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1.  Global transcriptome analysis of formalin-fixed prostate cancer specimens identifies biomarkers of disease recurrence 
Cancer research  2014;74(12):3228-3237.
Prostate cancer remains the second leading cause of cancer death in American men and there is an unmet need for biomarkers to identify patients with aggressive disease. In an effort to identify biomarkers of recurrence, we performed global RNA sequencing on 106 formalin-fixed, paraffin-embedded (FFPE) prostatectomy samples from 100 patients at three independent sites, defining a 24-gene signature panel. The 24 genes in this panel function in cell cycle progression, angiogenesis, hypoxia, apoptosis, PI3K signaling, steroid metabolism, translation, chromatin modification and transcription. Sixteen genes have been associated with cancer with five specifically associated with prostate cancer (BTG2, IGFBP3, SIRT1, MXI1 and FDPS). Validation was performed on an independent publicly available dataset of 140 patients, where the new signature panel outperformed markers published previously in terms of predicting biochemical recurrence (BCR). Our work also identified differences in gene expression between Gleason Pattern 4+3 and 3+4 tumors, including several genes involved in the epithelial to mesenchymal transition and developmental pathways. Overall, this study defines a novel biomarker panel that has the potential to improve the clinical management of prostate cancer.
PMCID: PMC4058362  PMID: 24713434
Prostate Cancer; Recurrence; Biomarkers
2.  Assessing Needs and Assets for Building a Regional Network Infrastructure to Reduce Cancer Related Health Disparities 
Significant cancer health disparities exist in the United States and Puerto Rico. While numerous initiatives have been implemented to reduce cancer disparities, regional coordination of these efforts between institutions is often limited. To address cancer health disparities nationwide, a series of regional transdisciplinary networks through the Geographic Management Program (GMaP) and the Minority Biospecimen/Biobanking Geographic Management Program (BMaP) were established in six regions across the country. This paper describes the development of the Region 3 GMaP/BMaP network composed of over 100 investigators from nine institutions in five Southeastern states and Puerto Rico to develop a state-of-the-art network for cancer health disparities research and training.
We describe a series of partnership activities that led to the formation of the infrastructure for this network, recount the participatory processes utilized to develop and implement a needs and assets assessment and implementation plan, and describe our approach to data collection. Completion, by all nine institutions, of the needs and assets assessment resulted in several beneficial outcomes for Region 3 GMaP/BMaP. This network entails ongoing commitment from the institutions and institutional leaders, continuous participatory and engagement activities, and effective coordination and communication centered on team science goals.
PMCID: PMC4360072  PMID: 24486917
cancer health disparities; needs assessment; evaluation; implementation plan; multi-institutional; biospecimen; biobank; team science
3.  Evaluating intra- and inter-individual variation in the human placental transcriptome 
Genome Biology  2015;16(1):54.
Gene expression variation is a phenotypic trait of particular interest as it represents the initial link between genotype and other phenotypes. Analyzing how such variation apportions among and within groups allows for the evaluation of how genetic and environmental factors influence such traits. It also provides opportunities to identify genes and pathways that may have been influenced by non-neutral processes. Here we use a population genetics framework and next generation sequencing to evaluate how gene expression variation is apportioned among four human groups in a natural biological tissue, the placenta.
We estimate that on average, 33.2%, 58.9%, and 7.8% of the placental transcriptome is explained by variation within individuals, among individuals, and among human groups, respectively. Additionally, when technical and biological traits are included in models of gene expression they each account for roughly 2% of total gene expression variation. Notably, the variation that is significantly different among groups is enriched in biological pathways associated with immune response, cell signaling, and metabolism. Many biological traits demonstrate correlated changes in expression in numerous pathways of potential interest to clinicians and evolutionary biologists. Finally, we estimate that the majority of the human placental transcriptome exhibits expression profiles consistent with neutrality; the remainder are consistent with stabilizing selection, directional selection, or diversifying selection.
