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The nuclear receptor TR3 regulates mTORC1 signaling in lung cancer cells expressing wild-type p53.

Lee SO, Andey T, Jin UH, Kim K, Singh M, Sachdeva M, Safe S - Oncogene (2011)

Bottom Line: The orphan nuclear receptor TR3 (NR41A and Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival.The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/ specificity protein 1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1).However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway, and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2, which subsequently activated the mTORC1 inhibitor AMP-activated protein kinase α (AMPKα) (mechanism 2).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioscience and Technology, Texas A&M Health Science Center, Houston, TX 77843-4466, USA.

ABSTRACT
The orphan nuclear receptor TR3 (NR41A and Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival. The function of TR3 was investigated in non-small-cell lung cancer A549 and H460 cells, and knockdown of TR3 by RNA interference (siTR3) inhibited cancer cell growth and induced apoptosis. The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/ specificity protein 1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1). However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway, and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2, which subsequently activated the mTORC1 inhibitor AMP-activated protein kinase α (AMPKα) (mechanism 2). This demonstrates that the pro-oncogenic activity of TR3 in lung cancer cells was due to inhibition of p53 and activation of mTORC1. 1,1-Bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) is a recently discovered inhibitor of TR3, which mimics the effects of siTR3. DIM-C-pPhOH inhibited growth and induced apoptosis in lung cancer cells and lung tumors in murine orthotopic and metastatic models, and this was accompanied by decreased expression of survivin and inhibition of mTORC1 signaling, demonstrating that inactivators of TR3 represent a novel class of mTORC1 inhibitors.

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Knockdown of TR3 inhibits mTORC1 signaling through sestrin2-dependent but LKB1-independent activation of AMPKα in NSCLC cells. (A, B, and D) Cells were transfected with indicated siRNAs for 72 hr, and whole cell lysates were analyzed by Western blot analysis. (C) Cells were transfected with either siScr or siTR3 for 72 hr (left panel) or for 60 hr (middle panel), and sestrin2 protein and mRNA levels were determined by western blot analysis and real-time PCR, respectively, as described in the Materials and Methods. β-Actin was used as a loading control and TBP was used as an internal control. Sestrin2 and TR3 mRNA levels are presented as means with SD of 3 experiments. #P<0.001 vs siScr. (C, right panel) Cells were cotransfected with each siRNA and pSESN2-Luc (−730/+190), and luciferase activity (relative to β-galactosidase activity) was determined. The corresponding empty vector was used as a control, and the results are presented as means with SD of 3 experiments. *P<0.005 and #P<0.001 vs siScr.
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Figure 3: Knockdown of TR3 inhibits mTORC1 signaling through sestrin2-dependent but LKB1-independent activation of AMPKα in NSCLC cells. (A, B, and D) Cells were transfected with indicated siRNAs for 72 hr, and whole cell lysates were analyzed by Western blot analysis. (C) Cells were transfected with either siScr or siTR3 for 72 hr (left panel) or for 60 hr (middle panel), and sestrin2 protein and mRNA levels were determined by western blot analysis and real-time PCR, respectively, as described in the Materials and Methods. β-Actin was used as a loading control and TBP was used as an internal control. Sestrin2 and TR3 mRNA levels are presented as means with SD of 3 experiments. #P<0.001 vs siScr. (C, right panel) Cells were cotransfected with each siRNA and pSESN2-Luc (−730/+190), and luciferase activity (relative to β-galactosidase activity) was determined. The corresponding empty vector was used as a control, and the results are presented as means with SD of 3 experiments. *P<0.005 and #P<0.001 vs siScr.

Mentions: Phosphorylated AKT and AMPKα are potential upstream activators and deactivators, respectively, of mTORC1 and transfection of siTR3 into A549 and A460 cells did not affect expression of phospho-AKT, whereas there was an increase in phospho-AMPKα in both cell lines (Fig. 3A). Moreover, in A549 cells transfected with siTR3, siTR3 increased phosphorylation of AMPKα and decreased phosphorylation of 4E-BP1 and p70S6K , whereas knockdown of AMPKα alone had minimal effects on phospho-p70S6K or 4E-BP1 (Figures 2C and 3A). However, siAMPKα inhibited the effects of siTR3 on phosphorylation of 70S6K and 4E-BP1 confirming that inactivation of TR3 resulted in activation of AMPKα and inhibition of the mTORC1 pathway.


