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Nuclear Receptor Expression and Function in Human Lung Cancer Pathogenesis.

Kim J, Sato M, Choi JW, Kim HW, Yeh BI, Larsen JE, Minna JD, Cha JH, Jeong Y - PLoS ONE (2015)

Bottom Line: Notably, PPARγ activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs.In fully tumorigenic HBECs with inducible expression of PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner.Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do, Republic of Korea; Institute of Lifestyle Medicine, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do, Republic of Korea; Nuclear Receptor Research Consortium, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do, Republic of Korea.

ABSTRACT
Lung cancer is caused by combinations of diverse genetic mutations. Here, to understand the relevance of nuclear receptors (NRs) in the oncogene-associated lung cancer pathogenesis, we investigated the expression profile of the entire 48 NR members by using QPCR analysis in a panel of human bronchial epithelial cells (HBECs) that included precancerous and tumorigenic HBECs harboring oncogenic K-rasV12 and/or p53 alterations. The analysis of the profile revealed that oncogenic alterations accompanied transcriptional changes in the expression of 19 NRs in precancerous HBECs and 15 NRs according to the malignant progression of HBECs. Amongst these, peroxisome proliferator-activated receptor gamma (PPARγ), a NR chosen as a proof-of-principle study, showed increased expression in precancerous HBECs, which was surprisingly reversed when these HBECs acquired full in vivo tumorigenicity. Notably, PPARγ activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs. In fully tumorigenic HBECs with inducible expression of PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner. Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression. This suggests that ligand-mediated sumoylation of PPARγ plays an important role in lung cancer pathogenesis by modulating prostaglandin metabolism.

No MeSH data available.


Related in: MedlinePlus

Characterization of wildtype and sumoylation mutant PPARγ.(A) Luciferase reporter assay of wildtype and sumoylation mutant PPARγ plasmids. RLU, relative luciferase unit. (B) Tetracycline-induced expression of PPARγ and EGFP protein in stably transfected HBEC-C1-wt-PPARγ clone. A microscopic view of tetracycline-induced EFGP expression (top in B). Immunoblot assays for the expression of lamin A/C and tetracycline-induced PPARγ (bottom in B). A bicistronic construct of PPARγ and EGFP was stably introduced into a tumorigenic HBEC clone to generate HBEC-C1-PPARγ cell lines as described in Materials and Methods. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.001 compared to HBEC-KT control.
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pone.0134842.g006: Characterization of wildtype and sumoylation mutant PPARγ.(A) Luciferase reporter assay of wildtype and sumoylation mutant PPARγ plasmids. RLU, relative luciferase unit. (B) Tetracycline-induced expression of PPARγ and EGFP protein in stably transfected HBEC-C1-wt-PPARγ clone. A microscopic view of tetracycline-induced EFGP expression (top in B). Immunoblot assays for the expression of lamin A/C and tetracycline-induced PPARγ (bottom in B). A bicistronic construct of PPARγ and EGFP was stably introduced into a tumorigenic HBEC clone to generate HBEC-C1-PPARγ cell lines as described in Materials and Methods. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.001 compared to HBEC-KT control.

