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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
3.  TGM2 A Cell Surface Marker in Esophageal Adenocarcinomas 
Introduction
Esophageal adenocarcinomas (EAC) are aggressive cancers that are increasing in incidence and associated with a poor prognosis. The identification of highly expressed genes in EAC relative to metaplastic Barrett’s esophagus (BE) may provide new targets for novel early cancer detection strategies using endoscopically administered, fluorescently labeled peptides.
Methods
Gene expression analysis of BE and EACs were used to identify the cell surface marker transglutaminase 2 (TGM2) as overexpressed in cancer. The expression of two major isoforms of TGM2 was determined by qRT-polymerase chain reaction in an independent cohort of 128 EACs. Protein expression was confirmed by tissue microarrays and immunoblot analysis of EAC cell lines. TGM2 DNA copy number was assessed using single nucleotide polymorphism microarrays and confirmed by qPCR. TGM2 expression in neoadjuvantly treated EACs and following small interfering RNA-mediated knockdown in cisplatin-treated EAC cells was used to determine its possible role in chemoresistance.
Results
TGM2 is overexpressed in 15 EACs relative to 26 BE samples. Overexpression of both TGM2 isoforms was confirmed in 128 EACs and associated with higher tumor stage, poor differentiation, and increased inflammatory and desmoplastic response. Tissue microarrays and immunohistochemistry confirmed elevated TGM2 protein expression in EAC. Single nucleotide polymorphism and qPCR analysis revealed increased TGM2 gene copy number as one mechanism underlying elevated TGM2 expression. TGM2 was highly expressed in resistant EAC after patient treatment with neoadjuvant chemotherapy/radiation suggesting a role for TGM2 in chemoresistance.
Conclusion
TGM2 may be a useful cell surface biomarker for early detection of EAC.
doi:10.1097/JTO.0000000000000229
PMCID: PMC4170218  PMID: 24828664
Esophageal adenocarcinoma; TGM2; Cell surface biomarker
4.  Comprehensive molecular characterization of gastric adenocarcinoma 
Bass, Adam J. | Thorsson, Vesteinn | Shmulevich, Ilya | Reynolds, Sheila M. | Miller, Michael | Bernard, Brady | Hinoue, Toshinori | Laird, Peter W. | Curtis, Christina | Shen, Hui | Weisenberger, Daniel J. | Schultz, Nikolaus | Shen, Ronglai | Weinhold, Nils | Kelsen, David P. | Bowlby, Reanne | Chu, Andy | Kasaian, Katayoon | Mungall, Andrew J. | Robertson, A. Gordon | Sipahimalani, Payal | Cherniack, Andrew | Getz, Gad | Liu, Yingchun | Noble, Michael S. | Pedamallu, Chandra | Sougnez, Carrie | Taylor-Weiner, Amaro | Akbani, Rehan | Lee, Ju-Seog | Liu, Wenbin | Mills, Gordon B. | Yang, Da | Zhang, Wei | Pantazi, Angeliki | Parfenov, Michael | Gulley, Margaret | Piazuelo, M. Blanca | Schneider, Barbara G. | Kim, Jihun | Boussioutas, Alex | Sheth, Margi | Demchok, John A. | Rabkin, Charles S. | Willis, Joseph E. | Ng, Sam | Garman, Katherine | Beer, David G. | Pennathur, Arjun | Raphael, Benjamin J. | Wu, Hsin-Ta | Odze, Robert | Kim, Hark K. | Bowen, Jay | Leraas, Kristen M. | Lichtenberg, Tara M. | Weaver, Stephanie | McLellan, Michael | Wiznerowicz, Maciej | Sakai, Ryo | Getz, Gad | Sougnez, Carrie | Lawrence, Michael S. | Cibulskis, Kristian | Lichtenstein, Lee | Fisher, Sheila | Gabriel, Stacey B. | Lander, Eric S. | Ding, Li | Niu, Beifang | Ally, Adrian | Balasundaram, Miruna | Birol, Inanc | Bowlby, Reanne | Brooks, Denise | Butterfield, Yaron S. N. | Carlsen, Rebecca | Chu, Andy | Chu, Justin | Chuah, Eric | Chun, Hye-Jung E. | Clarke, Amanda | Dhalla, Noreen | Guin, Ranabir | Holt, Robert A. | Jones, Steven J.M. | Kasaian, Katayoon | Lee, Darlene | Li, Haiyan A. | Lim, Emilia | Ma, Yussanne | Marra, Marco A. | Mayo, Michael | Moore, Richard A. | Mungall, Andrew J. | Mungall, Karen L. | Nip, Ka Ming | Robertson, A. Gordon | Schein, Jacqueline E. | Sipahimalani, Payal | Tam, Angela | Thiessen, Nina | Beroukhim, Rameen | Carter, Scott L. | Cherniack, Andrew D. | Cho, Juok | Cibulskis, Kristian | DiCara, Daniel | Frazer, Scott | Fisher, Sheila | Gabriel, Stacey B. | Gehlenborg, Nils | Heiman, David I. | Jung, Joonil | Kim, Jaegil | Lander, Eric S. | Lawrence, Michael S. | Lichtenstein, Lee | Lin, Pei | Meyerson, Matthew | Ojesina, Akinyemi I. | Pedamallu, Chandra Sekhar | Saksena, Gordon | Schumacher, Steven E. | Sougnez, Carrie | Stojanov, Petar | Tabak, Barbara | Taylor-Weiner, Amaro | Voet, Doug | Rosenberg, Mara | Zack, Travis I. | Zhang, Hailei | Zou, Lihua | Protopopov, Alexei | Santoso, Netty | Parfenov, Michael | Lee, Semin | Zhang, Jianhua | Mahadeshwar, Harshad S. | Tang, Jiabin | Ren, Xiaojia | Seth, Sahil | Yang, Lixing | Xu, Andrew W. | Song, Xingzhi | Pantazi, Angeliki | Xi, Ruibin | Bristow, Christopher A. | Hadjipanayis, Angela | Seidman, Jonathan | Chin, Lynda | Park, Peter J. | Kucherlapati, Raju | Akbani, Rehan | Ling, Shiyun | Liu, Wenbin | Rao, Arvind | Weinstein, John N. | Kim, Sang-Bae | Lee, Ju-Seog | Lu, Yiling | Mills, Gordon | Laird, Peter W. | Hinoue, Toshinori | Weisenberger, Daniel J. | Bootwalla, Moiz S. | Lai, Phillip H. | Shen, Hui | Triche, Timothy | Van Den Berg, David J. | Baylin, Stephen B. | Herman, James G. | Getz, Gad | Chin, Lynda | Liu, Yingchun | Murray, Bradley A. | Noble, Michael S. | Askoy, B. Arman | Ciriello, Giovanni | Dresdner, Gideon | Gao, Jianjiong | Gross, Benjamin | Jacobsen, Anders | Lee, William | Ramirez, Ricardo | Sander, Chris | Schultz, Nikolaus | Senbabaoglu, Yasin | Sinha, Rileen | Sumer, S. Onur | Sun, Yichao | Weinhold, Nils | Thorsson, Vésteinn | Bernard, Brady | Iype, Lisa | Kramer, Roger W. | Kreisberg, Richard | Miller, Michael | Reynolds, Sheila M. | Rovira, Hector | Tasman, Natalie | Shmulevich, Ilya | Ng, Santa Cruz Sam | Haussler, David | Stuart, Josh M. | Akbani, Rehan | Ling, Shiyun | Liu, Wenbin | Rao, Arvind | Weinstein, John N. | Verhaak, Roeland G.W. | Mills, Gordon B. | Leiserson, Mark D. M. | Raphael, Benjamin J. | Wu, Hsin-Ta | Taylor, Barry S. | Black, Aaron D. | Bowen, Jay | Carney, Julie Ann | Gastier-Foster, Julie M. | Helsel, Carmen | Leraas, Kristen M. | Lichtenberg, Tara M. | McAllister, Cynthia | Ramirez, Nilsa C. | Tabler, Teresa R. | Wise, Lisa | Zmuda, Erik | Penny, Robert | Crain, Daniel | Gardner, Johanna | Lau, Kevin | Curely, Erin | Mallery, David | Morris, Scott | Paulauskis, Joseph | Shelton, Troy | Shelton, Candace | Sherman, Mark | Benz, Christopher | Lee, Jae-Hyuk | Fedosenko, Konstantin | Manikhas, Georgy | Potapova, Olga | Voronina, Olga | Belyaev, Smitry | Dolzhansky, Oleg | Rathmell, W. Kimryn | Brzezinski, Jakub | Ibbs, Matthew | Korski, Konstanty | Kycler, Witold | ŁaŸniak, Radoslaw | Leporowska, Ewa | Mackiewicz, Andrzej | Murawa, Dawid | Murawa, Pawel | Spychała, Arkadiusz | Suchorska, Wiktoria M. | Tatka, Honorata | Teresiak, Marek | Wiznerowicz, Maciej | Abdel-Misih, Raafat | Bennett, Joseph | Brown, Jennifer | Iacocca, Mary | Rabeno, Brenda | Kwon, Sun-Young | Penny, Robert | Gardner, Johanna | Kemkes, Ariane | Mallery, David | Morris, Scott | Shelton, Troy | Shelton, Candace | Curley, Erin | Alexopoulou, Iakovina | Engel, Jay | Bartlett, John | Albert, Monique | Park, Do-Youn | Dhir, Rajiv | Luketich, James | Landreneau, Rodney | Janjigian, Yelena Y. | Kelsen, David P. | Cho, Eunjung | Ladanyi, Marc | Tang, Laura | McCall, Shannon J. | Park, Young S. | Cheong, Jae-Ho | Ajani, Jaffer | Camargo, M. Constanza | Alonso, Shelley | Ayala, Brenda | Jensen, Mark A. | Pihl, Todd | Raman, Rohini | Walton, Jessica | Wan, Yunhu | Demchok, John A. | Eley, Greg | Mills Shaw, Kenna R. | Sheth, Margi | Tarnuzzer, Roy | Wang, Zhining | Yang, Liming | Zenklusen, Jean Claude | Davidsen, Tanja | Hutter, Carolyn M. | Sofia, Heidi J. | Burton, Robert | Chudamani, Sudha | Liu, Jia
