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P3.02-071 Statins May Improve the Prognosis of Patients with Lung Adenocarcinoma by Suppressing Mutant p53-Induced EMT
November 2017 1a inhibitors demonstrated anti-NSCLC activity in vitro and a selectively synergistic effect with gefitinib in EGFR-TKI-insensitive NSCLC cell line (H460). Our study suggested a potential treatment approach using HIF-1a inhibitors plus gefitinib. Keywords: Hypoxia-inducible factor-1a inhibitor, Epidermal growth factor receptor tyrosine kinase inhibitor, non-small cell lung cancer
P3.02-071 Statins May Improve the Prognosis of Patients with Lung Adenocarcinoma by Suppressing Mutant p53Induced EMT S. Nishikawa, T. Menju, T. Soawa, K. Takahashi, R. Miyata, H. Ishikawa, M. Hamaji, H. Motoyama, A. Aoyama, T. Sato, C. Fengshi, M. Sonobe, H. Date Thoracic Surgery, Graduate School of Medicine, Kyoto University, Kyoto/JP Background: Epithelial-mesenchymal transition (EMT) is known to be pivotal for driving metastasis and recurrence in lung cancer. Some reports have shown statins suppressed EMT by inactivating mutant p53 functions in vitro. Although several clinical trials regarding conventional treatments with statins have been performed, the effect of statins on the prognosis is still controversial. The purpose of the present study is to clarify the impact of statins on EMT and the prognosis of patients with lung adenocarcinoma harboring p53 mutation. Method: Firstly, we transfected wild type p53 or mutant p53 (R175H, R273H) to H1650 cells and administrated simvastatin. We evaluated morphological changes by microscopic measurement and analyzed EMT markers (Ecadherin, vimentin) through western blotting of whole cell lysate. We also analyzed their invasive ability through Matrigel invasion assay. Secondly, a total of 282 lung adenocarcinoma specimens were collected from patients who underwent surgery in our institute from January 2001 to December 2007. We analyzed EMT markers through immunostaining of tumor specimens and we determined p53 mutation by single stranded conformational polymorphism followed by direct sequencing. The association between EMT, p53 mutation status, and statin use as well as the patients’ clinical information was statistically analyzed. Correlations were compared using Pearson’s chi-square test and overall survival were compared using the log-rank test. Result: When mutant p53 (R175H, R273H) were transfected, H1650 cells showed EMT like morphological changes. E-cadherin expression was decreased and vimentin expression was increased in H1650 harboring mutant p53 (H1650mut.p53). Additionally, H1650mut.p53 obtained more aggressive invasiveness compared to H1650 harboring wild type p53. However, simvastatin-treated H1650mut.p53 lost EMT character changes and aggressive invasiveness. According to the medical records, about 20% of the patients took statins (simvastatin, pravastatin, and so on) as a treatment of hyperlipidemia or coronary artery disease. Consistent with the results of in vitro experiments, IHC showed that statin administration was correlated to fewer EMT only in the patients with mutant p53. Moreover, the statin-administrated group showed significantly better survival compared to the non-statin group (p¼0.04), which was observed only in the patients with mutant p53. Conclusion: Statins suppressed EMT and improved the prognosis of patients with lung adenocarcinoma in a p53 mutation-dependent manner. Keywords: p53 mutation, statin, lung adenocarcinoma
P3.02-072 MiR-33b Inhibits Lung Adenocarcinoma Cell Epithelial-Mesenchymal Transition Through CeRNA Regulatory Network Y. Tan, M. Li, C. Hu Department of Respiratory Medicine, Xiangya Hospital Central South University, Changsha/CN
Abstracts
S2263
Background: Lung cancer is a significant health problem worldwide and the mechanism of lung cancer cell metastasis has not been fully elucidated. Epithelial-mesenchymal transition (EMT) is not only an important biological mechanism for natural physiological process but also has been proved to be related to cancer metastasis. Further studies have pointed out that EMT plays a indispensable role in the entire process of metastasis. In recent years, many miRNAs have been demonstrated to target EMT-related proteins, such as E-cadherin and Vimentin to regulate the initiation and progression of EMT. The preliminary study of my research group has indicated that miR-33b