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1.
Oncotarget ; 9(86): 35639-35654, 2018 Nov 02.
Article in English | MEDLINE | ID: mdl-30479694

ABSTRACT

The CYP19A1 gene encodes aromatase, an enzyme that converts androgens into estrogens and consequently directly contributes to both the depletion of androgens and the synthesis of estrogens in several organs. Aromatase is critical for diverse biological processes such as proliferation, regulation of fat metabolism and hormone signaling. Additionally, it is also overexpressed in diverse cancers and drives hormone-dependent tumor progression and increases 17-ß-estradiol (E2) within tumors and the tumor microenvironment. Although the inhibition of E2 production via aromatase inhibitors represents a major therapeutic paradigm in clinical oncology, fundamental questions regarding how cancer cells gain the capacity to overexpress aromatase remain unanswered. Multiple tissue-specific CYP19A1 promoters are known to be aberrantly active in tumors, yet how this occurs is unclear. Here, for the first time, we report that Dishevelled (DVL) proteins, which are key mediators of Wnt signaling, regulate aromatase expression in multiple breast cancer cell lines. We also report that DVL enters the nucleus and localizes to at least two different CYP19A1 promoters (pII and I.4) previously reported to drive overexpression in breast tumors and to a very distal CYP19A1 placental promoter (I.1) that remains poorly characterized. We go on to demonstrate that DVL-1 and DVL-3 loss of function leads to differential changes in various aromatase transcripts and in E2 production. The report, herein, uncovers a new regulator of CYP19A1 transcription and for the first time demonstrates that DVL, a critical mediator of WNT signaling, contributes to aberrant breast cancer-associated estrogen production.

2.
Int J Mol Sci ; 16(1): 950-65, 2015 Jan 05.
Article in English | MEDLINE | ID: mdl-25569080

ABSTRACT

SIRT1, an NAD(+)-dependent deacetylase, has been described in the literature as a major player in the regulation of cellular stress responses. Its expression has been shown to be altered in cancer cells, and it targets both histone and non-histone proteins for deacetylation and thereby alters metabolic programs in response to diverse physiological stress. Interestingly, many of the metabolic pathways that are influenced by SIRT1 are also altered in tumor development. Not only does SIRT1 have the potential to regulate oncogenic factors, it also orchestrates many aspects of metabolism and lipid regulation and recent reports are beginning to connect these areas. SIRT1 influences pathways that provide an alternative means of deriving energy (such as fatty acid oxidation and gluconeogenesis) when a cell encounters nutritive stress, and can therefore lead to altered lipid metabolism in various pathophysiological contexts. This review helps to show the various connections between SIRT1 and major pathways in cellular metabolism and the consequence of SIRT1 deregulation on carcinogenesis and lipid metabolism.


Subject(s)
Lipid Metabolism/physiology , Neoplasms/pathology , Sirtuin 1/metabolism , AMP-Activated Protein Kinase Kinases , Energy Metabolism , Fatty Acids/biosynthesis , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Humans , Neoplasms/metabolism , PPAR gamma/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Protein Serine-Threonine Kinases/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Transcription Factors/metabolism
3.
CBE Life Sci Educ ; 7(4): 410-21, 2008.
Article in English | MEDLINE | ID: mdl-19047427

ABSTRACT

Research based laboratory courses have been shown to stimulate student interest in science and to improve scientific skills. We describe here a project developed for a semester-long research-based laboratory course that accompanies a genetics lecture course. The project was designed to allow students to become familiar with the use of bioinformatics tools and molecular biology and genetic approaches while carrying out original research. Students were required to present their hypotheses, experiments, and results in a comprehensive lab report. The lab project concerned the yeast casein kinase 1 (CK1) protein kinase Yck2. CK1 protein kinases are present in all organisms and are well conserved in primary structure. These enzymes display sequence features that differ from other protein kinase subfamilies. Students identified such sequences within the CK1 subfamily, chose a sequence to analyze, used available structural data to determine possible functions for their sequences, and designed mutations within the sequences. After generating the mutant alleles, these were expressed in yeast and tested for function by using two growth assays. The student response to the project was positive, both in terms of knowledge and skills increases and interest in research, and several students are continuing the analysis of mutant alleles as summer projects.


Subject(s)
Cell Physiological Phenomena , Computational Biology/education , Curriculum , Laboratories , Molecular Biology/education , Research/education , Amino Acid Sequence , Casein Kinase I/chemistry , Conserved Sequence , Learning , Mutagenesis, Site-Directed , Mutation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces/enzymology , Task Performance and Analysis , Time Factors
4.
Biochim Biophys Acta ; 1640(1): 61-8, 2003 Apr 07.
Article in English | MEDLINE | ID: mdl-12676355

ABSTRACT

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.


Subject(s)
Adaptor Proteins, Vesicular Transport , Blood Proteins/metabolism , Carrier Proteins/metabolism , Phosphoproteins/metabolism , cdc42 GTP-Binding Protein/metabolism , 3T3 Cells , Animals , Blood Proteins/chemistry , Blood Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Signal Transduction
5.
J Biol Chem ; 277(35): 31808-17, 2002 Aug 30.
Article in English | MEDLINE | ID: mdl-12082106

ABSTRACT

Activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, supporting the importance of Raf-independent pathways in mediating Ras transformation. The p38 and JNK mitogen-activated protein kinase cascades are activated by Ras via Raf-independent effector function. Therefore, we determined whether p38 and JNK activation are involved in Ras transformation of RIE-1 epithelial cells. Rather surprisingly, we found that pharmacologic inhibition of p38, together with Raf activation of ERK, was sufficient to mimic the morphologic and growth transformation caused by oncogenic Ras. p38 inhibition together with ERK activation also caused the same alterations in cyclin D1 and p21(CIP1) expression caused by Ras and induced an autocrine growth factor loop important for transformation. Finally, in contrast to p38, we found that JNK activation promoted Ras transformation, and that Ras deregulation of p38 and JNK was not mediated by activation of the Rac small GTPase. We conclude that a key action of Raf-independent effector pathways important for Ras transformation may involve inhibition of p38 and activation of JNK.


Subject(s)
Cell Division/physiology , Cell Transformation, Neoplastic , Genes, ras , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Animals , Cell Cycle , Cell Line , Cell Transformation, Neoplastic/drug effects , Cloning, Molecular , Culture Media, Conditioned , Cyclin D1/genetics , Cyclins/metabolism , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Imidazoles/pharmacology , Intestinal Mucosa , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mutagenesis, Site-Directed , Phosphorylation , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/pharmacology , Rats , Recombinant Proteins/metabolism , Transforming Growth Factor alpha/pharmacology , p38 Mitogen-Activated Protein Kinases
6.
Nat Struct Biol ; 9(6): 468-75, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12006984

ABSTRACT

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.


Subject(s)
Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Proto-Oncogene Proteins/chemistry , rho GTP-Binding Proteins/chemistry , rho GTP-Binding Proteins/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Enzyme Activation , Guanosine Triphosphate/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/chemistry , Proteins/metabolism , Sequence Alignment , Structure-Activity Relationship , Substrate Specificity , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , cdc42 GTP-Binding Protein/chemistry , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/chemistry , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/chemistry , rhoA GTP-Binding Protein/metabolism
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