Nature  2014;513(7517):202-209.
Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.
doi:10.1038/nature13480
PMCID: PMC4170219  PMID: 25079317
5.  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
6.  Reconstructing targetable pathways in lung cancer by integrating diverse omics data 
Nature communications  2013;4:2617.
Global ‘multi-omics’ profiling of cancer cells harbours the potential for characterizing the signaling networks associated with specific oncogenes. Here we profile the transcriptome, proteome and phosphoproteome in a panel of non-small cell lung cancer (NSCLC) cell lines in order to reconstruct targetable networks associated with KRAS dependency. We develop a two-step bioinformatics strategy addressing the challenge of integrating these disparate data sets. We first define an ‘abundance-score’ combining transcript, protein and phospho-protein abundances to nominate differentially abundant proteins and then use the Prize Collecting Steiner Tree algorithm to identify functional sub-networks. We identify three modules centered on KRAS and MET, LCK and PAK1 and b-Catenin. We validate activation of these proteins in KRAS-dependent (KRAS-Dep) cells and perform functional studies defining LCK as a critical gene for cell proliferation in KRAS-Dep but not KRAS-independent NSCLCs. These results suggest that LCK is a potential druggable target protein in KRAS-Dep lung cancers.
doi:10.1038/ncomms3617
PMCID: PMC4107456  PMID: 24135919
7.  Telomerase Variant A279T Induces Telomere Dysfunction and Inhibits Non-Canonical Telomerase Activity in Esophageal Carcinomas 
PLoS ONE  2014;9(7):e101010.
Background
Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.
Methods
Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.
Results
Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage.
Conclusions
A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.
doi:10.1371/journal.pone.0101010
PMCID: PMC4077737  PMID: 24983628
8.  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
9.  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
10.  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
11.  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
12.  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
13.  Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: First in-human results 
Science translational medicine  2013;5(184):10.1126/scitranslmed.3004733.
Esophageal adenocarcinoma is rising rapidly in incidence, and usually develops from Barrett’s esophagus, a precursor condition commonly found in patients with chronic acid reflux. Pre-malignant lesions are challenging to detect on conventional screening endoscopy because of their flat appearance. Molecular changes can be used to improve detection of early neoplasia. We have developed a peptide that binds specifically to high-grade dysplasia and adenocarcinoma. We first applied the peptide ex vivo to esophageal specimens from 17 patients to validate specific binding. Next, we performed confocal endomicroscopy in vivo in 25 human subjects after topical peptide administration and found 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett’s esophagus and squamous epithelium with 75% sensitivity and 97% specificity. No toxicity was attributed to the peptide in either animal or patient studies. Therefore, our first-in-humans results show that this targeted imaging agent is safe, and may be useful for guiding tissue biopsy and for early detection of esophageal neoplasia and potentially other cancers of epithelial origin, such as bladder, colon, lung, pancreas, and stomach.
doi:10.1126/scitranslmed.3004733
PMCID: PMC3859345  PMID: 23658246
14.  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
15.  Exome and whole genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity 
Nature genetics  2013;45(5):10.1038/ng.2591.
