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M. Noguchi
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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.A. Boyle, M.G. Kris
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 2c-3c
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MINI35.12 - Stratifin Accelerates Progression of Lung Adenocarcinoma at an Early Stage (ID 506)
18:30 - 20:00 | Author(s): M. Noguchi
- Abstract
- Presentation
Background:
Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation (Aya Shiba-Ishii, IJC. 2011). We also found that SFN expression was totally suppressed in normal lung tissue, whereas demethylation of its promoter triggered aberrant SFN overexpression in eIAs in a p53-independent manner (Aya Shiba-Ishii, AJP. 2012). SFN has been linked to cancer most directly, possibly having tissue-specific functions and regulating progression of the cell cycle. Here, we performed an in vivo study to clarify the role of SFN in progression of lung adenocarcinoma.
Methods:
We induced stable knockdown of SFN using two individual shRNAs (shSFN). To evaluate the oncogenic activity of SFN, we injected A549-shSFN intrabronchially or intravenously into SCID mice. Additionally, we generated SFN-transgenic mice (Tg-SPC-SFN[+/-]) showing lung-specific expression of human SFN (hSFN) under the control of a tissue-specific enhancer, the SPC promoter. In order to observe the tumorigenic activity of SFN, Tg-SPC-SFN[+/-] and WT ICR mice were intraperitoneally administered 4 mg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a pulmonary carcinogen) or saline as a control, and tumorigenicity was assessed for 20 weeks. Lungs of representative mice were periodically examined using animal CT.
Results:
Although control A549 cells formed advanced tumors in the lungs of SCID mice after intrabronchial and/or intravenous injection, we also found pleural dissemination in the control group (in 75% after intravenous injection and in 25% after intrabronchial injection). However, A549-shSFN did not form any tumors. Next, we confirmed the lung-specific expression hSFN in Tg-SPC-SFN[+/-] using RT-PCR and IHC. In a chemical carcinogenesis experiment, animal CT revealed several pulmonary tumors in some Tg-SPC-SFN[+/-] from 15 weeks after NNK administration, and at 20 weeks 47.8% of Tg-SPC-SFN[+/-] (11/23) had developed lung tumors, whereas only 11.1% of WT ICR (3/27) had done so (statistically significant). Surprisingly, two of seven Tg-SPC-SFN[+/-] mice (28.6%) developed tumors even though they were not administered NNK. All of the tumors that developed in Tg-SPC-SFN[+/-] lung expressed hSFN abundantly.
Conclusion:
Here, we showed that suppression of SFN expression in lung adenocarcinoma A549 cells was significantly reduced in terms of not only lung tumor formation but also metastatic potential. Additionally, it was found that Tg-SPC-SFN[+/-] mice developed lung tumors at a significantly higher rate than control mice after NNK administration. Interestingly, several Tg-SPC-SFN[+/-] mice developed lung tumors without carcinogen. Because these tumors showed high hSFN expression, SFN was thought to facilitate not only tumor progression but also tumor initiation, and to work as an oncogene. Soda et al. found that 100% of Tg-EML4-ALK mice developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth (Nature 2007). Although the oncogenic activity of SFN is weaker than that of EML4-ALK fusion kinase, SFN might also have the potential to initiate peripheral-type lung adenocarcinoma.
