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

Oncogenic-Dbl signals to c-Jun to upregulate GLS in MEFs.(a) Western blot analysis of whole-cell lysates of uninduced (+Dox) or induced for 48 h (−Dox) MEFs±10 μM selective inhibitors of ROCK (Y-27632), p38 (SB203580) or JNK (SP600125). Inhibition of JNK largely blocks the upregulation of GLS downstream of oncogenic-Dbl. (b) Western blot analysis showing that inhibition of mTORC1 by rapamycin has little effect on the upregulation of GLS downstream of oncogenic-Dbl. (c) Glutaminase activity assay using mitochondria isolated from uninduced (+Dox) MEFs and from MEFs that had been induced for 24 h (−Dox) in the absence or presence of 10 μM SP600125 (JNK inhibitor). Activity is expressed per mg of total cellular protein and data presented are the mean±s.d. of triplicate assays. (d) RT–PCR analysis of uninduced (+Dox) and 24 h induced (−Dox) MEFs±10 μM SP600125, 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. (e) Western blot analysis showing that the AP-1 inhibitor SR11302 (10 μM) completely blocks GLS expression in oncogenic-Dbl-induced MEFs. (f) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with constitutively activated JNK/MKK fusion constructs leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (g) Western blot analysis of the samples from the previous panel, showing that ectopic expression of constitutively activated JNK fusion constructs in uninduced MEFs leads to increased phosphorylation of c-Jun and upregulated GLS protein levels. (h) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with a construct for expressing the JUN proto-oncogene leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (i) Western blot analysis of the samples from the previous panel, showing that ectopic expression of JUN in uninduced MEFs leads to increased levels of phospho-c-Jun and upregulated GLS protein levels. Note that the ectopically expressed c-Jun contains a V5-tag, and consequently runs at a slightly higher molecular weight than endogenous c-Jun. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Oncogenic-Dbl signals to c-Jun to upregulate GLS in MEFs.(a) Western blot analysis of whole-cell lysates of uninduced (+Dox) or induced for 48 h (−Dox) MEFs±10 μM selective inhibitors of ROCK (Y-27632), p38 (SB203580) or JNK (SP600125). Inhibition of JNK largely blocks the upregulation of GLS downstream of oncogenic-Dbl. (b) Western blot analysis showing that inhibition of mTORC1 by rapamycin has little effect on the upregulation of GLS downstream of oncogenic-Dbl. (c) Glutaminase activity assay using mitochondria isolated from uninduced (+Dox) MEFs and from MEFs that had been induced for 24 h (−Dox) in the absence or presence of 10 μM SP600125 (JNK inhibitor). Activity is expressed per mg of total cellular protein and data presented are the mean±s.d. of triplicate assays. (d) RT–PCR analysis of uninduced (+Dox) and 24 h induced (−Dox) MEFs±10 μM SP600125, 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. (e) Western blot analysis showing that the AP-1 inhibitor SR11302 (10 μM) completely blocks GLS expression in oncogenic-Dbl-induced MEFs. (f) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with constitutively activated JNK/MKK fusion constructs leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (g) Western blot analysis of the samples from the previous panel, showing that ectopic expression of constitutively activated JNK fusion constructs in uninduced MEFs leads to increased phosphorylation of c-Jun and upregulated GLS protein levels. (h) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with a construct for expressing the JUN proto-oncogene leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (i) Western blot analysis of the samples from the previous panel, showing that ectopic expression of JUN in uninduced MEFs leads to increased levels of phospho-c-Jun and upregulated GLS protein levels. Note that the ectopically expressed c-Jun contains a V5-tag, and consequently runs at a slightly higher molecular weight than endogenous c-Jun. 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: Since ROCK, p38 and JNK signalling were all activated downstream of oncogenic-Dbl, we tested whether selective inhibition of these kinases (by the small-molecule inhibitors Y-27632, SB203580 and SP600125, respectively) impacted the ability of oncogenic-Dbl to upregulate GLS. Cells were induced for 48 h, either in the absence or presence of 10 μM of each inhibitor, and whole-cell lysates were then analysed by western blot. Treatment of induced cells with the ROCK inhibitor Y-27632 or the p38 inhibitor SB203580 did not significantly impact GLS levels, whereas the JNK inhibitor SP600125 largely blocked the upregulation of GLS (Fig. 3a). Although rapamycin-sensitive mTORC1 can influence GLS levels by increasing MYC translation32, we found that regulation of GLS downstream of the Rho GTPases was independent of mTORC1 activity (Fig. 3b), and changes in c-Myc (and phospho-c-Myc) levels did not fully correspond to changes in GLS (Supplementary Fig. 2e). Treatment of induced cells with the c-Myc inhibitor 10058-F4 severely impacted viability in low-serum (0.5% FBS) culture medium (no viable cells remained after 24 h treatment with 10 μM 10058-F4), and it is possible that c-Myc contributes to the regulation of GLS expression by Rho GTPases under these conditions. In high-serum (10% FBS) medium, treatment with 10058-F4 at concentrations up to 60 μM had little impact on GLS levels in induced cells (Supplementary Fig. 2f), whereas inhibition of JNK by 10 μM SP600125 still suppressed GLS expression (Supplementary Fig. 2e).


