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1.  PTEN Is a Negative Regulator of NK Cell Cytolytic Function 
Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56dim NK cell efficiently kills malignant targets at rest, whereas the less mature CD56bright NK cells cannot. In this study, we show that resting CD56bright NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56dim NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56bright NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell–activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell’s ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell’s PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56bright NK cell to the cytolytic CD56dim NK cells.
doi:10.4049/jimmunol.1401224
PMCID: PMC4319309  PMID: 25595786
2.  Mouse Development with a Single E2F Activator 
Nature  2008;454(7208):1137-1141.
The E2F family is conserved from C. elegans to mammals with some family members having transcription activation functions and others having repressor functions1, 2. Whereas C. elegans3 and Drosophila melanogaster4, 5 have a single E2F activator and repressor proteins, mammals evolved to have at least three activator and five repressor proteins1, 2, 6. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a3bki; E2f3a1ki) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.
doi:10.1038/nature07066
PMCID: PMC4288824  PMID: 18594513
E2F3a; E2F3b; Rb; development; proliferation; transcription and apoptosis
3.  Identifying Nuclear Phenotypes Using Semi-supervised Metric Learning 
In systems–based approaches for studying processes such as cancer and development, identifying and characterizing individual cells within a tissue is the first step towards understanding the large–scale effects that emerge from the interactions between cells. To this end, nuclear morphology is an important phenotype to characterize the physiological and differentiated state of a cell. This study focuses on using nuclear morphology to identify cellular phenotypes in thick tissue sections imaged using 3D fluorescence microscopy. The limited label information, heterogeneous feature set describing a nucleus, and existence of sub-populations within cell-types makes this a difficult learning problem. To address these issues, a technique is presented to learn a distance metric from labeled data which is locally adaptive to account for heterogeneity in the data. Additionally, a label propagation technique is used to improve the quality of the learned metric by expanding the training set using unlabeled data. Results are presented on images of tumor stroma in breast cancer, where the framework is used to identify fibroblasts, macrophages and endothelial cells – three major stromal cells involved in carcinogenesis.
PMCID: PMC4285701  PMID: 21761673
Nuclear Morphometry; Metric Learning; Microscopy
4.  HO-3867, a Safe STAT3 Inhibitor, Is Selectively Cytotoxic to Ovarian Cancer 
Cancer research  2014;74(8):2316-2327.
STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (−NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated.
doi:10.1158/0008-5472.CAN-13-2433
PMCID: PMC4286190  PMID: 24590057
5.  A Retinoblastoma Allele That Is Mutated at Its Common E2F Interaction Site Inhibits Cell Proliferation in Gene-Targeted Mice 
Molecular and Cellular Biology  2014;34(11):2029-2045.
The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1ΔG) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1ΔG/ΔG mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1ΔG/ΔG MEFs display a magnitude of E2F target gene derepression similar to that of Rb1−/− cells, even though Rb1ΔG/ΔG cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1ΔG/ΔG MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1ΔG/ΔG mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.
doi:10.1128/MCB.01589-13
PMCID: PMC4019062  PMID: 24662053
6.  Stat5 Regulates the Phosphatidylinositol 3-Kinase/Akt1 Pathway during Mammary Gland Development and Tumorigenesis 
Molecular and Cellular Biology  2014;34(7):1363-1377.
Stat5 (signal transducer and activator of transcription 5) is an essential mediator of cytokine receptor signaling and plays important roles in the proliferation of alveolar progenitors and the survival of functionally differentiated epithelial cells in the mammary gland. A deregulated expression and activation of Stat5 leads to precocious alveolar development in the absence of pregnancy hormones, impaired mammary gland remodeling following the cessation of lactation, and mammary tumor formation. We reported previously that Stat5 induces the transcription of the Akt1 gene from a novel promoter. In this report, we provide experimental evidence that Akt1 is an essential mediator for the biological function of Stat5 as a survival factor. Additionally, Stat5 controls the expression of the regulatory and catalytic subunits of the phosphatidylinositol 3-kinase (PI3K) (p85α and p110α), thereby greatly augmenting signaling through the prosurvival PI3K/Akt pathway. In agreement with this model, we observed that the constitutive activation of Stat5 cooperates with the loss of function of the tumor suppressor PTEN by accelerating the formation of preneoplastic lesions and mammary tumors. The mammary gland-specific ablation of Stat5 is sufficient to prevent mammary carcinogenesis in a genuine mouse model for Cowden syndrome. Therefore, targeting the Jak2/Stat5 pathway might be a suitable strategy to prevent breast cancer in patients that carry a mutant PTEN allele.
