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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

Oncogenic-Dbl potently activates the MKK4-JNK-c-Jun signalling axis in MEFs.Western blot analysis of whole-cell lysates of MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox) under low-serum (0.5% FBS) conditions. The extent of activation of different signalling pathways was assessed using phospho-specific antibodies that recognize activated kinases or their downstream phosphorylation targets (left panels). Any changes in total protein levels were also assessed (right panels). (a) The MAPK signalling axis involving MKK4-JNK-c-Jun is potently activated downstream of oncogenic-Dbl. Total c-Jun levels are also elevated, consistent with previous reports showing auto-regulation of JUN expression. (b) The MAPK p38 is moderately activated, whereas ERK is only slightly activated on oncogenic-Dbl induction. (c) ROCK activity, as read-out by MLC 2 phosphorylation, is activated on induction. (d) Akt is slightly inhibited, and AMPK and mTORC1 activity (the latter read-out by S6K phosphorylation) are largely unaffected by oncogenic-Dbl induction. Relative densitometry data are the mean±s.d. of triplicate blots.
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f2: Oncogenic-Dbl potently activates the MKK4-JNK-c-Jun signalling axis in MEFs.Western blot analysis of whole-cell lysates of MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox) under low-serum (0.5% FBS) conditions. The extent of activation of different signalling pathways was assessed using phospho-specific antibodies that recognize activated kinases or their downstream phosphorylation targets (left panels). Any changes in total protein levels were also assessed (right panels). (a) The MAPK signalling axis involving MKK4-JNK-c-Jun is potently activated downstream of oncogenic-Dbl. Total c-Jun levels are also elevated, consistent with previous reports showing auto-regulation of JUN expression. (b) The MAPK p38 is moderately activated, whereas ERK is only slightly activated on oncogenic-Dbl induction. (c) ROCK activity, as read-out by MLC 2 phosphorylation, is activated on induction. (d) Akt is slightly inhibited, and AMPK and mTORC1 activity (the latter read-out by S6K phosphorylation) are largely unaffected by oncogenic-Dbl induction. Relative densitometry data are the mean±s.d. of triplicate blots.

Mentions: The results above show that oncogenic-Dbl signals to upregulate GLS at the transcript and protein level, and that cellular transformation driven by oncogenic-Dbl is dependent on GLS activity and an exogenous supply of glutamine. We next wanted to identify the signalling pathway downstream of the Rho GTPases that was responsible for regulating GLS expression. We first used phospho-specific antibodies to assess the effects of oncogenic-Dbl induction on key cellular signalling proteins. Here, and throughout the study, low-serum conditions (0.5% FBS) were used for signalling experiments to minimize ‘background' cellular signalling activity. Western blot analysis of whole-cell lysates revealed an extremely potent activation of the MKK4/c-Jun N-terminal kinase (JNK) signalling axis on induction (24 h), and downstream activating phosphorylation of the oncogenic transcription factor c-Jun (Fig. 2a). c-Jun is known to autoregulate its own gene expression in MEFs2930 and, consistent with this, we found that total c-Jun levels increased as well as phosphorylated c-Jun levels (2-fold and 26-fold, respectively) (Fig. 2a and Supplementary Fig. 2a). The JNK kinase MKK7 and the Jun-family members JunB and JunD were expressed at low levels, and phosphorylated forms could not be detected (Supplementary Fig. 2b,c). Another target of MKK4, p38, was phosphorylated downstream of oncogenic-Dbl, whereas the activation status of ERK showed only a subtle change (Fig. 2b). Rho-associated coiled-coil containing protein kinase (ROCK) was likely activated, as indicated by increased phosphorylation of its substrate myosin light chain 2 (MLC 2) (Fig. 2c). Consistent with earlier reports that ROCK signals to inhibit Akt31, a modest decrease in Akt phosphorylation at residue T308 occurred on induction (Fig. 2d), and this could be rescued by inhibition of ROCK (Supplementary Fig. 2d). The mTORC1-S6K signalling axis showed little change on oncogenic-Dbl induction, and we also detected no change in the phosphorylation level of AMPKα (Fig. 2d).