We apportion placental gene expression variation into individual, population, and biological trait factors and identify how each influence the transcriptome. Additionally, we advance methods to associate expression profiles with different forms of selection.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0627-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4404591  PMID: 25887593
4.  A Bayesian Model for the Analysis of Transgenerational Epigenetic Variation 
G3: Genes|Genomes|Genetics  2015;5(4):477-485.
Epigenetics has become one of the major areas of biological research. However, the degree of phenotypic variability that is explained by epigenetic processes still remains unclear. From a quantitative genetics perspective, the estimation of variance components is achieved by means of the information provided by the resemblance between relatives. In a previous study, this resemblance was described as a function of the epigenetic variance component and a reset coefficient that indicates the rate of dissipation of epigenetic marks across generations. Given these assumptions, we propose a Bayesian mixed model methodology that allows the estimation of epigenetic variance from a genealogical and phenotypic database. The methodology is based on the development of a T matrix of epigenetic relationships that depends on the reset coefficient. In addition, we present a simple procedure for the calculation of the inverse of this matrix (T−1) and a Gibbs sampler algorithm that obtains posterior estimates of all the unknowns in the model. The new procedure was used with two simulated data sets and with a beef cattle database. In the simulated populations, the results of the analysis provided marginal posterior distributions that included the population parameters in the regions of highest posterior density. In the case of the beef cattle dataset, the posterior estimate of transgenerational epigenetic variability was very low and a model comparison test indicated that a model that did not included it was the most plausible.
PMCID: PMC4390564  PMID: 25617408
epigenetics; Bayesian analysis; genetic variance; resemblance between relatives
5.  E2F Activators Signal and Maintain Centrosome Amplification in Breast Cancer Cells 
Molecular and Cellular Biology  2014;34(14):2581-2599.
Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2+ cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2+ cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2+ breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2.
PMCID: PMC4097658  PMID: 24797070
6.  Mutation of Murine Sox4 Untranslated Regions Results in Partially Penetrant Perinatal Lethality 
In vivo (Athens, Greece)  2014;28(5):709-718.
Sox4 is an essential gene, and genetic deletion results in embryonic lethality. In an effort to develop mice with tissue-specific deletion, we bred conditional knockout mice bearing LoxP recombination sites flanking the Sox4 gene, with the LoxP sites located in the Sox4 5’UTR and 3’UTR.
The number of mice homozygous for this LoxP-flanked conditional knockout allele was far below the expected number, suggesting embryonic lethality with reduced penetrance. From over 200 animals bred, only 11% were homozygous Sox4flox/flox mice compared to the expected Mendelian ratio of 25% (p < 0.001). Moreover, there was a significant reduction in the number of female Sox4flox/flox mice (26%) relative to male Sox4flox/flox mice (p = 0.0371). Reduced Sox4 expression in homozygous embryos was confirmed by in-situ hybridization and Quantitative real-time polymerase chain reaction (QPCR).
LoxP sites in the 5’ and 3’ UTR of both alleles of Sox4 resulted in reduced, but variable expression of Sox4 message.
PMCID: PMC4237010  PMID: 25189881
Mouse; Sox4; perinatal lethality; transcription
7.  Integrated Analysis of Whole-Genome Paired-End and Mate-Pair Sequencing Data for Identifying Genomic Structural Variations in Multiple Myeloma 
Cancer Informatics  2014;13(Suppl 2):49-53.
We present a pipeline to perform integrative analysis of mate-pair (MP) and paired-end (PE) genomic DNA sequencing data. Our pipeline detects structural variations (SVs) by taking aligned sequencing read pairs as input and classifying these reads into properly paired and discordantly paired categories based on their orientation and inferred insert sizes. Recurrent SV was identified from the discordant read pairs. Our pipeline takes into account genomic annotation and genome repetitive element information to increase detection specificity. Application of our pipeline to whole-genome MP and PE sequencing data from three multiple myeloma cell lines (KMS11, MM.1S, and RPMI8226) recovered known SVs, such as heterozygous TRAF3 deletion, as well as a novel experimentally validated SPI1 – ZNF287 inter-chromosomal rearrangement in the RPMI8226 cell line.
