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1.  Identification of a cyclin D1 network in prostate cancer that antagonizes epithelial-mesenchymal restraint 
Cancer research  2013;74(2):508-519.
Improved clinical management of prostate cancer (PCa) has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human PCa. The TGFβ/BMP-SMAD4 signaling pathway, which induces epithelial mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene-transformed prostate cancer cell lines. The in vivo cyclin D1-mediated molecular signature predicted poor outcome of recurrence free survival for prostate cancer patients (K-means hazard ratio 3.75, P-value=0.02) and demonstrated that endogenous cyclin D1 restrains TGFβ, Snail, Twist and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In ChIP-Seq, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical free recurrence in human prostate cancer.
doi:10.1158/0008-5472.CAN-13-1313
PMCID: PMC3914674  PMID: 24282282
Cyclin D1; gene network; prostate cancer; siRNA; shRNA
2.  Growth of v-src-Transformed Cells in Serum-Free Medium Through the Induction of Growth Factors 
Journal of cellular physiology  2013;228(7):1482-1486.
The v-src oncogene is one of only two oncogenes capable of transforming mouse embryo fibroblasts (MEFs) lacking the IGF-IR gene (R-cells). R-/v-src cells grow robustly in the absence of serum, suggesting the hypothesis that they may produce one or more growth factors that would sustain their ability to proliferate in serum-free condition. Using proteomic approaches on serum-free conditioned media derived from v-src-transformed cells, we have identified two growth promoting factors: ostepontin and proliferin. Subsequent experiments have indicated that osteopontin plays a prevalent role in promoting growth of v-src-transformed cells in serum-deprived condition.
doi:10.1002/jcp.24303
PMCID: PMC4062974  PMID: 23254450
3.  DACHSHUND BINDS P53 TO BLOCK THE GROWTH OF LUNG ADENOCARCINOMA CELLS 
Cancer research  2013;73(11):3262-3274.
Hyperactive EGFR and mutant p53 are common genetic abnormalities driving the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer deaths in the world. The Drosophila gene Dachshund (Dac) was originally cloned as an inhibitor of hyperactive EGFR alleles. Given the importance of EGFR signaling in lung cancer etiology, we examined the role of DACH1 expression in lung cancer development. DACH1 protein and mRNA expression was reduced in human NSCLC. Re-expression of DACH1 reduced NSCLC colony formation and tumor growth in vivo via p53. Endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar location, and shared occupancy of -15% of p53 bound genes in ChIP Seq. The C-terminus of DACH1 was necessary for direct p53 binding, contributing to the inhibition of colony formation and cell cycle arrest. Expression of the stem cell factor SOX2 was repressed by DACH1, and SOX2 expression was inversely correlated with DACH1 in NSCLC. We conclude that DACH1 binds p53 to inhibit NSCLC cellular growth.
doi:10.1158/0008-5472.CAN-12-3191
PMCID: PMC3674204  PMID: 23492369
4.  Acetylation of the Cell-Fate Factor Dachshund Determines p53 Binding and Signaling Modules in Breast Cancer 
Oncotarget  2013;4(6):923-935.
Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.
PMCID: PMC3757249  PMID: 23798621
p53; breast cancer; cell fate; stem cells; dach
5.  The canonical NF-κB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion 
Cancer research  2010;70(24):10464-10473.
The role of mammary tumor epithelial cell (MEC) NF-κB in tumor progression in vivo is unknown as murine NF-κB components and kinases are either required for murine survival or interfere with normal mammary gland development. As NF-κB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-κB, the importance of MEC NF-κB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-κB (IκBαSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-κB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Co-culture experiments demonstrated MEC NF-κB enhanced TAM recruitment. Genome wide expression and proteomic analysis demonstrated IκBαSR inhibited tumor stem cell pathways. IκBαSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. Mammary epithelial cell NF-κB contributes to mammary tumorigenesis. As we show NF-κB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-κB dependent mammary tumorigenesis.
doi:10.1158/0008-5472.CAN-10-0732
PMCID: PMC3010731  PMID: 21159656
ErbB2; NF-κB; inducible transgenics; Ponasterone A
6.  An Integrated Framework to Model Cellular Phenotype as a Component of Biochemical Networks 
Advances in Bioinformatics  2011;2011:608295.
Identification of regulatory molecules in signaling pathways is critical for understanding cellular behavior. Given the complexity of the transcriptional gene network, the relationship between molecular expression and phenotype is difficult to determine using reductionist experimental methods. Computational models provide the means to characterize regulatory mechanisms and predict phenotype in the context of gene networks. Integrating gene expression data with phenotypic data in transcriptional network models enables systematic identification of critical molecules in a biological network. We developed an approach based on fuzzy logic to model cell budding in Saccharomyces cerevisiae using time series expression microarray data of the cell cycle. Cell budding is a phenotype of viable cells undergoing division. Predicted interactions between gene expression and phenotype reflected known biological relationships. Dynamic simulation analysis reproduced the behavior of the yeast cell cycle and accurately identified genes and interactions which are essential for cell viability.
doi:10.1155/2011/608295
PMCID: PMC3235418  PMID: 22190923
7.  Cyclin D1/cyclin dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells 
Cancer research  2010;70(5):2105-2114.
