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1.  Reliable in vitro studies require appropriate ovarian cancer cell lines 
Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future.
PMCID: PMC4058698  PMID: 24936210
Epithelial ovarian cancer; Tubal cancer; Peritoneal cancer; Primary cultures; Immortalization
2.  Lung Cancer: Are we up to the Challenge? 
Current Genomics  2010;11(7):513-518.
Lung cancer is the leading cause of cancer deaths worldwide among both men and women, with more than 1 million deaths annually. Non–small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers.
Although recent advances have been made in diagnosis and treatment strategies, the prognosis of NSCLC patients is poor and it is basically due to a lack of early diagnostic tools.
However, in the last years genetic and biochemical studies have provided more information about the protein and gene’s mutations involved in lung tumors. Additionally, recent proteomic and microRNA’s approaches have been introduced to help biomarker discovery.
Here we would like to discuss the most recent discoveries in lung cancer pathways, focusing on the genetic and epigenetic factors that play a crucial role in malignant cell proliferation, and how they could be helpful in diagnosis and targeted therapy.
PMCID: PMC3048313  PMID: 21532835
Lung cancer; oncosuppressors; oncogenes; epigenetics of lung cancer; diagnostic tools for lung cancer.
3.  Stimulation of neoplastic mouse lung cell proliferation by alveolar macrophage-derived, insulin-like growth factor-1 can be blocked by inhibiting MEK and PI3K activation 
Molecular Cancer  2011;10:76.
Worldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear.
After culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection.
Primary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth.
Macrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.
PMCID: PMC3135566  PMID: 21699731
Lung cancer; macrophages; proliferation; IGF-1; cytokines
4.  Verification and Unmasking of Widely Used Human Esophageal Adenocarcinoma Cell Lines 
For decades, hundreds of different human tumor type–specific cell lines have been used in experimental cancer research as models for their respective tumors. The veracity of experimental results for a specific tumor type relies on the correct derivation of the cell line. In a worldwide effort, we verified the authenticity of all available esophageal adenocarcinoma (EAC) cell lines. We proved that the frequently used cell lines SEG-1 and BIC-1 and the SK-GT-5 cell line are in fact cell lines from other tumor types. Experimental results based on these contaminated cell lines have led to ongoing clinical trials recruiting EAC patients, to more than 100 scientific publications, and to at least three National Institutes of Health cancer research grants and 11 US patents, which emphasizes the importance of our findings. Widespread use of contaminated cell lines threatens the development of treatment strategies for EAC.
PMCID: PMC2902814  PMID: 20075370
5.  Lung cancer cell lines: Useless artifacts or invaluable tools for medical science? 
Multiple cell lines (estimated at 300–400) have been established from human small cell (SCLC) and non-small cell lung cancers (NSCLC). These cell lines have been widely dispersed to and used by the scientific community worldwide, with over 8000 citations resulting from their study. However, there remains considerable skepticism on the part of the scientific community as to the validity of research resulting from their use. These questions center around the genomic instability of cultured cells, lack of differentiation of cultured cells and absence of stromal–vascular–inflammatory cell compartments. In this report we discuss the advantages and disadvantages of the use of cell lines, address the issues of instability and lack of differentiation. Perhaps the most important finding is that every important, recurrent genetic and epigenetic change including gene mutations, deletions, amplifications, translocations and methylation-induced gene silencing found in tumors has been identified in cell lines and vice versa. These “driver mutations” represented in cell lines offer opportunities for biological characterization and application to translational research. Another potential shortcoming of cell lines is the difficulty of studying multistage pathogenesis in vitro.To overcome this problem, we have developed cultures from central and peripheral airways that serve as models for the multistage pathogenesis of tumors arising in these two very different compartments. Finally the issue of cell line contamination must be addressed and safeguarded against. A full understanding of the advantages and shortcomings of cell lines is required for the investigator to derive the maximum benefit from their use.
PMCID: PMC3110769  PMID: 20079948
Lung cancer; Cell lines; Preneoplasia; Oncogenes; Tumor suppressor genes; Genetic instability
6.  Characterization of the cell of origin for small cell lung cancer 
Cell Cycle  2011;10(16):2806-2815.
Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.
