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1.  A Preliminary Study on Racial Differences in HMOX1, NFE2L2 and TGFβ1 Gene Polymorphisms and Radiation Induced Late Normal Tissue Toxicity 
Purpose
This study tests whether racial differences in genetic polymorphisms of four genes involved in wound repair and response to radiation can be used to predict the occurrence of normal tissue late effects of radiotherapy and indicate potential therapeutic targets.
Methods and Materials
This prospective study examines genetic polymorphisms that modulate the expression of four genes involved in inflammation and fibrosis and response to radiation (HMOX1, NFE2L2, NOS3 and TGFβ1). DNA from blood samples of 179 patients (~80% breast and head and neck) collected at the time of diagnosis by their radiation oncologist as exhibiting late normal tissue toxicity was used for the analysis. Patient demographics were: 56% Caucasian, 43% African-American, 1% other. Allelic frequencies of the different polymorphisms of the participants were compared to those of the general American population stratified by race. Twenty-six additional patients treated with radiation, but without toxicity at 3 months or later post-therapy, were also analyzed.
Results
Increased frequency of a long GT repeat in the HMOX1 promoter was associated with late effects in both African-American and Caucasian populations. The single nucleotide polymorphisms (SNP) rs1800469 in the TGFβ1 promoter and the rs6721961 SNP in the NFE2L2 promoter were also found to significantly associate with late effects in African-Americans but not Caucasians. A combined analysis of these polymorphisms revealed that >90% of African-American patients with late effects had at least one and 58% two or more of these minor alleles. No statistical significance was found relating the studied NOS3 polymorphisms and normal tissue toxicity.
Conclusions
These results support a strong association between wound repair and late toxicities of radiation. The presence of these genetic risk factors can vary significantly among different ethnic groups, as demonstrated for some of the SNPs. Future studies should account for the possibility of such ethnic heterogeneity in the late toxicities of radiation.
doi:10.1016/j.ijrobp.2015.05.049
PMCID: PMC4575610  PMID: 26238954
2.  The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression 
Molecular cancer research : MCR  2015;13(6):1034-1043.
Here evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast to normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on HPLC analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin: dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid and head and neck tumors compared to normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling including increased cGMP-dependent protein kinase (PKG) activity, decreased β-catenin expression and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by clonogenic assay, Ki67 staining and 18F-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and as a consequence nitric oxide synthase activity generates more peroxynitrite and superoxide anion than nitric oxide resulting in important tumor growth promoting and anti-apoptotic signaling properties.
Implications
The synthetic BH4, Kuvan®, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.
doi:10.1158/1541-7786.MCR-15-0057-T
PMCID: PMC4470720  PMID: 25724429
Nitric Oxide; tetrahydrobiopterin; sepiapterin; cGMP; breast cancer
3.  Astrocyte elevated gene-1 (AEG-1) and c-Myc cooperate to promote hepatocarcinogenesis 
Hepatology (Baltimore, Md.)  2015;61(3):915-929.
Astrocyte elevated gene-1 (AEG-1) and c-Myc are overexpressed in human hepatocellular carcinoma (HCC) functioning as oncogenes. AEG-1 is transcriptionally regulated by c-Myc and AEG-1 itself induces c-Myc by activating Wnt/β-catenin signaling pathway. We now document cooperation of AEG-1 and c-Myc in promoting hepatocarcinogenesis by analyzing hepatocyte-specific transgenic mice expressing either AEG-1 (Alb/AEG-1), c-Myc (Alb/c-Myc) or both (Alb/AEG-1/c-Myc). WT and Alb/AEG-1 mice did not develop spontaneous HCC. Alb/c-Myc mice developed spontaneous HCC without distant metastasis while Alb/AEG-1/c-Myc mice developed highly aggressive HCC with frank metastasis to the lungs. Induction of carcinogenesis by N-nitrosodiethylamine (DEN) significantly accelerated the kinetics of tumor formation in all groups. However, in Alb/AEG-1/c-Myc the effect was markedly pronounced with lung metastasis. In vitro analysis showed that Alb/AEG-1/c-Myc hepatocytes acquired increased proliferation and transformative potential with sustained activation of pro-survival and epithelialmesenchymal transition (EMT) signaling pathways. RNA-sequencing analysis identified a unique gene signature in livers of Alb/AEG-1/c-Myc mice that was not observed when either AEG-1 or c-Myc was overexpressed. Specifically Alb/AEG-1/c-Myc mice overexpressed maternally imprinted non-coding RNAs, such as Rian, Meg-3 and Migr, which are implicated in hepatocarcinogenesis. Knocking down these ncRNAs significantly inhibited proliferation and invasion by Alb/AEG-1/c-Myc hepatocytes.