PMCID: PMC4179644  PMID: 25288879
structural variations; multiple myeloma; whole-genome sequencing; variant detection
8.  Tumor-infiltrating lymphocytes in glioblastoma are associated with specific genomic alterations and related to transcriptional class 
Tumor-infiltrating lymphocytes (TILs) have prognostic significance in many cancers, yet their roles in glioblastoma (GBM) have not been fully defined. We hypothesized TILs in GBM are associated with molecular alterations, histologies and survival.
Experimental Design
We used data from The Cancer Genome Atlas (TCGA) to investigate molecular, histologic and clinical correlates of TILs in GBMs. Lymphocytes were categorized as absent, present or abundant in histopathologic images from 171 TCGA GBMs. Associations were examined between lymphocytes and histologic features, mutations, copy number alterations, CpG island methylator phenotype, transcriptional class and survival. We validated histologic findings using CD3G gene expression.
We found a positive correlation between TILs and GBMs with gemistocytes, sarcomatous cells, epithelioid cells and giant cells. Lymphocytes were enriched in the mesenchymal transcriptional class and strongly associated with mutations in NF1 and RB1. These mutations are frequent in the mesenchymal class and characteristic of gemistocytic, sarcomatous, epithelioid and giant cell histologies. Conversely, TILs were rare in GBMs with small cells and oligodendroglioma components. Lymphocytes were depleted in the classical transcriptional class and in EGFR-amplified and homozygous PTEN-deleted GBMs. These alterations are characteristic of GBMs with small cells and GBMs of the classical transcriptional class. No association with survival was demonstrated.
TILs were enriched in GBMs of the mesenchymal class, strongly associated with mutations in NF1 and RB1 and typical of histologies characterized by these mutations. Conversely, TILs were depleted in the classical class, EGFR-amplified and homozygous PTEN-deleted tumors and rare in histologies characterized by these alterations.
PMCID: PMC3865611  PMID: 23864165
Glioblastoma; Lymphocytes; The Cancer Genome Atlas; Transcriptional class; Mesenchymal; Classical; Neurofibromatosis 1 (NF1); Retinoblastoma 1 (RB1); Epidermal growth factor receptor (EGFR); Tumor protein 53 (TP53); Phosphatase and tensin homolog (PTEN)
9.  Docking Analysis of Transient Complexes: Interaction of Ferredoxin-NADP+ Reductase with Ferredoxin and Flavodoxin 
Proteins  2008;72(3):848-862.
Ferredoxin (Fd) interacts with ferredoxin-NADP+ reductase (FNR) to transfer two electrons to the latter, one by one, which will finally be used to reduce NADP+ to NADPH. The formation of a transient complex between Fd and FNR is required for the electron transfer, and extensive mutational and crystallographic studies have been reported to characterize such protein-protein interaction. However, some aspects of the association mechanism still remain unclear. Moreover, in spite of their structural differences, flavodoxin (Fld) can replace Fd in its function and interact with FNR to transfer electrons with only slightly lower efficiency. Although crystallographic structures for the FNR:Fd association have been reported, experimental structural data for the FNR:Fld interaction are highly elusive. We have modeled here the interactions between FNR and both of its protein partners, Fd and Fld, using surface energy analysis, computational rigid-body docking simulations, and interface side-chain refinement. The results, consistent with previous experimental data, suggest the existence of alternative binding modes in these electron transfer proteins.
