ABSTRACT
Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The Km values for the intrinsic as well as protein-stimulated ATPase were increased whereas the kcat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.
Subject(s)
Mitochondria/enzymology , Protease La/chemistry , Protease La/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , B-Lymphocytes/enzymology , Biocatalysis , Craniofacial Abnormalities/enzymology , Craniofacial Abnormalities/genetics , Enzyme Stability/genetics , Eye Abnormalities/enzymology , Eye Abnormalities/genetics , Growth Disorders/enzymology , Growth Disorders/genetics , Hip Dislocation, Congenital/enzymology , Hip Dislocation, Congenital/genetics , Humans , Kinetics , Mice , Models, Molecular , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Osteochondrodysplasias/enzymology , Osteochondrodysplasias/genetics , Protease La/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity , Tooth Abnormalities/enzymology , Tooth Abnormalities/geneticsABSTRACT
BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.
Subject(s)
Gene Expression Regulation, Neoplastic , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , RNA-Binding Proteins/genetics , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Transcription Factors/genetics , Biomarkers, Tumor , Breast Neoplasms/etiology , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic/drug effects , Histones/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Kaplan-Meier Estimate , Models, Molecular , Neoplasm Staging , Nuclear Proteins/chemistry , Promoter Regions, Genetic , Protein Binding , RNA-Binding Proteins/chemistry , Recurrence , Repressor Proteins/chemistry , Structure-Activity Relationship , Transcription Factors/chemistryABSTRACT
A new series of 1,3,4-oxadiazole/chalcone hybrids was designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as inhibitors of EGFR, Src, and IL-6. The synthesized compounds showed promising anticancer activity, particularly against leukemia, with 8v being the most potent. The synthesized compounds exhibited strong to moderate cytotoxic activities against K-562, KG-1a, and Jurkat leukemia cell lines in MTT assays. Compound 8v showed the strongest cytotoxic activity with IC50 of 1.95⯵M, 2.36⯵M and 3.45⯵M against K-562, Jurkat and KG-1a leukemia cell lines, respectively. Moreover; the synthesized compounds inhibited EGFR, Src, and IL-6. Compound 8v was most effective at inhibiting EGFR (IC50â¯=â¯0.24⯵M), Src (IC50â¯=â¯0.96⯵M), and IL-6 (% of controlâ¯=â¯20%). Additionally, most of the compounds decreased STAT3 activation.
Subject(s)
Antineoplastic Agents/pharmacology , Chalcone/pharmacology , Interleukin-6/antagonists & inhibitors , Oxadiazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Chalcone/chemistry , Dose-Response Relationship, Drug , Drug Design , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Humans , Interleukin-6/metabolism , Molecular Structure , Oxadiazoles/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , STAT3 Transcription Factor/metabolism , Structure-Activity RelationshipABSTRACT
A series of quinoline-chalcone hybrids was designed as potential anti-cancer agents, synthesized and evaluated. Different cytotoxic assays revealed that compounds experienced promising activity. Compounds 9i and 9j were the most potent against all the cell lines tested with IC50â¯=â¯1.91-5.29⯵M against A549 and K-562 cells. Mechanistically, 9i and 9j induced G2/M cell cycle arrest and apoptosis in both A549 and K562 cells. Moreover, all PI3K isoforms were inhibited non selectively with IC50s of 52-473â¯nM when tested against the two mentioned compounds with 9i being most potent against PI3K-γ (IC50â¯=â¯52â¯nM). Docking of 9i and 9j showed a possible formation of H-bonding with essential valine residues in the active site of PI3K-γ isoform. Meanwhile, Western blotting analysis revealed that 9i and 9j inhibited the phosphorylation of PI3K, Akt, mTOR, as well as GSK-3ß in both A549 and K562 cells, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings support the antitumor potential of quinoline-chalcone derivatives for NSCLC and CML by inhibiting the PI3K/Akt/mTOR pathway.