Conclusion
Our studies reveal a novel cooperative oncogenic effect of AEG-1 and c-Myc that might explain the mechanism of aggressive HCC. Alb/AEG-1/c-Myc mice provide a useful model to understand the molecular mechanism of cooperation between these two oncogenes and other molecules involved in hepatocarcinogenesis. This model might also be of use for evaluating novel therapeutic strategies targeting HCC.
doi:10.1002/hep.27339
PMCID: PMC4309751  PMID: 25065684
AEG-1; c-Myc; hepatocarcinogenesis; metastasis; ncRNA
4.  Multivariate Granger Causality Analysis of Obesity Related Variables 
Obesity is a complex health outcome that is a combination of multiple health indicators. Here we attempt to explore the dependence network among multiple aspects of obesity. Two longitudinal cohort studies across multiple decades have been used. The concept of causality is defined similar to Granger causality among multiple time series, however, modified to accommodate multivariate time series as the nodes of the network. Our analysis reveals relatively central position of physical measurements and blood chemistry measures in the overall network across both genders. Also there are some patterns specific to only male or female population. The geometry of the causality network is expected to help in our strategy to control the increasing trend of obesity rate.
PMCID: PMC4743668  PMID: 26855968
Obesity; Granger causality; Network; Canonical correlation
5.  High dimensional data analysis using multivariate generalized spatial quantiles 
Journal of multivariate analysis  2010;102(4):768-780.
High dimensional data routinely arises in image analysis, genetic experiments, network analysis, and various other research areas. Many such datasets do not correspond to well-studied probability distributions, and in several applications the data-cloud prominently displays non-symmetric and non-convex shape features. We propose using spatial quantiles and their generalizations, in particular, the projection quantile, for describing, analyzing and conducting inference with multivariate data. Minimal assumptions are made about the nature and shape characteristics of the underlying probability distribution, and we do not require the sample size to be as high as the data-dimension. We present theoretical properties of the generalized spatial quantiles, and an algorithm to compute them quickly. Our quantiles may be used to obtain multidimensional confidence or credible regions that are not required to conform to a pre-determined shape. We also propose a new notion of multidimensional order statistics, which may be used to obtain multidimensional outliers. Many of the features revealed using a generalized spatial quantile-based analysis would be missed if the data was shoehorned into a well-known probabilistic configuration.
doi:10.1016/j.jmva.2010.12.002
PMCID: PMC4659409  PMID: 26617421
Multivariate quantile; Spatial quantile; Projection quantile; Generalized spatial quantile; Multidimensional coverage sets; Multivariate order statistics; Brain imaging; High dimensional data visualization
6.  AEG-1 regulates retinoid X receptor and inhibits retinoid signaling 
Cancer research  2014;74(16):4364-4377.
Retinoid X Receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including Hepatocellular Carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of Astrocyte Elevated Gene-1 (AEG-1)/Metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/Retinoic Acid Receptor (RAR)-mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myeloid leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In non-tumorigenic cells and primary hepatocytes, AEG-1/RXR co-localizes in the nucleus where AEG-1 interferes with recruitment of transcriptional co-activators to RXR preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, precluding its nuclear translocation. Additionally, ERK, activated by AEG-1, phosphorylates RXR which leads to its functional inactivation and attenuation of ligand-dependent transactivation. In nude mice models, combination of all-trans retinoic acid (ATRA) and AEG-1 knockdown synergistically inhibited growth of human HCC xenografts. The present study establishes AEG-1 as a novel homeostatic regulator of RXR and RXR/RAR that might contribute to hepatocarcinogenesis. Targeting AEG-1 could sensitize HCC and AML patients to retinoid- and rexinoid-based therapeutics.