PMCID: PMC4162409  PMID: 18260112
Protein-protein association; electron transfer; binding energy landscapes; computational docking
10.  High-Performance Computational Analysis of Glioblastoma Pathology Images with Database Support Identifies Molecular and Survival Correlates 
In this paper, we present a novel framework for microscopic image analysis of nuclei, data management, and high performance computation to support translational research involving nuclear morphometry features, molecular data, and clinical outcomes. Our image analysis pipeline consists of nuclei segmentation and feature computation facilitated by high performance computing with coordinated execution in multi-core CPUs and Graphical Processor Units (GPUs). All data derived from image analysis are managed in a spatial relational database supporting highly efficient scientific queries. We applied our image analysis workflow to 159 glioblastomas (GBM) from The Cancer Genome Atlas dataset. With integrative studies, we found statistics of four specific nuclear features were significantly associated with patient survival. Additionally, we correlated nuclear features with molecular data and found interesting results that support pathologic domain knowledge. We found that Proneural subtype GBMs had the smallest mean of nuclear Eccentricity and the largest mean of nuclear Extent, and MinorAxisLength. We also found gene expressions of stem cell marker MYC and cell proliferation maker MKI67 were correlated with nuclear features. To complement and inform pathologists of relevant diagnostic features, we queried the most representative nuclear instances from each patient population based on genetic and transcriptional classes. Our results demonstrate that specific nuclear features carry prognostic significance and associations with transcriptional and genetic classes, highlighting the potential of high throughput pathology image analysis as a complementary approach to human-based review and translational research.
PMCID: PMC4120024  PMID: 25098236
Glioblastoma; large-scale image analysis; survival analysis; phenotype-genotype integration; translational research
11.  Interobserver reliability of echocardiography for prognostication of normotensive patients with pulmonary embolism 
To evaluate the interobserver reliability of echocardiographic findings of right ventricle (RV) dysfunction for prognosticating normotensive patients with pulmonary embolism (PE).
A central panel of cardiologists evaluated echocardiographic studies of 75 patients included in the PROTECT study for the following signs: RV diameter, RV/left ventricular (LV) diameter ratio, hypokinesis of the RV free wall, and tricuspid plane systolic excursion (TAPSE). Investigators used intraclass correlation to assess agreement between the measurements of the central panel and each of the local cardiologists. Investigators used the single weighted kappa statistic to test for agreement between readers of interpretation of RV enlargement and RV hypokinesis.
The two observers had fair agreement (k = 0.45) for RV enlargement assessed by the RV diameter, and good agreement (k = 0.65) for RV enlargement assessed by the RV/LV diameter ratio. The interobserver reliability of the assessment whether hypokinesis of the RV free wall is present was good (к = 0.70), and whether RV dysfunction (assessed by TAPSE measurement) is present was very good (k = 0.86). The intraclass correlation for the RV/LV diameter ratio was fair (0.55; 95% confidence interval [CI], 0.37-0.69), for the RV diameter was good (0.70; 95% CI, 0.56-0.80), and for the TAPSE measurement was very good (0.85; 95% CI, 0.77-0.90). On Bland-Altman analysis, the mean differences for RV diameter, RV/LV diameter ratio and TAPSE measurement were 2.33 (±5.38), 0.06 (±0.23) and 0.08 (±2.20), respectively.
TAPSE measurement is the least user dependent and most reproducible echocardiographic finding of RV dysfunction in normotensive patients with PE.
PMCID: PMC4126908  PMID: 25092465
Pulmonary embolism; Prognosis; Echocardiography; Interobserver reliability; Reproducibility
12.  Reinforced Feedback in Virtual Environment for Rehabilitation of Upper Extremity Dysfunction after Stroke: Preliminary Data from a Randomized Controlled Trial 
BioMed Research International  2014;2014:752128.
Objectives. To study whether the reinforced feedback in virtual environment (RFVE) is more effective than traditional rehabilitation (TR) for the treatment of upper limb motor function after stroke, regardless of stroke etiology (i.e., ischemic, hemorrhagic). Design. Randomized controlled trial. Participants. Forty-four patients affected by stroke. Intervention. The patients were randomized into two groups: RFVE (N = 23) and TR (N = 21), and stratified according to stroke etiology. The RFVE treatment consisted of multidirectional exercises providing augmented feedback provided by virtual reality, while in the TR treatment the same exercises were provided without augmented feedbacks. Outcome Measures. Fugl-Meyer upper extremity scale (F-M UE), Functional Independence Measure scale (FIM), and kinematics parameters (speed, time, and peak). Results. The F-M UE (P = 0.030), FIM (P = 0.021), time (P = 0.008), and peak (P = 0.018), were significantly higher in the RFVE group after treatment, but not speed (P = 0.140). The patients affected by hemorrhagic stroke significantly improved FIM (P = 0.031), time (P = 0.011), and peak (P = 0.020) after treatment, whereas the patients affected by ischemic stroke improved significantly only speed (P = 0.005) when treated by RFVE. Conclusion. These results indicated that some poststroke patients may benefit from RFVE program for the recovery of upper limb motor function. This trial is registered with NCT01955291.
