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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

Functional evaluation of PPARγ in immortalized HBECs.(A) PPARγ and COX2 mRNA expression was measured using QPCR assay in HBEC lines with or without troglitazone treatment. (B) Immunoblots using antibodies against COX2, cyclin D1, PPARγ or beta-actin were used to measure the corresponding protein expression in the HBEC panel when treated or not treated with 1 μM of troglitazone. (C) Growth response of HBEC-KT cell lines to the combined treatment of PPARγ ligand troglitazone (3 μM) and RXR ligand LG268 (100 nM). Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to HBEC-KT control, ###P < 0.001 compared to K-rasV12 control, +++P < 0.001 compared to K-rasV12+p53shRNA control.
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pone.0134842.g003: Functional evaluation of PPARγ in immortalized HBECs.(A) PPARγ and COX2 mRNA expression was measured using QPCR assay in HBEC lines with or without troglitazone treatment. (B) Immunoblots using antibodies against COX2, cyclin D1, PPARγ or beta-actin were used to measure the corresponding protein expression in the HBEC panel when treated or not treated with 1 μM of troglitazone. (C) Growth response of HBEC-KT cell lines to the combined treatment of PPARγ ligand troglitazone (3 μM) and RXR ligand LG268 (100 nM). Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to HBEC-KT control, ###P < 0.001 compared to K-rasV12 control, +++P < 0.001 compared to K-rasV12+p53shRNA control.

Mentions: Since the PPARγ has been relatively well-studied amongst other NRs in diverse physiological functions (e.g., adipocyte differentiation, inflammation control) and its ligand TZDs available in the clinic of type II diabetes, we next decided to investigate the molecular study of PPARγ, as a proof-of-concept, out of the 48 NRs in the molecular pathogenesis of lung cancer [11, 34, 35]. Consistent with previous reports showing that COX2 expression is associated with lung cancer progression [36, 37], we observed a dramatic increase in COX2 expression in HBECs modified to contain oncogenic K-rasV12 (Fig 3A and 3B). While PPARγ mRNA increased in parallel with COX2 expression, PPARγ protein expressions were comparable in all 4 HBEC cells suggesting posttranscriptional regulation of PPARγ mRNA (Fig 3B). Given that PPARγ plays a significant role in anti-inflammatory response [34, 35], we wanted to test if PPARγ activation inhibits the pro-inflammatory COX2 expression in the lung cancer pathogenesis model. Indeed, the PPARγ agonist troglitazone reversed the increased expression of COX2 mRNA and protein (Fig 3A and 3B). Together with the PPARγ inhibition of lung cancer proliferation as previously reported [38, 39], this result suggests that PPARγ suppression of COX2 expression might be important in modulating oncogene-induced malignant transformation of HBECs into lung cancer. With loss of p53 function, the expression of cyclin D1 protein, a key factor in cell cycle progression from G1 to S phase, decreased in HBECs with p53 knockdown and decreased even more in HBECs with dual p53 and K-rasV12 oncogenic alterations coupled with troglitazone treatment (Fig 3B). By contrast, cyclin D1 exhibited no change in HBECs with only K-rasV12 expression with or without troglitazone. These data suggest that during precancerous lesions (typified by the HBEC cells with p53 loss and K-rasV12 changes), PPARγ is expressed and its ligand-dependent activation can lead to dramatic changes in COX-2 and cyclin D1 expression (Fig 3A and 3B). However, troglitazone treatment showed equal inhibition of HBEC-KT proliferation across the isogenic panel (Fig 3C). This suggests that additional factors, along with COX-2 and cyclin D1, might be involved in PPARγ-mediated growth suppression of HBEC cells.


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)

Functional evaluation of PPARγ in immortalized HBECs.(A) PPARγ and COX2 mRNA expression was measured using QPCR assay in HBEC lines with or without troglitazone treatment. (B) Immunoblots using antibodies against COX2, cyclin D1, PPARγ or beta-actin were used to measure the corresponding protein expression in the HBEC panel when treated or not treated with 1 μM of troglitazone. (C) Growth response of HBEC-KT cell lines to the combined treatment of PPARγ ligand troglitazone (3 μM) and RXR ligand LG268 (100 nM). Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to HBEC-KT control, ###P < 0.001 compared to K-rasV12 control, +++P < 0.001 compared to K-rasV12+p53shRNA control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526668&req=5

pone.0134842.g003: Functional evaluation of PPARγ in immortalized HBECs.(A) PPARγ and COX2 mRNA expression was measured using QPCR assay in HBEC lines with or without troglitazone treatment. (B) Immunoblots using antibodies against COX2, cyclin D1, PPARγ or beta-actin were used to measure the corresponding protein expression in the HBEC panel when treated or not treated with 1 μM of troglitazone. (C) Growth response of HBEC-KT cell lines to the combined treatment of PPARγ ligand troglitazone (3 μM) and RXR ligand LG268 (100 nM). Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to HBEC-KT control, ###P < 0.001 compared to K-rasV12 control, +++P < 0.001 compared to K-rasV12+p53shRNA control.
Mentions: Since the PPARγ has been relatively well-studied amongst other NRs in diverse physiological functions (e.g., adipocyte differentiation, inflammation control) and its ligand TZDs available in the clinic of type II diabetes, we next decided to investigate the molecular study of PPARγ, as a proof-of-concept, out of the 48 NRs in the molecular pathogenesis of lung cancer [11, 34, 35]. Consistent with previous reports showing that COX2 expression is associated with lung cancer progression [36, 37], we observed a dramatic increase in COX2 expression in HBECs modified to contain oncogenic K-rasV12 (Fig 3A and 3B). While PPARγ mRNA increased in parallel with COX2 expression, PPARγ protein expressions were comparable in all 4 HBEC cells suggesting posttranscriptional regulation of PPARγ mRNA (Fig 3B). Given that PPARγ plays a significant role in anti-inflammatory response [34, 35], we wanted to test if PPARγ activation inhibits the pro-inflammatory COX2 expression in the lung cancer pathogenesis model. Indeed, the PPARγ agonist troglitazone reversed the increased expression of COX2 mRNA and protein (Fig 3A and 3B). Together with the PPARγ inhibition of lung cancer proliferation as previously reported [38, 39], this result suggests that PPARγ suppression of COX2 expression might be important in modulating oncogene-induced malignant transformation of HBECs into lung cancer. With loss of p53 function, the expression of cyclin D1 protein, a key factor in cell cycle progression from G1 to S phase, decreased in HBECs with p53 knockdown and decreased even more in HBECs with dual p53 and K-rasV12 oncogenic alterations coupled with troglitazone treatment (Fig 3B). By contrast, cyclin D1 exhibited no change in HBECs with only K-rasV12 expression with or without troglitazone. These data suggest that during precancerous lesions (typified by the HBEC cells with p53 loss and K-rasV12 changes), PPARγ is expressed and its ligand-dependent activation can lead to dramatic changes in COX-2 and cyclin D1 expression (Fig 3A and 3B). However, troglitazone treatment showed equal inhibition of HBEC-KT proliferation across the isogenic panel (Fig 3C). This suggests that additional factors, along with COX-2 and cyclin D1, might be involved in PPARγ-mediated growth suppression of HBEC cells.

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