doi:10.1158/0008-5472.CAN-14-0421
PMCID: PMC4135401  PMID: 25125681
Hepatocellular Carcinoma; Protein-protein interaction; Transcriptional regulation; Retinoic acid; Cancer therapeutics
7.  Respiratory motion variability of primary tumors and lymph nodes during radiotherapy of locally advanced non-small-cell lung cancers 
Background and purpose
The need for target adjustment due to respiratory motion variation and the value of carina as a motion surrogate is evaluated for locally advanced non-small-cell lung cancer.
Material and methods
Using weekly 4D CTs (with audio-visual biofeedback) of 12 patients, respiratory motion variation of primary tumors (PT), lymph nodes (LN) and carina (C) were determined.
Results
Mean (SD) 3D respiratory motion ranges of PT, LN and C were 4 (3), 5 (3) and 5 (3) mm. PT and LN (p = 0.003), and LN and C motion range were correlated (p = 0.03). Only 20 %/5 % of all scans had variations >3 mm/5 mm. Large respiratory motion range on the initial scan was associated with larger during-treatment variations for PT (p = 0.03) and LN (p = 0.001).
Mean (SD) 3D relative displacements of PT-C, LN-C and PT-LN were each 6 (2) mm. Variations of displacements >3 mm/5 mm were observed in 28 %/6 % of scans for PT-LN, 20 %/9 % for PT-C, and 20 %/8 % for LN-C.
Conclusions
Motion reassessment is recommended in patients with large initial motion range. Relative motion-related displacements between PT and LN were larger than PT and LN motion alone. Both PT and C appear to be comparable surrogates for LN respiratory motion.
doi:10.1186/s13014-015-0435-3
PMCID: PMC4476088  PMID: 26071910
Non-small-cell lung cancer; Respiratory motion; Primary tumor; Lymph nodes
8.  Induction of MicroRNA-21 with Exogenous Hydrogen Sulfide Attenuates Myocardial Ischemic and Inflammatory Injury in Mice 
Background
Maintaining physiological levels of hydrogen sulfide (H2S) during ischemia is necessary to limit injury to the heart. Due to the anti-inflammatory effects of H2S, we proposed that the H2S donor, Na2S, would attenuate myocardial injury through upregulation of ‘protective’ microRNA (miR)-21 and suppression of the inflammasome, a macromolecular structure that amplifies inflammation and mediates further injury.
Methods and Results
Na2S-induced miR-21 expression was measured by qPCR in adult primary rat cardiomyocytes and in the mouse heart. We measured inflammasome formation and activity in cardiomyocytes challenged with lipopolysaccharide (LPS) and adenosine-tri-phosphate (ATP) or simulated ischemia/reoxygenation; and in the heart following regional myocardial ischemia/reperfusion (I/R), in the presence or absence of Na2S. To assess the direct anti-inflammatory effects of H2S in vivo, we utilized a peritonitis model by way of intraperitoneal injection of zymosan A. Na2S attenuated inflammasome formation and activity - measured by counting cytoplasmic aggregates of the scaffold protein Apoptosis Speck-like protein containing a Caspase-recruitment domain (ASC; −57%) and caspase-1 activity (−50%) in isolated cardiomyocytes and in the mouse heart (all P<0.05). Na2S also inhibited apoptosis (−38%) and necrosis (−43%) in cardiomyocytes in vitro and reduced myocardial infarct size (−63%) following I/R injury in vivo (all P<0.05). These protective effects were absent in cells treated with antagomiR-21 and in miR-21 KO mice. Na2S also limited the severity of inflammasome-dependent inflammation in the model of peritonitis (P<0.05) in wild-type but not in miR-21 KO mice.