plays an inhibitory role in the EMT of lung cancer and two genes, ZEB1 and Twist1, are downstream target of miR-33b.Then snail1 which is EMTrelated proteins like ZEB1 has also been founded that a potential correlation with miR-33b.A ceRNA hypothesis tells that If different transcripts have the same miRNA binding site, it can combine with miRNA completely and influence its inhibitory effect on the target, and then adjust the expression of each other. Snial1, ZEB1 and Twist1 three genes would bind to the same miRNA which is miR-33b，but the relations among this three genes and miR-33b still need to be clarified. Method: Western blotting, RT-pcr, Immunofluorescence, Immunohistochemistry and Luciferase assay were used to explore the relations among Snial1, ZEB1, Twist1 and miR-33b.The effects of miR-33b and Snail1 on lung adenocarcinoma cancer cell functions were investigated by using migration and invasion assays. Construct gene expression vectors and transfect the cells to research the regulative relations among Snial1, ZEB1, Twist1 and miR-33b.Tumor formation in nude mice to certify the relations between Snail1 and miR-33b in vitro. Result: miR-33b suppresses EMT and lung adenocarcinoma cell proliferation and migration in vitro.miR-33b directly targets SNAIL1.miR33b regulates lung adenocarcinoma cells by down-regulating Snial1 expression.miR-33b inhibits tumor growth through the SNAIL1 in vivo.Snial1, ZEB1, Twist1 influence each other. Conclusion: Snial1, ZEB1 and Twist1 function as ceRNA for each other.MiR-33b inhibits lung adenocarcinoma cell epithelial-mesenchymal transition through ceRNA regulatory network Keywords: miR-33b, ceRNA, SNIAL1
P3.02-073 Stromal Hedgehog Pathway Activation Suppresses Growth and Metastasis of Lung Adenocarcinoma S. Kasiri,1 B. Chen,1 A. Wilson,2 U. Barrie,2 U. Marriam,2 Z. Zeng,3 L. Girard,4 J. Kim5 1Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern, Dallas, TX/US, 2Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern, Dallas, TX/US, 3Dept. of Molecular Biology, University of Texas Southwestern, Dallas, TX/US, 4Hamon Center for Therapeutic Oncology Research and the Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX/US, 5Internal Medicine, Div. of Hematology-Oncology, University of Texas Southwestern, Dallas, TX/US Background: Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung adenocarcinoma. Method: Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss, and SHH loss compared to wild-type SHH. Result: In human lung
P3.02-071 Statins May Improve the Prognosis of Patients with Lung Adenocarcinoma by Suppressing Mutant p53Induced EMT S. Nishikawa, T. Menju, T. Soawa, K. Takahashi, R. Miyata, H. Ishikawa, M. Hamaji, H. Motoyama, A. Aoyama, T. Sato, C. Fengshi, M. Sonobe, H. Date Thoracic Surgery, Graduate School of Medicine, Kyoto University, Kyoto/JP Background: Epithelial-mesenchymal transition (EMT) is known to be pivotal for driving metastasis and recurrence in lung cancer. Some reports have shown statins suppressed EMT by inactivating mutant p53 functions in vitro. Although several clinical trials regarding conventional treatments with statins have been performed, the effect of statins on the prognosis is still controversial. The purpose of the present study is to clarify the impact of statins on EMT and the prognosis of patients with lung adenocarcinoma harboring p53 mutation. Method: Firstly, we transfected wild type p53 or mutant p53 (R175H, R273H) to H1650 cells and administrated simvastatin. We evaluated morphological changes by microscopic measurement and analyzed EMT markers (Ecadherin, vimentin) through western blotting of whole cell lysate. We also analyzed their invasive ability through Matrigel invasion assay. Secondly, a total of 282 lung adenocarcinoma specimens were collected from patients who underwent surgery in our institute from January 2001 to December 2007. We analyzed EMT markers through immunostaining of tumor specimens and we determined p53 mutation by single stranded conformational polymorphism followed by direct sequencing. The association between EMT, p53 mutation status, and statin use as well as the patients’ clinical information was statistically analyzed. Correlations were compared using Pearson’s chi-square test and overall survival were compared using the log-rank test. Result: When mutant p53 (R175H, R273H) were transfected, H1650 cells showed EMT like morphological changes. E-cadherin