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)

Oncogenic-Dbl potently activates the MKK4-JNK-c-Jun signalling axis in MEFs.Western blot analysis of whole-cell lysates of MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox) under low-serum (0.5% FBS) conditions. The extent of activation of different signalling pathways was assessed using phospho-specific antibodies that recognize activated kinases or their downstream phosphorylation targets (left panels). Any changes in total protein levels were also assessed (right panels). (a) The MAPK signalling axis involving MKK4-JNK-c-Jun is potently activated downstream of oncogenic-Dbl. Total c-Jun levels are also elevated, consistent with previous reports showing auto-regulation of JUN expression. (b) The MAPK p38 is moderately activated, whereas ERK is only slightly activated on oncogenic-Dbl induction. (c) ROCK activity, as read-out by MLC 2 phosphorylation, is activated on induction. (d) Akt is slightly inhibited, and AMPK and mTORC1 activity (the latter read-out by S6K phosphorylation) are largely unaffected by oncogenic-Dbl induction. Relative densitometry data are the mean±s.d. of triplicate blots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Oncogenic-Dbl potently activates the MKK4-JNK-c-Jun signalling axis in MEFs.Western blot analysis of whole-cell lysates of MEFs in which oncogenic-Dbl expression was either uninduced (+Dox) or induced for 24 h (−Dox) under low-serum (0.5% FBS) conditions. The extent of activation of different signalling pathways was assessed using phospho-specific antibodies that recognize activated kinases or their downstream phosphorylation targets (left panels). Any changes in total protein levels were also assessed (right panels). (a) The MAPK signalling axis involving MKK4-JNK-c-Jun is potently activated downstream of oncogenic-Dbl. Total c-Jun levels are also elevated, consistent with previous reports showing auto-regulation of JUN expression. (b) The MAPK p38 is moderately activated, whereas ERK is only slightly activated on oncogenic-Dbl induction. (c) ROCK activity, as read-out by MLC 2 phosphorylation, is activated on induction. (d) Akt is slightly inhibited, and AMPK and mTORC1 activity (the latter read-out by S6K phosphorylation) are largely unaffected by oncogenic-Dbl induction. Relative densitometry data are the mean±s.d. of triplicate blots.
Mentions: The results above show that oncogenic-Dbl signals to upregulate GLS at the transcript and protein level, and that cellular transformation driven by oncogenic-Dbl is dependent on GLS activity and an exogenous supply of glutamine. We next wanted to identify the signalling pathway downstream of the Rho GTPases that was responsible for regulating GLS expression. We first used phospho-specific antibodies to assess the effects of oncogenic-Dbl induction on key cellular signalling proteins. Here, and throughout the study, low-serum conditions (0.5% FBS) were used for signalling experiments to minimize ‘background' cellular signalling activity. Western blot analysis of whole-cell lysates revealed an extremely potent activation of the MKK4/c-Jun N-terminal kinase (JNK) signalling axis on induction (24 h), and downstream activating phosphorylation of the oncogenic transcription factor c-Jun (Fig. 2a). c-Jun is known to autoregulate its own gene expression in MEFs2930 and, consistent with this, we found that total c-Jun levels increased as well as phosphorylated c-Jun levels (2-fold and 26-fold, respectively) (Fig. 2a and Supplementary Fig. 2a). The JNK kinase MKK7 and the Jun-family members JunB and JunD were expressed at low levels, and phosphorylated forms could not be detected (Supplementary Fig. 2b,c). Another target of MKK4, p38, was phosphorylated downstream of oncogenic-Dbl, whereas the activation status of ERK showed only a subtle change (Fig. 2b). Rho-associated coiled-coil containing protein kinase (ROCK) was likely activated, as indicated by increased phosphorylation of its substrate myosin light chain 2 (MLC 2) (Fig. 2c). Consistent with earlier reports that ROCK signals to inhibit Akt31, a modest decrease in Akt phosphorylation at residue T308 occurred on induction (Fig. 2d), and this could be rescued by inhibition of ROCK (Supplementary Fig. 2d). The mTORC1-S6K signalling axis showed little change on oncogenic-Dbl induction, and we also detected no change in the phosphorylation level of AMPKα (Fig. 2d).

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