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MS 25 - Lung Carcinogenesis (ID 43)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:W.A. Franklin, H. Kato
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 2a-3b
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MS25.04 - Molecular Pathology of Alveolar Premalignancy (ID 1961)
14:15 - 15:45 | Author(s): M. Noguchi
- Abstract
- Presentation
Abstract:
Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We examined epigenetical and genetic abnormalities of very early adenocarcinoma and compared them to early but advanced adenocarcinoma. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation. Here, we performed an in vivo study to clarify the role of SFN in progression of lung adenocarcinoma. Suppression of SFN expression in A549 (a human lung adenocarcinoma cell line) by siSFN significantly reduced cell proliferation activity and the S-phase subpopulation. In vivo, tumor development or metastasis to the lung was reduced in shSFN-transfected A549 cells. Moreover, we generated SFN-transgenic mice (Tg-SPC-SFN[+/-]) showing lung-specific expression of human SFN under the control of a tissue-specific enhancer, the SPC promoter. We found that Tg-SPC-SFN[+/-] mice developed lung tumors at a significantly higher rate than control mice after administration of chemical carcinogen, NNK (Fig 1). Interestingly, several Tg-SPC-SFN+/- mice developed tumors without NNK. These tumor cells showed high hSFN expression. These results suggest that SFN facilitates lung tumor development and progression. SFN appears to be a novel oncogene with potential as a therapeutic target. Next, gnetic abnormality in early-stage lung adenocarcinoma was examined. Six in situ lung adenocarcinomas and nine small but invasive adenocarcinomas were examined by array-comparative genomic hybridization (array-CGH), and candidate genes of interest were screened. To examine gene abnormalities, 83 cases of various types of lung carcinoma were examined by quantitative real-time genomic PCR (qPCR) and immunohistochemistry (IHC). The results were then finally verified using another set of early-stage adenocarcinomas. Array-CGH demonstrated frequent amplification at chromosome 3q26, and among the 7 genes located in this region, we focused on the epithelial cell transforming sequence 2 (ECT2) oncogene, as ECT2 amplification was detected only in invasive adenocarcinoma, and not in in situ carcinoma. FISH and IHC analyses also detected amplification and overexpression of ECT2 in invasive adenocarcinoma (Fig 2), and this was correlated with both the Ki-67 labeling index and mitotic index. In addition, it was associated with disease-free survival and overall survival of patients with lung adenocarcinoma. These results were verified using another set of early-stage adenocarcinomas resected at another hospital. Abnormality of the ECT2 gene occurs at a relatively early stage of lung adenocarcinogenesis and would be applicable as a new biomarker for prognostication of patients with lung adenocarcinoma. Figure 1Figure 2
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ORAL 24 - CT Detected Nodules - Predicting Biological Outcome (ID 122)
- Event: WCLC 2015
- Type: Oral Session
- Track: Screening and Early Detection
- Presentations: 2
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ORAL24.01 - Natural History of Pulmonary Subsolid Nodules: A Prospective Multicenter Study (ID 1245)
10:45 - 12:15 | Author(s): M. Noguchi
- Abstract
- Presentation
Background:
The purpose of this prospective multicenter study was to evaluate the natural course of progression of pulmonary subsolid nodules.
Methods:
Eight facilities participated in this prospective study. This study was conducted with the approval of the institutional review board of each of the participating institutions. Written informed consent was obtained from all the patients. A total of 845 patients with 1325 pulmonary subsolid nodules were registered, of whom 795 patients (341 men, 454 women; mean age, 62 years [range, 31-88]) with 1238 subsolid nodules were selected as being eligible for this study. In this study, the pulmonary subsolid nodules were classified into three categories: pure ground-glass nodules (hereafter abbreviated as PGGNs), heterogeneous GGNs (solid component detected only in the lung window setting; hereafter abbreviated as HGGN), and part-solid nodules (solid component also detected in the mediastinal window setting). The CT images of the nodules that showed progression were reviewed by an expert radiologists’ panel. Pathological specimens of the resected nodules were reviewed by an expert pathologists’ panel.
