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P3.01-061 Prognostic Significance of Stem Cell-Related Marker Expression and Its Correlation with Histologic Subtypes in Lung Adenocarcinoma
S1156
Journal of Thoracic Oncology
Vol. 12 No. 1S
Results: CSC from A549 were expanded in vitro as tumorspheres and stemness marker expression profiles analyzed by flow cytometry. Flow cytometry comparing the cells labeled with the initial library or with the selected aptamers, showed in the latter a large increase in the labeled population. This highly fluorescence-tagged aptamer-labeled cell population was also positive for CD90, described as a marker for cancer stem cells. This doublelabeled population was isolated by cell sorting with the aptamers being purified from the cells, sequenced and grouped into families based on homology between sequences. Eight aptamers were identified, whose affinity and specificity are currently being analyzed.
Conclusion: A stem cell oriented phylogeny of lung cancers objectively orients cancers by level of differentiation in a clinically coherent fashion. Lung cancers most similar to stem cells in expression are associated with a poorer prognosis after treatment.
Topic: Stem Cells in Lung Cancer 2
Keywords: Cancer Stem Cells, Phenotypic markers, Aptamers, Molecular Signature of Membrane
P3.01-061 Prognostic Significance of Stem Cell-Related Marker Expression and Its Correlation with Histologic Subtypes in Lung Adenocarcinoma
P3.01-060 Aptamers as a Tool to Detect Lung Cancer Stem Cells
1
Conclusion: The cell line A549 has a population of cells with stem cell characteristics. Furthermore, the selected aptamers are able to identify a subpopulation of cells, which express stem cell markers. Financial support: FAPESP; CNPq (Brazil).
Topic: Stem Cells in Lung Cancer 2
Isis Nascimento, Jan Mavri, Tina Smuc, Arthur Nery,1 Matjaz Peterka,2 Henning Ulrich1 1 Department of Biochemistry, University of São Paulo, São Paulo/Brazil, 2Centre of Excellence for Biosensors, Instrumentation and Process Control (Cobik), Ajdovscina/ Slovenia Background: Cancer stem cells (CSC) are a subpopulation of cells in the tumor with capacity for self-renewal and differentiation. Due to these characteristics, CSC are referred to as tumor initiating cells. Several studies suggest that CSC might be responsible for metastasis and resistance to conventional therapies leading to tumor recurrence. A challenge in cancer biology is to discover the biomarkers for specific types of cancer and the development of probes capable of identifying these targets. Thus, the objective of this study is the development of DNA aptamers for selective identification of the molecular signature of lung CSC. Methods: A549 lung carcinoma cells were used as target to perform the isolation of aptamers from a random library of DNA through the cell SELEX technique. For negative cycles for removal of DNA molecules binding to common epitopes between different cell types, blood cells were used.
Hyojin Kim,1 Eunhyang Park,2 Soo Young Park,3 Ping-Li Sun,4 Yan Jin,5 Ji Eun Kim,6 Sanghoon Jheon,3 Kwhanmien Kim,3 Choon Taek Lee,3 Jin-Haeng Chung1 1seoul National University Bundang Hospital, seongnam/Korea, Republic of, 2Pathology, Seoul National University College of Medicine, Seoul/Korea, Republic of, 3Seoul National University Bundang Hospital, Seongnam/Korea, Republic of, 4Jilin University Second Hospital, Changchun/China, 5Fudan Univesity Shanghai Cancer Center, Shanghai/China, 6Seoul National University Boramae Hospital, Seoul/Korea, Republic of Background: Cancer stem cells (CSCs) are a small subset of tumor cells that exhibit stem cell-like properties and contribute in treatment failure. CSCs can be distinguished from other cancer cells on the basis of specific markers. Although the clinical impact of these markers is unclear, they may have a prognostic or predictive value in NSCLC. Methods: To clarify the expression and prognostic significance of several CSC markers in non-small cell lung cancer, we retrospectively analyzed 368 patients with adenocarcinoma (n ¼ 226) or squamous cell carcinoma (n ¼ 142). We correlated the expression of six CSC
January 2017
Abstracts
S1157
markers e CD133, CD44, aldehyde dehydrogenase 1 (ALDH1), sex determining region Y-box 2 (SOX2), octamer binding transcription factor 4 (OCT4), and Nanog e with clinicopathologic and molecular variables and survival outcomes.
the reprogramming process; here, using the lung CSC/ stromal cells co-culture model, integrating with both genome-wide transcriptome and DNA methylome, the gene expression and DNA methylation patterns were analyzed in lung CSCs and the differentiated clones.
