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author:("Chen, guan")
1.  Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model 
Oncotarget  2014;5(23):12383-12397.
The potential utility of circulating tumor cells (CTCs) to guide clinical care in oncology patients has gained momentum with emerging micro- and nanotechnologies. Establishing the role of CTCs in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of CTCs for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. Recent attempts in the literature to culture CTCs isolated from metastatic patients using monoculture have had limited success rates of less than 20%. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully expanded CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis.
PMCID: PMC4323004  PMID: 25474037
expansion of CTCs; early stage lung cancer; microfluidic co-culture
2.  A Novel Energy Recovery System for Parallel Hybrid Hydraulic Excavator 
The Scientific World Journal  2014;2014:184909.
Hydraulic excavator energy saving is important to relieve source shortage and protect environment. This paper mainly discusses the energy saving for the hybrid hydraulic excavator. By analyzing the excess energy of three hydraulic cylinders in the conventional hydraulic excavator, a new boom potential energy recovery system is proposed. The mathematical models of the main components including boom cylinder, hydraulic motor, and hydraulic accumulator are built. The natural frequency of the proposed energy recovery system is calculated based on the mathematical models. Meanwhile, the simulation models of the proposed system and a conventional energy recovery system are built by AMESim software. The results show that the proposed system is more effective than the conventional energy saving system. At last, the main components of the proposed energy recovery system including accumulator and hydraulic motor are analyzed for improving the energy recovery efficiency. The measures to improve the energy recovery efficiency of the proposed system are presented.
doi:10.1155/2014/184909
PMCID: PMC4227448  PMID: 25405215
3.  Epigenetic inactivation of microRNA-34b/c predicts poor disease-free survival in early stage lung adenocarcinoma 
Purpose
The microRNA-34b/c (miR-34b/c) has been considered a tumor suppressor in different tumor types and it is a known transcriptional target of the tumor suppressor gene TP53. The main objectives of this study were to investigate the clinical implications of miR-34b/c methylation in early stage lung adenocarcinoma (AC) patients and to determine the functional role of miR-34b/c re-expression in lung AC cell lines.
Experimental Design
Aberrant methylation and expression of miR-34b/c were assessed in 15 lung AC cell lines and a cohort of 140 early stage lung AC. Lung AC cell lines were transfected with miR-34b/c and the effects upon cell proliferation, migration, invasion and apoptosis were investigated.
Results
Aberrant methylation of miR-34b/c was detected in 6 (40%) of 15 lung AC cell lines and 64 out of 140 (46%) primary lung adenocarcinomas. Expression of miR-34b/c was significantly reduced in all methylated cell lines and primary tumors, especially in those harboring a TP53 mutation. Patients with high levels of miR-34b/c methylation had significantly shorter disease-free survival and overall survival as compared to patients with unmethylated miR-34b/c or low level of miR-34b/c methylation. Ectopic expression of miR-34b/c in lung AC cell lines decreased cell proliferation, migration and invasion.
Conclusions
Epigenetic inactivation of miR-34b/c by DNA methylation has independent prognostic value in early stage lung AC patients with surgically resected tumors. Re-expression of miR-34b/c leads to a less aggressive phenotype in lung AC cell lines.
doi:10.1158/1078-0432.CCR-13-0736
PMCID: PMC4161219  PMID: 24130071
microRNA; DNA methylation; microRNA-34b/c; lung adenocarcinoma; TP53
4.  Checkpoint kinase 1 protein expression indicates sensitization to therapy by checkpoint kinase 1 inhibition in non–small cell lung cancer 
Background
When presenting with advanced stage disease, lung cancer patients have <5% 5-y survival. The overexpression of checkpoint kinase 1 (CHK1) is associated with poorer outcomes and may contribute to therapy resistance. Targeting CHK1 with small-molecule inhibitors in p53 mutant tumors might improve the effectiveness of chemotherapy and radiotherapy in non–small cell lung cancer (NSCLC).
Methods
We evaluatedCHK1 messenger RNA and protein levels in multiple NSCLC cell lines. We assessed cell line sensitization to gemcitabine, pemetrexed, and radiotherapy by CHK1 inhibition with the small molecule AZD7762 using proliferation and clonogenic cell survival assays. We analyzed CHK1 signaling by Western blotting to confirm that AZD7762 inhibits CHK1.
