Journal Thoracic Oncology

Abstracts Journal of Thoracic Oncology • Volume 12 Issue S1 January 2017
POSTER SESSION 3 – P3.01: BIOLOGY/PATHOLOGY
FUNCTIONAL BIOLOGY IN LUNG CANCER –
WEDNESDAY, DECEMBER 7, 2016
P3.01-048 CIGARETTE SMOKING IS ASSOCIATED WITH EPITHELIO-
MESENCHYMAL TRANSITION IN HUMAN ADENOCARCINOMA
Toshi Menju 1 , Ryo Miyata 1 , Shigeto Nishikawa 1 , Koji Takahashi 1 , Hiroyuki Cho 1 ,
Shinya Neri 1 , Takao Nakanishi 1 , Masatsugu Hamaji 1 , Hideki Motoyama 1 , Kyoko
Hijiya 1 , Akihiro Aoyama 1 , Toyofumi Chen-Yoshikawa 1 , Toshihiko Sato 1 , Makoto
Sonobe 1 , Akihiko Yoshizawa 2 , Hironori Haga 3 , Hiroshi Date 1
1 Thoracic Surgery, Kyoto University, Graduate School of Medicine, Kyoto/Japan,
2 Diagnostic Pathology, Kyoto University Hospital, Kyoto/Japan, 3 Department of
Diagnostic Pathology, Kyoto University Hospital, Kyoto/Japan
Background: Cigarette smoking (CS) is well known to cause lung cancer. In
addition to the mechanisms of tumorigenesis of lung cancer with CS, a lot of
evidences are currently accumulating that CS induces epithelio-mesenchymal
transition (EMT) in tumor cells. The correlation of CS with the malignant
properties of lung cancer remained elusive in clinical settings. Here we
examined the clinical significance of CS with regard to EMT and malignant
progression in human lung adenocarcinoma. Methods: Clinical samples
were obtained from the 239 cases of resected lung adenocarcinoma which
were consecutively operated from January 2001 to December 2007 in our
institution. Pathological stage distribution of the cases by TNM classification
(WHO, 7th edition) was below: 1A: 118, 1B: 71, 2A: 22, 2B: 4, 3A: 23, 3B: 1.
Smoking history was taken from all the patients, then their smoking status
were classified into 3 groups according to the Brinkman Index (Non;0, n=109:
Light;1~400, n=29: Heavy;401~, n=101). The samples were immunostained
against E-cadherin and Vimentin using tissue microarrays of resected
specimens to assess the activation level of EMT. Then, we classified into 3
groups: the group ‘N’, E-cadherin(E+) and Vimentin(V-), “null” EMT activation;
the group ‘F’, E-/V+, ‘full’ EMT; the group ‘P’, E-/V- or E+/V+, ‘partial’ EMT. The
numbers of the group F/ P/ N were 38/ 93/ 108, respectively. Furthermore, DNA
samples were extracted from frozen surgical samples and the mutations for
the hot-spot exons of EGFR, K-ras, and p53 were detected by SSCP or direct
sequencing methods. The differences of survival duration, pathological
invasive factors, DNA mutations and EMT activation level were statistically
analysed among smoking groups. Results: Significant difference was found in
5-year survival rate among 3 smoking groups: Non, 89.1%; Light, 89.5%; Heavy,
61.7%. Smoking status was significantly associated with EMT activation, DNA
mutation status, local invasive factors, and lymph-node metastasis. In the
tumors harboring either wild-type K-ras or wild-type p53, heavy smoking was
associated with EMT activation (p70% of
cases (p=1.64E-21). However, mutation of known upstream regulators was
not sufficient to explain the frequency of ELF3 overexpression. Instead, the
ELF3 locus underwent frequent (>80%) genetic alteration including focal
amplification and promoter hypomethylation, which corresponded with
increased expression. ELF3 was predominantly localized to the nucleus,
consistent with its transcription factor function. Analysis of PPI networks
indicated highly LUAD-specific ELF3 interactions whereby loss and gain
of interactions lead to reprogramming of LUAD transcriptional networks,
including loss of TNFα pathway, and gain of TGFβ pathway, PI3K pathway, and
translesion (DNA repair) pathway interactions. Furthermore, EGFR, KRAS,
and MYC transgenic models of LUAD tumourigenesis all displayed a marked
increase (6 to 8-fold) in ELF3 expression signifying its importance to LUAD of
varied genetic backgrounds. In culture, ELF3 regulated proliferation, viability
and anchorage-independent growth. In animal models, ELF3 knock-down cells
underwent negative clonal selection, suggesting ELF3 expression is beneficial
to tumour growth. Clinically, high expression of ELF3 was associated with
poor survival regardless of tumour stage. Conclusion: Overexpression of ELF3
reprograms protein-protein-interactions in LUAD leading to the activation of
cancer-specific pathways, and producing oncogenic phenotypes. Depletion
of ELF3 with shRNAs reverses tumour cell growth, suggesting ELF3 is a
promising therapeutic target. In addition to ELF3, interruption of cancerspecific
PPIs also represents a therapeutically actionable strategy.