Subject(s)
Antineoplastic Agents/pharmacology , Chalcones/pharmacology , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Quinolines/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Catalytic Domain , Cell Line, Tumor , Cell Proliferation/drug effects , Chalcones/chemical synthesis , Chalcones/chemistry , Drug Design , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , G2 Phase Cell Cycle Checkpoints/drug effects , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Molecular Docking Simulation , Molecular Structure , Phosphatidylinositol 3-Kinase/chemistry , Proto-Oncogene Proteins c-akt/metabolism , Quinolines/chemical synthesis , Quinolines/chemistry , Signal Transduction/drug effects , Structure-Activity Relationship , TOR Serine-Threonine Kinases/metabolismABSTRACT
BACKGROUND: Prostate ductal branching morphogenesis involves a complex spatiotemporal regulation of cellular proliferation and remodeling of the extracellular matrix (ECM) around the developing ducts. Decorin (Dcn) is a small leucine-rich proteoglycan known to sequester several growth factors and to act as a tumor suppressor in prostate cancer. RESULTS: Dcn expression in the developing prostate paralleled branching morphogenesis and was dynamically regulated by androgen and Hedgehog (Hh) signaling. DCN colocalized with collagen in the periductal stroma and acellular interstitium. Exogenous DCN decreased epithelial proliferation in ex vivo organ cultures of developing prostate, whereas genetic ablation of Dcn resulted in increased epithelial proliferation in the developing prostate. CONCLUSIONS: Dcn expression and localization in the developing prostate is consistent with a primary role in organizing collagen around the developing ducts. Regulation of Dcn expression appears to be complex, involving both androgen and Hh signaling. The growth inhibitory effect of Dcn suggests a unique linkage between a structural proteoglycan and epithelial growth regulation. This may serve to coordinate two elements of the morphogenetic process: ductal growth and organization of the collagen matrix around the nascent duct. Developmental Dynamics 247:679-685, 2018. © 2018 Wiley Periodicals, Inc.
Subject(s)
Decorin/metabolism , Prostate/metabolism , Signal Transduction/physiology , Animals , Decorin/genetics , Female , Male , Mice , Morphogenesis/physiology , Organ Culture Techniques , Organogenesis/physiology , Prostate/embryology , Prostate/growth & developmentABSTRACT
The mouse prostate is a male sex-accessory gland comprised of a branched ductal network arranged into three separate bilateral lobes: the anterior, dorsolateral, and ventral lobes. Prostate ductal development is the primary morphogenetic event in prostate development and requires a complex regulation of spatiotemporal factors. This review provides an overview of prostate development and the major genetic regulators and signaling pathways involved. To identify new areas for further study, we briefly highlight the likely important, but relatively understudied, role of the extracellular matrix (ECM). Finally, we point out the potential importance of the ECM in influencing the behavior and prognosis of prostate cancer. Developmental Dynamics 246:89-99, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Morphogenesis/genetics , Prostate/growth & development , Animals , Extracellular Matrix/physiology , Humans , Male , Mice , Organogenesis , Prostatic Neoplasms/pathology , Signal TransductionABSTRACT
INTRODUCTION: The reoperation is considered as the access to the abdominal cavity before complete healing of the surgical wound from a previous operation within the first 60 days after the first procedure. It occurs in 0.5 to 15% of patients undergoing abdominal surgery and generates significant increase in morbidity and mortality in patients undergoing abdominal surgery. OBJECTIVES: Identify the number of unplanned abdominal surgical reoperations and identify the causes of these unplanned reoperations were performed in our department. METHODOLOGY: This is a retrospective study conducted at the University Hospital of Puebla in the period between April 2009 to February 2012, a total of 1,709 abdominal surgeries performed by the Service of General Surgery were included. RESULTS: Ninety-seven cases of reoperation of which 50 cases were not planned surgery cases were identified; 72% (36 cases) from emergency operations, and 28% of elective surgery. CONCLUSIONS: The incidence found in our study is low compared to similar studies. Prospective studies and focus on risk factors and causes of unplanned reoperations are required, in order to know them in detail and, consequently, reduce its incidence and morbidity and mortality they add.