Conclusions
Na2S induces cardioprotective effects through miR-21-dependent attenuation of ischemic and inflammatory injury in cardiomyocytes.
doi:10.1161/CIRCGENETICS.113.000381
PMCID: PMC4090021  PMID: 24825878
microRNA; myocardial infarction; cardiac function; hydrogen sulfide; inflammasome
9.  Interfraction Displacement of Primary Tumor and Involved Lymph Nodes Relative to Anatomical Landmarks in Image–guided Radiotherapy of Locally Advanced Lung Cancer 
Purpose
Image-guided radiotherapy for patients with locally advanced lung cancer relies on bony landmarks and carina or - if visible - the primary tumor (PT) for daily patient alignment, neglecting potential variations in the relative position of PT and involved lymph nodes (LN). This study analyzes PT and LN position changes relative to each other and relative to anatomical landmarks during conventionally fractionated radiotherapy.
Methods and Materials
In 12 patients with locally advanced non-small cell lung cancer PT, LN, carina and one thoracic vertebra were manually contoured on weekly 4D fan beam CTs. Systematic and random interfraction displacements of all contoured structures were identified in the three cardinal directions, resulting setup margins were calculated. Time trends and the effect of volume changes on displacements were analyzed.
Results
Three-dimensional displacement vectors and systematic/random interfraction displacements were smaller for carina than vertebra both for PT and LN. For PT, mean 3D displacement vectors with carina-based alignment were 7 mm/SD 4 mm versus 9 mm/SD 5 mm with bony anatomy (p<0.0001). For LN, smaller displacements were found with carina- (5 mm/SD 3 mm, p<0.0001) and vertebra-based (6 mm/SD 3 mm, p=0.002) alignment compared to using PT for setup (8 mm/SD 5 mm). Primary tumor and LN displacements relative to bone and carina were independent (p>0.05). Displacements between PT and bone (p=0.04), and between PT and LN (p=0.01) were significantly correlated with PT volume regression. Displacements between LN and carina were correlated with LN volume change (p=0.03).
Conclusions
Carina-based setup results in a more reproducible PT and LN alignment than bony anatomy setup. Considering the independence of PT and LN displacement and the impact of volume regression on displacements over time, repeated CT imaging even with primary tumorbased alignment is recommended in locally advanced disease.
doi:10.1016/j.ijrobp.2013.09.050
PMCID: PMC3895914  PMID: 24239387
Lung cancer; Mediastinal lymph nodes; Interfraction displacement; Conventional radiotherapy; Image guidance
10.  Novel Role of MDA-9/Syntenin in Regulating Urothelial Cell Proliferation by Modulating EGFR Signaling 
Purpose
Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression.
Experimental Design
Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knock down was examined in multiple cells lines and key findings were validated in primary tumors.
Results
Significantly higher (p= 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28/44) of primary tumors and an association was evident with stage (p=0.01), grade (p=0.03) and invasion status (p=0.02). MDA-9/Syntenin overexpression in non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and co-localization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin: AKT expressions with stage (p=0.04, EGFR), (p=0.01, AKT). A correlation between MDA-9/Syntenin: β-catenin co-expression with stage (p=0.03) and invasion (p=0.04) was also evident.
Conclusions
Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring and therapeutic strategies for managing UCC.
doi:10.1158/1078-0432.CCR-13-0585
PMCID: PMC3872137  PMID: 23873690
Urothelial cancer; MDA-9/Syntenin; invasion; EGFR signaling
11.  ATM kinase inhibition preferentially sensitizes p53 mutant glioma to ionizing radiation 
Purpose
Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12–15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of GBM patients is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. ATM, ataxia telangiectasia (A-T) mutated, is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, growth, and potently radiosensitized human glioma cells in vitro.
Experimental Design
Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intra-cranially, and KU-60019 was administered intra-tumorally by convection-enhanced delivery or osmotic pump.
Results
Our results demonstrate that the combined effect of KU-60019 and radiation significantly increased survival of mice 2–3 fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma.
Conclusions
Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.
doi:10.1158/1078-0432.CCR-12-3408
PMCID: PMC3687028  PMID: 23620409
12.  Staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion via angiotensin II type 1 receptor (AT1R) and TGFβ signaling 
FEBS Open Bio  2014;4:353-361.
Highlights
•SND1 augments AT1R receptor level by posttranscriptional regulation.•SND1 activates TGFβ signaling which promotes the epithelial–mesenchymal transition.•Migration and invasion by human hepatocellular carcinoma (HCC) cells are augmented by SND1.•A correlation is observed between SND1 and AT1R expression in HCC patients.