Keywords: protein-protein interactions, oncogene, Genomics, genetic
mechanisms
POSTER SESSION 3 – P3.01: BIOLOGY/PATHOLOGY
FUNCTIONAL BIOLOGY IN LUNG CANCER –
WEDNESDAY, DECEMBER 7, 2016
P3.01-050 ISOLATION AND CHARCTERIZATION OF LYMPHATIC
ENDOTHELIAL CELLS FROM NEOPLASTIC AND NORMAL HUMAN
LUNG
Federico Quaini 1 , Bruno Lorusso 1 , Angela Falco 1 , Denise Madeddu 1 , Costanza
Lagrasta 2 , Giovanni Bocchialini 1 , Giulia Mazzaschi 1 , Andrea Gervasi 1 , Stefano
Cavalli 1
1 Clinical and Experimental Medicine, University of Parma, Parma/Italy, 2 S.Bi.Bi.T,
University of Parma, Parma/Italy
Background: Cell culture models may be crucial to study lung microvascular
endothelium and its role in cancer progression and treatment. In addition, a
high heterogeneity among endothelial cells from various districts has been
demonstrated, with greater difference on the lymphatic circulatory system.
Organ-specific endothelial cells are essential to elucidate signalling pathways
involved in the pathogenetic mechanisms of neoplastic lung diseases and to
provide novel approaches to reach the goal of a true personalized therapy.
The aim of the present study was to isolate and characterize lymphatic
endothelial cells from neoplastic and healthy human lung. Methods: A simple
and unexpensive method, requiring minimal equipment and accessories
was utilized to harvest, isolate and expand lymphatic endothelial cells from
human lung (Lu-LECs). Specifically, samples from lung cancer (T) and spared
distal lung (D) of 46 patients undergoing lobectomy or pneumonectomy for
NSCLC, were processed. To obtain a pure population of Lu-LEC, a two-step
purification tool based on sorting with monoclonal antibody to CD31 and
podoplanin coated paramagnetic beads was employed. Immunohistochemical
analysis on harvested pulmonary tissues was performed to assess the
presence or absence of neoplastic cells and to identify blood and lymphatic
vasculature. Results: The purity of cultured endothelial cells was ascertained
by morphologic criteria including TEM analysis, immunocytochemistry, flow
cytometry and functional assays. T and D Lu-LECs were positive for CD31.
Moreover, to define the lymphatic phenotype, we examined the specific
markers podoplanin, LYVE-1 and Prox-1. No significant immunophenotypic
differences between D and T Lu-LEC were detected by FACS. Although T
Lu-LEC were larger than D Lu-LEC, morphologic features as cytoplasmic
microvescicles, Weibel-Palade Bodies and aggregate of parallel intermediate
filaments were equally observed. Cells were characterized in vitro for the
ability to express several receptor tyrosine kinases (RTKs) implicated in cell
survival and proliferation and in the development and progression of cancer.
Cultured lymphatic endothelial cells variably expressed VEGFR-2, VEGFR-3,
PDGFR-beta, c-met, and IGF-1R according to D or T origin. Moreover, FGFR-1
and EGFR-1 were present in a large fraction of T and D Lu-LEC. Matrigel assay
documented that T Lu-LEC more efficiently organized in tubular structures at
early time point when compared to D counterpart. Conversely, wound healing
assay revealed that D Lu-LEC had a superior migratory ability. Conclusion:
Primary lines of LECs from the human lung have been consistently obtained
Copyright © 2016 by the International Association for the Study of Lung Cancer
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