Subject(s)
Abdomen/surgery , Reoperation/statistics & numerical data , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Hospitals, University , Humans , Incidence , Infant , Male , Mexico , Middle Aged , Retrospective Studies , Risk Factors , Young AdultABSTRACT
Clear cell renal cell carcinomas (ccRCCs) are characterized by biallelic loss of the von Hippel-Lindau tumor suppressor and subsequent constitutive activation of the hypoxia-inducible factors, whose transcriptional programs dictate major phenotypic attributes of kidney tumors. We recently described a role for the macrophage migration inhibitory factor (MIF) in ccRCC as an autocrine-signaling molecule with elevated expression in tumor tissues and in the circulation of patients that has potent tumor cell survival effects. MIF is a pleiotropic cytokine implicated in a variety of diseases and cancers and is the target of both small molecule and antibody-based therapies currently in clinical trials. Recent work by others has described D-dopachrome tautomerase (DDT) as a functional homologue of MIF with a similar genomic structure and expression patterns. Thus, we sought to determine a role for DDT in renal cancer. We find that DDT expression mirrors MIF expression in ccRCC tumor sections with high correlation and that, mechanistically, DDT is a novel hypoxia-inducible gene and direct target of HIF1α and HIF2α. Functionally, DDT and MIF demonstrate a significant overlap in controlling cell survival, tumor formation, and tumor and endothelial cell migration. However, DDT inhibition consistently displayed more severe effects on most phenotypes. Accordingly, although dual inhibition of DDT and MIF demonstrated additive effects in vitro, DDT plays a dominant role in tumor growth in vivo. Together, our findings identify DDT as a functionally redundant but more potent cytokine to MIF in cancer and suggest that current attempts to inhibit MIF signaling may fail because of DDT compensation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Carcinoma, Renal Cell/metabolism , Cell Transformation, Neoplastic/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Intramolecular Oxidoreductases/metabolism , Kidney Neoplasms/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Neoplasm Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Survival/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation, Neoplastic/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Intramolecular Oxidoreductases/genetics , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Macrophage Migration-Inhibitory Factors/genetics , Neoplasm Proteins/genetics , Signal Transduction/geneticsABSTRACT
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.
Subject(s)
Hydro-Lyases/metabolism , Plant Proteins/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , Chloroplasts/metabolism , Cross-Linking Reagents/chemistry , DNA Transposable Elements , Hydro-Lyases/genetics , Immunoprecipitation , Mutation , Plant Leaves/chemistry , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Protein Structure, Tertiary , Ribulose-Bisphosphate Carboxylase/genetics , Zea mays/geneticsABSTRACT
We show that HEXIM1 (hexamethylene bis-acetamide inducible 1) functions as an AR (androgen receptor) co-repressor as it physically interacts with the AR and is required for the ability of anti-androgens to inhibit androgen-induced target gene expression and cell proliferation. Oncomine™ database and IHC (immunohistochemistry) analyses of human prostate tissues revealed that expression of HEXIM1 mRNA and protein are down-regulated during the development and progression of prostate cancer. Enforced down-regulation of HEXIM1 in parental hormone-dependent LNCaP cells results in resistance to the inhibitory action of anti-androgens. Conversely, ectopic expression of HEXIM1 in the CRPC (castration-resistant prostate cancer) cell line, C4-2, enhances their sensitivity to the repressive effects of the anti-androgen bicalutamide. Novel insight into the mechanistic basis for HEXIM1 inhibition of AR activity is provided by the present studies showing that HEXIM1 induces expression of the histone demethylase KDM5B (lysine-specific demethylase 5B) and inhibits histone methylation, resulting in the inhibition of FOXA1 (forkhead box A1) licensing activity. This is a new mechanism of action attributed to HEXIM1, and distinct from what has been reported so far to be involved in HEXIM1 regulation of other nuclear hormone receptors, including the oestrogen receptor.
Subject(s)
Androgen Antagonists/pharmacology , Prostatic Neoplasms/metabolism , RNA-Binding Proteins/metabolism , Receptors, Androgen/metabolism , Anilides/pharmacology , Cell Line, Tumor , Enhancer Elements, Genetic , Epithelial Cells/metabolism , Gene Expression/drug effects , Histones/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/metabolism , Male , Metribolone/pharmacology , Nitriles/pharmacology , Nuclear Proteins/metabolism , Prostate/metabolism , Prostatic Neoplasms, Castration-Resistant/metabolism , Protein Transport , Repressor Proteins/metabolism , Tosyl Compounds/pharmacology , Transcription Factors , Transcriptional Elongation Factors/metabolismABSTRACT
The potency of a series of Hexamethylene bis-acetamide (HMBA) derivatives inducing Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) was determined in LNCaP prostate cancer cells. Several compounds with unsymmetrical structures showed significantly improved activity. Distinct from HMBA, these analogs have increased hydrophobicity and can improve the short half-life of HMBA, which is one of the factors that have limited the application of HMBA in clinics. The unsymmetrical scaffolds of the new analogs provide the basis for further lead optimization of the compounds using combinatorial chemistry strategy.