Results:
The mean prospective follow-up period was 4.3 ± 2.5 years (range, 0.2–12.1; median, 3.5 [IQR, 2.4–6.0]). After exclusion of 9 resected nodules (2 no-lung-cancer nodules and 7 lung cancers not reviewed by the expert pathologists’ panel), the pulmonary subsolid nodules were classified as follows at the baseline: 1046 PGGNs, 81 HGGNs, and 102 part-solid nodules. Among the 1047 PGGNs, 13 (13/1046; 1.2%) developed into HGGNs, and 56 (56/1046; 5.4%) developed into part-solid nodules. Among the 81 HGGNs, 16 (16/81; 19.8%) developed into part-solid nodules. Thus, the subsolid nodules were classified as follows at the time of the final follow-up: 977 PGGNs, 78 HGGNs and 174 part-solid nodules. Of the 977 PGGNs, 35 (3.6%) were resected; from the histopathologic standpoint, the 35 resected PGGNs consisted of 9 minimally invasive adenocarcinomas (MIAs), 21 adenocarcinomas in situ (AISs), and 5 atypical adenomatous hyperplasias (AAHs). Of the 78 HGGNs, 7 (9%) were resected; from the histopathologic standpoint, the 7 HGGNs consisted of 5 MIAs and 2 AISs. Of the 174 part-solid nodules, 49 (28.2%) were resected; from the histopathologic standpoint, the 49 part-solid nodules consisted of 12 invasive adenocarcinomas, 26 MIAs, 10 AISs, and 1 AAHs. In total, 12 (12/1229, 1%) invasive adenocarcinomas, 40 (40/1229; 3.3%) MIAs, 33 (33/1229; 2.7%) AISs, and 6 (6/1229; 0.5%) AAHs were resected as of December 31, 2013; For the PGGNs, the mean period to development into part-solid nodules was 3.8 ± 2.0 years (range, 0.5-8.7; median, 3.4 [IQR, 2.0–5.2]); for the HGGNs, the mean period to development into part-solid nodules was 2.1 ± 2.3 years (range, 0.2–8.8; median, 1.0 [IQR, 0.7–3.4]) (P=0.0004).
Conclusion:
Our prospective multicenter study revealed the frequency and period of development from PGGNs and HGGNs into part-solid nodules. Invasive adenocarcinomas were only diagnosed in the part-solid nodules. The findings of the study may contribute to the development of guidelines for follow-up of pulmonary subsolid nodules.
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ORAL24.08 - Discussant for ORAL24.05, ORAL24.06, ORAL24.07 (ID 3359)
10:45 - 12:15 | Author(s): M. Noguchi
- Abstract
- Presentation
Abstract not provided
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-066 - Site-Selected Chromatin-Immunoprecipitation (ChIP) Analysis by Laser Captured Microdissection (ID 1752)
09:30 - 17:00 | Author(s): M. Noguchi
- Abstract
Background:
High throughput sequencing methods such as exome sequencing, RNA sequencing, Chromatin–immunoprecipitation (ChIP) sequencing are essential tools for cancer research. However, these fine and delicate analyses contain several methodological problems. For example, although tumor mass may be suitable for mutation analysis, histological heterogeneity of the tumor tissue causes insufficient results especially for epigenetic or RNA analyses. Besides, the cancer-associated stromal cells and immune cells in the tumor will also affect the results. In this study, we tried ChIP for tiny but pure tumor samples which were selected by laser captured microdissection and verified its availability for ChIP sequence analysis.
Methods:
We used a lung adenocarcinoma frozen tissue harboring EGFR L858R mutation. After formalin fixation (1%, 10min), tumor cells, stroma cells and immune cells were microdissected separately by LMD4000 (Leica) and ChIP was performed to using H3K4me3 anti-body. Then, the quality was confirmed by real-time PCR for CCR7 which is one of the tumor specific markers and CD3 which is representative T lymphocyte marker. Sanger sequence for EGFR L858R mutation was also analyzed for confirmation that each sample was dissected and extracted correctly.
Results:
Only from the sample of tumor cells, we detected EGFR L858R mutation by Sanger sequence but from stromal cells and immune cells, we did not detect EGFR mutation. The result showed that we extracted samples correctly. And H3K4me3 mark at CCR7 gene was detected only from tumor cells and was not detected from the other samples. Moreover, H3K4me3 mark at CD3 gene was detected from stroma cells and immune cells but not tumor cells. These results indicated that microdissection method is useful and necessary method for ChIP analysis.