expression was decreased and vimentin expression was increased in H1650 harboring mutant p53 (H1650mut.p53). Additionally, H1650mut.p53 obtained more aggressive invasiveness compared to H1650 harboring wild type p53. However, simvastatin-treated H1650mut.p53 lost EMT character changes and aggressive invasiveness. According to the medical records, about 20% of the patients took statins (simvastatin, pravastatin, and so on) as a treatment of hyperlipidemia or coronary artery disease. Consistent with the results of in vitro experiments, IHC showed that statin administration was correlated to fewer EMT only in the patients with mutant p53. Moreover, the statin-administrated group showed significantly better survival compared to the non-statin group (p¼0.04), which was observed only in the patients with mutant p53. Conclusion: Statins suppressed EMT and improved the prognosis of patients with lung adenocarcinoma in a p53 mutation-dependent manner. Keywords: p53 mutation, statin, lung adenocarcinoma
P3.02-072 MiR-33b Inhibits Lung Adenocarcinoma Cell Epithelial-Mesenchymal Transition Through CeRNA Regulatory Network Y. Tan, M. Li, C. Hu Department of Respiratory Medicine, Xiangya Hospital Central South University, Changsha/CN
Abstracts
S2263
Background: Lung cancer is a significant health problem worldwide and the mechanism of lung cancer cell metastasis has not been fully elucidated. Epithelial-mesenchymal transition (EMT) is not only an important biological mechanism for natural physiological process but also has been proved to be related to cancer metastasis. Further studies have pointed out that EMT plays a indispensable role in the entire process of metastasis. In recent years, many miRNAs have been demonstrated to target EMT-related proteins, such as E-cadherin and Vimentin to regulate the initiation and progression of EMT. The preliminary study of my research group has indicated that miR-33b plays an inhibitory role in the EMT of lung cancer and two genes, ZEB1 and Twist1, are downstream target of miR-33b.Then snail1 which is EMTrelated proteins like ZEB1 has also been founded that a potential correlation with miR-33b.A ceRNA hypothesis tells that If different transcripts have the same miRNA binding site, it can combine with miRNA completely and influence its inhibitory effect on the target, and then adjust the expression of each other. Snial1, ZEB1 and Twist1 three genes would bind to the same miRNA which is miR-33b，but the relations among this three genes and miR-33b still need to be clarified. Method: Western blotting, RT-pcr, Immunofluorescence, Immunohistochemistry and Luciferase assay were used to explore the relations among Snial1, ZEB1, Twist1 and miR-33b.The effects of miR-33b and Snail1 on lung adenocarcinoma cancer cell functions were investigated by using migration and invasion assays. Construct gene expression vectors and transfect the cells to research the regulative relations among Snial1, ZEB1, Twist1 and miR-33b.Tumor formation in nude mice to certify the relations between Snail1 and miR-33b in vitro. Result: miR-33b suppresses EMT and lung adenocarcinoma cell proliferation and migration in vitro.miR-33b directly targets SNAIL1.miR33b regulates lung adenocarcinoma cells by down-regulating Snial1 expression.miR-33b inhibits tumor growth through the SNAIL1 in vivo.Snial1, ZEB1, Twist1 influence each other. Conclusion: Snial1, ZEB1 and Twist1 function as ceRNA for each other.MiR-33b inhibits lung adenocarcinoma cell epithelial-mesenchymal transition through ceRNA regulatory network Keywords: miR-33b, ceRNA, SNIAL1
P3.02-073 Stromal Hedgehog Pathway Activation Suppresses Growth and Metastasis of Lung Adenocarcinoma S. Kasiri,1 B. Chen,1 A. Wilson,2 U. Barrie,2 U. Marriam,2 Z. Zeng,3 L. Girard,4 J. Kim5 1Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern, Dallas, TX/US, 2Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern, Dallas, TX/US, 3Dept. of Molecular Biology, University of Texas Southwestern, Dallas, TX/US, 4Hamon Center for Therapeutic Oncology Research and the Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX/US, 5Internal Medicine, Div. of Hematology-Oncology, University of Texas Southwestern, Dallas, TX/US Background: Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung adenocarcinoma. Method: Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss, and SHH loss compared to wild-type SHH. Result: In human lung