Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of TR3 regulates sestrin2-AMPKα-mTORC1 through induction of p53 transcriptional activity. (A and B) Cells were transfected with the indicated siRNAs for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control and the experiment was repeated three times with similar results. (C) Cells were cotransfected with each siRNA and p53x14-Luc, 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<0.001 vs siScr. (D) ChIP assay. Cells were transfected with either siScr or siTR3 for 60 hr, and binding of p53 with the sestrin2 promoter region containing p53 binding site as indicated was determined as described in the Materials and Methods. The experiment was repeated three times with similar results. (E) Schematic diagram summarizing knockdown of TR3 inhibits cell growth through dual-targeting of mTORC1 signaling and Sp1 in human lung cancer cells expressing wild-type p53.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3299891&req=5

Figure 4: Knockdown of TR3 regulates sestrin2-AMPKα-mTORC1 through induction of p53 transcriptional activity. (A and B) Cells were transfected with the indicated siRNAs for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control and the experiment was repeated three times with similar results. (C) Cells were cotransfected with each siRNA and p53x14-Luc, 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<0.001 vs siScr. (D) ChIP assay. Cells were transfected with either siScr or siTR3 for 60 hr, and binding of p53 with the sestrin2 promoter region containing p53 binding site as indicated was determined as described in the Materials and Methods. The experiment was repeated three times with similar results. (E) Schematic diagram summarizing knockdown of TR3 inhibits cell growth through dual-targeting of mTORC1 signaling and Sp1 in human lung cancer cells expressing wild-type p53.

Mentions: p53 activates sestrin 2 (Budanov and Karin, 2008), and TR3 directly interacts with p53 and inhibits the transcriptional activity by blocking p53 acetylation (Budanov and Karin, 2008). Functional interactions between TR3 and p53 were examined in A549 cells (p53 wild-type) transfected with siTR3 and sip53 alone or in combination (Fig. 4A). Transfection with siTR3 induced sestrin 2 and only slightly increased p53 protein levels and affected phosphorylation of AMPKα, p70S6K and S6RP as indicated in Figures 2C, 3B and 3D. sip53 alone decreased p70S6K and S6RP phosphorylation and, in combination with siTR3, sip53 decreased the effects of siTR3 on sestrin 2 protein expression and phosphorylation of AMPKα, 70S6K and S6RP. In contrast, transfection of siTR3 into p53- H1299 lung cancer cells did not affect expression of sestrin 2 or the phospho-proteins associated with AMPKα-dependent inhibition of mTORC1 (Fig. 4B), whereas p53-independent inactivation of the TR3/p300/Sp1 complex by siTR3 resulted in downregulation of survivin and induction of apoptosis (Supplemental Fig. S4A). However, we observed that overexpression of p53 in H1299 cells cotransfected with siTR3 increased SESN2/phospho-AMPKα and inhibited mTORC1 signaling, whereas this was not observed in cells cotransfected with siTR3 overexpressing mutant p53 (Supplemental Fig. S4B). Inactivation of TR3 by RNA interference also significantly induced luciferase activity in cells transfected with a p53-responsive construct (Fig. 4C), and in A549 cells transfected with siTR3, results of a ChIP assay showed enhanced recruitment of p53 to the p53 response element in the sestrin 2 promoter (Fig. 4D). Supplemental Figure S5A confirms that both p53 and TR3 are coimmunoprecipitated in A549 and H460 cells as previously reported (Zhao et al., 2006). We also demonstrate that TR3 overexpression (a) decreased luciferase activity in A549 and H460 cells transfected with the p53-luc construct, (b) decreased p53 binding to the SESN2 promoter in a ChIP assay in H460 cells, and (c) decreased luciferase activity in H460 cells transfected with the SESN2-luc construct (Supplemental Figs. S5B, S5C and S5D). Thus, TR3 is not only required for basal expression of survivin and survival genes via a p300/TR3/Sp1 complex but also activates the mTORC1 pathway through inactivation of p53 (Fig. 4E), suggesting that agents or drugs that inactivate or block TR3 signaling will be highly effective as inhibitors of cancer cell and tumor growth and survival.