PMCID: PMC3972918  PMID: 24745024
13.  Computational identification of conserved transcription factor binding sites upstream of genes induced in rat brain by transient focal ischemic stroke 
Brain research  2012;1495:10.1016/j.brainres.2012.11.052.
Microarray analysis has been used to understand how gene regulation plays a critical role in neuronal injury, survival and repair following ischemic stroke. To identify the transcriptional regulatory elements responsible for ischemia-induced gene expression, we examined gene expression profiles of rat brains following focal ischemia and performed computational analysis of consensus transcription factor binding sites (TFBS) in the genes of the dataset. In this study, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) stroke and gene transcription in brain tissues following ischemia/reperfusion was examined using Affymetrix GeneChip technology. The CONserved transcription FACtor binding site (CONFAC) software package was used to identify over-represented TFBS in the upstream promoter regions of ischemia-induced genes compared to control datasets. CONFAC identified 12 TFBS that were statistically over-represented from our dataset of ischemia-induced genes, including three members of the Ets-1 family of transcription factors (TFs). Microarray results showed that mRNA for Ets-1 was increased following tMCAO but not pMCAO. Immunohistochemical analysis of Ets-1 protein in rat brains following MCAO showed that Ets-1 was highly expressed in neurons in the brain of sham control animals. Ets-1 protein expression was virtually abolished in injured neurons of the ischemic brain but was unchanged in peri-infarct brain areas. These data indicate that TFs, including Ets-1, may influence neuronal injury following ischemia. These findings could provide important insights into the mechanisms that lead to brain injury and could provide avenues for the development of novel therapies.
PMCID: PMC3816791  PMID: 23246490
Ischemia; Microarray; Reperfusion; Stroke; Transcription Factors; Rat
14.  Machine-Based Morphologic Analysis of Glioblastoma Using Whole-Slide Pathology Images Uncovers Clinically Relevant Molecular Correlates 
PLoS ONE  2013;8(11):e81049.
Pathologic review of tumor morphology in histologic sections is the traditional method for cancer classification and grading, yet human review has limitations that can result in low reproducibility and inter-observer agreement. Computerized image analysis can partially overcome these shortcomings due to its capacity to quantitatively and reproducibly measure histologic structures on a large-scale. In this paper, we present an end-to-end image analysis and data integration pipeline for large-scale morphologic analysis of pathology images and demonstrate the ability to correlate phenotypic groups with molecular data and clinical outcomes. We demonstrate our method in the context of glioblastoma (GBM), with specific focus on the degree of the oligodendroglioma component. Over 200 million nuclei in digitized pathology slides from 117 GBMs in the Cancer Genome Atlas were quantitatively analyzed, followed by multiplatform correlation of nuclear features with molecular and clinical data. For each nucleus, a Nuclear Score (NS) was calculated based on the degree of oligodendroglioma appearance, using a regression model trained from the optimal feature set. Using the frequencies of neoplastic nuclei in low and high NS intervals, we were able to cluster patients into three well-separated disease groups that contained low, medium, or high Oligodendroglioma Component (OC). We showed that machine-based classification of GBMs with high oligodendroglioma component uncovered a set of tumors with strong associations with PDGFRA amplification, proneural transcriptional class, and expression of the oligodendrocyte signature genes MBP, HOXD1, PLP1, MOBP and PDGFRA. Quantitative morphologic features within the GBMs that correlated most strongly with oligodendrocyte gene expression were high nuclear circularity and low eccentricity. These findings highlight the potential of high throughput morphologic analysis to complement and inform human-based pathologic review.