Results
We selected two p53 mutant NSCLC cell lines with either high (H1299) or low (H1993) CHK1 levels for further analysis. We found that AZD7762 sensitized both cell lines to gemcitabine, pemetrexed, and radiotherapy. Chemosensitization levels were greater, however, for the higher CHK1 protein expressing cell line, H1299, when compared with H1993. Furthermore, analysis of the CHK1 signaling pathway showed that H1299 cells have an increased dependence on the CHK1 pathway in response to chemotherapy. There was no increased sensitization to radiation in H1299 versus H1993.
Conclusions
CHK1 inhibition by AZD7762 preferentially sensitizes high CHK1 expressing cells, H1299, to anti-metabolite chemotherapy as compared with low CHK1 expressing H1993 cells. Thus, CHK1 inhibitors may improve the efficacy of standard lung cancer therapies, especially for those subgroups of tumors harboring higher expression levels of CHK1 protein.
doi:10.1016/j.jss.2013.12.016
PMCID: PMC4073630  PMID: 24418519
CHK1; NSCLC cell lines; Combination therapies; AZD7762; Chemosensitivity; Radiosensitivity
5.  CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells 
Objective
Overexpression of checkpoint kinase 1 (CHK1) is associated with poorer patient outcome and therapeutic resistance in multiple tumor models. Inhibition of CHK1 has been proposed as a strategy to increase the effectiveness of chemotherapeutic agents, especially in p53-deficient tumors. In this study, we evaluated the effects of a novel CHK1 inhibitor, MK-8776, in combination with pemetrexed (PMX) on cell proliferation and survival in a panel of p53 mutant non-small cell lung cancer (NSCLC) cell lines.
Methods
We examined CHK1 expression in 442 resected lung adenocarcinoma specimens using Affymetrix U133A gene expression arrays. We correlated CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. We evaluated CHK1 levels in NSCLC cell lines and identified four p53 mutant cell lines with variable CHK1 expression (H1993, H23, H1437 and H1299) based on publicly available gene expression data. We confirmed differential CHK1 mRNA and CHK1 protein levels by qRT-PCR, ELISA, Western Blot analysis (WB) and immunohistochemistry. We examined cell line sensitization to PMX in response to CHK1 inhibition with MK-8776 using WST-1 and clonogenic survival assays.
Results
We found that elevated CHK1 expression in primary lung adenocarcinomas correlates with poor tumor differentiation and significantly worse patient survival. Tumors with elevated CHK1 mRNA levels have a higher number of gene mutations and DNA copy number gain or amplifications. CHK1 inhibition by MK-8776 enhances sensitivity of NSCLC cell lines to PMX. CHK1 mRNA and protein expression are variable among NSCLC cell lines, and cells expressing higher levels of CHK1 protein are more sensitive to the CHK1 inhibition by MK-8776 as compared to low CHK1 expressing cells.
Conclusions
These findings suggest that CHK1 levels may not only serve as a biomarker of poor prognosis in surgically-resected lung adenocarcinomas, but could also be a predictive marker for CHK1 inhibitor sensitivity, pending in vivo and clinical confirmation.
doi:10.1016/j.lungcan.2013.09.010
PMCID: PMC4073640  PMID: 24113549
CHK1; Lung; Chemosensitivity; NSCLC; Patient survival; Genomic complexity
6.  Bond Graph Modeling and Validation of an Energy Regenerative System for Emulsion Pump Tests 
The Scientific World Journal  2014;2014:289839.
The test system for emulsion pump is facing serious challenges due to its huge energy consumption and waste nowadays. To settle this energy issue, a novel energy regenerative system (ERS) for emulsion pump tests is briefly introduced at first. Modeling such an ERS of multienergy domains needs a unified and systematic approach. Bond graph modeling is well suited for this task. The bond graph model of this ERS is developed by first considering the separate components before assembling them together and so is the state-space equation. Both numerical simulation and experiments are carried out to validate the bond graph model of this ERS. Moreover the simulation and experiments results show that this ERS not only satisfies the test requirements, but also could save at least 25% of energy consumption as compared to the original test system, demonstrating that it is a promising method of energy regeneration for emulsion pump tests.
doi:10.1155/2014/289839
PMCID: PMC4055536  PMID: 24967428
7.  Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma 
Purpose
The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation and tumor-initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells.
