Limits...
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

Glutamine-dependent transformation by oncogenic-Dbl.(a) Western blot analysis showing timecourse of oncogenic-Dbl expression in an inducible MEF system, and downstream elevation of GLS levels. Cells were induced by plating in doxycycline-free growth medium, and samples were collected at time-points up to 72 h. (b) Glutaminase activity assay using mitochondria isolated from MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox). 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 uninduced and induced (24 h) MEFs, showing relative levels of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Saturation density analysis showing the effect of glutamine withdrawal on oncogenic-Dbl inducible MEFs. Dishes of uninduced (+Dox) and induced (−Dox) cells cultured in 4 mM glutamine, or in glutamine-free medium ±2 mM dimethyl α-ketoglutarate, were fixed and then stained with crystal violet. (e) Cell death analysis for uninduced or induced cells after 6 days culture in 4.0 or 0.2 mM glutamine. Data presented are the mean±s.d. of triplicate assays. (f) Cell proliferation assays showing the effect of glutamine depletion on proliferation of uninduced and induced MEFs over 6 days. Data presented are the mean±s.d. of triplicate assays. (g) Saturation density analysis showing the effect of the GLS inhibitor BPTES on oncogenic-Dbl induced MEFs. Induced (−Dox) cells cultured in the absence or presence of BPTES (30 or 40 μM)±2 mM dimethyl α-ketoglutarate were fixed and then stained with crystal violet. (h) BPTES dose curves showing the effect of different BPTES concentrations on proliferation over 6 days of uninduced or induced MEFs. Fractional proliferation relative to untreated cells is shown. Assays were carried out in 10% FBS medium, and data presented are the mean±s.d. of triplicate assays. (i) Anchorage-independent growth assay for uninduced (+Dox) cells, and for induced (−Dox) cells cultured under increasing BPTES concentrations. Data presented are the mean±s.d. of triplicate assays. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4837472&req=5

f1: Glutamine-dependent transformation by oncogenic-Dbl.(a) Western blot analysis showing timecourse of oncogenic-Dbl expression in an inducible MEF system, and downstream elevation of GLS levels. Cells were induced by plating in doxycycline-free growth medium, and samples were collected at time-points up to 72 h. (b) Glutaminase activity assay using mitochondria isolated from MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox). 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 uninduced and induced (24 h) MEFs, showing relative levels of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Saturation density analysis showing the effect of glutamine withdrawal on oncogenic-Dbl inducible MEFs. Dishes of uninduced (+Dox) and induced (−Dox) cells cultured in 4 mM glutamine, or in glutamine-free medium ±2 mM dimethyl α-ketoglutarate, were fixed and then stained with crystal violet. (e) Cell death analysis for uninduced or induced cells after 6 days culture in 4.0 or 0.2 mM glutamine. Data presented are the mean±s.d. of triplicate assays. (f) Cell proliferation assays showing the effect of glutamine depletion on proliferation of uninduced and induced MEFs over 6 days. Data presented are the mean±s.d. of triplicate assays. (g) Saturation density analysis showing the effect of the GLS inhibitor BPTES on oncogenic-Dbl induced MEFs. Induced (−Dox) cells cultured in the absence or presence of BPTES (30 or 40 μM)±2 mM dimethyl α-ketoglutarate were fixed and then stained with crystal violet. (h) BPTES dose curves showing the effect of different BPTES concentrations on proliferation over 6 days of uninduced or induced MEFs. Fractional proliferation relative to untreated cells is shown. Assays were carried out in 10% FBS medium, and data presented are the mean±s.d. of triplicate assays. (i) Anchorage-independent growth assay for uninduced (+Dox) cells, and for induced (−Dox) cells cultured under increasing BPTES concentrations. Data presented are the mean±s.d. of triplicate assays. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.

Mentions: We previously reported that oncogenic-Dbl, a guanine nucleotide exchange factor and potent activator of Rho GTPases, signals to upregulate mitochondrial GLS activity in NIH/3T3 cells20. This is an essential event for maintaining Dbl-induced cellular transformation. To explore further the signalling connections that link Rho GTPases with GLS, we utilized an inducible, tetracycline-off, system to control the expression of oncogenic-Dbl in mouse embryonic fibroblasts (MEFs). When doxycycline (0.6 μg ml−1) is present in the culture medium, HA-tagged oncogenic-Dbl is undetectable by western blot analysis of whole-cell lysates. Removal of doxycycline triggers a robust expression of oncogenic-Dbl within 10 h that remains elevated through 72 h (Fig. 1a). This is accompanied by a corresponding increase in GLS protein levels, which peak 24–48 h following induction (Fig. 1a). We isolated mitochondria from uninduced and induced MEFs and assayed the preparations for glutaminase activity as described previously28. This confirmed that induction of oncogenic-Dbl results in elevated glutaminase activity (Fig. 1b). We then tested whether GLS was upregulated at the transcriptional level. Cells that were either uninduced or induced (24 h) were analysed by real-time PCR (RT–PCR), which revealed that induction of oncogenic-Dbl expression leads to an ∼12-fold increase of the GLS transcript (Fig. 1c).


