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A proteomic profiling of laser-microdissected lung adenocarcinoma cells of early lepidic-types.

ABSTRACT: BACKGROUND:In the new pathologic classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011, lepidic type adenocarcinomas are constituted by three subtypes; adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPIA). Although these subtypes are speculated to show sequential progression from preinvasive lesion to invasive lung cancer, changes of protein expressions during these processes have not been fully studied yet. This study aims to glimpse a proteomic view of the early lepidic type lung adenocarcinomas. METHODS:A total of nine formalin-fixed and paraffin-embedded (FFPE) lepidic type lung adenocarcinoma tissues were selected from our archives, three tissues each in AIS, MIA and LPIA. The tumor and peripheral non-tumor cells in these FFPE tissues were collected with laser microdissection (LMD). Using liquid chromatography-tandem mass spectrometry (MS/MS), protein compositions were compared with respect to the peptide separation profiles among tumors collected from three types of tissues, AIS, MIA and LPIA. Proteins identified were semi-quantified by spectral counting-based or identification-based protein-based approach, and statistical evaluation was performed by pairwise G-tests. RESULTS:A total of 840 proteins were identified. Spectral counting-based semi-quantitative comparisons of all identified proteins through AIS to LPIA have revealed that the protein expression profile of LPIA was significantly differentiated from other subtypes. 70 proteins including HPX, CTTN, CDH1, EGFR, MUC1 were found as LPIA-type marker candidates, 15 protein candidates for MIA-type marker included CRABP2, LMO7, and NPEP, and 26 protein candidates for AIS-type marker included LTA4H and SOD2. The STRING gene set enrichment resulted from the protein-protein interaction (PPI) network analysis suggested that AIS was rather associated with pathways of focal adhesion, adherens junction, tight junction, that MIA had a strong association predominantly with pathways of proteoglycans in cancer and with PI3K-Akt. In contrast, LPIA was associated broadly with numerous tumor-progression pathways including ErbB, Ras, Rap1 and HIF-1 signalings. CONCLUSIONS:The proteomic profiles obtained in this study demonstrated the technical feasibility to elucidate protein candidates differentially expressed in FFPE tissues of LPIA. Our results may provide candidates of disease-oriented proteins which may be related to mechanisms of the early-stage progression of lung adenocarcinoma.

SUBMITTER: Kato Y

PROVIDER: S-EPMC4501340 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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A proteomic profiling of laser-microdissected lung adenocarcinoma cells of early lepidic-types.

Kato Yasufumi Y Nakamura Haruhiko H Tojo Hiromasa H Nomura Masaharu M Nagao Toshitaka T Kawamura Takeshi T Kodama Tatsuhiko T Ohira Tatsuo T Ikeda Norihiko N Fehniger Thomas T Marko-Varga György G Nishimura Toshihide T Kato Harubumi H

Clinical and translational medicine 20150703 1

<h4>Background</h4>In the new pathologic classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011, lepidic type adenocarcinomas are constituted by three subtypes; adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPIA). Although these subtypes are speculated to show sequential progression from preinvasive lesion to invasive lung cancer, changes of protein expressions during these processes have not been fully stu ...[more]

PMID: 26162278

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Project description:BACKGROUND: Non-mucinous bronchioloalveolar carcinoma (BAC) is considered the early stage of lung adenocarcinoma and is classified as the lung adenocarcioma in situ (AIS) by the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society. This study was designed to investigate the gene expression differences between AIS (formerly non-mucinous BAC) and invasive lepidic predominant adenocarcinoma (LPA, formerly non-mucinous BAC pattern with >5 mm invasion, mixed type adenocarcinoma with BAC features) and to investigate the mechanism of the progression of lung adenocarcinoma in situ to invasive adenocarcinoma. METHODS: Gene expression analysis was performed by using Agilent 4?×?44 K Whole Human Genome Oligo Microarray on 10 fresh frozen tissue samples of AIS and LPA, respectively. Real time RT-PCR was used to validate the differential expression of 13 genes selected by cDNA microarray on fresh frozen tissue samples from 41 patients with lung adenocarcinoma and 4 genes were confirmed. These 4 genes were then validated by western blotting. Immunohistochemical staining for these validated genes was performed on formalin-fixed, paraffin-embedded tissue samples from 81 cases of lung adenocarcinomna. RESULTS: We identified a 13 gene expression signature by comparative analysis of gene expression. Expression of these genes strongly differed between AIS and LPA. Four genes (MMP-2, c-fos, claudin 1 (CLDN1) and claudin 10(CLDN10)) were correlated with the results of microarray and real time RT-PCR analyses for the gene-expression data in samples from 41 patients with lung adenocarcinoma. As confirmed by western blotting, the expression levels of MMP-2 and c-fos were higher in LPA than those in AIS; the expression levels of CLDN1 and CLDN10 in LPA were lower than those in AIS. Immunohistochemical staining for these genes in samples from 81 cases of lung adenocarcinoma demonstrated the expressions of CLDN1 and CLDN10 were correlated with overall survival of patients with lung adenocarcinoma. CONCLUSIONS: CLDN1 and CLDN10 may play important roles in the development of AIS to LPA. Overexpression of CLDN1 and CLDN10 indicates a favorable prognosis for overall survival in some patients with lung adenocarcinoma. Expression of CLDN10 may be regulated by the c-fos pathway.

Project description:Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown.To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA.Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs.Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.

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A proteomic profiling of laser-microdissected lung adenocarcinoma cells of early lepidic-types.

Publications

A proteomic profiling of laser-microdissected lung adenocarcinoma cells of early lepidic-types.

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