PMCID: PMC3827469  PMID: 24236209
15.  Wnt signaling in triple negative breast cancer is associated with metastasis 
BMC Cancer  2013;13:537.
Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies.
We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease.
The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/β-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or β-catenin (functional read out of Wnt/β-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from β-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/β-catenin classifier had a greater risk of lung and brain, but not bone metastases.
These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.
PMCID: PMC4226307  PMID: 24209998
Breast cancer; Triple negative; Wnt; FFPE; Microarray
16.  Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells 
Triple-negative breast cancer (TNBC) is an aggressive clinical subtype of breast cancer that is characterized by the lack of estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) overexpression. The TNBC subtype constitutes approximately 10%–20% of all breast cancers, but has no effective molecular targeted therapies. Previous meta-analysis of gene expression profiles of 587 TNBC cases from 21 studies demonstrated high expression of Wnt signaling pathway-associated genes in basal-like 2 and mesenchymal subtypes of TNBC. In this study, we investigated the potential of Wnt pathway inhibitors in effective treatment of TNBC.
Activation of Wnt pathway was assessed in four TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937), and the ER+ cell line MCF-7 using confocal microscopy and Western blot analysis of pathway components. Effectiveness of five different Wnt pathway inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4 and XAV-939) on cell proliferation and apoptosis were tested in vitro. The inhibitory effects of iCRT-3 on canonical Wnt signaling in TNBC was evaluated by quantitative real-time RT-PCR analysis of Axin2 and dual-luciferase reporter assays. The effects of shRNA knockdown of SOX4 in combination with iCRT-3 and/or genistein treatments on cell proliferation, migration and invasion on BT-549 cells were also evaluated.
Immunofluorescence staining of β-catenin in TNBC cell lines showed both nuclear and cytoplasmic localization, indicating activation of Wnt pathway in TNBC cells. iCRT-3 was the most effective compound for inhibiting proliferation and antagonizing Wnt signaling in TNBC cells. In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro. Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.
These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.
PMCID: PMC4228255  PMID: 24188694
Triple-negative breast cancer; Wnt signaling pathway; iCRT-3; SOX4
17.  The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import 
Retrovirology  2013;10:25.
HIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M.
We find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α.
Our study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms.
PMCID: PMC3598646  PMID: 23448277
18.  Characterization of primary prostate carcinoma by anti-1-amino-2-[18F] -fluorocyclobutane-1-carboxylic acid (anti-3-[18F] FACBC) uptake 
Anti-1-amino-3-[18F] fluorocyclobutane-1-carboxylic acid (anti-3-[18F] FACBC) is a synthetic amino acid positron emission tomography (PET) radiotracer with utility in the detection of recurrent prostate carcinoma. The aim of this study is to correlate uptake of anti-3-[18F] FACBC with histology of prostatectomy specimens in patients undergoing radical prostatectomy and to determine if uptake correlates to markers of tumor aggressiveness such as Gleason score. Ten patients with prostate carcinoma pre-radical prostatectomy underwent 45 minute dynamic PET-CT of the pelvis after IV injection of 347.8 ± 81.4 MBq anti-3-[18F] FACBC. Each prostate was co-registered to a separately acquired MR, divided into 12 sextants, and analyzed visually for abnormal focal uptake at 4, 16, 28, and 40 min post-injection by a single reader blinded to histology. SUVmax per sextant and total sextant activity (TSA) was also calculated. Histology and Gleason scores were similarly recorded by a urologic pathologist blinded to imaging. Imaging and histologic analysis were then compared. In addition, 3 representative sextants from each prostate were chosen based on highest, lowest and median SUVmax for immunohistochemical (IHC) analysis of Ki67, synaptophysin, P504s, chromogranin A, P53, androgen receptor, and prostein. 79 sextants had malignancy and 41 were benign. Highest combined sensitivity and specificity was at 28 min by visual analysis; 81.3% and 50.0% respectively. SUVmax was significantly higher (p<0.05) for malignant sextants (5.1±2.6 at 4 min; 4.5±1.6 at 16 min; 4.0±1.3 at 28 min; 3.8±1.0 at 40 min) compared to non-malignant sextants (4.0±1.9 at 4 min; 3.5±0.8 at 16 min; 3.4±0.9 at 28 min; 3.3±0.9 at 40 min), though there was overlap of activity between malignant and non-malignant sextants. SUVmax also significantly correlated (p<0.05) with Gleason score at all time points (r=0.28 at 4 min; r=0.42 at 16 min; r=0.46 at 28 min; r=0.48 at 40 min). There was no significant correlation of anti-3-[18F] FACBC SUVmax with Ki-67 or other IHC markers. Since there was no distinct separation between malignant and non-malignant sextants or between Gleason score levels, we believe that anti-3-[18F] FACBC PET should not be used alone for radiation therapy planning but may be useful to guide biopsy to the most aggressive lesion.