Mentions: As ligand-mediated PPARγ sumoylation suppresses the expression of inducible nitric oxide synthase (iNOS), a well-known proinflammatory enzyme that generates nitric oxide, and the anti-inflammatory role of PPARγ is believed to contribute to its tumor suppressive function of that receptor [34], we tested whether PPARγ sumoylation is critical for the anti-tumorigenic function of that receptor in the HBEC progression series. To do this, we established tumorigenic stable cell lines (HBEC-C1) expressing the wild-type (wt-PPARγ) or sumoylation mutant (SUMO-PPARγ) form of PPARγ to study if gain-of-function of different forms of PPARγ (wt-PPARγ vs. SUMO-PPARγ) could reverse the growth resistance of the tumorigenic HBEC-C1 to ligand treatment. We identified that both PPARγ forms showed no difference in ligand-mediated trans-activation function for target gene expression using a luciferase assay (Fig 6A). HBEC-C1 cells tightly regulated the expression of wt-PPARγ or SUMO-PPARγ under the control of tetracycline-inducible operating promoter (Tet/ON) (Fig 6B). HBEC-C1 cells with induced expression of wt-PPARγ showed significant growth inhibition by more than 50% when treated with pioglitazone or troglitazone (TZDs) (Fig 7A). However, this growth inhibitory response was greatly diminished in HBEC-C1 cells with inducible expression of SUMO-PPARγ under the same treatment conditions as the corresponding HBEC-C1 cells expressing wt-PPARγ (Fig 7A and 7B). Similarly, liquid colony formation assay showed that PPARγ ligand treatments inhibited clonogenecity of HBECs expressing wt-PPARγ and other lung cancer cell lines such as calu6 and H2347 expressing endogenouse PPARγ, but not of the one with SUMO-PPARγ (Fig 7B and 7C). However, treatment of PPARα ligand WY-14643 showed no colonogenic effect in both wt-PPARγ and SUMO-PPARγ expressing HBECs (Fig 7C). This suggests that inhibitory effect of TZDs is specifically dependent on PPARγ sumoylation. PPARγ activation also inhibited cell migration in a sumoylation-dependent manner (Fig 7D). Consistent with this, the expression of both cyclin A and cyclin D1 decreased in HBEC-C1 cells expressing wt-PPARγ, but was not changed in HBEC-C1 cells expressing SUMO-PPARγ, when treated with TZDs (Fig 8A and S5A Fig). Note that the expression of cyclin-dependent kinase inhibitors, p16 and p21, were not significantly changed in the same treatment conditions (Fig 8A and S5A Fig). In addition, we investigated several inflammatory signaling pathways involved in nitric oxide production, prostaglandin biochemistry, and TNFα signal transduction. Intriguingly, we found that TZD activation of wt-PPARγ decreased the proinflammatory COX2 expression by three-fold, whereas SUMO-PPARγ activation, notably, increased the expression of COX2 protein by six-fold in HBEC-C1 cells (Fig 8B). Further, the expression of 15-hydroxyprostaglandin dehydrogenase (HPGD), a prostaglandin-metabolizing enzyme, was dramatically induced by eighteen-fold when the wt-PPARγ was activated by TZDs. However, HBEC-C1-SUMO-PPARγ cells induced the HPGD expression significantly less (five- to seven-fold) when treated with TZDs. PPARγ activation also significantly suppressed TNFα expression, but not other NFκB signaling factors, in a sumoylation-dependent manner (S5B Fig). Note that iNOS was not expressed in tumorigenic HBEC clones (S5C Fig). Taken together, this suggests that ligand-induced PPARγ sumoylation is specifically involved in the suppression of inflammatory COX2 and TNFα signaling pathways, but not the iNOS pathway, in lung cancer pathogenesis.


Nuclear Receptor Expression and Function in Human Lung Cancer Pathogenesis.

Kim J, Sato M, Choi JW, Kim HW, Yeh BI, Larsen JE, Minna JD, Cha JH, Jeong Y - PLoS ONE (2015)

Characterization of wildtype and sumoylation mutant PPARγ.(A) Luciferase reporter assay of wildtype and sumoylation mutant PPARγ plasmids. RLU, relative luciferase unit. (B) Tetracycline-induced expression of PPARγ and EGFP protein in stably transfected HBEC-C1-wt-PPARγ clone. A microscopic view of tetracycline-induced EFGP expression (top in B). Immunoblot assays for the expression of lamin A/C and tetracycline-induced PPARγ (bottom in B). A bicistronic construct of PPARγ and EGFP was stably introduced into a tumorigenic HBEC clone to generate HBEC-C1-PPARγ cell lines as described in Materials and Methods. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.001 compared to HBEC-KT control.
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Related In: Results  -  Collection