PMCID: PMC3219544  PMID: 21822053
Rb; p53; SCLC; cell of origin; cancer; lung; neuroendocrine
7.  Bone Morphogenetic Protein Type I Receptor Antagonists Decrease Growth and Induce Cell Death of Lung Cancer Cell Lines 
PLoS ONE  2013;8(4):e61256.
Bone morphogenetic proteins (BMPs) are highly conserved morphogens that are essential for normal development. BMP-2 is highly expressed in the majority of non-small cell lung carcinomas (NSCLC) but not in normal lung tissue or benign lung tumors. The effects of the BMP signaling cascade on the growth and survival of cancer cells is poorly understood. We show that BMP signaling is basally active in lung cancer cell lines, which can be effectively inhibited with selective antagonists of the BMP type I receptors. Lung cancer cell lines express alk2, alk3, and alk6 and inhibition of a single BMP receptor was not sufficient to decrease signaling. Inhibition of more than one type I receptor was required to decrease BMP signaling in lung cancer cell lines. BMP receptor antagonists and silencing of BMP type I receptors with siRNA induced cell death, inhibited cell growth, and caused a significant decrease in the expression of inhibitor of differentiation (Id1, Id2, and Id3) family members, which are known to regulate cell growth and survival in many types of cancers. BMP receptor antagonists also decreased clonogenic cell growth. Knockdown of Id3 significantly decreased cell growth and induced cell death of lung cancer cells. H1299 cells stably overexpressing Id3 were resistant to growth suppression and induction of cell death induced by the BMP antagonist DMH2. These studies suggest that BMP signaling promotes cell growth and survival of lung cancer cells, which is mediated through its regulation of Id family members. Selective antagonists of the BMP type I receptors represents a potential means to pharmacologically treat NSCLC and other carcinomas with an activated BMP signaling cascade.
PMCID: PMC3625205  PMID: 23593444
8.  Diallyl Trisulfide Selectively Causes Bax and Bak Mediated Apoptosis in Human Lung Cancer Cells 
Garlic-derived organosulfur compounds (OSCs) are highly effective in affording protection against chemically-induced pulmonary carcinogenesis in animal models. We now demonstrate that garlic constituent diallyl trisulfide (DATS) suppresses viability of cultured human lung cancer cell lines H358 (a non-small cell lung cancer cell line) and H460 (a large cell lung cancer cell line) by causing G2-M phase cell cycle arrest and apoptotic cell death. On the other hand, a normal human bronchial epithelial cell line BEAS-2B was significantly more resistant to growth inhibition and apoptosis induction by DATS compared with lung cancer cells. We also found that even a subtle change in the OSC structure could have a significant impact on its biological activity. For example, DATS was significantly more effective than either diallyl sulfide or diallyl disulfide against proliferation of lung cancer cells. The DATS-mediated G2-M phase cell cycle arrest was explained by down-regulation of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C protein expression leading to accumulation of Tyr15 phosphorylated (inactive) Cdk1. The DATS-induced apoptosis correlated with induction of proapoptotic proteins Bax, Bak, and BID, and a decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL in lung cancer cells but not in BEAS-2B. Knockdown of Bax and Bak proteins conferred significant protection against DATS-induced apoptotic cytoplasmic histone-associated DNA fragmentation. On the other hand, BID protein was dispensable for DATS-induced apoptosis. In conclusion, the present study indicates that Bax and Bak proteins are critical targets of DATS-induced apoptosis in human lung cancer cells.
PMCID: PMC2656596  PMID: 18800351
Diallyl trisulfide; lung cancer; Bax; Bak; apoptosis
9.  Assessing the effectiveness and safety of liposomal paclitaxel in combination with cisplatin as first-line chemotherapy for patients with advanced NSCLC with regional lymph-node metastasis: study protocol for a randomized controlled trial (PLC-GC trial) 
Trials  2013;14:45.