doi:10.1128/MCB.01220-13
PMCID: PMC3993568  PMID: 24469394
7.  Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer 
Molecular cancer therapeutics  2009;8(11):3117-3129.
Cannabinoids have been reported to possess antitumorogenic activity. Not much is known, however, about the effects and mechanism of action of synthetic nonpsychotic cannabinoids on breast cancer growth and metastasis. We have shown that the cannabinoid receptors CB1 and CB2 are overexpressed in primary human breast tumors compared with normal breast tissue. We have also observed that the breast cancer cell lines MDA-MB231, MDA-MB231-luc, and MDA-MB468 express CB1 and CB2 receptors. Furthermore, we have shown that the CB2 synthetic agonist JWH-133 and the CB1 and CB2 agonist WIN-55,212-2 inhibit cell proliferation and migration under in vitro conditions. These results were confirmed in vivo in various mouse model systems. Mice treated with JWH-133 or WIN-55,212-2 showed a 40% to 50% reduction in tumor growth and a 65% to 80% reduction in lung metastasis. These effects were reversed by CB1 and CB2 antagonists AM 251 and SR144528, respectively, suggesting involvement of CB1 and CB2 receptors. In addition, the CB2 agonist JWH-133 was shown to delay and reduce mammary gland tumors in the polyoma middle T oncoprotein (PyMT) transgenic mouse model system. Upon further elucidation, we observed that JWH-133 and WIN-55,212-2 mediate the breast tumor-suppressive effects via a coordinated regulation of cyclooxygenase-2/ prostaglandin E2 signaling pathways and induction of apoptosis. These results indicate that CB1 and CB2 receptors could be used to develop novel therapeutic strategies against breast cancer growth and metastasis.
doi:10.1158/1535-7163.MCT-09-0448
PMCID: PMC4128286  PMID: 19887554
8.  Loss of fibroblast HIF-1α accelerates tumorigenesis 
Cancer research  2012;72(13):3187-3195.
Solid tumors consist of malignant cells and associated stromal components, including fibroblastic cells that contribute to tumor growth and progression. Although tumor fibrosis and aberrant vascularization contribute to the hypoxia often found in advanced tumors, the contribution of hypoxic signaling within tumor-associated fibroblasts to tumorigenesis remains unknown. In this study, we used a fibroblast-specific promoter to create mice in which key hypoxia regulatory genes, including VHL, HIF-1α, HIF-2α and VEGF-A, were knocked out specifically in tumor stromal fibroblasts. We found that loss of HIF-1α and its target gene VEGF-A accelerated tumor growth in murine model of mammary cancer. HIF-1α and VEGF-A loss also led to a reduction in vascular density and myeloid cell infiltration, which correlated with improved tumor perfusion. Together, our findings indicate that the fibroblast HIF-1α response is a critical component of tumor vascularization.
doi:10.1158/0008-5472.CAN-12-0534
PMCID: PMC4089958  PMID: 22556263
fibroblast; hypoxia; hypoxia-inducible factor-1
9.  Proliferation in the Absence of E2F1-3 
Developmental biology  2010;351(1):10.1016/j.ydbio.2010.12.025.
