLS inhibition (the IC50 for BPTES shifts from 1.8 to 12 μM). Curves were fitted using SigmaPlot, with data from triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots.

Mentions: We then carried out chromatin immunoprecipitations (ChIPs) to test whether c-Jun binds directly to the GLS promoter. For these experiments, we used MDA-MB-231 breast cancer cells, which have high endogenous levels of both GLS and c-Jun (Fig. 4a). Briefly, cross-linked chromatin was digested to a length of ∼150–900 bp, and an antibody against endogenous c-Jun was used to immunoprecipitate c-Jun/DNA complexes. A parallel immunoprecipitation was carried out using IgG, as a negative control. Protein-DNA cross-links were then reversed, and RT–PCR was performed using primers designed to amplify a 196-bp fragment centred on the putative c-Jun binding site at position −188 bp relative to the TSS (Supplementary Fig. 7a). This yielded a strong signal from the c-Jun ChIP relative to the IgG ChIP, indicating that c-Jun binds to this region of the GLS promoter (Fig. 5a). Similar results were obtained using two additional sets of primers to amplify slightly shorter fragments also centred on the predicted c-Jun binding site (Supplementary Fig. 8).


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)

Inhibition of c-Jun suppresses GLS expression and BPTES sensitivity in human breast cancer cell lines.(a) ChIP analysis showing that c-Jun binds to the GLS promoter. Complexes containing c-Jun were immunoprecipitated from cross-linked, digested, chromatin isolated from MDA-MB-231 cells. A parallel immunoprecipitation using rabbit IgG was carried out as a negative control. Following reversal of cross-links and purification of DNA, RT–PCR was run using primers designed to amplify a 196-bp fragment centred on the putative c-Jun binding site at position −188 bp relative to the TSS. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (b) Western blot analysis showing that treatment of MDA-MB-231 or TSE cells with the JNK inhibitor SP600125 (15 μM) leads to decreased phosphorylation of c-Jun, and decreased GLS levels. (c) Treatment of MDA-MB-231 cells with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h results in a dose-dependent decrease in GLS. (d) Western blot analysis showing that transient transfection of MDA-MB-231 cells with a constitutively activated JNK fusion construct results in increased c-Jun phosphorylation and upregulated GLS levels. Cells were collected 48 h after transfection. (e) Western blot analysis showing that in the drug-resistant breast cancer cell line BT-549, treatment with 15 μM SP600125 has little effect on c-Jun phosphorylation and does not lead to decreased GLS levels (left panels). However, knockdown of JUN expression using siRNAs leads to decreased GLS levels. Relative band intensities are indicated. (f) Representative BPTES dose curves showing the effect of BPTES on the proliferation of breast cancer cell lines over 6 days. Curves were fitted using SigmaPlot, with data from triplicate assays. (g) Sensitivity of breast cancer cell lines to GLS inhibition, as indicated by inhibition of proliferation over 6 days by 2 μM BPTES. Of the high-c-Jun lines, only the drug-resistant BT-549 cells were not highly sensitive to BPTES. None of the low-c-Jun lines were highly sensitive. Data presented are the mean±s.d. of triplicate assays. (h) BPTES dose curves for MDA-MB-231 cells±15 μM SP600125, showing that inhibition of JNK desensitizes cells to GLS inhibition (the IC50 for BPTES shifts from 1.8 to 12 μM). Curves were fitted using SigmaPlot, with data from triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots.
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f5: Inhibition of c-Jun suppresses GLS expression and BPTES sensitivity in human breast cancer cell lines.(a) ChIP analysis showing that c-Jun binds to the GLS promoter. Complexes containing c-Jun were immunoprecipitated from cross-linked, digested, chromatin isolated from MDA-MB-231 cells. A parallel immunoprecipitation using rabbit IgG was carried out as a negative control. Following reversal of cross-links and purification of DNA, RT–PCR was run using primers designed to amplify a 196-bp fragment centred on the putative c-Jun binding site at position −188 bp relative to the TSS. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (b) Western blot analysis showing that treatment of MDA-MB-231 or TSE cells with the JNK inhibitor SP600125 (15 μM) leads to decreased phosphorylation of c-Jun, and decreased GLS levels. (c) Treatment of MDA-MB-231 cells with the AP-1 inhibitor SR11302 (1–10 μM) for 48 h results in a dose-dependent decrease in GLS. (d) Western blot analysis showing that transient transfection of MDA-MB-231 cells with a constitutively activated JNK fusion construct results in increased c-Jun phosphorylation and upregulated GLS levels. Cells were collected 48 h after transfection. (e) Western blot analysis showing that in the drug-resistant breast cancer cell line BT-549, treatment with 15 μM SP600125 has little effect on c-Jun phosphorylation and does not lead to decreased GLS levels (left panels). However, knockdown of JUN expression using siRNAs leads to decreased GLS levels. Relative band intensities are indicated. (f) Representative BPTES dose curves showing the effect of BPTES on the proliferation of breast cancer cell lines over 6 days. Curves were fitted using SigmaPlot, with data from triplicate assays. (g) Sensitivity of breast cancer cell lines to GLS inhibition, as indicated by inhibition of proliferation over 6 days by 2 μM BPTES. Of the high-c-Jun lines, only the drug-resistant BT-549 cells were not highly sensitive to BPTES. None of the low-c-Jun lines were highly sensitive. Data presented are the mean±s.d. of triplicate assays. (h) BPTES dose curves for MDA-MB-231 cells±15 μM SP600125, showing that inhibition of JNK desensitizes cells to GLS inhibition (the IC50 for BPTES shifts from 1.8 to 12 μM). Curves were fitted using SigmaPlot, with data from triplicate assays. Relative densitometry data are the mean±s.d. of triplicate blots.
Mentions: We then carried out chromatin immunoprecipitations (ChIPs) to test whether c-Jun binds directly to the GLS promoter. For these experiments, we used MDA-MB-231 breast cancer cells, which have high endogenous levels of both GLS and c-Jun (Fig. 4a). Briefly, cross-linked chromatin was digested to a length of ∼150–900 bp, and an antibody against endogenous c-Jun was used to immunoprecipitate c-Jun/DNA complexes. A parallel immunoprecipitation was carried out using IgG, as a negative control. Protein-DNA cross-links were then reversed, and RT–PCR was performed using primers designed to amplify a 196-bp fragment centred on the putative c-Jun binding site at position −188 bp relative to the TSS (Supplementary Fig. 7a). This yielded a strong signal from the c-Jun ChIP relative to the IgG ChIP, indicating that c-Jun binds to this region of the GLS promoter (Fig. 5a). Similar results were obtained using two additional sets of primers to amplify slightly shorter fragments also centred on the predicted c-Jun binding site (Supplementary Fig. 8).

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