The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a five-year survival rate of 15%, identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole exome sequencing of 149 EAC tumors/normal pairs, 15 of which have also been subjected to whole genome sequencing. We identify a mutational signature defined by a high prevalence of A to C transversions at AA dinucleotides. Statistical analysis of exome data identified significantly mutated 26 genes. Of these genes, four (TP53, CDKN2A, SMAD4, and PIK3CA) have been previously implicated in EAC. The novel significantly mutated genes include chromatin modifying factors and candidate contributors: SPG20, TLR4, ELMO1, and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 reveal increased cellular invasion. Therefore, we suggest a new hypothesis about the potential activation of the RAC1 pathway to be a contributor to EAC tumorigenesis.
doi:10.1038/ng.2591
PMCID: PMC3678719  PMID: 23525077
16.  Gastrointestinal adenocarcinomas of the esophagus, stomach and colon exhibit distinct patterns of genome instability and oncogenesis 
Cancer research  2012;72(17):4383-4393.
A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations (SCNAs) in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential utility of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared to colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions.
doi:10.1158/0008-5472.CAN-11-3893
PMCID: PMC3432726  PMID: 22751462
Esophagus; stomach; colon; adenocarcinoma; copy-number
17.  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
18.  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
19.  Signaling pathway-based identification of extensive prognostic gene signatures for lung adenocarcinoma 
Tumor recurrence is the major cause of death in lung cancer treatment. To date, there is no clinically applied gene expression-based model to predict the risk for tumor recurrence in non-small cell lung cancer (NSCLC). We sought to embed crosstalk with major signaling pathways into biomarker identification. Three approaches were used to identify prognostic gene signatures from 442 lung adenocarcinoma samples. Candidate genes co-expressed with 6 or 7 major NSCLC signaling hallmarks were identified from genome-wide coexpression networks specifically associated with different prognostic groups. From these candidate genes, the first approach selected genes significantly associated with disease-specific survival using univariate Cox model. The second approach used random forests to refine the gene signatures; and the third approach used Relief algorithm to form the final gene sets. A total of 21 gene signatures were identified using these three approaches. These gene signatures generated significant prognostic stratifications (log-rank P < 0.05 in Kaplan-Meier analyses; Hazard Ratio >1, P< 0.05) in all tumors, stage I only, and in stage I patients not receiving chemotherapy in all training and test sets. In multivariate analyses with age, gender, race, smoking history, cancer stage, and tumor differentiation, a 10-gene signature had a hazard ratio of 3.23 (95% CI: [1.48, 7.06]), which was a more significant prognostic factor than other clinical factors, except cancer stage (III vs. I; with no significant difference). All identified 21 gene signatures outperformed other lung cancer signatures evaluated in the Director's Challenge Study. This study is an important step toward personalized prognosis of tumor recurrence and patient selection for adjuvant chemotherapy, with significant impact on down-stream clinical applications.
doi:10.1016/j.lungcan.2011.09.016
PMCID: PMC3274612  PMID: 22047960
lung adenocarcinoma; gene co-expression networks; biomarker identification; signaling pathways; prognostic stratification; tumor recurrence; metastasis; non-small cell lung cancer
20.  Integrative Genomics Identified RFC3 as an Amplified Candidate Oncogene in Esophageal Adenocarcinoma 
Purpose
Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett’s esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers.
Experimental Design
Whole genome tiling-path array CGH was used to identify significant regions of copy number (CN) alteration in 20 EACs and 10 matching BE tissues. CN and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes.
Results
We identified RFC3 as a novel, candidate oncogene activated by amplification in ~25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification, and was differentially expressed between non-malignant and EAC tissues. CN gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased CN, but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types.
Conclusions
RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker.
doi:10.1158/1078-0432.CCR-11-1431
PMCID: PMC3523177  PMID: 22328562
RFC3; esophageal adenocarcinoma; Barrett’s esophagus; DNA amplification
21.  Epidermal Growth Factor Receptor Tyrosine Kinase Defines Critical Prognostic Genes of Stage I Lung Adenocarcinoma 
PLoS ONE  2012;7(9):e43923.
Purpose
To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy.
Patients and Methods
Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). “Gefitinib-sensitive” genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer.
Results
The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively.
Conclusion
The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis.