E2F transcription factors regulate the progression of the cell cycle by repression or transactivation of genes that encode cyclins, cyclin dependent kinases, checkpoint regulators, and replication proteins. Although some E2F functions are independent of the Retinoblastoma tumor suppressor (Rb) and related family members, p107 and p130, much of E2F-mediated repression of S phase entry is dependent upon Rb. We previously showed in cultured mouse embryonic fibroblasts that concomitant loss of three E2F activators with overlapping functions (E2F1, E2F2, and E2F3) triggered the p53-p21Cip1 response and caused cell cycle arrest. Here we report on a dramatic difference in the requirement for E2F during development and in cultured cells by showing that cell cycle entry occurs normally in E2f1-3 triply-deficient epithelial stem cells and progenitors of the developing lens. Sixteen days after birth, however, massive apoptosis in differentiating epithelium leads to a collapse of the entire eye. Prior to this collapse, we find that expression of cell cycle-regulated genes in E2F-deficient lenses is aberrantly high. In a second set of experiments, we demonstrate that E2F3 ablation alone does not cause abnormalities in lens development but rescues phenotypic defects caused by loss of Rb, a binding partner of E2F known to recruit histone deacetylases, SWI/SNF and CtBP-polycomb complexes, methyltransferases, and other co-repressors to gene promoters. Together, these data implicate E2F1-3 in mediating transcriptional repression by Rb during cell cycle exit and point to a critical role for their repressive functions in cell survival.
doi:10.1016/j.ydbio.2010.12.025
PMCID: PMC3868453  PMID: 21185283
Proliferation; Cell cycle; E2F; Rb; Lens; Repression; Cell survival; Transcription
10.  QChIPat: a quantitative method to identify distinct binding patterns for two biological ChIP-seq samples in different experimental conditions 
BMC Genomics  2013;14(Suppl 8):S3.
Background
Many computational programs have been developed to identify enriched regions for a single biological ChIP-seq sample. Given that many biological questions are often asked to compare the difference between two different conditions, it is important to develop new programs that address the comparison of two biological ChIP-seq samples. Despite several programs designed to address this question, these programs suffer from some drawbacks, such as inability to distinguish whether the identified differential enriched regions are indeed significantly enriched, lack of distinguishing binding patterns, and neglect of the normalization between samples.
Results
In this study, we developed a novel quantitative method for comparing two biological ChIP-seq samples, called QChIPat. Our method employs a new global normalization method: nonparametric empirical Bayes (NEB) correction normalization, utilizes pre-defined enriched regions identified from single-sample peak calling programs, uses statistical methods to define differential enriched regions, then defines binding (histone modification) pattern information for those differential enriched regions. Our program was tested on a benchmark data: histone modifications data used by ChIPDiffs. It was then applied on two study cases: one to identify differential histone modification sites for ChIP-seq of H3K27me3 and H3K9me2 data in AKT1-transfected MCF10A cells; the other to identify differential binding sites for ChIP-seq of TCF7L2 data in MCF7 and PANC1 cells.
Conclusions
Several advantages of our program include: 1) it considers a control (or input) experiment; 2) it incorporates a novel global normalization strategy: nonparametric empirical Bayes correction normalization; 3) it provides the binding pattern information among different enriched regions. QChIPat is implemented in R, Perl and C++, and has been tested under Linux. The R package is available at http://motif.bmi.ohio-state.edu/QChIPat.
doi:10.1186/1471-2164-14-S8-S3
PMCID: PMC4042236  PMID: 24564479
11.  Ets2 in Tumor Fibroblasts Promotes Angiogenesis in Breast Cancer 
PLoS ONE  2013;8(8):e71533.
Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.
doi:10.1371/journal.pone.0071533
PMCID: PMC3745457  PMID: 23977064
12.  Canonical and Atypical E2Fs Regulate the Mammalian Endocycle 
Nature cell biology  2012;14(11):1192-1202.
SUMMARY
The endocycle is a variant cell cycle consisting of successive DNA synthesis and Gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using novel lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals.
doi:10.1038/ncb2595
PMCID: PMC3616487  PMID: 23064266
13.  Atypical E2F Repressors and Activators Coordinate Placental Development 
Developmental cell  2012;22(4):849-862.