Lung cancer is still the leading cause of cancer-related mortality worldwide. Around 80 to 85% of lung cancers are non-small cell lung cancer (NSCLC). Regional lymphatic metastasis is a frequent occurrence in NSCLC, and the extent of lymphatic dissemination significantly determines the prognosis of patients with NSCLC. Hence, identification of alternative treatments for these patients should be considered a priority. Liposomal paclitaxel is a new formulation composed of paclitaxel and liposomes, with favorable pharmacokinetic properties. In particular, it produces dramatically higher drug concentrations in the lymph nodes than occurs with the current formulations of paclitaxel, thus we believe that patients with NSCLC with regional lymphatic metastasis may benefit from this new drug. Cisplatin-based doublet chemotherapy is recommended as the first-line treatment for patients with advanced NSCLC. We have designed a trial to assess whether first-line chemotherapy using liposomal paclitaxel combined with cisplatin (LP regimen) is superior to gemcitabine combined with cisplatin (GP regimen) in efficacy (both short-term and long-term efficacy) and safety (adverse events; AEs).
This is a prospective, open-label, controlled randomized clinical trial (RCT) to assess the therapeutic effects and safety of liposomal paclitaxel. The study aims to enroll 126 patients, who will be randomly allocated to one of the two treatment groups (LP and GP), with 63 patients in each group. Patients will receive four to six cycles of the assigned chemotherapy, and primary outcome will be assessed every two cycles. Patients will be recommended for surgery if the tumor becomes resectable. All participants will be followed up for at least 12 months. The objective response rate (ORR), changes in regional lymphatic metastasis (including number and size) and TNM (tumor, node, metastasis) staging will be the primary outcome measures. Progression-free survival, objective survival, median survival time, 1-year survival rate, toxicity, and time to disease progression will be the secondary outcome measures.
A systematic search has indicated that this proposed study will be the first RCT to evaluate whether liposomal paclitaxel plus cisplatin will have beneficial effects, compared with gemcitabine plus cisplatin, on enhancing ORR, changing TNM staging, improving long-term survival, and reducing the frequency of AEs for patients with NSCLC with regional lymphatic metastasis.
Trial registration Identifier: ChiCTR-TRC-12602105
PMCID: PMC3599280  PMID: 23413951
Liposomal paclitaxel; Cisplatin; Gemcitabine; Regional lymph node metastasis; Trials
10.  Synchronous lung and gastric cancers successfully treated with carboplatin and pemetrexed: a case report 
Lung and gastric cancers are the first and second leading causes of death from cancer worldwide, and are especially prevalent in Eastern Asia. Relatively few reports are available in relation to the treatment and outcome of synchronous lung and gastric cancers, although there are increasing numbers of patients with these cancers. Efforts to develop more effective drugs for the treatment of synchronous cancers, without serious adverse effects, have been intensifying. Pemetrexed, a multi-targeted antifolate enzyme inhibitor, was approved by the United States Food and Drug Administration as a first-line chemotherapy for advanced non-squamous non-small cell lung cancer in 2007. Although clinical activity against several tumor types of adenocarcinoma, including gastric cancer, has been demonstrated, the efficacy of pemetrexed for gastric cancer remains to be fully evaluated.
Case presentation
We report a case involving a 62-year-old Japanese woman with synchronous locally-advanced poorly-differentiated lung adenocarcinoma and poorly-differentiated gastric adenocarcinoma, containing signet-ring cells distinguished by immunohistochemical profiles. She had been treated with carboplatin and pemetrexed as a first-line chemotherapy for lung cancer, and had achieved partial responses for both lung and gastric cancers. These responses led to a favorable 12-month progression-free survival after the initiation of chemotherapy, and the patient is still alive more than 33 months after diagnosis.
This case suggests a new chemotherapeutic regimen for patients with synchronous multiple primary cancers that have an adenocarcinoma background.
PMCID: PMC3441851  PMID: 22938085
11.  An Integrated Inspection of the Somatic Mutations in a Lung Squamous Cell Carcinoma Using Next-Generation Sequencing 
PLoS ONE  2013;8(11):e78823.
Squamous cell carcinoma (SCC) of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding) of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.
PMCID: PMC3823931  PMID: 24244370
12.  Doubling Times and CT Screen–Detected Lung Cancers in the Pittsburgh Lung Screening Study 
Rationale: As computed tomography (CT) screening for lung cancer becomes more widespread, volumetric analyses, including doubling times, of CT-screen detected lung nodules and lung cancers may provide useful information in the follow-up and management of CT-detected lung nodules and cancers.
Objectives: To analyze doubling times in CT screen detected lung cancers and compare prevalent and nonprevalent cancers and different cell types on non small cell lung cancer.
