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author:("Chen, guan")
1.  A unified Bayesian semiparametric approach to assess discrimination ability in survival analysis 
Biometrics  2015;72(2):554-562.
The discriminatory ability of a marker for censored survival data is routinely assessed by the time-dependent ROC curve and the c-index. The time-dependent ROC curve evaluates the ability of a biomarker to predict whether a patient lives past a particular time t. The c-index measures the global concordance of the marker and the survival time regardless of the time point. We propose a Bayesian semiparametric approach to estimate these two measures. The proposed estimators are based on the conditional distribution of the survival time given the biomarker and the empirical biomarker distribution. The conditional distribution is estimated by a linear dependent Dirichlet process mixture model. The resulting ROC curve is smooth as it is estimated by a mixture of parametric functions. The proposed c-index estimator is shown to be more efficient than the commonly used Harrell's c-index since it uses all pairs of data rather than only informative pairs. The proposed estimators are evaluated through simulations and illustrated using a lung cancer dataset.
PMCID: PMC4899301  PMID: 26676324
AUC; c-index; Concordance; Diagnostic test; Linear dependent Dirichlet process; Time-dependent ROC; ROC
2.  Non-coding RNA LINC00857 is predictive of poor patient survival and promotes tumor progression via cell cycle regulation in lung cancer 
Oncotarget  2016;7(10):11487-11499.
We employed next generation RNA sequencing analysis to reveal dysregulated long non-coding RNAs (lncRNAs) in lung cancer utilizing 461 lung adenocarcinomas (LUAD) and 156 normal lung tissues from 3 separate institutions. We identified 281 lncRNAs with significant differential-expression between LUAD and normal lung tissue. LINC00857, a top deregulated lncRNAs, was overexpressed in tumors and significantly associated with poor survival in LUAD. knockdown of LINC00857 with siRNAs decreased tumor cell proliferation, colony formation, migration and invasion in vitro, as well as tumor growth in vivo. Overexpression of LINC00857 increased cancer cell proliferation, colony formation and invasion. Mechanistic analyses indicated that LINC00857 mediates tumor progression via cell cycle regulation. Our study highlights the diagnostic/prognostic potential of LINC00857 in LUAD besides delineating the functional and mechanistic aspects of its aberrant disease specific expression and potentially using as a new therapeutic target.
PMCID: PMC4905488  PMID: 26862852
non-coding RNA; LINC00857; lung adenocarcinoma; prognosis; diagnosis
3.  Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer 
Oncotarget  2015;6(27):23445-23461.
Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.
PMCID: PMC4695129  PMID: 26140362
lung adenocarcinoma; amplification; purine biosynthesis; glutamine; xenograft
4.  Heat Shock Protein 90β Stabilizes Focal Adhesion Kinase and Enhances Cell Migration and Invasion in Breast Cancer Cells 
Experimental cell research  2014;326(1):78-89.
Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer.
PMCID: PMC4120946  PMID: 24880126
HSP90β; FAK; breast cancer; metastasis; ubiquitin
5.  Transcriptome Meta-Analysis of Lung Cancer Reveals Recurrent Aberrations in NRG1 and Hippo Pathway Genes 
Nature communications  2014;5:5893.
Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here, we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyze 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumors without known driver mutations. In addition, we observe exon skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.
PMCID: PMC4274748  PMID: 25531467
6.  MAP3K3 expression in tumor cells and tumor-infiltrating lymphocytes is correlated with favorable patient survival in lung cancer 
Scientific Reports  2015;5:11471.
MAP3K3 is involved in both the immune response and in tumor progression. Its potential biological role in vitro in lung cancer cell lines and the association of mRNA/protein expression patterns with clinical outcome of primary lung tumors were investigated in this study. Silencing MAP3K3 using siRNA in lung cancer cell lines resulted in decreased cell proliferation, migration and invasion. These effects were associated with down-regulation of the JNK, p38, AKT, and GSK3β pathways as determined using phospho-protein and gene expression array analyses. However, MAP3K3 mRNA and protein overexpression in primary lung tumors correlated significantly with favorable patient survival. Gene cluster and pathway analyses of primary tumor datasets indicated that genes positively-correlated with MAP3K3 are significantly involved in immune response rather than the cell cycle regulators observed using in vitro analyses. These results indicate that although MAP3K3 overexpression has an oncogenic role in vitro, in primary lung adenocarcinomas it correlates with an active immune response in the tumor environment that correlates with improved patient survival. MAP3K3 may potentially not only serve as diagnostic/prognostic markers for patients with lung cancer but also provide an indicator for future investigations into immunomodulatory therapies for lung cancer.
PMCID: PMC4650617  PMID: 26088427
7.  Epithelial–mesenchymal transition-associated secretory phenotype predicts survival in lung cancer patients 
Carcinogenesis  2014;35(6):1292-1300.
A gene signature derived from EASP strongly correlated with nodal metastasis, advanced tumor stage and poor differentiation and predicted survival in three independent lung cancer data sets, including early-stage patients. It may serve as a prognostic signature to identify high-risk early-stage patients.