Cyclin D1 belongs to the family of proteins that regulates progression through the G1-S phase of the cell cycle through binding to cyclin dependent kinase 4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon and prostate over-express the cyclin D1 gene. However, the correlation between cyclin D1 over-expression with E2F target gene regulation or cyclin dependent kinase-dependent cyclin D1 activity with tumor development have not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A, a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. Filamin A has also been associated with a variety of cancers including lung, prostate, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. Using mass spectrometry, we found that cyclin D1 and Filamin A co-immunoprecipitate and that lower levels of cyclin D1 are associated with decreased phosphorylation of FLNa at serine 2152 and 1459. We also identify many proteins related to cytoskeletal function, biomolecular synthesis, organelle biogenesis, and calcium regulation whose levels of expression change concomitant with decreased cell motility induced by decreased cyclin D1 and cyclin D1-cdk4/6 activity.
doi:10.1158/0008-5472.CAN-08-1108
PMCID: PMC2917898  PMID: 20179208
8.  Hormonal Control of Androgen Receptor Function through SIRT1▿  
Molecular and Cellular Biology  2006;26(21):8122-8135.
The NAD-dependent histone deacetylase Sir2 plays a key role in connecting cellular metabolism with gene silencing and aging. The androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens, including dihydrotestosterone (DHT). Here, SIRT1 antagonists induce endogenous AR expression and enhance DHT-mediated AR expression. SIRT1 binds and deacetylates the AR at a conserved lysine motif. Human SIRT1 (hSIRT1) repression of DHT-induced AR signaling requires the NAD-dependent catalytic function of hSIRT1 and the AR lysine residues deacetylated by SIRT1. SIRT1 inhibited coactivator-induced interactions between the AR amino and carboxyl termini. DHT-induced prostate cancer cellular contact-independent growth is also blocked by SIRT1, providing a direct functional link between the AR, which is a critical determinant of progression of human prostate cancer, and the sirtuins.
doi:10.1128/MCB.00289-06
PMCID: PMC1636736  PMID: 16923962
9.  Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate 
The Journal of Cell Biology  2006;173(4):533-544.
The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions.
doi:10.1083/jcb.200512059
PMCID: PMC2063863  PMID: 16717128
10.  Cyclin D1 Determines Mitochondrial Function In Vivo†  
Molecular and Cellular Biology  2006;26(14):5449-5469.
The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.
doi:10.1128/MCB.02074-05
PMCID: PMC1592725  PMID: 16809779
11.  Cyclin D1 Regulates Cellular Migration through the Inhibition of Thrombospondin 1 and ROCK Signaling 
Molecular and Cellular Biology  2006;26(11):4240-4256.
Cyclin D1 is overexpressed in human tumors, correlating with cellular metastasis, and is induced by activating Rho GTPases. Herein, cyclin D1-deficient mouse embryo fibroblasts (MEFs) exhibited increased adhesion and decreased motility compared with wild-type MEFs. Retroviral transduction of cyclin D1 reversed these phenotypes. Mutational analysis of cyclin D1 demonstrated that its effects on cellular adhesion and migration were independent of the pRb and p160 coactivator binding domains. Genomewide expression arrays identified a subset of genes regulated by cyclin D1, including Rho-activated kinase II (ROCKII) and thrombospondin 1 (TSP-1). cyclin D1−/− cells showed increased Rho GTP and ROCKII activity and signaling, with increased phosphorylation of LIM kinase, cofilin (Ser3), and myosin light chain 2 (Thr18/Ser19). Cyclin D1 repressed ROCKII and TSP-1 expression, and the migratory defect of cyclin D1−/− cells was reversed by ROCK inhibition or TSP-1 immunoneutralizing antibodies. cyclin E knockin to the cyclin D1−/− MEFs rescued the DNA synthesis defect of cyclin D1−/− MEFs but did not rescue either the migration defect or the abundance of ROCKII. Cyclin D1 promotes cellular motility through inhibiting ROCK signaling and repressing the metastasis suppressor TSP-1.
doi:10.1128/MCB.02124-05
PMCID: PMC1489104  PMID: 16705174
12.  Linear fuzzy gene network models obtained from microarray data by exhaustive search 
BMC Bioinformatics  2004;5:108.
Background
Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome. Gene network models are needed to interpret the resulting large and complex data sets. Rationally designed perturbations (e.g., gene knock-outs) can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis. We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling.
Results
We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle. Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance. Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes.
Conclusion
Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation. Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system. Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems.
doi:10.1186/1471-2105-5-108
PMCID: PMC514698  PMID: 15304201

Results 1-12 (12)