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S. Sakashita
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P1.05 - Poster Session with Presenters Present (ID 457)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Early Stage NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.05-006 - Identification of miRNAs and mRNAs Associated with Metastasis in Early-Stage Non-Small Cell Lung Cancer (NSCLC) (ID 5829)
14:30 - 14:30 | Author(s): S. Sakashita
- Abstract
Background:
Early-stage NSCLC patients whose tumours can form primary xenografts (XG) in immune deficient mice have significantly shorter disease-free survival and are at a greater risk of early metastasis compared with patients whose tumours do not form xenografts (non-XG). Genomic and proteomic characterization of XG and non-XG-forming primary patient tumours may reveal clinically relevant genetic aberrations that are associated with early metastasis.
Methods:
miRNA-seq and RNA-seq data of 100 early-stage NSCLC patients with known engraftment status were acquired. The cohort includes 62% adenocarcinoma (ADC) and 38% squamous cell carcinoma (SQCC). Least absolute shrinkage and selection operator (LASSO) was applied to identify features associated with XG status using integrated miRNA and mRNA abundance profiles. Gene Ontology (GO) annotation was subsequently performed to elucidate biological processes that may be altered between the two patient groups.
Results:
Using miRNA and mRNA data alone, ADC patients were classified as XG and non-XG with 88.7% and 95.2% accuracy. The integration of these two data types classified the patients with 100% accuracy using 20 features (7 miRNAs and 13 mRNAs). While less is known regarding the roles of the identified miRNAs in lung ADC, several of the genes have been suggested to affect the metastatic ability of lung cancer cells; these include PITX1, GPNMB and KRT14. In SQCC, both the miRNA and mRNA data alone and the integrated profiles were able to classify patients into XG and non-XG-forming groups with 100% accuracy. However, the roles of the selected features (1 miRNA and 11 mRNAs) in the metastasis of SQCC are not well defined. GO annotation of the identified mRNAs in ADC revealed enrichment of biological processes related to B cell differentiation, wound healing and regulation of the immune response and signalling pathway, while catabolic and metabolic processes were enriched in SQ.
Conclusion:
The use of single-dimensional data to classify patients into different prognostic groups may not be sufficient in the presence of heterogeneous patient populations. Integrative analysis of multi-omic data can provide greater insights into clinically relevant genetic aberrations, which can be used to improve the molecular classification of NSCLC.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-014 - miR-3941: A Novel microRNA That Controls IGBP1 Expression and is Associated with Malignant Progression of Lung Adenocarcinoma (ID 4429)
14:30 - 14:30 | Author(s): S. Sakashita
- Abstract
Background:
Immunoglobulin (CD79A) binding protein 1 (IGBP1) binds to PP2Ac and exerts an anti-apoptotic effect. We have already reported that IGBP1 overexpression occurs during the course of malignant progression of lung adenocarcinoma (Sakashita S et al., Pathol Int. 2011). However, the molecular mechanism of IGBP1 overexpression is still unclear. A few reports have documented mutation, hypomethylation, or amplification of IGBP1, but only one study has suggested that down-regulation of miR-34b leads to high expression of IGBP1 (L-P Chen et al. Oncogene. 2011). In this study, we have detected miR-3941 as another functional microRNA that influences the expression status of IGBP1.
Methods:
We performed microRNA array analysis using total RNA extracted from fresh specimens of invasive lung adenocarcinoma (IGBP1+) and minimally invasive adenocarcinoma (IGBP1-). We compared the results of microRNA array with microRNAs listed in TargetScan (a microRNA database) that would potentially bind to IGBP1. Using reverse transcription-quantitative PCR (RT-qPCR), we analyzed the expression levels of candidate microRNAs in frozen specimens of lung adenocarcinoma. We also validated these microRNAs by checking IGBP1 expression and cell proliferation after they had been transfected into lung adenocarcinoma cell lines (A549, PC-9) and confirmed the direct effect of the microRNAs by luciferase reporter assay.
Results:
Using microRNA array and TargetScan, we selected 6 microRNAs (miR-34b, miR-138, miR-374a, miR-374b, miR-1909, miR-3941). RT-qPCR analysis showed that these microRNAs were down-regulated in invasive adenocarcinoma (IGPB1+) relative to adjacent normal lung tissue (IGBP1-) (Fig1A). We transfected these microRNAs into lung adenocarcinoma cell lines, and all of the microRNAs suppressed IGBP1 expression. Among these microRNAs, miR-34b and miR-3941 depressed luciferase activity by targeting 3’UTR-IGBP1 in the luciferase vector (Fig1B). Figure 1
Conclusion:
We have found that miR-3941 targets IGBP1 in addition to miR-34b. Down-regulation of both microRNAs can lead to high expression of IGBP1, and this is thought to be associated with progression of lung adenocarcinoma.
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P2.01-071 - Biological Implication of Cytoplasmic ECT2 in Malignant Progression of Lung Adenocarcinoma (ID 4361)
14:30 - 14:30 | Author(s): S. Sakashita
- Abstract
Background:
Epithelial cell transforming 2 (ECT2) is a guanine nucleotide exchange factor (GEF) for Rho family GTPases including RhoA, Rac1, and Cdc42. In normal cells, ECT2 is localized in the nucleus,where it regulates dynamic processes including the cell cycle and cytokinesis. On the other hand, several studies have suggested that ECT2 signaling promotes tumor proliferation, migration, and invasion in non-small cell lung cancer. Recently, Murata et al. demonstrated that ECT2 is amplified in early invasive adenocarcinoma but not in situ adenocarcinoma (Cancer Sci, 105:490, 2014). However, the oncogenic mechanism whereby ECT2 drives cell transformation in lung adenocarcinoma is still unknown
Methods:
Cellular fractionation assay was conducted using nine lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, LC-2/ad, and HCC827. Immunoblotting, Immunofluorescence, and Immunohistochemistry assays were used to evaluate the expression and localization of ECT2. For ECT2 amplification, nine lung adenocarcinoma were genetically examined using Quantitative Real-Time PCR. Immunoprecipitation was used to examine the interaction between ECT2 and PKCι. And ECT2 siRNA was confirmed the effect of ECT2 on the downstream singling pathway.
Results:
In this study, we showed that ECT2 was localized predominantly in the nucleus of normal lung epithelial cells, whereas tumor cells in nine lung adenocarcinoma cell lines expressed ECT2 protein to differing degrees in their cytoplasm. Importantly, high expression of cytoplasmic ECT2 in surgically resected materials was significantly associated with poor outcome. Moreover, our data showed that overexpression of ECT2 mRNA was roughly correlated with ECT2 amplification in lung adenocarcinoma cell lines. We then investigated the mechanism underlying the cytoplasmic localization of ECT2 and its oncogenic activity in lung adenocarcinoma using the lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, LC-2/ad, and HCC827. We found that the cytoplasmic ECT2 was phosphorylated and bound to protein kinase C iota (PKCι) in the cytoplasm. We also observed that the overexpression of cytoplasmic ECT2 greatly increased its degree of phosphorylation and enhanced its interaction with PKCι, resulting in significant promotion of tumor growth through activation of the Mek1,2/Erk1,2 cytoplasmic downstream signaling pathway.
Conclusion:
These results indicate that aberrant cytoplasmic localization of ECT2 is a specific feature of lung adenocarcinoma and important for its malignant progression. This finding offers new insight into the molecular mechanism responsible for aberrant cytoplasmic localization of ECT2, which is correlated with the progression of malignancy, and highlights cytoplasmic ECT2 expression as a new prognostic biomarker in lung adenocarcinoma.