Experimental Design
We investigated the role of Notch activity in lung adenocarcinoma utilizing a Notch GFP-reporter construct and a gamma-secretase inhibitor (GSI), which inhibits Notch pathway activity.
Results
Transduction of lung cancer cells with Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media, and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 441 lung adenocarcinoma patients. This correlation was further confirmed in an independent group of 89 adenocarcinoma patients where Hes-1 overexpression correlated with poor overall survival.
Conclusions
Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma.
doi:10.1158/1078-0432.CCR-12-0370
PMCID: PMC3630232  PMID: 23444212
Cancer Stem cells; Notch pathway; Lung cancer
8.  KRAS Protein Stability Is Regulated through SMURF2: UBCH5 Complex-Mediated β-TrCP1 Degradation12 
Neoplasia (New York, N.Y.)  2014;16(2):115-128.
Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutant of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.
PMCID: PMC3978392  PMID: 24709419
9.  Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis  
Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine KrasG12D-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of KrasG12D-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers.
doi:10.1172/JCI70297
PMCID: PMC3904615  PMID: 24430184
10.  Drug resistance in multiple myeloma: latest findings and new concepts on molecular mechanisms 
Oncotarget  2013;4(12):2186-2207.
In the era of new and mostly effective therapeutic protocols, multiple myeloma still tends to be a hard-to-treat hematologic cancer. This hallmark of the disease is in fact a sequel to drug resistant phenotypes persisting initially or emerging in the course of treatment. Furthermore, the heterogeneous nature of multiple myeloma makes treating patients with the same drug challenging because finding a drugable oncogenic process common to all patients is not yet feasible, while our current knowledge of genetic/epigenetic basis of multiple myeloma pathogenesis is outstanding. Nonetheless, bone marrow microenvironment components are well known as playing critical roles in myeloma tumor cell survival and environment-mediated drug resistance happening most possibly in all myeloma patients. Generally speaking, however; real mechanisms underlying drug resistance in multiple myeloma are not completely understood. The present review will discuss the latest findings and concepts in this regard. It reviews the association of important chromosomal translocations, oncogenes (e.g. TP53) mutations and deranged signaling pathways (e.g. NFκB) with drug response in clinical and experimental investigations. It will also highlight how bone marrow microenvironment signals (Wnt, Notch) and myeloma cancer stem cells could contribute to drug resistance in multiple myeloma.
PMCID: PMC3926819  PMID: 24327604
multiple myeloma; drug resistance; signaling pathways; oncogenes
11.  Characterization of vitamin D receptor (VDR) in lung adenocarcinoma 
Purpose
The anti-proliferative effects of 1α,25-dihydroxyvitamin D3 (1,25-D3, calcitriol, the active form of vitamin D) are mediated by the nuclear vitamin D receptor (VDR). In the present study, we characterized VDR expression in lung adenocarcinoma (AC).
Experimental Design
We examined VDR mRNA expression using a quantitative real-time PCR (qRT-PCR) in 100 patients who underwent surgery for lung AC. In a subset of these patients (n = 89), we examined VDR protein expression using immunohistochemistry. We also examined the association of VDR protein expression with circulating serum levels of 25-hydroxyvitamin D3 (25-D3) and 1,25-D3. The antiproliferative effects and cell cycle arrest of 1,25-D3 were examined using lung cancer cell lines with high (SKLU-1) as well as low (A549) expression of VDR mRNA.
Results
Higher VDR expression correlates with longer survival after adjusting for age, sex, disease stage and tumor grade (HR 0.73, 95% CI 0.58–0.91). In addition, there was a positive correlation (r = 0.38) between serum 1,25-D3 and tumor VDR protein expression. A greater anti-proliferative effect of 1,25-D3 was observed in high compared to low VDR-expressing cell lines; these effects corresponded to G1 cell cycle arrest; this was associated with a decline in cyclin D1, S-phase kinase protein 2 (Skp2), retinoblastoma (Rb) and minichromosome maintenance 2 (MCM2) proteins involved in S-phase entry.