SUMMARY
The evolutionarily ancient arm of the E2f family of transcription factors consisting of the two atypical members E2f7 and E2f8 is essential for murine embryonic development. However, the critical tissues, cellular processes and molecular pathways regulated by these two factors remain unknown. Using a series of fetal and placental lineage-specific cre mice we show that E2F7/E2F8 functions in extra-embryonic trophoblast lineages are both necessary and sufficient to carry fetuses to term. Expression profiling and biochemical approaches exposed the canonical E2F3a activator as a key family member that antagonizes E2F7/E2F8 functions. Remarkably, the concomitant loss of E2f3a normalized placental gene expression programs, corrected placental defects and fostered the survival of E2f7/E2f8 deficient embryos to birth. In summary, we identified a placental transcriptional network tightly coordinated by activation and repression through two distinct arms of the E2F family that is essential for extra-embryonic cell proliferation, placental development and fetal viability.
doi:10.1016/j.devcel.2012.01.013
PMCID: PMC3483796  PMID: 22516201
14.  A Positive Feedback Loop Involving Gcm1 and Fzd5 Directs Chorionic Branching Morphogenesis in the Placenta 
PLoS Biology  2013;11(4):e1001536.
Placenta formation during pregnancy requires chorioallantoic branching morphogenesis that involves establishing an amplifying feedback loop between Frizzled5 and Gcm1 to regulate branching initiation and trophoblast differentiation.
Chorioallantoic branching morphogenesis is a key milestone during placental development, creating the large surface area for nutrient and gas exchange, and is therefore critical for the success of term pregnancy. Several Wnt pathway molecules have been shown to regulate placental development. However, it remains largely unknown how Wnt-Frizzled (Fzd) signaling spatiotemporally interacts with other essential regulators, ensuring chorionic branching morphogenesis and angiogenesis during placental development. Employing global and trophoblast-specific Fzd5-null and Gcm1-deficient mouse models, combining trophoblast stem cell lines and tetraploid aggregation assay, we demonstrate here that an amplifying signaling loop between Gcm1 and Fzd5 is essential for normal initiation of branching in the chorionic plate. While Gcm1 upregulates Fzd5 specifically at sites where branching initiates in the basal chorion, this elevated Fzd5 expression via nuclear β-catenin signaling in turn maintains expression of Gcm1. Moreover, we show that Fzd5-mediated signaling induces the disassociation of cell junctions for branching initiation via downregulating ZO-1, claudin 4, and claudin 7 expressions in trophoblast cells at the base of the chorion. In addition, Fzd5-mediated signaling is also important for upregulation of Vegf expression in chorion trophoblast cells. Finally, we demonstrate that Fzd5-Gcm1 signaling cascade is operative during human trophoblast differentiation. These data indicate that Gcm1 and Fzd5 function in an evolutionary conserved positive feedback loop that regulates trophoblast differentiation and sites of chorionic branching morphogenesis.
Author Summary
Abnormal placental development during pregnancy is associated with conditions such as preeclampsia, intrauterine growth restriction, and even fetal death in humans. Here we focus on the earliest steps of placenta formation, which involves the development of the labyrinthine layer, a specialized epithelium that sits between the maternal blood and fetal blood vessels and facilitates the exchange of nutrients, gases, and wastes between the mother and fetus. Pivotal to the development of a functional labyrinth layer are the processes of folding and branching of a flat sheet of trophoblast cells (originally the outer layer of the blastocyst), and of trophoblast cell differentiation. Here, we show in mice that Frizzled5, a receptor component of the Wnt signaling pathway, and Gcm1, an important transcription factor for labyrinth development, form a positive feedback loop that directs normal placental development. We find that Gcm1 up-regulates Fzd5 specifically at branching sites and that elevated Fzd5 expression in turn maintains expression of Gcm1. Moreover, Fzd5-mediated signaling is required for the disassociation of cell junctions and for the up-regulation of Vegf expression in trophoblast cells. Finally, with implications for human disease, we demonstrate that the FZD5-GCM1 signaling cascade operates in primary cultures of human trophoblasts undergoing differentiation.
doi:10.1371/journal.pbio.1001536
PMCID: PMC3627642  PMID: 23610556
15.  Emerging roles of E2Fs in cancer: an exit from cell cycle control 
Nature reviews. Cancer  2009;9(11):785-797.
Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation.
doi:10.1038/nrc2696
PMCID: PMC3616489  PMID: 19851314
16.  S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways 
Cancer Research  2011;72(3):604-615.
S100A7/Psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive ER-negative (ERα-) breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homolog mS100a7a15, enhanced cell proliferation and upregulated various pro-inflammatory molecules in ERα- breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared to uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in pro-metastatic gene expression and recruitment of tumor-associated macrophages (TAMs). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Further, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work employed a powerful new model system to demonstrate that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways.
doi:10.1158/0008-5472.CAN-11-0669
PMCID: PMC3271140  PMID: 22158945
18.  Evidence for a stepwise program of extrathymic T cell development within the human tonsil 
The Journal of Clinical Investigation  2012;122(4):1403-1415.
The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34+CD38dimLin– cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34+CD38brightLin– cells; (c) CD34+CD1a+CD11c– cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34–CD1a+CD3–CD11c– cells, which resemble CD4+CD8+ double-positive T cells in the thymus; and (e) CD34–CD1a+CD3+CD11c– cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT+ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre–T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.
doi:10.1172/JCI46125
PMCID: PMC3314444  PMID: 22378041
19.  Pten in the Breast Tumor Microenvironment: Modeling Tumor-Stroma Co-Evolution 
Cancer research  2011;71(4):1203-1207.
Solid human tumors and their surrounding microenvironment are hypothesized to co-evolve in a manner that promotes tumor growth, invasiveness and spread. Mouse models of cancer have focused on genetic changes in the epithelial tumor cells and therefore have not robustly tested this hypothesis. We have recently developed a murine breast cancer model that ablates the PTEN tumor suppressor pathway in stromal fibroblasts. Remarkably, the model resembles human breast tumors both at morphologic and molecular levels. We propose that such models reflect subtypes of tumor-stromal co-evolution relevant to human breast cancer, and will therefore be useful in defining the mechanisms that underpin tumor-stroma crosstalk. Additionally, these models should also aid in molecularly classifying human breast tumors based on both the microenvironment subtypes they contain as well as on the tumor subtype.
doi:10.1158/0008-5472.CAN-10-3263
PMCID: PMC3075554  PMID: 21303970
20.  The Retinoblastoma Tumor Suppressor Regulates a Xenobiotic Detoxification Pathway 
PLoS ONE  2011;6(10):e26019.
The retinoblastoma tumor suppressor (pRb) regulates cell cycle entry, progression and exit by controlling the activity of the E2F-family of transcription factors. During cell cycle exit pRb acts as a transcriptional repressor by associating with E2F proteins and thereby inhibiting their ability to stimulate the expression of genes required for S phase. Indeed, many tumors harbor mutations in the RB gene and the pRb-E2F pathway is compromised in nearly all types of cancers. In this report we show that both pRb and its interacting partners, the transcriptional factors E2F1-2-3, act as positive modulators of detoxification pathways important for metabolizing and clearing xenobiotics—such as toxins and drugs—from the body. Using a combination of conventional molecular biology techniques and microarray analysis of specific cell populations, we have analyzed the detoxification pathway in murine samples in the presence or absence of pRb and/or E2F1-2-3. In this report, we show that both pRb and E2F1-2-3 act as positive modulators of detoxification pathways in mice, challenging the conventional view of E2F1-2-3 as transcriptional repressors negatively regulated by pRb. These results suggest that mutations altering the pRb-E2F axis may have consequences beyond loss of cell cycle control by altering the ability of tissues to remove toxins and to properly metabolize anticancer drugs, and might help to understand the formation and progression rates of different types of cancer, as well as to better design appropriate therapies based on the particular genetic composition of the tumors.
doi:10.1371/journal.pone.0026019
PMCID: PMC3192141  PMID: 22022495
21.  Targeted Deletion of Prkar1a Reveals a Role for Protein Kinase A in Mesenchymal-to-Epithelial Transition 
Cancer research  2008;68(8):2671-2677.