Subject(s)
Acetamides/chemistry , RNA-Binding Proteins/metabolism , Acetamides/metabolism , Acetamides/pharmacology , Cell Line, Tumor , Drug Design , Gene Expression Regulation/drug effects , Half-Life , Humans , Transcription FactorsABSTRACT
We have previously reported on the inhibition of HIF-1α (hypoxia-inducible factor α)-regulated pathways by HEXIM1 [HMBA (hexamethylene-bis-acetamide)-inducible protein 1]. Disruption of HEXIM1 activity in a knock-in mouse model expressing a mutant HEXIM1 protein resulted in increased susceptibility to the development of mammary tumours, partly by up-regulation of VEGF (vascular endothelial growth factor) expression, HIF-1α expression and aberrant vascularization. We now report on the mechanistic basis for HEXIM1 regulation of HIF-1α. We observed direct interaction between HIF-1α and HEXIM1, and HEXIM1 up-regulated hydroxylation of HIF-1α, resulting in the induction of the interaction of HIF-1α with pVHL (von Hippel-Lindau protein) and ubiquitination of HIF-1α. The up-regulation of hydroxylation involves HEXIM1-mediated induction of PHD3 (prolyl hydroxylase 3) expression and interaction of PHD3 with HIF-1α. Acetylation of HIF-1α has been proposed to result in increased interaction of HIF-1α with pVHL and induced pVHL-mediated ubiquitination, which leads to the proteasomal degradation of HIF-1α. HEXIM1 also attenuated the interaction of HIF-1α with HDAC1 (histone deacetylase 1), resulting in acetylation of HIF-1α. The consequence of HEXIM1 down-regulation of HIF-1α protein expression is attenuated expression of HIF-1α target genes in addition to VEGF and inhibition of HIF-1α-regulated cell invasion.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , RNA-Binding Proteins/physiology , Acetylation , Breast Neoplasms , Cell Movement , Down-Regulation , Gene Expression Regulation, Neoplastic , Histone Deacetylase 1/metabolism , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , MCF-7 Cells , Protein Processing, Post-Translational , Protein Stability , Transcription FactorsABSTRACT
FOXA1, estrogen receptor alpha (ERalpha) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERalpha and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERalpha expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERalpha and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERalpha expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERalpha-positive breast cancers, at least in part, through its control of ERalpha expression.
Subject(s)
Breast Neoplasms/genetics , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Hepatocyte Nuclear Factor 3-alpha/genetics , Morphogenesis/genetics , Breast/metabolism , Breast/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Epithelium/metabolism , Epithelium/pathology , Female , Hepatocyte Nuclear Factor 3-alpha/metabolism , HumansABSTRACT
We have previously shown that estrogen receptor ß (ERß)-mediated up-regulation of quinone reductase (QR) is involved in the protection against estrogen-induced mammary tumorigenesis. Our present study provides evidence that the ERß agonist, 2,3-bis-(4-hydroxy-phenyl)-propionitrile (DPN), and the selective estrogen receptor modulator tamoxifen (Tam), inhibit estrogen-induced DNA damage and mammary tumorigenesis in the aromatase transgenic (Arom) mouse model. We also show that either DPN or Tam treatment increases QR levels and results in a decrease in ductal hyperplasia, proliferation, oxidative DNA damage (ODD), and an increase in apoptosis. To corroborate the role of QR, we provide additional evidence in triple transgenic MMTV/QR/Arom mice, wherein the QR expression is induced in the mammary glands via doxycycline, causing a decrease in ductal hyperplasia and ODD. Overall, we provide evidence that up-regulation of QR through induction by Tam or DPN can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a novel mechanism of prevention against breast cancer. Thus, our data have important clinical implications in the management of breast cancer; our findings bring forth potentially new therapeutic strategies involving ERß agonists.
Subject(s)
Estrogen Antagonists/pharmacology , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Animal/metabolism , Mammary Neoplasms, Animal/prevention & control , NAD(P)H Dehydrogenase (Quinone)/metabolism , Nitriles/pharmacology , Tamoxifen/pharmacology , Animals , Aromatase/genetics , Aromatase/metabolism , Blotting, Western , Estrogen Receptor beta/agonists , Estrogen Receptor beta/antagonists & inhibitors , Estrogen Receptor beta/metabolism , Immunohistochemistry , In Situ Nick-End Labeling , Mammary Neoplasms, Animal/genetics , Mice , Mice, Transgenic , NAD(P)H Dehydrogenase (Quinone)/geneticsABSTRACT
Our studies indicate that expression of antioxidative stress enzymes is upregulated by Selective Estrogen Receptor Modulators (SERMs) in breast epithelial cell lines, providing protection against the genotoxic effects of estrogens and against estrogen-induced mammary tumorigenesis. This upregulation of antioxidative stress enzymes requires Estrogen Receptor beta (ERß) and human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). Further studies indicate that hPMC2 has a functional exonuclease domain that is required for upregulation of antioxidative stress enzymes by SERMs and repair of estrogen-induced abasic sites.