Conclusions
Increased VDR expression in lung AC is associated with improved survival. This may relate to a lower proliferative status and G1 arrest in high VDR-expressing tumors.
doi:10.1016/j.lungcan.2012.04.010
PMCID: PMC3396768  PMID: 22564539
VDR; Vitamin D; 1,25-D3; Lung Adenocarcinoma; Survival
12.  Development and Validation of a qRT-PCR Classifier for Lung Cancer Prognosis 
Purpose
This prospective study aimed to develop a robust and clinically-applicable method to identify high-risk early stage lung cancer patients and then to validate this method for use in future translational studies.
Patients and Methods
Three published Affymetrix microarray data sets representing 680 primary tumors were used in the survival-related gene selection procedure using clustering, Cox model and random survival forest (RSF) analysis. A final set of 91 genes was selected and tested as a predictor of survival using a qRT-PCR-based assay utilizing an independent cohort of 101 lung adenocarcinomas.
Results
The RSF model built from 91 genes in the training set predicted patient survival in an independent cohort of 101 lung adenocarcinomas, with a prediction error rate of 26.6%. The mortality risk index (MRI) was significantly related to survival (Cox model p < 0.00001) and separated all patients into low, medium, and high-risk groups (HR = 1.00, 2.82, 4.42). The MRI was also related to survival in stage 1 patients (Cox model p = 0.001), separating patients into low, medium, and high-risk groups (HR = 1.00, 3.29, 3.77).
Conclusions
The development and validation of this robust qRT-PCR platform allows prediction of patient survival with early stage lung cancer. Utilization will now allow investigators to evaluate it prospectively by incorporation into new clinical trials with the goal of personalized treatment of lung cancer patients and improving patient survival.
doi:10.1097/JTO.0b013e31822918bd
PMCID: PMC3167380  PMID: 21792073
Lung cancer; qRT-PCR; Prognosis
13.  Stromal LRP1 in lung adenocarcinoma predicts clinical outcome 
Purpose
LRP1 is a broadly-expressed receptor that binds multiple extracellular ligands and participates in protein clearance. LRP1 is expressed numerous cancers, but its role in lung cancer has not been characterized. Here, we investigate the relationship between LRP1 and lung cancer.
Experimental Design
LRP1 mRNA levels were determined in lung tumors from several large, multicenter studies. LRP1 protein localization was determined by immunohistochemical analysis of lung tumor microarrays. Normal fibroblasts, fibroblasts treated with the LRP1 inhibitor RAP, and LRP1 null fibroblasts were co-cultured with three independent lung cancer cell lines to investigate the role of LRP1 on tumor cell proliferation.
Results
LRP1 mRNA levels are significantly decreased in lung tumors relative to non-tumorous lung tissue. Lower expression of LRP1 in lung adenocarcinomas correlates with less favorable clinical outcome in a cohort of 439 patients. Immunohistochemical analysis demonstrates that LRP1 is primarily expressed in stromal cells in 94/111 lung cancers, with very little protein found in cancer cells. A growth suppressive function of mouse embryonic fibroblast cells (MEF) was observed in three lung cancer cell lines tested (H460, H2347, and HCC4006 cells); growth suppression was blocked by the LRP1 inhibitor, RAP. LRP1 deletion in fibroblasts reduced the ability of MEF cells to suppress tumor cell mitosis. In a validation set of adenocarcinomas, we confirmed a significant positive correlation between both LRP1 mRNA and protein levels and favorable clinical outcomes.
Conclusions
LRP1 expression is associated with improved lung cancer outcomes. Mechanistically, stromal LRP1 may non-cell autonomously suppress lung tumor cell proliferation.
doi:10.1158/1078-0432.CCR-10-2385
PMCID: PMC3079007  PMID: 21325077
14.  CYP24A1 Is an Independent Prognostic Marker of Survival in Patients with Lung Adenocarcinoma 
Purpose
The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25-D3) exerts antiproliferative effects in cancers, including lung adenocarcinoma (AC). CYP24A1 is overexpressed in many cancers and catabolizes 1,25-D3. The purpose of our study was to assess CYP24A1 as a prognostic marker and to study its relevance to antiproliferative activity of 1,25-D3 in lung AC cells.