Methods: We performed volumetric and doubling time analysis on 63 non–small cell lung cancers detected as part of the Pittsburgh Lung Screening Study using a commercially available VITREA 2 workstation and VITREA VITAL nodule segmentation software.
Measurements and Main Results: Doubling times (DT) were divided into three groups: rapid (DT < 183 d), typical (DT 183–365 d), and slow (DT > 365 d). Adenocarcinoma/bronchioloalveolar carcinoma comprised 86.7% of the slow DT group compared with 20% of the rapid DT group. Conversely, squamous cell cancer comprised 60% of the rapid DT group compared with 3.3% of the slow DT group. Twenty-eight of 42 (67%) prevalent and 2 of 21 (10%) nonprevalent cancers were in the slow DT group (P < 0.0001; Fisher's exact test). Twenty-four of 32 (75%) prevalent and 1 of 11 (9%) nonprevalent adenocarcinomas were in the slow DT group (P < 0.0002; Fisher's exact test).
Conclusions: Volumetric analysis of CT-detected lung cancers is particularly useful in AC/BAC. Prevalent cancers have a significantly slower DT than nonprevalent cancers and a higher percentage of adenocarcinoma/bronchioloalveolar carcinoma. These results should affect the management of indeterminant lung nodules detected on screening CT scans.
PMCID: PMC3262038  PMID: 21997335
lung cancer; doubling times; lung cancer screening
13.  Integrative Proteomics and Tissue Microarray Profiling Indicate the Association between Overexpressed Serum Proteins and Non-Small Cell Lung Cancer 
PLoS ONE  2012;7(12):e51748.
Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.
PMCID: PMC3526638  PMID: 23284758
14.  The Transcriptional Consequences of Somatic Amplifications, Deletions, and Rearrangements in a Human Lung Squamous Cell Carcinoma12 
Neoplasia (New York, N.Y.)  2012;14(11):1075-1086.
Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC). As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1) and its matched lymphocyte cell line (AGLCL) to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1), resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA), in a strand-directional manner. We also captured small RNA (<30 bp). We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA). We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.
PMCID: PMC3514744  PMID: 23226101
15.  Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines 
PLoS ONE  2013;8(3):e57020.
Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population.
PMCID: PMC3587631  PMID: 23469181
16.  Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues 
Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.
PMCID: PMC2965362  PMID: 20614197
Cell authentication; STR profiling; Consensus standard; Quality control
17.  Evaluation of safety and efficacy of tivantinib in the treatment of inoperable or recurrent non-small-cell lung cancer 
Tivantinib is a selective, oral, non-ATP-competitive, small molecule inhibitor of the c-Met receptor, tyrosine kinase, which is implicated at different levels of tumor cell migration, invasion, proliferation, and metastasis. Tivantinib has shown antitumor activity in various human tumor cell lines and in xenograft models of human cancers, including non-small-cell lung cancer. Few therapeutic options are available at present for advanced non-small-cell lung cancer, so there is a pressing need for new therapeutic strategies to improve response and survival. Amplification of Met has been reported in more than 20% of lung tumors that have acquired resistance to epidermal growth factor receptor inhibitors, implying that treatment of these tumors with a c-Met inhibitor should overcome resistance. Tivantinib has shown interesting and promising results in advanced non-small-cell lung cancer and appears to be well tolerated, either alone or in combination with other drugs. An interesting additional feature is the ability of the drug to delay development of new metastasis, in agreement with the proposed role of Met in this particular setting.
PMCID: PMC3559079  PMID: 23378782
non-small-cell lung cancer; Met inhibitors; tivantinib; biomarkers
18.  Overexpression of Pygopus-2 is required for canonical Wnt activation in human lung cancer 
Oncology Letters  2013;7(1):233-238.