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)

Glutamine-dependent transformation by oncogenic-Dbl.(a) Western blot analysis showing timecourse of oncogenic-Dbl expression in an inducible MEF system, and downstream elevation of GLS levels. Cells were induced by plating in doxycycline-free growth medium, and samples were collected at time-points up to 72 h. (b) Glutaminase activity assay using mitochondria isolated from MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox). 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 uninduced and induced (24 h) MEFs, showing relative levels of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Saturation density analysis showing the effect of glutamine withdrawal on oncogenic-Dbl inducible MEFs. Dishes of uninduced (+Dox) and induced (−Dox) cells cultured in 4 mM glutamine, or in glutamine-free medium ±2 mM dimethyl α-ketoglutarate, were fixed and then stained with crystal violet. (e) Cell death analysis for uninduced or induced cells after 6 days culture in 4.0 or 0.2 mM glutamine. Data presented are the mean±s.d. of triplicate assays. (f) Cell proliferation assays showing the effect of glutamine depletion on proliferation of uninduced and induced MEFs over 6 days. Data presented are the mean±s.d. of triplicate assays. (g) Saturation density analysis showing the effect of the GLS inhibitor BPTES on oncogenic-Dbl induced MEFs. Induced (−Dox) cells cultured in the absence or presence of BPTES (30 or 40 μM)±2 mM dimethyl α-ketoglutarate were fixed and then stained with crystal violet. (h) BPTES dose curves showing the effect of different BPTES concentrations on proliferation over 6 days of uninduced or induced MEFs. Fractional proliferation relative to untreated cells is shown. Assays were carried out in 10% FBS medium, and data presented are the mean±s.d. of triplicate assays. (i) Anchorage-independent growth assay for uninduced (+Dox) cells, and for induced (−Dox) cells cultured under increasing BPTES concentrations. Data presented are the mean±s.d. of triplicate assays. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837472&req=5

f1: Glutamine-dependent transformation by oncogenic-Dbl.(a) Western blot analysis showing timecourse of oncogenic-Dbl expression in an inducible MEF system, and downstream elevation of GLS levels. Cells were induced by plating in doxycycline-free growth medium, and samples were collected at time-points up to 72 h. (b) Glutaminase activity assay using mitochondria isolated from MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox). 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 uninduced and induced (24 h) MEFs, showing relative levels of the GLS transcript. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (d) Saturation density analysis showing the effect of glutamine withdrawal on oncogenic-Dbl inducible MEFs. Dishes of uninduced (+Dox) and induced (−Dox) cells cultured in 4 mM glutamine, or in glutamine-free medium ±2 mM dimethyl α-ketoglutarate, were fixed and then stained with crystal violet. (e) Cell death analysis for uninduced or induced cells after 6 days culture in 4.0 or 0.2 mM glutamine. Data presented are the mean±s.d. of triplicate assays. (f) Cell proliferation assays showing the effect of glutamine depletion on proliferation of uninduced and induced MEFs over 6 days. Data presented are the mean±s.d. of triplicate assays. (g) Saturation density analysis showing the effect of the GLS inhibitor BPTES on oncogenic-Dbl induced MEFs. Induced (−Dox) cells cultured in the absence or presence of BPTES (30 or 40 μM)±2 mM dimethyl α-ketoglutarate were fixed and then stained with crystal violet. (h) BPTES dose curves showing the effect of different BPTES concentrations on proliferation over 6 days of uninduced or induced MEFs. Fractional proliferation relative to untreated cells is shown. Assays were carried out in 10% FBS medium, and data presented are the mean±s.d. of triplicate assays. (i) Anchorage-independent growth assay for uninduced (+Dox) cells, and for induced (−Dox) cells cultured under increasing BPTES concentrations. Data presented are the mean±s.d. of triplicate assays. Differences were analysed with Student's t-test. *P<0.05, **P<0.01.
Mentions: We previously reported that oncogenic-Dbl, a guanine nucleotide exchange factor and potent activator of Rho GTPases, signals to upregulate mitochondrial GLS activity in NIH/3T3 cells20. This is an essential event for maintaining Dbl-induced cellular transformation. To explore further the signalling connections that link Rho GTPases with GLS, we utilized an inducible, tetracycline-off, system to control the expression of oncogenic-Dbl in mouse embryonic fibroblasts (MEFs). When doxycycline (0.6 μg ml−1) is present in the culture medium, HA-tagged oncogenic-Dbl is undetectable by western blot analysis of whole-cell lysates. Removal of doxycycline triggers a robust expression of oncogenic-Dbl within 10 h that remains elevated through 72 h (Fig. 1a). This is accompanied by a corresponding increase in GLS protein levels, which peak 24–48 h following induction (Fig. 1a). We isolated mitochondria from uninduced and induced MEFs and assayed the preparations for glutaminase activity as described previously28. This confirmed that induction of oncogenic-Dbl results in elevated glutaminase activity (Fig. 1b). We then tested whether GLS was upregulated at the transcriptional level. Cells that were either uninduced or induced (24 h) were analysed by real-time PCR (RT–PCR), which revealed that induction of oncogenic-Dbl expression leads to an ∼12-fold increase of the GLS transcript (Fig. 1c).

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