Experimental Design
Tumors and corresponding normal specimens from 86 patients with lung AC (stages I–III) were available. AffymetrixR array data and subsequent confirmation by quantitative real time-PCR were used to determine CYP24A1 mRNA expression. A subsequent validation set of 101 lung AC was used to confirm CYP24A1 mRNA expression and its associations with clinical variables. The antiproliferative effects of 1,25-D3 were examined using lung cancer cell lines with high as well as low expression of CYP24A1 mRNA.
Results
CYP24A1 mRNA was elevated 8–50 fold in lung AC (compared to normal nonneoplastic lung) and significantly higher in poorly-differentiated cancers. At 5 years of follow-up, the probability of survival was 42% (high CYP24A1, n = 29) versus 81% (low CYP24A1, n = 57) (P = 0.007). The validation set of 101 tumors showed that CYP24A1 was independently prognostic of survival (multivariate Cox model adjusted for age, gender and stage, P = 0.001). A549 cells (high CYP24A1) were more resistant to antiproliferative effects of 1,25-D3 compared with SKLU-1 cells (low CYP24A1).
Conclusions
CYP24A1 overexpression is associated with poorer survival in lung AC. This may relate to abrogation of antiproliferative effects of 1,25-D3 in high CYP24A1 expressing lung AC.
doi:10.1158/1078-0432.CCR-10-1789
PMCID: PMC3058389  PMID: 21169243
15.  Signatures of Drug Sensitivity in Nonsmall Cell Lung Cancer 
We profiled receptor tyrosine kinase pathway activation and key gene mutations in eight human lung tumor cell lines and 50 human lung tumor tissue samples to define molecular pathways. A panel of eight kinase inhibitors was used to determine whether blocking pathway activation affected the tumor cell growth. The HER1 pathway in HER1 mutant cell lines HCC827 and H1975 were found to be highly activated and sensitive to HER1 inhibition. H1993 is a c-MET amplified cell line showing c-MET and HER1 pathway activation and responsiveness to c-MET inhibitor treatment. IGF-1R pathway activated H358 and A549 cells are sensitive to IGF-1R inhibition. The downstream PI3K inhibitor, BEZ-235, effectively inhibited tumor cell growth in most of the cell lines tested, except the H1993 and H1650 cells, while the MEK inhibitor PD-325901 was effective in blocking the growth of KRAS mutated cell line H1734 but not H358, A549 and H460. Hierarchical clustering of primary tumor samples with the corresponding tumor cell lines based on their pathway signatures revealed similar profiles for HER1, c-MET and IGF-1R pathway activation and predict potential treatment options for the primary tumors based on the tumor cell lines response to the panel of kinase inhibitors.
doi:10.1155/2011/215496
PMCID: PMC3200133  PMID: 22091388
16.  Regulation of EGFR Protein Stability by the HECT-type Ubiquitin Ligase SMURF212 
Neoplasia (New York, N.Y.)  2011;13(7):570-578.
Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA)-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nudemice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expressionmicroarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.
PMCID: PMC3132843  PMID: 21750651
17.  An integrative approach to reveal driver gene fusions from paired-end sequencing data in cancer 
Nature biotechnology  2009;27(11):1005-1011.
Cancer genomes contain many aberrant gene fusions—a few that drive disease and many more that are nonspecific passengers. We developed an algorithm (the concept signature or ‘ConSig’ score) that nominates biologically important fusions from high-throughput data by assessing their association with ‘molecular concepts’ characteristic of cancer genes, including molecular interactions, pathways and functional annotations. Copy number data supported candidate fusions and suggested a breakpoint principle for intragenic copy number aberrations in fusion partners. By analyzing lung cancer transcriptome sequencing and genomic data, we identified a novel R3HDM2-NFE2 fusion in the H1792 cell line. Lung tissue microarrays revealed 2 of 76 lung cancer patients with genomic rearrangement at the NFE2 locus, suggesting recurrence. Knockdown of NFE2 decreased proliferation and invasion of H1792 cells. Together, these results present a systematic analysis of gene fusions in cancer and describe key characteristics that assist in new fusion discovery.
doi:10.1038/nbt.1584
PMCID: PMC3086882  PMID: 19881495
18.  Decreased selenium binding protein-1 (SELENBP1) in esophageal adenocarcinoma results from post-transcriptional and epigenetic regulation and affects chemosensitivity 
Purpose
The chemopreventive effects of selenium have been extensively examined but its role in cancer development or as a chemotherapeutic agent have only recently been explored. Because Selenium Binding Protein 1 (SELENBP1, SBP1, hSP56) has been shown to bind selenium covalently and selenium deficiency has been associated with esophageal adenocarcinoma (EAC), we examined its role in EAC development and its potential effect on chemosensitivity in the presence of selenium.