In cancer cells, the process of epithelial–mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival. Utilizing a transforming growth factor-β-induced cell culture model of EMT, we quantitatively profiled differentially secreted proteins, by GeLC-tandem mass spectrometry. Integrating with the corresponding transcriptome, we derived an EMT-associated secretory phenotype (EASP) comprising of proteins that were differentially upregulated both at protein and mRNA levels. Four independent primary tumor-derived gene expression data sets of lung cancers were used for survival analysis by the random survival forests (RSF) method. Analysis of 97-gene EASP expression in human lung adenocarcinoma tumors revealed strong positive correlations with lymph node metastasis, advanced tumor stage and histological grade. RSF analysis built on a training set (n = 442), including age, sex and stage as variables, stratified three independent lung cancer data sets into low-, medium- and high-risk groups with significant differences in overall survival. We further refined EASP to a 20 gene signature (rEASP) based on variable importance scores from RSF analysis. Similar to EASP, rEASP predicted survival of both adenocarcinoma and squamous carcinoma patients. More importantly, it predicted survival in the early-stage cancers. These results demonstrate that integrative analysis of the critical biological process of EMT provides mechanism-based and clinically relevant biomarkers with significant prognostic value.
PMCID: PMC4043235  PMID: 24510113
8.  APCCdc20 Suppresses Apoptosis through Targeting Bim for Ubiquitination and Destruction 
Developmental cell  2014;29(4):377-391.
APCCdc20 plays pivotal roles in governing mitotic progression. By suppressing APCCdc20, anti-mitotic agents activate the spindle-assembly-checkpoint (SAC), and induce apoptosis after prolonged-treatment, while depletion of endogenous Cdc20 suppresses in vivo tumorigenesis in part by triggering mitotic arrest and subsequent apoptosis. However, the molecular mechanism(s) underlying apoptosis induced by Cdc20 abrogation remains poorly understood. Here we report that the BH3-only pro-apoptotic protein Bim is an APCCdc20 target, as such depletion of Cdc20 sensitizes cells to apoptotic stimuli. Strikingly, Cdc20 and multiple APC-core components were identified in an siRNA screen that upon knockdown sensitizes otherwise resistant cancer cells to chemo-radiation therapies in a Bim-dependent manner. Consistently, human Adult-T-cell-Leukemia (ATL) cells that acquire elevated APCCdc20 activity via expressing the Tax-viral-oncoprotein, exhibit reduced Bim levels and resistance to anti-cancer agents. These results reveal an important role for APCCdc20 in governing apoptosis, strengthening the rationale for developing specific Cdc20 inhibitors as effective anti-cancer agents.
PMCID: PMC4081014  PMID: 24871945
9.  Multiple myeloma acquires resistance to EGFR inhibitor via induction of pentose phosphate pathway 
Scientific Reports  2015;5:9925.
Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM.
PMCID: PMC4403500  PMID: 25894462
11.  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
12.  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.
PMCID: PMC4227448  PMID: 25405215
13.  Epigenetic inactivation of microRNA-34b/c predicts poor disease-free survival in early stage lung adenocarcinoma 
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.
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.
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.
PMCID: PMC4161219  PMID: 24130071
microRNA; DNA methylation; microRNA-34b/c; lung adenocarcinoma; TP53
14.  Checkpoint kinase 1 protein expression indicates sensitization to therapy by checkpoint kinase 1 inhibition in non–small cell lung cancer 
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).
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.
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.
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.
PMCID: PMC4073630  PMID: 24418519
CHK1; NSCLC cell lines; Combination therapies; AZD7762; Chemosensitivity; Radiosensitivity
15.  CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells 
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.
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.
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.
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.
PMCID: PMC4073640  PMID: 24113549
CHK1; Lung; Chemosensitivity; NSCLC; Patient survival; Genomic complexity
16.  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.
PMCID: PMC4055536  PMID: 24967428
17.  Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma 
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.
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.
Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma.
PMCID: PMC3630232  PMID: 23444212
Cancer Stem cells; Notch pathway; Lung cancer
18.  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
19.  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.
PMCID: PMC3904615  PMID: 24430184
20.  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
21.  Characterization of vitamin D receptor (VDR) in lung adenocarcinoma 
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.
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.
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.
PMCID: PMC3396768  PMID: 22564539
VDR; Vitamin D; 1,25-D3; Lung Adenocarcinoma; Survival
22.  Development and Validation of a qRT-PCR Classifier for Lung Cancer Prognosis 
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.
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).
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.
PMCID: PMC3167380  PMID: 21792073
Lung cancer; qRT-PCR; Prognosis
23.  Stromal LRP1 in lung adenocarcinoma predicts clinical outcome 
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.
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.
LRP1 expression is associated with improved lung cancer outcomes. Mechanistically, stromal LRP1 may non-cell autonomously suppress lung tumor cell proliferation.
PMCID: PMC3079007  PMID: 21325077
24.  CYP24A1 Is an Independent Prognostic Marker of Survival in Patients with Lung Adenocarcinoma 
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.
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).
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.
PMCID: PMC3058389  PMID: 21169243
25.  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.
PMCID: PMC3200133  PMID: 22091388

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