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 regulates sestrin2-AMPKα-mTORC1 through induction of p53 transcriptional activity. (A and B) Cells were transfected with the indicated siRNAs for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control and the experiment was repeated three times with similar results. (C) Cells were cotransfected with each siRNA and p53x14-Luc, 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<0.001 vs siScr. (D) ChIP assay. Cells were transfected with either siScr or siTR3 for 60 hr, and binding of p53 with the sestrin2 promoter region containing p53 binding site as indicated was determined as described in the Materials and Methods. The experiment was repeated three times with similar results. (E) Schematic diagram summarizing knockdown of TR3 inhibits cell growth through dual-targeting of mTORC1 signaling and Sp1 in human lung cancer cells expressing wild-type p53.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3299891&req=5

Figure 4: Knockdown of TR3 regulates sestrin2-AMPKα-mTORC1 through induction of p53 transcriptional activity. (A and B) Cells were transfected with the indicated siRNAs for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control and the experiment was repeated three times with similar results. (C) Cells were cotransfected with each siRNA and p53x14-Luc, 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<0.001 vs siScr. (D) ChIP assay. Cells were transfected with either siScr or siTR3 for 60 hr, and binding of p53 with the sestrin2 promoter region containing p53 binding site as indicated was determined as described in the Materials and Methods. The experiment was repeated three times with similar results. (E) Schematic diagram summarizing knockdown of TR3 inhibits cell growth through dual-targeting of mTORC1 signaling and Sp1 in human lung cancer cells expressing wild-type p53.
Mentions: p53 activates sestrin 2 (Budanov and Karin, 2008), and TR3 directly interacts with p53 and inhibits the transcriptional activity by blocking p53 acetylation (Budanov and Karin, 2008). Functional interactions between TR3 and p53 were examined in A549 cells (p53 wild-type) transfected with siTR3 and sip53 alone or in combination (Fig. 4A). Transfection with siTR3 induced sestrin 2 and only slightly increased p53 protein levels and affected phosphorylation of AMPKα, p70S6K and S6RP as indicated in Figures 2C, 3B and 3D. sip53 alone decreased p70S6K and S6RP phosphorylation and, in combination with siTR3, sip53 decreased the effects of siTR3 on sestrin 2 protein expression and phosphorylation of AMPKα, 70S6K and S6RP. In contrast, transfection of siTR3 into p53- H1299 lung cancer cells did not affect expression of sestrin 2 or the phospho-proteins associated with AMPKα-dependent inhibition of mTORC1 (Fig. 4B), whereas p53-independent inactivation of the TR3/p300/Sp1 complex by siTR3 resulted in downregulation of survivin and induction of apoptosis (Supplemental Fig. S4A). However, we observed that overexpression of p53 in H1299 cells cotransfected with siTR3 increased SESN2/phospho-AMPKα and inhibited mTORC1 signaling, whereas this was not observed in cells cotransfected with siTR3 overexpressing mutant p53 (Supplemental Fig. S4B). Inactivation of TR3 by RNA interference also significantly induced luciferase activity in cells transfected with a p53-responsive construct (Fig. 4C), and in A549 cells transfected with siTR3, results of a ChIP assay showed enhanced recruitment of p53 to the p53 response element in the sestrin 2 promoter (Fig. 4D). Supplemental Figure S5A confirms that both p53 and TR3 are coimmunoprecipitated in A549 and H460 cells as previously reported (Zhao et al., 2006). We also demonstrate that TR3 overexpression (a) decreased luciferase activity in A549 and H460 cells transfected with the p53-luc construct, (b) decreased p53 binding to the SESN2 promoter in a ChIP assay in H460 cells, and (c) decreased luciferase activity in H460 cells transfected with the SESN2-luc construct (Supplemental Figs. S5B, S5C and S5D). Thus, TR3 is not only required for basal expression of survivin and survival genes via a p300/TR3/Sp1 complex but also activates the mTORC1 pathway through inactivation of p53 (Fig. 4E), suggesting that agents or drugs that inactivate or block TR3 signaling will be highly effective as inhibitors of cancer cell and tumor growth and survival.

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