Results: In adenocarcinoma, CD133, ALDH1 and CD44 expression was associated with low pathologic stage and absence of lymphovascular invasion, while Nanog expression correlated with high histologic grade, lymphatic invasion and increased expression of Snail-1, a transcription factor associated with epithelial-mesenchymal transition. CSC marker expression was also associated with histologic subtypes in adenocarcinoma. Multivariate analysis showed that high Nanog expression was an independent factor associated with a poor prognosis in adenocarcinoma.
Results: We found that the stromal cells-incubated lung CSCs (StriCLS1) were significantly characterized as the CSC enriched population; with highly expressed stemness makers, nanog, oct3/4, sox2, and klf4, and showed relatively higher percentages of side population, ALDH activity and tumorigeniety than differentiated cells in both in vitro tumor sphere forming ability and in vivo xenograft model (only 10 StriCLS1 cells could generate tumor formation in NSG mice). We found that these stemness characteristics diminished as removing the feeder stroma cells and could be restored after re-co-culture with the tumorous stroma cells. Although the whole exon-seq data showed the comparability of StriCLS1 and differentiated CLS1 CSCs on the DNA sequences; the gene expression and DNA methylation patterns revealed significantly changes. The results showed that stemness and its reprogramming were related to paracrine/autocrine networks (IGFBPs, WNTs/Hedgehog, HGF/Met, LIF/ LIFR), metabolism shift (PDKs and LDHs), and other drug-resistance or stress-response signaling (ABCG2 and AKTs), indicating that these key factors were regulated via the niche which may be affected by the DNA methylation patterning.
Conclusion: In conclusion, we have shown that CSC markers may be prognostic factors in NSCLC, and high Nanog expression is an independent prognostic factor for poor survival that may be associated with EMT features in ADC patients. In addition, the clinicopathologic implication of CSC markers in lung ADC differed from those in tumors arising from other organs. Thus, the impact of CSC marker expression should be considered in a tumor/organ specific manner. Keywords: Cancer stem cell marker, lung adenocarcinoma, NANOG, non-small cell lung cancer
P3.01-062 Profiling DNA Methylation and Gene Expression on Cancer Stemness Reprogramming in Lung Adenocarcinoma Topic: Stem Cells in Lung Cancer Sheng-Fang Su,1 Hsuan-Yu Chen,1 Jeremy Chen,2 Huei-Wen Chen3 1Academia Sinica, Taipei/Taiwan, 2 National Chung-Hsing University, Taichung/Taiwan, 3 National Taiwan University, Taipei/Taiwan Background: The cancer stemness niche that could promote and induce the reprogramming of cancer stem cells (CSCs), is considered as one of the key factors in cancer metastasis, tumor recurrence and drug resistance. The behavior of this specific tumorous microenvironment may correlate with poor prognosis of the disease. However, the contribution of the cancer stemness niche on regulating the differentiation or dedifferentiation of CSCs remains unclear. Methods: To investigate the mystery of the niche and to discover the genetic and epigenetic machineries along
Conclusion: We conclude that the cancer stemness reprogramming is a well-regulatory process via the paracrine/autocrine connective networks in the tumor microenvironment. This process may contribute to cancer progression, and assist the tumor growth and evolution under different stresses. This study provides new insight into the importance of crosstalk between CSCs and the cancerous microenvironment that could be targeted as potential genetic/epigenetic signaling regulators for anticancer therapy. Keywords: Cancer Stem Cells, Transcriptomics/ Epigenomics, microenvironment, Cancer Stemness Niche
P3.01-063 XIAP Inhibits Mature Smac Induced Apoptosis by Degrading It through Ubiquitination in NSCLC Topic: Apoptosis in Lung Cancer Sida Qin,1 Chengcheng Yang,2 Boxiang Zhang,1 Xiang Li,1 Xin Sun,1 Gang Li,1 Jing Zhang,1 Guodong Xiao,1 Xiao Gao,1 Guanghong Huang,1
Journal of Thoracic Oncology
Vol. 12 No. 1S
Results: CSC from A549 were expanded in vitro as tumorspheres and stemness marker expression profiles analyzed by flow cytometry. Flow cytometry comparing the cells labeled with the initial library or with the selected aptamers, showed in the latter a large increase in the labeled population. This highly fluorescence-tagged aptamer-labeled cell population was also positive for CD90, described as a marker for cancer stem cells. This doublelabeled population was isolated by cell sorting with the aptamers being purified from the cells, sequenced and grouped into families based on homology between sequences. Eight aptamers were identified, whose affinity and specificity are currently being analyzed.