Experimental Design
SELENBP1 expression level and copy number variation were determined by oligonucleotide microarrays, real-time RT-PCR, tissue microarrays, immunoblotting and SNP arrays. Bisulfite sequencing and sequence analysis of RT-PCR-amplified products explored epigenetic and post-transcriptional regulation of SELENBP1 expression, respectively. WST-1 cell proliferation assays, senescence-associated β-galactosidase staining, immunoblotting, and flow cytometry were performed to evaluate the biological significance of SELENBP1 overexpression in selenium-supplemented EAC cells.
Results
SELENBP1 expression decreased significantly in Barrett's esophagus to adenocarcinoma progression. Both epigenetic and post-transcriptional mechanisms appeared to modulate SELENBP1 expression. Stable overexpression of SELENBP1 in methylseleninic acid-supplemented Flo-1 cells resulted in enhanced apoptosis, increased cellular senescence, and enhanced cisplatin cytotoxicity. Although inorganic sodium selenite similarly enhanced cisplatin cytotoxicity, these 2 forms of selenium elicited different cellular responses.
Conclusions
SELENBP1 expression may be an important predictor of response to chemoprevention or chemosensitization with certain forms of selenium in esophageal tissues.
doi:10.1158/1078-0432.CCR-09-2801
PMCID: PMC2953959  PMID: 20332323
19.  The neuronal repellent SLIT2 is a target for repression by EZH2 in prostate cancer 
Oncogene  2010;29(39):5370-5380.
The neuronal repellent SLIT2 is repressed in a number of cancer types primarily through promoter hypermethylation. SLIT2, however, has not been studied in prostate cancer. Through genome-wide location analysis we identified SLIT2 as a target of Polycomb group (PcG) protein EZH2. The EZH2-containing Polycomb repressive complexes bound to the SLIT2 promoter inhibiting its expression. SLIT2 was down-regulated in a majority of metastatic prostate tumors exhibiting a negative correlation with EZH2. This repressed expression could be restored by methylation inhibitors or EZH2-suppressing compounds. In addition, a low level of SLIT2 expression was associated with aggressive prostate, breast and lung cancers. Functional assays showed that SLIT2 inhibited prostate cancer cell proliferation and invasion. Thus, this study demonstrated for the first time epigenetic silencing of SLIT2 in prostate tumors, and supported SLIT2 as a potential biomarker for aggressive solid tumors. Importantly, PcG-mediated repression may serve as a precursor for the silencing of SLIT2 by DNA methylation in cancer.
doi:10.1038/onc.2010.269
PMCID: PMC2948081  PMID: 20622896
Polycomb group proteins; EZH2; SLIT2; prostate cancer; epigenetic silencing; DNA hypermethylation
20.  An embryonic stem cell-like signature identifies poorly-differentiated lung adenocarcinoma, but not squamous cell carcinoma 
Purpose
An embryonic stem cell profile correlates with poorly differentiated breast, bladder and glioma cancers. In this manuscript, we assess the correlation between the embryonic stem cell profile and clinical variables in lung cancer.
Experimental Design
Microarray gene expression analysis was done using Affymetrix Human Genome U133A on 443 samples of human lung adenocarcinoma and 130 samples of squamous cell carcinoma. To identify gene-set enrichment patterns we used the Genomica software.
Results
Our analysis showed that an increased expression of the embryonic stem cell gene set and decreased expression of Polycomb target gene set identified poorly-differentiated lung adenocarcinoma. In addition, this gene expression signature was associated with markers of poor prognosis and worse overall survival in lung adenocarcinoma. However, there was no correlation between this embryonic stem cell gene signature and any histological or clinical variable assessed in lung squamous cell carcinoma.