Lung cancer is the most common cause of cancer-related mortality worldwide. It is necessary to improve the understanding of the molecular mechanisms involved in lung cancer in order to develop more effective therapeutics for the treatment of this fatal disease. The canonical Wnt signaling pathway has been known to be important in a number of cancer types, including lung cancer. Pygopus (Pygo) is a recently identified downstream component of the Wnt signaling pathway required for β-catenin/T-cell factor (TCF)-dependent transcription. However, the role of Pygo in lung cancer remains to be elucidated. The present study showed that Pygo2 is overexpressed in human lung cancer tissue samples and cell lines. Expression levels of Pygo2 were found to correlate with cytosolic β-catenin protein levels in the samples examined. Co-immunofluorescent staining showed that Pygo2 protein accumulated in the nuclei and colocalized with nuclear β-catenin in lung cancer cell lines expressing Pygo2. To investigate the functional importance of the Pygo2 overexpression in lung cancer, short hairpin RNA (shRNA) was used to knockdown Pygo2 mRNA in lung cancer cells expressing the gene. Pygo2 shRNA was observed to inhibit cell proliferation and decrease β-catenin/TCF-dependent transcriptional activity in vitro. Furthermore, Pygo2 shRNA significantly suppressed lung cancer xenograft models in vivo (P<0.05). These results suggested that Pygo2 is a putative therapeutic target for human lung cancer and overexpression of Pygo2 may be important for aberrant Wnt activation in lung cancer.
PMCID: PMC3861596  PMID: 24348855
Pygopus-2; Wnt activation; lung cancer; RNAi; inhibition
19.  CC-Chemokine Ligand 18 Induces Epithelial to Mesenchymal Transition in Lung Cancer A549 Cells and Elevates the Invasive Potential 
PLoS ONE  2013;8(1):e53068.
Lung cancer is one of the leading causes of cancer related death worldwide with more than a million deaths per year. The poor prognosis is due to its high aggressiveness and its early metastasis. Although the exact mechanisms are still unknown, the process of epithelial to mesenchymal transition (EMT) seems to be involved in these neoplastic processes. We already demonstrated that serum levels of CCL18, a primate specific chemokine, are highly elevated in patients with lung cancer and correlate with their survival time of patients with adenocarcinoma of the lung. Therefore, we hypothesized that CCL18 may be directly involved in pathological processes of lung cancer, e.g. EMT. We investigated the effect of CCL18 on A549, an adenocarcinoma cell line of the lung, on EMT and other cell functions like proliferation, chemotaxis, invasion, chemoresistance and proliferation. Exposure of A549 lung cancer cells to CCL18 in various concentrations decreases the epithelial marker E-cadherin, whereas FSP-1, a marker of the mesenchymal phenotype increases. Accordingly, CCL18 induced the transcriptional EMT regulator SNAIL1 in a dose dependent fashion. In contrast, an increasing CCL18 concentration was associated with a decline of cell proliferation rate. In addition, CCL18 induced chemotaxis of these cells and increased their chemoresistance. Therefore, CCL18 may be an interesting therapeutic target for NSCLC.
PMCID: PMC3548837  PMID: 23349697
20.  Prognostic Significance of Twist and N-Cadherin Expression in NSCLC 
PLoS ONE  2013;8(4):e62171.
Metastasis is the most common cause of disease failure and mortality for non-small cell lung cancer after surgical resection. Twist has been recently identified as a putative oncogene and a key regulator of carcinoma metastasis. N-cadherin is associated with a more aggressive behavior of cell lines and tumors. The aim of this study was to evaluate the clinical relevance of Twist and N-cadherin expression in NSCLC, and the effects of Twist1 knockdown on lung cancer cells.
We examined the expressions of Twist and N-cadherin by immunohistochemistry in 120 cases of non-small cell lung cancer (including 68 cases with follow-up records). We also analyzed Twist1 and N-cadherin mRNA expression in 30 non-small cell lung cancer tissues using quantitative reverse transcription polymerase chain reaction. The functional roles of Twist1 in lung cancer cell lines were evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell apoptosis and invasion.
In lung cancer tissues, the overexpression rate of Twist was 38.3% in lung cancer tissues. Overexpression of N-cadherin was shown in 40.83% of primary tumors. Moreover, Twist1 mRNA expression levels correlated with N-cadherin mRNA levels. Furthermore, overexpression of Twist1 or N-cadherin in primary non-small cell lung cancers was associated with a shorter overall survival (P<0.01, P<0.01, respectively). Depleting Twist expression inhibited cell invasion and increased apoptosis in lung cancer cell lines.
The overexpression of Twist and N-cadherin could be considered as useful biomarkers for predicting the prognosis of NSCLC. Twist1 could inhibit apoptosis and promote the invasion of lung cancer cells, and depletion of Twist1 in lung cancer cells led to inhibition of N-cadherin expression.