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 signals to c-Jun to upregulate GLS in MEFs.(a) Western blot analysis of whole-cell lysates of uninduced (+Dox) or induced for 48 h (−Dox) MEFs±10 μM selective inhibitors of ROCK (Y-27632), p38 (SB203580) or JNK (SP600125). Inhibition of JNK largely blocks the upregulation of GLS downstream of oncogenic-Dbl. (b) Western blot analysis showing that inhibition of mTORC1 by rapamycin has little effect on the upregulation of GLS downstream of oncogenic-Dbl. (c) Glutaminase activity assay using mitochondria isolated from uninduced (+Dox) MEFs and from MEFs that had been induced for 24 h (−Dox) in the absence or presence of 10 μM SP600125 (JNK inhibitor). Activity is expressed per mg of total cellular protein and data presented are the mean±s.d. of triplicate assays. (d) RT–PCR analysis of uninduced (+Dox) and 24 h induced (−Dox) MEFs±10 μM SP600125, 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. (e) Western blot analysis showing that the AP-1 inhibitor SR11302 (10 μM) completely blocks GLS expression in oncogenic-Dbl-induced MEFs. (f) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with constitutively activated JNK/MKK fusion constructs leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (g) Western blot analysis of the samples from the previous panel, showing that ectopic expression of constitutively activated JNK fusion constructs in uninduced MEFs leads to increased phosphorylation of c-Jun and upregulated GLS protein levels. (h) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with a construct for expressing the JUN proto-oncogene leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (i) Western blot analysis of the samples from the previous panel, showing that ectopic expression of JUN in uninduced MEFs leads to increased levels of phospho-c-Jun and upregulated GLS protein levels. Note that the ectopically expressed c-Jun contains a V5-tag, and consequently runs at a slightly higher molecular weight than endogenous c-Jun. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Oncogenic-Dbl signals to c-Jun to upregulate GLS in MEFs.(a) Western blot analysis of whole-cell lysates of uninduced (+Dox) or induced for 48 h (−Dox) MEFs±10 μM selective inhibitors of ROCK (Y-27632), p38 (SB203580) or JNK (SP600125). Inhibition of JNK largely blocks the upregulation of GLS downstream of oncogenic-Dbl. (b) Western blot analysis showing that inhibition of mTORC1 by rapamycin has little effect on the upregulation of GLS downstream of oncogenic-Dbl. (c) Glutaminase activity assay using mitochondria isolated from uninduced (+Dox) MEFs and from MEFs that had been induced for 24 h (−Dox) in the absence or presence of 10 μM SP600125 (JNK inhibitor). Activity is expressed per mg of total cellular protein and data presented are the mean±s.d. of triplicate assays. (d) RT–PCR analysis of uninduced (+Dox) and 24 h induced (−Dox) MEFs±10 μM SP600125, 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. (e) Western blot analysis showing that the AP-1 inhibitor SR11302 (10 μM) completely blocks GLS expression in oncogenic-Dbl-induced MEFs. (f) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with constitutively activated JNK/MKK fusion constructs leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (g) Western blot analysis of the samples from the previous panel, showing that ectopic expression of constitutively activated JNK fusion constructs in uninduced MEFs leads to increased phosphorylation of c-Jun and upregulated GLS protein levels. (h) RT–PCR analysis showing that transient transfection of uninduced (+Dox) MEFs with a construct for expressing the JUN proto-oncogene leads to increased GLS mRNA abundance. The data presented are the RQ values, with error bars marking RQ max and RQ min, from triplicate reactions. (i) Western blot analysis of the samples from the previous panel, showing that ectopic expression of JUN in uninduced MEFs leads to increased levels of phospho-c-Jun and upregulated GLS protein levels. Note that the ectopically expressed c-Jun contains a V5-tag, and consequently runs at a slightly higher molecular weight than endogenous c-Jun. 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: Since ROCK, p38 and JNK signalling were all activated downstream of oncogenic-Dbl, we tested whether selective inhibition of these kinases (by the small-molecule inhibitors Y-27632, SB203580 and SP600125, respectively) impacted the ability of oncogenic-Dbl to upregulate GLS. Cells were induced for 48 h, either in the absence or presence of 10 μM of each inhibitor, and whole-cell lysates were then analysed by western blot. Treatment of induced cells with the ROCK inhibitor Y-27632 or the p38 inhibitor SB203580 did not significantly impact GLS levels, whereas the JNK inhibitor SP600125 largely blocked the upregulation of GLS (Fig. 3a). Although rapamycin-sensitive mTORC1 can influence GLS levels by increasing MYC translation32, we found that regulation of GLS downstream of the Rho GTPases was independent of mTORC1 activity (Fig. 3b), and changes in c-Myc (and phospho-c-Myc) levels did not fully correspond to changes in GLS (Supplementary Fig. 2e). Treatment of induced cells with the c-Myc inhibitor 10058-F4 severely impacted viability in low-serum (0.5% FBS) culture medium (no viable cells remained after 24 h treatment with 10 μM 10058-F4), and it is possible that c-Myc contributes to the regulation of GLS expression by Rho GTPases under these conditions. In high-serum (10% FBS) medium, treatment with 10058-F4 at concentrations up to 60 μM had little impact on GLS levels in induced cells (Supplementary Fig. 2f), whereas inhibition of JNK by 10 μM SP600125 still suppressed GLS expression (Supplementary Fig. 2e).

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