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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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DIM-C-pPhOH inhibits tumor growth and lung metastasis in vivo. (A and B) The orthotopic mouse model of lung cancer. A549 cells were orthotopically implanted into athymic nude mice, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks starting 7 days after implantation. Median tumor weights and volumes (A, left panel) were calculated as described in the Materials and Methods. The data are presented as means with SD (n=10 per group). *P<0.05 and #P<0.001 vs control group. (A, right panel) TUNEL staining. Tumor sections were stained using the DeadEnd colorimetric kit as described in Materials and Methods. The apoptotic tumor cells are stained. Images were collected at high (×100) magnification. (B) Protein expression in tumor lysates. Tumor lysates from tumor samples were further analyzed by western blot analysis, and β-actin was used as a loading control. (C and D) The metastatic mouse model of lung cancer. A549 (2 × 106) cells were inoculated into athymic nude mice via the tail vein for 4 weeks before treatment, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks. (C) Lung micrographs show development of multiple tumor foci (arrows). (D) Metastatic tumor weight, volume, and burden were calculated.
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Figure 6: DIM-C-pPhOH inhibits tumor growth and lung metastasis in vivo. (A and B) The orthotopic mouse model of lung cancer. A549 cells were orthotopically implanted into athymic nude mice, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks starting 7 days after implantation. Median tumor weights and volumes (A, left panel) were calculated as described in the Materials and Methods. The data are presented as means with SD (n=10 per group). *P<0.05 and #P<0.001 vs control group. (A, right panel) TUNEL staining. Tumor sections were stained using the DeadEnd colorimetric kit as described in Materials and Methods. The apoptotic tumor cells are stained. Images were collected at high (×100) magnification. (B) Protein expression in tumor lysates. Tumor lysates from tumor samples were further analyzed by western blot analysis, and β-actin was used as a loading control. (C and D) The metastatic mouse model of lung cancer. A549 (2 × 106) cells were inoculated into athymic nude mice via the tail vein for 4 weeks before treatment, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks. (C) Lung micrographs show development of multiple tumor foci (arrows). (D) Metastatic tumor weight, volume, and burden were calculated.

Mentions: The effects of DIM-C-pPhOH on the growth of orthotopic lung tumors derived from A549 cells was carried out to complement the in vitro studies with siTR3 and DIM-C-pPhOH in lung cancer cells (Figs. 2-5). DIM-C-pPhOH (30 mg/kg/d) decreased lung tumor weights and volumes and this was accompanied by increased apoptosis (TUNEL staining) in the tumors from animals treated with DIM-C-pPhOH compared to tumors from the control (corn oil) mice (Fig. 6A and Supplemental Table S3). Treatment with DIM-C-pPhOH also decreased survivin and increased cleavage of caspases 3 and 7 and PARP (Fig. 6B) which is associated with inactivation of the p300/TR3/Sp1 complex (Figs. 2, S2 and S3). DIM-C-pPhOH also inhibited mTORC1 signaling through activation of sestrin 2 and AMPKα and this was accompanied by decreased phosphorylation of 4E-BP1 and p7056K (Fig. 6C). The effects of DIM-C-pPhOH (30 mg/kg/d) were also investigated in a metastatic mouse model for lung cancer where cells were introduced by tail vein injection (Figs. 6C and 6D). In this study, DIM-C-pPhOH also decreased tumor weights and volumes and tumor burden (Fig. 6D and Supplemental Table S4). These data clearly demonstrate that in vivo deactivation of TR3 by DIM-C-pPhOH results in tumor growth inhibition by inhibiting at least two TR3-mediated pro-oncogenic pathways (Fig. 4E).


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)

DIM-C-pPhOH inhibits tumor growth and lung metastasis in vivo. (A and B) The orthotopic mouse model of lung cancer. A549 cells were orthotopically implanted into athymic nude mice, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks starting 7 days after implantation. Median tumor weights and volumes (A, left panel) were calculated as described in the Materials and Methods. The data are presented as means with SD (n=10 per group). *P<0.05 and #P<0.001 vs control group. (A, right panel) TUNEL staining. Tumor sections were stained using the DeadEnd colorimetric kit as described in Materials and Methods. The apoptotic tumor cells are stained. Images were collected at high (×100) magnification. (B) Protein expression in tumor lysates. Tumor lysates from tumor samples were further analyzed by western blot analysis, and β-actin was used as a loading control. (C and D) The metastatic mouse model of lung cancer. A549 (2 × 106) cells were inoculated into athymic nude mice via the tail vein for 4 weeks before treatment, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks. (C) Lung micrographs show development of multiple tumor foci (arrows). (D) Metastatic tumor weight, volume, and burden were calculated.
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Figure 6: DIM-C-pPhOH inhibits tumor growth and lung metastasis in vivo. (A and B) The orthotopic mouse model of lung cancer. A549 cells were orthotopically implanted into athymic nude mice, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks starting 7 days after implantation. Median tumor weights and volumes (A, left panel) were calculated as described in the Materials and Methods. The data are presented as means with SD (n=10 per group). *P<0.05 and #P<0.001 vs control group. (A, right panel) TUNEL staining. Tumor sections were stained using the DeadEnd colorimetric kit as described in Materials and Methods. The apoptotic tumor cells are stained. Images were collected at high (×100) magnification. (B) Protein expression in tumor lysates. Tumor lysates from tumor samples were further analyzed by western blot analysis, and β-actin was used as a loading control. (C and D) The metastatic mouse model of lung cancer. A549 (2 × 106) cells were inoculated into athymic nude mice via the tail vein for 4 weeks before treatment, and each mouse was dosed 3 times a week by oral gavage with either corn oil (control) or DIM-C-pPhOH (30 mg/kg/day) for 4 weeks. (C) Lung micrographs show development of multiple tumor foci (arrows). (D) Metastatic tumor weight, volume, and burden were calculated.
Mentions: The effects of DIM-C-pPhOH on the growth of orthotopic lung tumors derived from A549 cells was carried out to complement the in vitro studies with siTR3 and DIM-C-pPhOH in lung cancer cells (Figs. 2-5). DIM-C-pPhOH (30 mg/kg/d) decreased lung tumor weights and volumes and this was accompanied by increased apoptosis (TUNEL staining) in the tumors from animals treated with DIM-C-pPhOH compared to tumors from the control (corn oil) mice (Fig. 6A and Supplemental Table S3). Treatment with DIM-C-pPhOH also decreased survivin and increased cleavage of caspases 3 and 7 and PARP (Fig. 6B) which is associated with inactivation of the p300/TR3/Sp1 complex (Figs. 2, S2 and S3). DIM-C-pPhOH also inhibited mTORC1 signaling through activation of sestrin 2 and AMPKα and this was accompanied by decreased phosphorylation of 4E-BP1 and p7056K (Fig. 6C). The effects of DIM-C-pPhOH (30 mg/kg/d) were also investigated in a metastatic mouse model for lung cancer where cells were introduced by tail vein injection (Figs. 6C and 6D). In this study, DIM-C-pPhOH also decreased tumor weights and volumes and tumor burden (Fig. 6D and Supplemental Table S4). These data clearly demonstrate that in vivo deactivation of TR3 by DIM-C-pPhOH results in tumor growth inhibition by inhibiting at least two TR3-mediated pro-oncogenic pathways (Fig. 4E).

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