Conclusion: A stem cell oriented phylogeny of lung cancers objectively orients cancers by level of differentiation in a clinically coherent fashion. Lung cancers most similar to stem cells in expression are associated with a poorer prognosis after treatment.
Topic: Stem Cells in Lung Cancer 2
Keywords: Cancer Stem Cells, Phenotypic markers, Aptamers, Molecular Signature of Membrane
P3.01-061 Prognostic Significance of Stem Cell-Related Marker Expression and Its Correlation with Histologic Subtypes in Lung Adenocarcinoma
P3.01-060 Aptamers as a Tool to Detect Lung Cancer Stem Cells
1
Conclusion: The cell line A549 has a population of cells with stem cell characteristics. Furthermore, the selected aptamers are able to identify a subpopulation of cells, which express stem cell markers. Financial support: FAPESP; CNPq (Brazil).
Topic: Stem Cells in Lung Cancer 2
Isis Nascimento, Jan Mavri, Tina Smuc, Arthur Nery,1 Matjaz Peterka,2 Henning Ulrich1 1 Department of Biochemistry, University of São Paulo, São Paulo/Brazil, 2Centre of Excellence for Biosensors, Instrumentation and Process Control (Cobik), Ajdovscina/ Slovenia Background: Cancer stem cells (CSC) are a subpopulation of cells in the tumor with capacity for self-renewal and differentiation. Due to these characteristics, CSC are referred to as tumor initiating cells. Several studies suggest that CSC might be responsible for metastasis and resistance to conventional therapies leading to tumor recurrence. A challenge in cancer biology is to discover the biomarkers for specific types of cancer and the development of probes capable of identifying these targets. Thus, the objective of this study is the development of DNA aptamers for selective identification of the molecular signature of lung CSC. Methods: A549 lung carcinoma cells were used as target to perform the isolation of aptamers from a random library of DNA through the cell SELEX technique. For negative cycles for removal of DNA molecules binding to common epitopes between different cell types, blood cells were used.
Hyojin Kim,1 Eunhyang Park,2 Soo Young Park,3 Ping-Li Sun,4 Yan Jin,5 Ji Eun Kim,6 Sanghoon Jheon,3 Kwhanmien Kim,3 Choon Taek Lee,3 Jin-Haeng Chung1 1seoul National University Bundang Hospital, seongnam/Korea, Republic of, 2Pathology, Seoul National University College of Medicine, Seoul/Korea, Republic of, 3Seoul National University Bundang Hospital, Seongnam/Korea, Republic of, 4Jilin University Second Hospital, Changchun/China, 5Fudan Univesity Shanghai Cancer Center, Shanghai/China, 6Seoul National University Boramae Hospital, Seoul/Korea, Republic of Background: Cancer stem cells (CSCs) are a small subset of tumor cells that exhibit stem cell-like properties and contribute in treatment failure. CSCs can be distinguished from other cancer cells on the basis of specific markers. Although the clinical impact of these markers is unclear, they may have a prognostic or predictive value in NSCLC. Methods: To clarify the expression and prognostic significance of several CSC markers in non-small cell lung cancer, we retrospectively analyzed 368 patients with adenocarcinoma (n ¼ 226) or squamous cell carcinoma (n ¼ 142). We correlated the expression of six CSC
January 2017
Abstracts
S1157
markers e CD133, CD44, aldehyde dehydrogenase 1 (ALDH1), sex determining region Y-box 2 (SOX2), octamer binding transcription factor 4 (OCT4), and Nanog e with clinicopathologic and molecular variables and survival outcomes.
the reprogramming process; here, using the lung CSC/ stromal cells co-culture model, integrating with both genome-wide transcriptome and DNA methylome, the gene expression and DNA methylation patterns were analyzed in lung CSCs and the differentiated clones.