Staphylococcal nuclease domain containing-1 (SND1) is overexpressed in human hepatocellular carcinoma (HCC) patients and promotes tumorigenesis by human HCC cells. We now document that SND1 increases angiotensin II type 1 receptor (AT1R) levels by increasing AT1R mRNA stability. This results in activation of ERK, Smad2 and subsequently the TGFβ signaling pathway, promoting epithelial–mesenchymal transition (EMT) and migration and invasion by human HCC cells. A positive correlation was observed between SND1 and AT1R expression levels in human HCC patients. Small molecule inhibitors of SND1, alone or in combination with AT1R blockers, might be an effective therapeutic strategy for late-stage aggressive HCC.
doi:10.1016/j.fob.2014.03.012
PMCID: PMC4050181  PMID: 24918049
ACE, angiotensin-I converting enzyme; ACE-I, ACE inhibitors; AT1R, angiotensin II type 1 receptor; EMT, epithelial–mesenchymal transition; FDR, false discovery rate; HCC, human hepatocellular carcinoma; LP, losartan potassium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NASH, non-alcoholic steatohepatitis; PAI-1, plasminogen activator inhibitor-1; RISC, RNA-induced silencing complex; SND1, Staphylococcal nuclease domain containing-1; SND1; AT1R; TGFβ; PAI-1; Invasion
13.  Astrocyte elevated gene-1 (AEG-1) promotes hepatocarcinogenesis: novel insights from a mouse model 
Hepatology (Baltimore, Md.)  2012;56(5):1782-1791.
Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not Wild type (WT) mice, with N-nitrosodiethylamine (DEN), resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of pro-survival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance towards senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media (CM) from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated association of Factor XII (FXII) mRNA with polysomes resulting in increased translation. siRNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis.
Conclusion
We uncover novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence and activation of coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.
doi:10.1002/hep.25868
PMCID: PMC3449036  PMID: 22689379
Astrocyte elevated gene-1 (AEG-1); transgenic; hepatocellular carcinoma (HCC); senescence; angiogenesis
14.  The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing 
Nucleic Acids Research  2013;42(2):926-940.
Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.
doi:10.1093/nar/gkt944
PMCID: PMC3902934  PMID: 24163099
15.  SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling 
The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p=0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced over-expression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p=0.0015–0.030) and in vivo (p=0.016) cellular growth, invasion (p=0.029–0.049), and colony formation (p=0.023–0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser473), STAT3 (Tyr705), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated β-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
doi:10.1007/s00109-012-0955-3
PMCID: PMC3691869  PMID: 22968441
Single nucleotide polymorphism array; Lung cancer; SH3GL2; Deletion
16.  Mitochondrial DNA Mutations in Respiratory Complex-I in Never-Smoker Lung Cancer Patients Contribute to Lung Cancer Progression and associated with EGFR gene mutation 
Journal of cellular physiology  2012;227(6):2451-2460.
Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with EGFR and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients’ samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P=0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P=0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients’ population. MtDNA mutation was significantly (P=0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P=0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
doi:10.1002/jcp.22980
PMCID: PMC3256258  PMID: 21830212
Lung cancer; never-smokers; MtDNA mutation; Respiratory Complex-I; EGFR mutation
17.  Tumor, Lymph node and Lymph Node-to-Tumor Displacements over a Radiotherapy Series: Analysis of Inter- and Intrafraction Variations using Active Breathing Control (ABC) in Lung Cancer 
Purpose
To estimate errors in soft tissue-based image guidance due to relative changes between primary tumor (PT) and affected lymph node (LN) position and volume, and to compare the results to bony anatomy-based displacements of PTs and LNs during radiotherapy of lung cancer.
Materials and Methods
Weekly repeated breath hold CT scans were acquired in 17 lung cancer patients undergoing radiotherapy. PTs and affected LNs were manually contoured on all scans after rigid registration. Inter- and intrafraction displacements in the centers of mass of PTs and LNs relative to bone, and LNs relative to PTs (LN-PT) were calculated.