Conclusion:
Microdissection can be applied for epigenetic analysis like ChIP method. Our results indicated that microdissection method is useful for tumor-cell-specific epigenome profiling by ChIP sequencing.
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P1.04-071 - Inhibition of EGFR Lysosomal Degradation in Lung Adenocarcinoma by Ubiquitin-Specific Protease 8 and Stratifin (ID 475)
09:30 - 17:00 | Author(s): M. Noguchi
- Abstract
Background:
The epidermal growth factor receptor (EGFR) is one of the best-known targets of therapy for non-small cell lung cancer (NSCLC). Our purpose was to investigate whether ubiquitin-specific protease 8 (USP8) and stratifin (14-3-3σ or SFN) inhibit or stimulate lysosomal degradation of EGFR in lung adenocarcinoma.
Methods:
Using Western blotting and immunofluorescence analysis, we examined the effect of USP8 or SFN knockdown by siRNA and overexpression of USP8. Expressions of USP8 and SFN in normal and tumorous lung tissue were examined by Western blotting and immunohistochemistry.
Results:
USP8 or SFN knockdown led to downregulation of cellular proliferation, receptor tyrosine kinases such as EGFR and proto-oncogenes (c-Met), and downstream signaling pathways such as the AKT, ERK, and STAT3 pathways, whereas it upregulated the accumulation of EGFR at lysosomes for degradation. However, overexpression of USP8 led to an increase of EGFR and downstream signaling after EGF stimulation. Moreover, USP8 and SFN expressions were increased in the tumorous lung tissue in comparison with normal lung tissue from the same patient.
Conclusion:
USP8 and SFN inhibit ubiquitination of EGFR for lysosomal degradation in lung adenocarcinoma cells, suggesting that USP8 and SFN could be potential therapeutic targets for NSCLC.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-091 - Expression of Cytoplasmic ECT2 as a New Prognostic Marker for Early-Stage Lung Adenocarcinoma (ID 2194)
09:30 - 17:00 | Author(s): M. Noguchi
- Abstract
Background:
We have examined genetic abnormalities in early-stage lung adenocarcinoma (LAd) using array-comparative genomic hybridization (array-CGH) and found that ECT2 amplification and overexpression can be a new prognostic marker (Cancer Science, 2014). In normal cells, ECT2 is localized in the nucleus, and its function is associated with cytokinesis. In cancer cells, however, ECT2 is thought to exist in the cytoplasm as well as the nucleus. In the cytoplasm, ECT2 is reported to bind to PKCi-Par6a and activate the Rac1 and MAPK pathway. Therefore, cytoplasmic ECT2 is thought to be associated with tumor growth and invasion. In the present study, we examined the clinicopathological implication of cytoplasmic ECT2 in terms of patient outcome, and also the biological significance of cytoplasmic ECT2 using lung adenocarcinoma cell lines.
Methods:
To examine the clinicopathological implication of cytoplasmic ECT2, 66 cases of various types of lung adenocarcinoma were examined using immunohistochemistry (IHC). Nine lung adenocarcinoma cell lines – A549, Calu-3, HCC827, LC-2/ad, NCI-H23, NCI-H1650, NCI-H1975, PC-9 and RERF-LC-KJ – were genetically examined for ECT2 amplification using FISH and for intracellular localization of ECT2 by Western blotting.
Results:
Overexpression of ECT2 in the nucleus was closely associated with the MIB-1 index (r=0.76) and was a strong prognostic factor of lung adenocarcinoma (OS; P=0.0096, DFS; P=0.019). On the other hand, cytoplasmic ECT2 was also associated with patient outcome (OS; P=0.02, DFS; P=0.023). Two of the nine lung adenocarcinoma cell lines, Calu-3 and A549, expressed ECT2 in the cytoplasm as well as the nucleus.Figure 1
Conclusion:
Cytoplasmic ECT2 is a prognostic factor of lung adenocarcinoma, and some lung adenocarcinoma cell lines show localization of ECT2 in the cytoplasm as well as the nucleus.