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Show All Figures
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pone.0134842.g006: Characterization of wildtype and sumoylation mutant PPARγ.(A) Luciferase reporter assay of wildtype and sumoylation mutant PPARγ plasmids. RLU, relative luciferase unit. (B) Tetracycline-induced expression of PPARγ and EGFP protein in stably transfected HBEC-C1-wt-PPARγ clone. A microscopic view of tetracycline-induced EFGP expression (top in B). Immunoblot assays for the expression of lamin A/C and tetracycline-induced PPARγ (bottom in B). A bicistronic construct of PPARγ and EGFP was stably introduced into a tumorigenic HBEC clone to generate HBEC-C1-PPARγ cell lines as described in Materials and Methods. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.001 compared to HBEC-KT control.
Mentions: As ligand-mediated PPARγ sumoylation suppresses the expression of inducible nitric oxide synthase (iNOS), a well-known proinflammatory enzyme that generates nitric oxide, and the anti-inflammatory role of PPARγ is believed to contribute to its tumor suppressive function of that receptor [34], we tested whether PPARγ sumoylation is critical for the anti-tumorigenic function of that receptor in the HBEC progression series. To do this, we established tumorigenic stable cell lines (HBEC-C1) expressing the wild-type (wt-PPARγ) or sumoylation mutant (SUMO-PPARγ) form of PPARγ to study if gain-of-function of different forms of PPARγ (wt-PPARγ vs. SUMO-PPARγ) could reverse the growth resistance of the tumorigenic HBEC-C1 to ligand treatment. We identified that both PPARγ forms showed no difference in ligand-mediated trans-activation function for target gene expression using a luciferase assay (Fig 6A). HBEC-C1 cells tightly regulated the expression of wt-PPARγ or SUMO-PPARγ under the control of tetracycline-inducible operating promoter (Tet/ON) (Fig 6B). HBEC-C1 cells with induced expression of wt-PPARγ showed significant growth inhibition by more than 50% when treated with pioglitazone or troglitazone (TZDs) (Fig 7A). However, this growth inhibitory response was greatly diminished in HBEC-C1 cells with inducible expression of SUMO-PPARγ under the same treatment conditions as the corresponding HBEC-C1 cells expressing wt-PPARγ (Fig 7A and 7B). Similarly, liquid colony formation assay showed that PPARγ ligand treatments inhibited clonogenecity of HBECs expressing wt-PPARγ and other lung cancer cell lines such as calu6 and H2347 expressing endogenouse PPARγ, but not of the one with SUMO-PPARγ (Fig 7B and 7C). However, treatment of PPARα ligand WY-14643 showed no colonogenic effect in both wt-PPARγ and SUMO-PPARγ expressing HBECs (Fig 7C). This suggests that inhibitory effect of TZDs is specifically dependent on PPARγ sumoylation. PPARγ activation also inhibited cell migration in a sumoylation-dependent manner (Fig 7D). Consistent with this, the expression of both cyclin A and cyclin D1 decreased in HBEC-C1 cells expressing wt-PPARγ, but was not changed in HBEC-C1 cells expressing SUMO-PPARγ, when treated with TZDs (Fig 8A and S5A Fig). Note that the expression of cyclin-dependent kinase inhibitors, p16 and p21, were not significantly changed in the same treatment conditions (Fig 8A and S5A Fig). In addition, we investigated several inflammatory signaling pathways involved in nitric oxide production, prostaglandin biochemistry, and TNFα signal transduction. Intriguingly, we found that TZD activation of wt-PPARγ decreased the proinflammatory COX2 expression by three-fold, whereas SUMO-PPARγ activation, notably, increased the expression of COX2 protein by six-fold in HBEC-C1 cells (Fig 8B). Further, the expression of 15-hydroxyprostaglandin dehydrogenase (HPGD), a prostaglandin-metabolizing enzyme, was dramatically induced by eighteen-fold when the wt-PPARγ was activated by TZDs. However, HBEC-C1-SUMO-PPARγ cells induced the HPGD expression significantly less (five- to seven-fold) when treated with TZDs. PPARγ activation also significantly suppressed TNFα expression, but not other NFκB signaling factors, in a sumoylation-dependent manner (S5B Fig). Note that iNOS was not expressed in tumorigenic HBEC clones (S5C Fig). Taken together, this suggests that ligand-induced PPARγ sumoylation is specifically involved in the suppression of inflammatory COX2 and TNFα signaling pathways, but not the iNOS pathway, in lung cancer pathogenesis.

Bottom Line: Notably, PPARγ activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs.In fully tumorigenic HBECs with inducible expression of PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner.Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do, Republic of Korea; Institute of Lifestyle Medicine, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do, Republic of Korea; Nuclear Receptor Research Consortium, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do, Republic of Korea.

ABSTRACT
Lung cancer is caused by combinations of diverse genetic mutations. Here, to understand the relevance of nuclear receptors (NRs) in the oncogene-associated lung cancer pathogenesis, we investigated the expression profile of the entire 48 NR members by using QPCR analysis in a panel of human bronchial epithelial cells (HBECs) that included precancerous and tumorigenic HBECs harboring oncogenic K-rasV12 and/or p53 alterations. The analysis of the profile revealed that oncogenic alterations accompanied transcriptional changes in the expression of 19 NRs in precancerous HBECs and 15 NRs according to the malignant progression of HBECs. Amongst these, peroxisome proliferator-activated receptor gamma (PPARγ), a NR chosen as a proof-of-principle study, showed increased expression in precancerous HBECs, which was surprisingly reversed when these HBECs acquired full in vivo tumorigenicity. Notably, PPARγ activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs. In fully tumorigenic HBECs with inducible expression of PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner. Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression. This suggests that ligand-mediated sumoylation of PPARγ plays an important role in lung cancer pathogenesis by modulating prostaglandin metabolism.

No MeSH data available.


Related in: MedlinePlus