Results
The mean volume after 5 weeks was 65% for PTs and 63% for LNs. Systematic/random interfraction displacements were 2.6 – 4.6 mm/2.7 – 2.9 mm for PTs, 2.4 – 3.8 mm/1.4 – 2.7 mm for LNs, and 2.3 – 3.9 mm/1.9 – 2.8 mm for LN-PT. Systematic/random intrafraction displacements were < 1 mm except in superior-inferior direction. Interfraction LN-PT displacements > 3 mm were observed in 67% of fractions and require a safety margin of 12 mm in lateral, 11 mm in anteroposterior and 9 mm in superior-inferior direction. LN-PT displacements displayed significant time trends (p<0.0001) and depended on the presence of pathoanatomical conditions of the ipsilateral lung, such as atelectasis.
Conclusion
Interfraction LN-PT displacements were mostly systematic and comparable to bony anatomy-based displacements of PTs or LNs alone. Time trends, large volume changes and the influence of pathoanatomical conditions underline the importance of soft tissue-based image guidance and the potential of plan adaptation.
doi:10.1016/j.ijrobp.2011.08.021
PMCID: PMC3294098  PMID: 22197237
lung cancer; active breathing control; interfraction motion; intrafraction motion; image-guided radiotherapy
18.  An inferential framework for biological network hypothesis tests 
BMC Bioinformatics  2013;14:94.
Background
Networks are ubiquitous in modern cell biology and physiology. A large literature exists for inferring/proposing biological pathways/networks using statistical or machine learning algorithms. Despite these advances a formal testing procedure for analyzing network-level observations is in need of further development. Comparing the behaviour of a pharmacologically altered pathway to its canonical form is an example of a salient one-sample comparison. Locating which pathways differentiate disease from no-disease phenotype may be recast as a two-sample network inference problem.
Results
We outline an inferential method for performing one- and two-sample hypothesis tests where the sampling unit is a network and the hypotheses are stated via network model(s). We propose a dissimilarity measure that incorporates nearby neighbour information to contrast one or more networks in a statistical test. We demonstrate and explore the utility of our approach with both simulated and microarray data; random graphs and weighted (partial) correlation networks are used to form network models. Using both a well-known diabetes dataset and an ovarian cancer dataset, the methods outlined here could better elucidate co-regulation changes for one or more pathways between two clinically relevant phenotypes.
Conclusions
Formal hypothesis tests for gene- or protein-based networks are a logical progression from existing gene-based and gene-set tests for differential expression. Commensurate with the growing appreciation and development of systems biology, the dissimilarity-based testing methods presented here may allow us to improve our understanding of pathways and other complex regulatory systems. The benefit of our method was illustrated under select scenarios.
doi:10.1186/1471-2105-14-94
PMCID: PMC3621801  PMID: 23496778
19.  Estimating statistical uncertainty of Monte Carlo efficiency-gain in the context of a correlated sampling Monte Carlo code for brachytherapy treatment planning with non-normal dose distribution 
Correlated sampling Monte Carlo methods can shorten computing times in brachytherapy treatment planning. Monte Carlo efficiency is typically estimated via efficiency gain, defined as the reduction in computing time by correlated sampling relative to conventional Monte Carlo methods when equal statistical uncertainties have been achieved. The determination of the efficiency gain uncertainty arising from random effects, however, is not a straightforward task specially when the error distribution is non-normal. The purpose of this study is to evaluate the applicability of the F distribution and standardized uncertainty propagation methods (widely used in metrology to estimate uncertainty of physical measurements) for predicting confidence intervals about efficiency gain estimates derived from single Monte Carlo runs using fixed-collision correlated sampling in a simplified brachytherapy geometry. A bootstrap based algorithm was used to simulate the probability distribution of the efficiency gain estimates and the shortest 95% confidence interval was estimated from this distribution. It was found that the corresponding relative uncertainty was as large as 37% for this particular problem. The uncertainty propagation framework predicted confidence intervals reasonably well; however its main disadvantage was that uncertainties of input quantities had to be calculated in a separate run via a Monte Carlo method. The F distribution noticeably underestimated the confidence interval. These discrepancies were influenced by several photons with large statistical weights which made extremely large contributions to the scored absorbed dose difference. The mechanism of acquiring high statistical weights in the fixed-collision correlated sampling method was explained and a mitigation strategy was proposed.