Conclusions
This work suggests that not all poorly-differentiated non-small cell lung cancers exhibit a gene expression profile similar to ESC, and that other characteristics may play a more important role in the determination of differentiation and survival in squamous cell carcinoma of the lung.
doi:10.1158/1078-0432.CCR-09-1105
PMCID: PMC2787085  PMID: 19808871
Embryonic genes; stem cell; Affymetrix; lung; cancer
21.  Clinically Relevant Characterization of Lung Adenocarcinoma Subtypes Based on Cellular Pathways: An International Validation Study 
PLoS ONE  2010;5(7):e11712.
Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient's prognosis. Additional results suggest the ability to use a similar approach to determine a patient's sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.
doi:10.1371/journal.pone.0011712
PMCID: PMC2908611  PMID: 20661423
22.  Application of serum SELDI proteomic patterns in diagnosis of lung cancer 
BMC Cancer  2005;5:83.
Background
Currently, no satisfactory biomarkers are available to screen for lung cancer. Surface-Enhanced Laser Desorption/ionization Time-of- Flight Mass Spectrometry ProteinChip system (SELDI-TOF-MS) is one of the currently used techniques to identify biomarkers for cancers. The aim of this study is to explore the application of serum SELDI proteomic patterns to distinguish lung cancer patients from healthy individuals.
Methods
A total of 208 serum samples, including 158 lung cancer patients and 50 healthy individuals, were randomly divided into a training set (including 11 sera from patients with stages I/II lung cancer, 63 from patients with stages III/IV lung cancer and 20 from healthy controls) and a blinded test set (including 43 sera from patients with stages I/II lung cancer, 41 from patients with stages III/IV lung cancer and 30 from healthy controls). All samples were analyzed by SELDI technology. The spectra were generated on weak cation exchange (WCX2) chips, and protein peaks clustering and classification analyses were made using Ciphergen Biomarker Wizard and Biomarker Pattern software, respectively. We additionally determined Cyfra21-1 and NSE in the 208 serum samples included in this study using an electrochemiluminescent immunoassay.
Results
Five protein peaks at 11493, 6429, 8245, 5335 and 2538 Da were automatically chosen as a biomarker pattern in the training set. When the SELDI marker pattern was tested with the blinded test set, it yielded a sensitivity of 86.9%, a specificity of 80.0% and a positive predictive value of 92.4%. The sensitivities provided by Cyfra21-1 and NSE used individually or in combination were significantly lower than that of the SELDI marker pattern (P < 0.005 or 0.05, respectively). Based on the results of the test set, we found that the SELDI marker pattern showed a sensitivity of 91.4% in the detection of non-small cell lung cancers (NSCLC), which was significantly higher than that in the detection of small cell lung cancers (P < 0.05); The pattern also had a sensitivity of 79.1% in the detection of lung cancers in stages I/II.
Conclusion
These results suggest that serum SELDI protein profiling can distinguish lung cancer patients, especially NSCLC patients, from normal subjects with relatively high sensitivity and specificity, and the SELDI-TOF-MS is a potential tool for the screening of lung cancer.
doi:10.1186/1471-2407-5-83
PMCID: PMC1183195  PMID: 16029516
23.  Proteomic Analysis of Cytokeratin Isoforms Uncovers Association with Survival in Lung Adenocarcinoma1 
Neoplasia (New York, N.Y.)  2002;4(5):440-448.
Abstract
Cytokeratins (CK) are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, and 19 occurred at significantly higher levels (P<.05) in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms (nos. 2165 and 2091), one of eight CK8 isoforms (no. 439), and one of three CK19 isoforms (no. 1955) were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.
PMCID: PMC1661678  PMID: 12192603
lung adenocarcinoma; isoforms; 2D PAGE; mass spectrometry; microarrays
24.  Upregulated INHBA Expression May Promote Cell Proliferation and Is Associated with Poor Survival in Lung Adenocarcinoma1 
Neoplasia (New York, N.Y.)  2009;11(4):388-396.
Introduction
The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated.
Methods
INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2′ deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression.
Results
Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin βA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2′ deoxycytidine.
Conclusions
INHBA is overexpressed in AD relative to controls. Inhibin βA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs.
PMCID: PMC2657883  PMID: 19308293

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