PMCID: PMC3633889  PMID: 23626784
21.  DNA Fingerprinting of the NCI-60 Cell Line Panel 
Molecular cancer therapeutics  2009;8(4):713-724.
The National Cancer Institute’s NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Since many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also demonstrate that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.
PMCID: PMC4020356  PMID: 19372543
DNA fingerprinting; NCI-60; cell contamination
22.  Can gene expression profiling predict survival for patients with squamous cell carcinoma of the lung? 
Molecular Cancer  2004;3:35.
Lung cancer remains to be the leading cause of cancer death worldwide. Patients with similar lung cancer may experience quite different clinical outcomes. Reliable molecular prognostic markers are needed to characterize the disparity. In order to identify the genes responsible for the aggressiveness of squamous cell carcinoma of the lung, we applied DNA microarray technology to a case control study. Fifteen patients with surgically treated stage I squamous cell lung cancer were selected. Ten were one-to-one matched on tumour size and grade, age, gender, and smoking status; five died of lung cancer recurrence within 24 months (high-aggressive group), and five survived more than 54 months after surgery (low-aggressive group). Five additional tissues were included as test samples. Unsupervised and supervised approaches were used to explore the relationship among samples and identify differentially expressed genes. We also evaluated the gene markers' accuracy in segregating samples to their respective group. Functional gene networks for the significant genes were retrieved, and their association with survival was tested.
Unsupervised clustering did not group tumours based on survival experience. At p < 0.05, 294 and 246 differentially expressed genes for matched and unmatched analysis respectively were identified between the low and high aggressive groups. Linear discriminant analysis was performed on all samples using the 27 top unique genes, and the results showed an overall accuracy rate of 80%. Tests on the association of 24 gene networks with study outcome showed that 7 were highly correlated with the survival time of the lung cancer patients.
The overall gene expression pattern between the high and low aggressive squamous cell carcinomas of the lung did not differ significantly with the control of confounding factors. A small subset of genes or genes in specific pathways may be responsible for the aggressive nature of a tumour and could potentially serve as panels of prognostic markers for stage I squamous cell lung cancer.
PMCID: PMC544571  PMID: 15579197
23.  Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer 
Molecular Cancer  2011;10:96.
Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.
The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo.
We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.
PMCID: PMC3199900  PMID: 21827695
Lung cancer; ALDH activity; cancer stem cells; telomerase
24.  Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression 
The complement system contributes to various immune and inflammatory diseases, including cancer. In this study we investigated the capacity of lung cancer cells to activate complement, and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in non-malignant bronchial epithelial cells. Interestingly, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL6, IL10, LAG3 and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.
PMCID: PMC3478398  PMID: 23028051
25.  Cyclin E Transgenic Mice: Discovery Tools for Lung Cancer Biology, Therapy, and Prevention 
Lung cancer is the leading cause of cancer-related mortality in the United States and many other countries. This fact underscores the need for clinically relevant models to increase our understanding of lung cancer biology and to help design and implement preventive and more-effective therapeutic interventions for lung cancer. New murine transgenic models of non-small cell lung cancer (NSCLC) have been engineered for this purpose. In one such model, overexpression of the cell-cycle regulator cyclin E is targeted to type II alveolar lung cells; dysplasia, hyperplasia and adenocarcinoma forming in this model have features recapitulating key features of carcinogenesis found in NSCLC patients. These features include the presence of chromosomal instability, pulmonary dysplasia and hyperplasia, hedgehog-pathway activation, single and multiple adenocarcinomas, and even metastases. Cell lines that expressed either a human wild-type or mutant (proteasome-degradation–resistant) form of cyclin E were derived from the transgenic mouse lung cancers. These cell lines are transplantable into syngeneic host mice, which rapidly develop lung tumors and thus facilitate the rapid testing of agents targeting lung carcinogenesis. These transgenic and transplantable models have already aided in the discovery of oncogenic and growth-suppressive microRNAs (miRNAs) and in the identification of a novel anti-neoplastic mechanism of action for inhibitors of cyclin-dependent kinase 2. This review discusses the general utility of murine carcinogen-induced and transgenic models of lung carcinogenesis and describes the optimization of cyclin E–overexpressing lung carcinogenesis models and their use in testing candidate agents for the prevention and therapy of lung cancer.
PMCID: PMC3058281  PMID: 21149327

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