ABSTRACT
DNA methylation, catalyzed by DNA methyltransferase (DNMT), is a well-characterized epigenetic modification in cancer cells. In particular, promoter hypermethylation of AR and ESR1 results in loss of expression on Androgen Receptor (AR) and Estrogen Receptor (ER), respectively, and is associated with a hormone refractory state. We now report that Glycogen Synthase Kinase 3 (GSK3) phosphorylates DNMT1 at S714, which is localized to a 62 amino acid region referred to as auto-inhibitory linker, which functions to occlude the DNA from the active site of DNMT1 to prevent the methylation of unmethylated DNA. Molecular Dynamics simulation indicates that phosphorylation at S714 resulted in conformational rearrangement of the autoinhibitory domain that inactivated its ability to block the methylation of unmethylated DNA and resulted in enhanced DNA binding. Treatment with a novel and more selective inhibitor of GSK3 resulted in decreased methylation of the promoter region of genes encoding the Androgen Receptor (AR) and Estrogen Receptor alpha (ERa) and re-expression of the AR and ERa in AR negative prostate cancer and ER negative breast cancer cells, respectively. As a result, concurrent treatment with the GSK3 inhibitor resulted in responsiveness of AR negative prostate cancer and ER negative breast cancer cells to inhibitors of the AR or ER, respectively, in in vitro and in vivo experimental models.
ABSTRACT
We have previously reported that hexamethylene bis-acetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound estrogen receptor α (ERα) and the androgen receptor (AR) by disrupting the interaction between these receptors and positive transcriptional elongation factor b (P-TEFb) and attenuating RNA polymerase II (RNAPII) phosphorylation at serine 2. Functional consequences of the inhibition of transcriptional activity of ERα and AR by HEXIM1 include the inhibition of ERα- and AR-dependent gene expression, respectively, and the resulting attenuation of breast cancer (BCa) and prostate cancer (PCa) cell proliferation and growth. In our present study, we determined that HEXIM1 inhibited AKR1C3 expression in BCa and PCa cells. AKR1C3, also known as 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 5, is a key enzyme involved in the synthesis of 17ß-estradiol (E2) and 5-dihydrotestosterone (DHT). Downregulation of AKR1C3 by HEXIM1 influenced E2 and DHT production, estrogen- and androgen-dependent gene expression, and cell proliferation. Our studies indicate that HEXIM1 has the unique ability to inhibit both the transcriptional activity of the ER and AR and the synthesis of the endogenous ligands of these receptors.
Subject(s)
Dihydrotestosterone/metabolism , Down-Regulation , Estradiol/metabolism , Estrogen Receptor alpha/metabolism , Gene Expression Regulation , RNA-Binding Proteins/biosynthesis , Receptors, Androgen/metabolism , Transcription Factors/biosynthesis , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Estrogens/metabolism , Female , Humans , Ligands , MCF-7 Cells , Male , Prostatic Neoplasms/metabolism , RNA, Small Interfering/metabolismABSTRACT
Our previous studies and those of others indicated that the transcription factor Hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) is a tumor suppressor and cyclin-dependent kinase inhibitor, and that these HEXIM1 functions are mainly dependent on its C-terminal region. We provide evidence here that the HEXIM1 C-terminal region is critical for cardiovascular development. HEXIM1 protein was detected in the heart during critical time periods in cardiac growth and chamber maturation. We created mice carrying an insertional mutation in the HEXIM1 gene that disrupted its C-terminal region and found that this resulted in prenatal lethality. Heart defects in HEXIM1(1 to 312) mice included abnormal coronary patterning and thin ventricular walls. The thin myocardium can be partly attributed to increased apoptosis. Platelet endothelial cell adhesion molecular precursor-1 staining of HEXIM1(1 to 312) heart sections revealed decreased vascularization of the myocardium despite the presence of coronary vasculature in the epicardium. The expression of vascular endothelial growth factor (VEGF), known to affect angioblast invasion and myocardial proliferation and survival, was decreased in HEXIM1(1 to 312) mice compared with control littermates. We also observed decreased fibroblast growth factor 9 (FGF9) expression, suggesting that effects of HEXIM1 in the myocardium are partly mediated through epicardial FGF9 signaling. Together our results suggest that HEXIM1 plays critical roles in coronary vessel development and myocardial growth. The basis for this role of HEXIM1 is that VEGF is a direct transcriptional target of HEXIM1, and involves attenuation a repressive effects of C/EBPalpha on VEGF gene transcription.