doi:10.1016/j.apradiso.2011.09.015
PMCID: PMC3242326  PMID: 21992844
Monte Carlo; correlated sampling; efficiency; uncertainty; bootstrap
20.  Clinical evaluation of soft tissue organ boundary visualization on cone-beam computed tomographic (CBCT) imaging 
Purpose
Cone-beam computed tomographic images (CBCTs) are increasingly used for set up correction, soft tissue targeting and image-guided adaptive radiotherapy (IGART). However, CBCT image quality is limited by low contrast and imaging artifacts. This analysis investigates the detectability of soft tissue boundaries in CBCT by performing a multiple-observer segmentation study.
Material and Methods
In 4 prostate cancer patients prostate, bladder and rectum were repeatedly delineated by 5 observers on CBCTs and fan beam CTs (FBCTs). A volumetric analysis of contouring variations was performed by calculating coefficients of variation (COV: standard deviation/average volume). The topographical distribution of contouring variations was analyzed using an average surface mesh-based method.
Results
Observer- and patient-averaged COVs for FBCT/CBCT were 0.09/0.19 for prostate, 0.05/0.08 for bladder and 0.09/0.08 for rectum. Contouring variations on FBCT were significantly smaller than on CBCT for prostate (p<0.03) and bladder (p<0.04), but not for rectum (p<0.37) (intermodality differences). Intraobserver variations from repeated contouring of the same image set were not significant for either FBCT or CBCT (p<0.05). Average standard deviations of individual observers’ contour differences from average surface meshes on FBCT versus CBCT were 1.5 versus 2.1 mm for prostate, 0.7 versus 1.4 mm for bladder, and 1.3 versus 1.5 mm for rectum. The topographical distribution of contouring variations was similar for FBCT and CBCT.
Conclusion
Contouring variations were larger on CBCT than FBCT, except for rectum. Given the well-documented uncertainty in soft tissue contouring in the pelvis, improvement of CBCT image quality and establishment of well-defined soft tissue identification rules are desirable for image-guided radiotherapy.
doi:10.1016/j.ijrobp.2010.02.007
PMCID: PMC2946461  PMID: 20542644
Cone-beam CT; Image guided adaptive radiotherapy; Prostate cancer; Image quality; Segmentation
21.  Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression 
Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/β-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-κB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.
doi:10.1172/JCI36460
PMCID: PMC2648696  PMID: 19221438
22.  Identification of genes for complex disease using longitudinal phenotypes 
BMC Genetics  2003;4(Suppl 1):S58.
Using the simulated data set from Genetic Analysis Workshop 13, we explored the advantages of using longitudinal data in genetic analyses. The weighted average of the longitudinal data for each of seven quantitative phenotypes were computed and analyzed. Genome screen results were then compared for these longitudinal phenotypes and the results obtained using two cross-sectional designs: data collected near a single age (45 years) and data collected at a single time point. Significant linkage was obtained for nine regions (LOD scores ranging from 5.5 to 34.6) for six of the phenotypes. Using cross-sectional data, LOD scores were slightly lower for the same chromosomal regions, with two regions becoming nonsignificant and one additional region being identified. The magnitude of the LOD score was highly correlated with the heritability of each phenotype as well as the proportion of phenotypic variance due to that locus. There were no false-positive linkage results using the longitudinal data and three false-positive findings using the cross-sectional data. The three false positive results appear to be due to the kurtosis in the trait distribution, even after removing extreme outliers. Our analyses demonstrated that the use of simple longitudinal phenotypes was a powerful means to detect genes of major to moderate effect on trait variability. In only one instance was the power and heritability of the trait increased by using data from one examination. Power to detect linkage can be improved by identifying the most heritable phenotype, ensuring normality of the trait distribution and maximizing the information utilized through novel longitudinal designs for genetic analysis.
doi:10.1186/1471-2156-4-S1-S58
PMCID: PMC1866495  PMID: 14975126

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