Trial Registration
The Gene Expression Omnibus (GEO) GSE31210
doi:10.1371/journal.pone.0043923
PMCID: PMC3446964  PMID: 23028479
22.  Comparative Genomics of Esophageal Adenocarcinoma and Squamous Cell Carcinoma 
The Annals of Thoracic Surgery  2012;93(4):1101-1106.
Background
Esophageal cancer consists of two major histologic types: esophageal squamous cell carcinoma (ESCC) predominant globally and esophageal adenocarcinoma (EAC) with a higher incidence in westernized countries. Five-year overall survival is 15%. Clinical trials frequently combine histologies although they are different diseases with distinct origins. In the evolving era of personalized medicine and targeted therapies, we hypothesized that ESCC and EAC have genomic differences important for developing new therapeutic strategies for esophageal cancer.
Methods
We explored DNA copy number abnormalities (CNAs) in 70 ESCCs with publicly available array data and 189 EAC from our group. All data was from Affymetrix single nucleotide polymorphism (SNP) arrays. Analysis was performed with Nexus 5.0 Copy number software using a SNPRank segmentation algorithm. Log ratio thresholds for copy number gain and loss were set at +/− 0.2 (approximately 2.3 and 1.7 copies respectively).
Results
ESCC and EAC genomes showed some CNAs with similar frequencies (e.g., CDKN2A, EGFR, KRAS, MYC, CDK6, MET) but also many CNAs with different frequencies between histologies, most of which were amplification events. Some of these regions harbor genes to which targeted therapies are currently available (VEGFA, ERBB2) or where agents are in clinical trials (PIK3CA, FGFR1). Other regions contain putative oncogenes that may be targeted in the future.
Conclusions
Using SNP arrays we compared genomic abnormalities in a large cohort of EAC and ESCC. We report here the similar and different frequencies of CNAs in ESCC and EAC. These results may allow development of histology-specific therapeutic agents for esophageal cancer.
doi:10.1016/j.athoracsur.2012.01.064
PMCID: PMC3401935  PMID: 22450065
Esophageal cancer; genetics; genomics
23.  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
24.  Affinity Peptide for Targeted Detection of Dysplasia in Barrett's Esophagus 
Gastroenterology  2010;139(5):1472-1480.
Background & Aims
Dysplasia is a pre-malignant condition in Barrett's esophagus that is difficult to detect on screening endoscopy because of its flat architecture and patchy distribution. Peptides are promising for use as novel molecular probes that identify cell surface targets unique to disease, and can be fluorescence-labeled for detection. We aim to select and validate an affinity peptide that binds to esophageal dysplasia for future clinical studies.
Methods
Peptide selection was performed using phage display by removing non-specific binders using Q-hTERT (intestinal metaplasia) cells and achieving specific binding against OE33 (esophageal adenocarcinoma) cells. Selective binding was confirmed on bound phage counts, ELISA, flow cytometry, competitive inhibition, and fluorescence microscopy. On stereomicroscopy, specific peptide binding to dysplasia on endoscopically resected specimens was assessed by rigorous registration of fluorescence intensity to histology in 1 mm intervals.
Results
The peptide sequence SNFYMPL was selected and demonstrated preferential binding to target cells on bound phage counts, ELISA, and flow cytometry. Reducing binding was observed on competition with unlabeled peptide in a dose dependent manner, an affinity of Kd = 164 nM was measured, and peptide binding to the surface of OE33 cells was validated on fluorescence microscopy. On esophageal specimens (n=12), the fluorescence intensity (mean±SEM) in 1 mm intervals classified histologically as squamous (n=145), intestinal metaplasia (n=83), dysplasia (n=61) and gastric mucosa (n=69) was 46.5±1.6, 62.3±5.8, 100.0±9.0, and 42.4±3.0 arb units, respectively.
Conclusions
The peptide sequence SNFYMPL binds specifically to dysplasia in Barrett's esophagus, and can be fluorescence-labeled to target pre-malignant mucosa on imaging.
doi:10.1053/j.gastro.2010.07.007
PMCID: PMC3319360  PMID: 20637198
peptide; Barrett's; esophagus; dysplasia; early detection
25.  Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer 
Cancer discovery  2011;1(1):78-89.
While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials.
doi:10.1158/2159-8274.CD-11-0005
PMCID: PMC3274752  PMID: 22328973
Squamous cell lung cancer; DDR2; dasatinib; tyrosine kinase inhibitors; lung cancer genomics

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