PMCID: PMC3545368  PMID: 23342303
Positron emission tomography (PET); prostate carcinoma; anti-3-[18F] FACBC
19.  Carboxymethylation of the PP2A Catalytic Subunit in Saccharomyces cerevisiae Is Required for Efficient Interaction with the B-type Subunits Cdc55p and Rts1p* 
The Journal of biological chemistry  2001;276(2):1570-1577.
Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine phosphatase known to play important roles in cell cycle regulation. Association of different B-type targeting subunits with the heterodimeric core (A/C) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. We have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approaches were taken. First, S. cerevisiae cells were generated whose survival depended on the expression of different carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PP2A C subunit carboxyl-terminal leucine was identified, and cells deleted for this methyltransferase were utilized for our studies. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p. Loss of Cdc55p association was accompanied in each case by a large reduction in binding of the yeast A subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylation also greatly reduced the association of another yeast B-type subunit, Rts1p. Thus, methylation of Pph21p is important for formation of PP2A trimeric and dimeric complexes, and consequently, for PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.
PMCID: PMC3508460  PMID: 11038366
20.  WD40 Repeat Proteins Striatin and S/G2 Nuclear Autoantigen Are Members of a Novel Family of Calmodulin-binding Proteins That Associate with Protein Phosphatase 2A* 
The Journal of biological chemistry  2000;275(8):5257-5263.
Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca2+-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G2 nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the postsynaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G2 phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca2+-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca2+-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.
PMCID: PMC3505218  PMID: 10681496
21.  A Mammalian Homolog of Yeast MOB1 Is Both a Member and a Putative Substrate of Striatin Family-Protein Phosphatase 2A Complexes* 
The Journal of biological chemistry  2001;276(26):24253-24260.
Striatin and S/G2 nuclear autoantigen (SG2NA) are related proteins that contain membrane binding domains and associate with protein phosphatase 2A (PP2A) and many additional proteins that may be PP2A regulatory targets. Here we identify a major member of these complexes as class II mMOB1, a mammalian homolog of the yeast protein MOB1, and show that its phosphorylation appears to be regulated by PP2A. Yeast MOB1 is critical for cytoskeletal reorganization during cytokinesis and exit from mitosis. We show that mMOB1 associated with PP2A is not detectably phosphorylated in asynchronous murine fibroblasts. However, treatment with the PP2A inhibitor okadaic acid induces phosphorylation of PP2A-associated mMOB1 on serine. Moreover, specific inhibition of PP2A also results in hyperphosphorylation of striatin, SG2NA, and three unidentified proteins, suggesting that these proteins may also be regulated by PP2A. Indirect immunofluorescence produced highly similar staining patterns for striatin, SG2NA, and mMOB1, with the highest concentrations for each protein adjacent to the nuclear membrane. We also present evidence that these complexes may interact with each other. These data are consistent with a model in which PP2A may regulate mMOB1, striatin, and SG2NA to modulate changes in the cytoskeleton or interactions between the cytoskeleton and membrane structures.