Dysregulation of protein kinase A (PKA) activity, caused by loss of function mutations in PRKAR1A, is known to induce tumor formation in the inherited tumor syndrome Carney complex (CNC) and is also associated with sporadic tumors of the thyroid and adrenal. We have previously shown that Prkar1a+/− mice develop schwannomas reminiscent of those seen in CNC and that similar tumors are observed in tissue-specific knockouts (KO) of Prkar1a targeted to the neural crest. Within these tumors, we have previously described the presence of epithelial islands, although the nature of these structures was unclear. In this article, we report that these epithelial structures are derived from KO cells originating in the neural crest. Analysis of the mesenchymal marker vimentin revealed that this protein was markedly down-regulated not only from the epithelial islands, but also from the tumor as a whole, consistent with mesenchymal-to-epithelial transition (MET). In vitro, Prkar1a null primary mouse embryonic fibroblasts, which display constitutive PKA signaling, also showed evidence for MET, with a loss of vimentin and up-regulation of the epithelial marker E-cadherin. Reduction of vimentin protein occurred at the posttranslational level and was rescued by proteasomal inhibition. Finally, this down-regulation of vimentin was recapitulated in the adrenal nodules of CNC patients, confirming an unexpected and previously unrecognized role for PKA in MET.
doi:10.1158/0008-5472.CAN-07-6002
PMCID: PMC3129546  PMID: 18413734
22.  An Ets2-Specific Transcriptional Program in Tumor Associated Macrophages Promotes Tumor Metastasis 
Cancer research  2010;70(4):1323.
Tumor associated macrophages (TAMs) are implicated in breast cancer progression and metastasis, but relatively little is known about the genes pathways in these cells that contribute to malignant phenotypes. The transcription factor Ets2 is a direct target of signaling pathways involved in regulating macrophage functions during inflammation. To test whether Ets2 in TAMs modulated mouse mammary tumor growth and metastasis a genetic approach was used to conditionally delete Ets2 in TAMs. Ets2 deletion in TAMs decreased the frequency and size of mammary tumor metastases to lung in three different metastatic models. Expression profiling and chromatin immunoprecipitation assays with isolated TAMs established that Ets2 repressed several well characterized inhibitors of angiogenesis. Consistent with these results, Ets2 ablation in TAMs led to decreased tumor angiogenesis and growth. An Ets2-TAM expression signature was identified within human breast cancer expression data and this signature could retrospectively predict overall survival of breast cancer patients in two independent sets of human breast cancer microarray data. In summary, we have identified Ets2 as a critical factor that acts to enhance mammary tumor growth and metastasis by regulating a transcriptional program in TAMs.
doi:10.1158/0008-5472.CAN-09-1474
PMCID: PMC2822898  PMID: 20145133
Tumor Macrophages; Breast Cancer; Ets2; Metastasis; Angiogenesis
23.  e-Science, caGrid, and Translational Biomedical Research 
Computer  2008;41(11):58-66.
Translational research projects target a wide variety of diseases, test many different kinds of biomedical hypotheses, and employ a large assortment of experimental methodologies. Diverse data, complex execution environments, and demanding security and reliability requirements make the implementation of these projects extremely challenging and require novel e-Science technologies.
doi:10.1109/MC.2008.459
PMCID: PMC3035203  PMID: 21311723
24.  Inactivation of E2F3 results in centrosome amplification 
Cancer cell  2003;3(4):333-346.
Summary
The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.
PMCID: PMC3033013  PMID: 12726860
25.  E2F3 Is a Mediator of DNA Damage-Induced Apoptosis▿  
Molecular and Cellular Biology  2009;30(2):524-536.
The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.
doi:10.1128/MCB.00938-09
PMCID: PMC2798461  PMID: 19917728

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