Subject(s)
Coronary Vessels/embryology , Heart Defects, Congenital/physiopathology , Neovascularization, Physiologic/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Apoptosis/physiology , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Coronary Circulation/physiology , Coronary Vessels/physiology , Down-Regulation/physiology , Endocardium/embryology , Endocardium/physiology , Female , Gene Expression Regulation, Developmental , Genes, Lethal , Heart Defects, Congenital/genetics , Heart Defects, Congenital/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mutagenesis/physiology , Myocardium/cytology , Pericardium/embryology , Pericardium/physiology , Phenotype , Phosphorylation , RNA Polymerase II/metabolism , RNA-Binding Proteins , Signal Transduction/physiologyABSTRACT
We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in breast and prostate cancer. The anti-cancer actions of HEXIM1 in melanomas and AML have been reported by other groups. Previous studies have shown that 5-Aza-2'deoxycytidine (5-AzadC), a DNMT1 inhibitor, induces re-expression of tumor suppressor genes by removing/erasing methylation marks from their promoters. Our studies highlighted another mechanism wherein 5-AzadC induced DNA damage, which then resulted in enhanced occupancy of NF-ĸB, P-TEFb, and serine 2 phosphorylated RNA Polymerase II on the HEXIM1 gene. As a consequence, 5-AzadC induced HEXIM1 expression in prostate cancer cell lines and triple negative breast cancers. 5-AzadC-induced DNA damage enhanced P-TEFb occupancy via a mechanism that involved activation of ATR and ATM and induction of NF-ĸB recruitment to the HEXIM1 promoter. Downregulation of NF-ĸB attenuated 5-AzadC-induced HEXIM1 expression in prostate and breast cancer cells. The functional relevance of 5-AzadC-induced HEXIM1 expression is revealed by studies showing the HEXIM1 is required for the induction of apoptosis. Collectively, our findings support a non-epigenetic mechanism for 5-AzadC-induced re-expression of HEXIM1 protein, and may contribute to the clinical efficacy of 5-AzadC.
Subject(s)
DNA (Cytosine-5-)-Methyltransferase 1/antagonists & inhibitors , Decitabine/pharmacology , Enzyme Inhibitors/pharmacology , Prostatic Neoplasms/metabolism , RNA-Binding Proteins/genetics , Transcription Factors/genetics , Triple Negative Breast Neoplasms/metabolism , Antimetabolites, Antineoplastic/pharmacology , Apoptosis , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , NF-kappa B/metabolism , Prostatic Neoplasms/genetics , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Triple Negative Breast Neoplasms/geneticsABSTRACT
Collagen remodeling occurs in many prostate pathologies; however, the underlying structural architecture in both normal and diseased prostatic tissues is largely unexplored. Here, we use second-harmonic generation (SHG) microscopy to specifically probe the role of the proteoglycan decorin (Dcn) on collagen assembly in a wild type (wt) and Dcn null mouse (Dcn - / - ). Dcn is required for proper organization of collagen fibrils as it regulates size by forming an arch-like structure at the end of the fibril. We have utilized SHG metrics based on emission directionality (forward-backward ratio) and relative conversion efficiency, which are both related to the SHG coherence length, and found more disordered fibril organization in the Dcn - / - . We have also used image analysis readouts based on entropy, multifractal dimension, and wavelet transforms to compare the collagen fibril/fiber architecture in the two models, where all these showed that the Dcn - / - prostate comprised smaller and more disorganized collagen structures. All these SHG metrics are consistent with decreased SHG phase matching in the Dcn - / - and are further consistent with ultrastructural analysis of collagen in this model in other tissues, which show a more random distribution of fibril sizes and their packing into fibers. As Dcn is a known tumor suppressor, this work forms the basis for future studies of collagen remodeling in both malignant and benign prostate disease.