Results: In adenocarcinoma, CD133, ALDH1 and CD44 expression was associated with low pathologic stage and absence of lymphovascular invasion, while Nanog expression correlated with high histologic grade, lymphatic invasion and increased expression of Snail-1, a transcription factor associated with epithelial-mesenchymal transition. CSC marker expression was also associated with histologic subtypes in adenocarcinoma. Multivariate analysis showed that high Nanog expression was an independent factor associated with a poor prognosis in adenocarcinoma.
Results: We found that the stromal cells-incubated lung CSCs (StriCLS1) were significantly characterized as the CSC enriched population; with highly expressed stemness makers, nanog, oct3/4, sox2, and klf4, and showed relatively higher percentages of side population, ALDH activity and tumorigeniety than differentiated cells in both in vitro tumor sphere forming ability and in vivo xenograft model (only 10 StriCLS1 cells could generate tumor formation in NSG mice). We found that these stemness characteristics diminished as removing the feeder stroma cells and could be restored after re-co-culture with the tumorous stroma cells. Although the whole exon-seq data showed the comparability of StriCLS1 and differentiated CLS1 CSCs on the DNA sequences; the gene expression and DNA methylation patterns revealed significantly changes. The results showed that stemness and its reprogramming were related to paracrine/autocrine networks (IGFBPs, WNTs/Hedgehog, HGF/Met, LIF/ LIFR), metabolism shift (PDKs and LDHs), and other drug-resistance or stress-response signaling (ABCG2 and AKTs), indicating that these key factors were regulated via the niche which may be affected by the DNA methylation patterning.
Conclusion: In conclusion, we have shown that CSC markers may be prognostic factors in NSCLC, and high Nanog expression is an independent prognostic factor for poor survival that may be associated with EMT features in ADC patients. In addition, the clinicopathologic implication of CSC markers in lung ADC differed from those in tumors arising from other organs. Thus, the impact of CSC marker expression should be considered in a tumor/organ specific manner. Keywords: Cancer stem cell marker, lung adenocarcinoma, NANOG, non-small cell lung cancer
P3.01-062 Profiling DNA Methylation and Gene Expression on Cancer Stemness Reprogramming in Lung Adenocarcinoma Topic: Stem Cells in Lung Cancer Sheng-Fang Su,1 Hsuan-Yu Chen,1 Jeremy Chen,2 Huei-Wen Chen3 1Academia Sinica, Taipei/Taiwan, 2 National Chung-Hsing University, Taichung/Taiwan, 3 National Taiwan University, Taipei/Taiwan Background: The cancer stemness niche that could promote and induce the reprogramming of cancer stem cells (CSCs), is considered as one of the key factors in cancer metastasis, tumor recurrence and drug resistance. The behavior of this specific tumorous microenvironment may correlate with poor prognosis of the disease. However, the contribution of the cancer stemness niche on regulating the differentiation or dedifferentiation of CSCs remains unclear. Methods: To investigate the mystery of the niche and to discover the genetic and epigenetic machineries along
Conclusion: We conclude that the cancer stemness reprogramming is a well-regulatory process via the paracrine/autocrine connective networks in the tumor microenvironment. This process may contribute to cancer progression, and assist the tumor growth and evolution under different stresses. This study provides new insight into the importance of crosstalk between CSCs and the cancerous microenvironment that could be targeted as potential genetic/epigenetic signaling regulators for anticancer therapy. Keywords: Cancer Stem Cells, Transcriptomics/ Epigenomics, microenvironment, Cancer Stemness Niche
P3.01-063 XIAP Inhibits Mature Smac Induced Apoptosis by Degrading It through Ubiquitination in NSCLC Topic: Apoptosis in Lung Cancer Sida Qin,1 Chengcheng Yang,2 Boxiang Zhang,1 Xiang Li,1 Xin Sun,1 Gang Li,1 Jing Zhang,1 Guodong Xiao,1 Xiao Gao,1 Guanghong Huang,1