PMCID: PMC3503316  PMID: 11319234
22.  Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells 
BMC Cancer  2012;12:145.
Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT), to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells.
Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined.
Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1). In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation.
Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways. In addition, genistein cooperates with vorinostat to induce cell death in prostate cancer cell lines with a greater effect on early stage prostate cancer.
PMCID: PMC3472186  PMID: 22494660
Prostate cancer; Soy; Natural compounds; Epigenetics; Apoptosis
23.  Identification of Candidate Genes for Histiocytoid Cardiomyopathy (HC) Using Whole Genome Expression Analysis: Analyzing Material from the HC Registry 
Histiocytoid cardiomyopathy (HC) is a rare but distinctive arrhythmogenic disorder characterized by incessant ventricular tachycardia, cardiomegaly, and often sudden death by age 2 years. The underlying genetic mechanism of HC has eluded researchers for decades. To reveal the molecular-genetic basis of HC, molecular analyses of HC hearts and hearts of age-matched controls were performed.
Total RNA and genomic DNA were prepared from formalin-fixed paraffin-embedded cardiac tissue from 12 cases of HC and 12 age-matched controls. To identify genes differentially expressed in HC, whole genome cDNA-mediated Annealing, Selection, extension and Ligation profiling was performed. TaqMan quantitative polymerase chain reaction confirmed changes in RNA expression. DNA copy number changes were measured by TaqMan copy number analysis.
Analysis of differential gene expression in HC cases identified two significantly down regulated gene sets aligned sequentially along the genome. The first gene cluster consisted of genes S100A8, S100A9, and S100A12 at 1q21.3c, and the second cluster consisted of genes IL1RL1 (ST2), IL18R1, and IL18RAP at 2q12.1a. Strong decreases in interleukin 33 expression were also observed. Decreases in copy number of the S100A genes were confirmed by TaqMan CNV assays. S100A genes are downstream of the p38-MAPK pathway that can be activated by interleukin 33 signaling.
These data suggest a model in which the interleukin 33-IL1RL1/p38-MAPK/S100A8-S100A9 axis is down regulated in HC cardiac tissue and provide several candidate genes on 1q21.3c and 2q12.1a for inherited mutations that may predispose individuals to HC.
PMCID: PMC3295543  PMID: 21585276
histiocytoid cardiomyopathy; arrhythmia; SIDS; whole genome DASL
24.  In Silico Analysis of Nuclei in Glioblastoma using Large-scale Microscopy Images Improves Prediction of Treatment Response 
Conference Proceedings  2011;2011:87-90.
In this paper, we present a complete and novel workflow for quantitative nuclear feature analysis of glioblastoma using high-throughput whole-slide microscopy image processing as it relates to treatment response and patient survival. With a complete suite of computer algorithms, large numbers of micro-anatomical structures, in this case nuclei, are analyzed and represented efficiently from whole-slide digitized images with numerical features. With regard to endpoints of treatment response, the computerized analysis presents a better discrimination than traditional neuropathologic review. As a result, this analysis method shows potential to facilitate a better understanding of disease progression and patients’ response to therapy for glioblastoma.
PMCID: PMC3292262  PMID: 22254257
25.  Integrative, Multi-modal Analysis of Glioblastoma Using TCGA Molecular Data, Pathology Images and Clinical Outcomes 
Multi-modal, multi-scale data synthesis is becoming increasingly critical for successful translational biomedical research. In this paper, we present a large-scale investigative initiative on glioblastoma, a high-grade brain tumor, with complementary data types using in silico approaches. We integrate and analyze data from The Cancer Genome Atlas Project on glioblastoma that includes novel nuclear phenotypic data derived from microscopic slides, genotypic signatures described by transcriptional class and genetic alterations, and clinical outcomes defined by response to therapy and patient survival. Our preliminary results demonstrate numerous clinically and biologically significant correlations across multiple data types, revealing the power of in silico multi-modal data integration for cancer research.
PMCID: PMC3292263  PMID: 21947516
Glioblastoma; multi-modal data process; in silico; cluster analysis; translational integration

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