The nuclear receptor TR3 regulates mTORC1 signaling in lung cancer cells expressing wild-type p53.

Lee SO, Andey T, Jin UH, Kim K, Singh M, Sachdeva M, Safe S - Oncogene (2011)

Knockdown of TR3 inhibits mTORC1 signaling through sestrin2-dependent but LKB1-independent activation of AMPKα in NSCLC cells. (A, B, and D) Cells were transfected with indicated siRNAs for 72 hr, and whole cell lysates were analyzed by Western blot analysis. (C) Cells were transfected with either siScr or siTR3 for 72 hr (left panel) or for 60 hr (middle panel), and sestrin2 protein and mRNA levels were determined by western blot analysis and real-time PCR, respectively, as described in the Materials and Methods. β-Actin was used as a loading control and TBP was used as an internal control. Sestrin2 and TR3 mRNA levels are presented as means with SD of 3 experiments. #P<0.001 vs siScr. (C, right panel) Cells were cotransfected with each siRNA and pSESN2-Luc (−730/+190), and luciferase activity (relative to β-galactosidase activity) was determined. The corresponding empty vector was used as a control, and the results are presented as means with SD of 3 experiments. *P<0.005 and #P<0.001 vs siScr.
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Related In: Results  -  Collection

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Figure 3: Knockdown of TR3 inhibits mTORC1 signaling through sestrin2-dependent but LKB1-independent activation of AMPKα in NSCLC cells. (A, B, and D) Cells were transfected with indicated siRNAs for 72 hr, and whole cell lysates were analyzed by Western blot analysis. (C) Cells were transfected with either siScr or siTR3 for 72 hr (left panel) or for 60 hr (middle panel), and sestrin2 protein and mRNA levels were determined by western blot analysis and real-time PCR, respectively, as described in the Materials and Methods. β-Actin was used as a loading control and TBP was used as an internal control. Sestrin2 and TR3 mRNA levels are presented as means with SD of 3 experiments. #P<0.001 vs siScr. (C, right panel) Cells were cotransfected with each siRNA and pSESN2-Luc (−730/+190), and luciferase activity (relative to β-galactosidase activity) was determined. The corresponding empty vector was used as a control, and the results are presented as means with SD of 3 experiments. *P<0.005 and #P<0.001 vs siScr.
Mentions: Phosphorylated AKT and AMPKα are potential upstream activators and deactivators, respectively, of mTORC1 and transfection of siTR3 into A549 and A460 cells did not affect expression of phospho-AKT, whereas there was an increase in phospho-AMPKα in both cell lines (Fig. 3A). Moreover, in A549 cells transfected with siTR3, siTR3 increased phosphorylation of AMPKα and decreased phosphorylation of 4E-BP1 and p70S6K , whereas knockdown of AMPKα alone had minimal effects on phospho-p70S6K or 4E-BP1 (Figures 2C and 3A). However, siAMPKα inhibited the effects of siTR3 on phosphorylation of 70S6K and 4E-BP1 confirming that inactivation of TR3 resulted in activation of AMPKα and inhibition of the mTORC1 pathway.

Bottom Line: The orphan nuclear receptor TR3 (NR41A and Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival.The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/ specificity protein 1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1).However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway, and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2, which subsequently activated the mTORC1 inhibitor AMP-activated protein kinase α (AMPKα) (mechanism 2).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioscience and Technology, Texas A&M Health Science Center, Houston, TX 77843-4466, USA.

ABSTRACT
The orphan nuclear receptor TR3 (NR41A and Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival. The function of TR3 was investigated in non-small-cell lung cancer A549 and H460 cells, and knockdown of TR3 by RNA interference (siTR3) inhibited cancer cell growth and induced apoptosis. The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/ specificity protein 1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1). However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway, and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2, which subsequently activated the mTORC1 inhibitor AMP-activated protein kinase α (AMPKα) (mechanism 2). This demonstrates that the pro-oncogenic activity of TR3 in lung cancer cells was due to inhibition of p53 and activation of mTORC1. 1,1-Bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) is a recently discovered inhibitor of TR3, which mimics the effects of siTR3. DIM-C-pPhOH inhibited growth and induced apoptosis in lung cancer cells and lung tumors in murine orthotopic and metastatic models, and this was accompanied by decreased expression of survivin and inhibition of mTORC1 signaling, demonstrating that inactivators of TR3 represent a novel class of mTORC1 inhibitors.

Show MeSH
Related in: MedlinePlus