Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 27
Filter
Add more filters










Publication year range
1.
J Med Chem ; 66(13): 8782-8807, 2023 07 13.
Article in English | MEDLINE | ID: mdl-37343272

ABSTRACT

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.


Subject(s)
Antineoplastic Agents , Neoplasms , Rats , Animals , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met , Drug Design , Adenosine Triphosphate , Antineoplastic Agents/pharmacology
2.
Nat Commun ; 11(1): 3520, 2020 07 14.
Article in English | MEDLINE | ID: mdl-32665551

ABSTRACT

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.


Subject(s)
DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Blotting, Western , Cell Survival/genetics , Cell Survival/physiology , Chromatin Immunoprecipitation , Computational Biology , DNA-Binding Proteins/genetics , Female , Flow Cytometry , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Humans , Lymphoma/genetics , Lymphoma/metabolism , Mice , Mice, SCID , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Random Allocation , Transcription Factors/genetics , Transcriptome/genetics
3.
Mol Brain ; 13(1): 66, 2020 05 04.
Article in English | MEDLINE | ID: mdl-32366277

ABSTRACT

Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.


Subject(s)
Alzheimer Disease/metabolism , Apolipoproteins E/metabolism , Astrocytes/drug effects , Astrocytoma/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , ATP Binding Cassette Transporter 1/metabolism , Animals , Apolipoproteins E/genetics , Astrocytoma/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Gene Knockdown Techniques , High-Throughput Screening Assays , Humans , Mice , Microglia/drug effects , Microglia/metabolism , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Axl Receptor Tyrosine Kinase
4.
ACS Med Chem Lett ; 10(9): 1322-1327, 2019 Sep 12.
Article in English | MEDLINE | ID: mdl-31531204

ABSTRACT

Many small molecule inhibitors of the cMET receptor tyrosine kinase have been evaluated in clinical trials for the treatment of cancer and resistance-conferring mutations of cMET are beginning to be reported for a number of such compounds. There is now a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition. Toward this end, we report here the first crystal structures of the recent clinically observed resistance-conferring D1228V cMET mutant in complex with small molecule inhibitors, along with a crystal structure of wild-type cMET bound by the clinical compound savolitinib and supporting cellular, biochemical, and biophysical data. Our findings indicate that the D1228V alteration induces conformational changes in the kinase, which could have implications for small molecule inhibitor design. The data we report here increases our molecular understanding of the D1228V cMET mutation and provides insight for future inhibitor design.

5.
Cancer Cell ; 36(2): 194-209.e9, 2019 08 12.
Article in English | MEDLINE | ID: mdl-31408619

ABSTRACT

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Ethylenediamines/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Pyrroles/pharmacology , RNA Splicing/drug effects , RNA, Neoplasm/metabolism , Animals , Antineoplastic Agents/pharmacokinetics , Catalysis , Enzyme Inhibitors/pharmacokinetics , Ethylenediamines/pharmacokinetics , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , K562 Cells , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Pyrroles/pharmacokinetics , RNA, Neoplasm/genetics , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , THP-1 Cells , Tumor Cells, Cultured , U937 Cells , Xenograft Model Antitumor Assays
6.
Drug Deliv Transl Res ; 8(6): 1635-1643, 2018 12.
Article in English | MEDLINE | ID: mdl-28849387

ABSTRACT

Capping is a common problem in the manufacture of some types of tablets and unless resolved, the tableting process cannot proceed. Hence, all factors that can help to lessen the likelihood of capping without unnecessarily reduce turret speed and/or compaction force would be tenable. This study investigated the influence of tablet punch configuration on mitigation of tablet capping. Tablets were prepared from high-dose paracetamol-potato starch granules in a rotary tablet press with flat face plain (FFP), flat face bevel edge (FFBE) and flat face radius edge (FFRE) punch configurations. The directly compressible (DC) fillers tested were microcrystalline cellulose (MCC), pre-gelatinised starch (PGS) and lactose. Design of experiments (DoE), a tool of quality by design (QbD) paradigm, was used and the interaction of input variables (compression force, tablet punch configuration and DC filler) affecting the response factors (tablet hardness and capping rating) were evaluated. FFP punches were able to mitigate capping best. FFRE punches showed more potential than FFBE punches at alleviating capping in a particular compression force range, without the limitations of the FFP punches that produce cylindrical tablets that were more friable. Incorporation of PGS in the tablet formulation was observed to be more efficient at mitigating capping than the other DC fillers when FFBE and FFRE punches were used. Overall, this study serves as a model for prospective product development based on the QbD framework and the optimal use of compaction tools.


Subject(s)
Acetaminophen/chemistry , Drug Compounding/methods , Excipients/chemistry , Solanum tuberosum/chemistry , Starch/chemistry , Tablets , Tensile Strength
7.
Sci Rep ; 6: 19943, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26887977

ABSTRACT

Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor--Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.


Subject(s)
DNA-Binding Proteins/metabolism , Epithelial-Mesenchymal Transition , Histones/metabolism , MicroRNAs/metabolism , Neoplasm Proteins/metabolism , Ovarian Neoplasms/metabolism , Protein Processing, Post-Translational , RNA, Neoplasm/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Zinc Finger E-box-Binding Homeobox 1/metabolism , Cell Line, Tumor , DNA-Binding Proteins/genetics , Female , Histones/genetics , Humans , MicroRNAs/genetics , Neoplasm Proteins/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , RNA, Neoplasm/genetics , Response Elements , Transcription Factors/genetics , Zinc Finger E-box-Binding Homeobox 1/genetics
8.
Bioessays ; 38(3): 266-75, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26778668

ABSTRACT

MYC is a transcription factor, which not only directly modulates multiple aspects of transcription and co-transcriptional processing (e.g. RNA-Polymerase II initiation, elongation, and mRNA capping), but also indirectly influences several steps of RNA metabolism, including both constitutive and alternative splicing, mRNA stability, and translation efficiency. As MYC is an oncoprotein whose expression is deregulated in multiple human cancers, identifying its critical downstream activities in tumors is of key importance for designing effective therapeutic strategies. With this knowledge and recent technological advances, we now have multiple angles to reach the goal of targeting MYC in tumors, ranging from the direct reduction of MYC levels, to the dampening of selected house-keeping functions in MYC-overexpressing cells, to more targeted approaches based on MYC-induced secondary effects.


Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Proto-Oncogene Proteins c-myc/physiology , Animals , Gene Expression/drug effects , Humans , Molecular Targeted Therapy , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism
9.
J Clin Invest ; 126(1): 68-84, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26595814

ABSTRACT

MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.


Subject(s)
Exons , Melanoma/therapy , Nuclear Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins/genetics , Animals , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , Humans , Melanoma/pathology , Mice , RNA-Binding Proteins/physiology , Serine-Arginine Splicing Factors , Tumor Suppressor Protein p53/physiology
10.
Nature ; 523(7558): 96-100, 2015 Jul 02.
Article in English | MEDLINE | ID: mdl-25970242

ABSTRACT

Deregulated expression of the MYC transcription factor occurs in most human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Overexpressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here we show that during lymphomagenesis in Eµ-myc transgenic mice, MYC directly upregulates the transcription of the core small nuclear ribonucleoprotein particle assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins. This coordinated regulatory effect is critical for the core biogenesis of small nuclear ribonucleoprotein particles, effective pre-messenger-RNA splicing, cell survival and proliferation. Our results demonstrate that MYC maintains the splicing fidelity of exons with a weak 5' donor site. Additionally, we identify pre-messenger-RNAs that are particularly sensitive to the perturbation of the MYC-PRMT5 axis, resulting in either intron retention (for example, Dvl1) or exon skipping (for example, Atr, Ep400). Using antisense oligonucleotides, we demonstrate the contribution of these splicing defects to the anti-proliferative/apoptotic phenotype observed in PRMT5-depleted Eµ-myc B cells. We conclude that, in addition to its well-documented oncogenic functions in transcription and translation, MYC also safeguards proper pre-messenger-RNA splicing as an essential step in lymphomagenesis.


Subject(s)
Gene Expression Regulation, Neoplastic , Lymphoma/physiopathology , Proto-Oncogene Proteins c-myc/metabolism , RNA Precursors/metabolism , RNA Splicing/physiology , Animals , Exons/genetics , HEK293 Cells , Humans , Introns/genetics , Mice , Oligonucleotides, Antisense/metabolism , Protein Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases , Proto-Oncogene Proteins c-myc/genetics
11.
J Clin Invest ; 125(5): 2109-22, 2015 May.
Article in English | MEDLINE | ID: mdl-25893605

ABSTRACT

Constitutively active MYC and reactivated telomerase often coexist in cancers. While reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with cofactors, confers several growth advantages to cancer cells. It is known that the reactivation of TERT, the catalytic subunit of telomerase, is limiting for reconstituting telomerase activity in tumors. However, while reactivation of TERT has been functionally linked to the acquisition of several "hallmarks of cancer" in tumors, the molecular mechanisms by which this occurs and whether these mechanisms are distinct from the role of telomerase on telomeres is not clear. Here, we demonstrated that first-generation TERT-null mice, unlike Terc-null mice, show delayed onset of MYC-induced lymphomagenesis. We further determined that TERT is a regulator of MYC stability in cancer. TERT stabilized MYC levels on chromatin, contributing to either activation or repression of its target genes. TERT regulated MYC ubiquitination and proteasomal degradation, and this effect of TERT was independent of its reverse transcriptase activity and role in telomere elongation. Based on these data, we conclude that reactivation of TERT, a direct transcriptional MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis.


Subject(s)
Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/genetics , Neoplasm Proteins/physiology , Proto-Oncogene Proteins c-myc/physiology , Telomerase/physiology , Animals , Cell Line, Tumor , Enzyme Activation , Feedback, Physiological , Genes, myc , Glycogen Synthase Kinase 3/physiology , Glycogen Synthase Kinase 3 beta , Heterografts , Humans , Lymphoma, Non-Hodgkin/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Phosphorylation , Promoter Regions, Genetic , Protein Processing, Post-Translational , Protein Stability , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA/genetics , RNA/physiology , RNA Interference , Telomerase/deficiency , Telomerase/genetics , Telomere Homeostasis/genetics , Time Factors , Transcription, Genetic , Ubiquitination
12.
Methods Enzymol ; 533: 15-21, 2013.
Article in English | MEDLINE | ID: mdl-24182914

ABSTRACT

Yeast and bacteria can be cryopreserved and stored almost indefinitely. It is useful to prepare stocks for archival purposes.


Subject(s)
Bacteria , Cryopreservation/methods , Microbiological Techniques/methods , Yeasts
13.
Methods Enzymol ; 533: 235-40, 2013.
Article in English | MEDLINE | ID: mdl-24182928

ABSTRACT

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Cells are immobilized on a solid support that is optically suitable for microscopy, and then fixed. When coupled with antibody-based immunofluorescent or immunocytochemical methods, the expression of specific proteins can be quantified and localized. Specialized stains and dyes can also be used to visualize nucleic acids or other cellular structures. See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using 'Cell Blocks' and for adherent cells on Preparation of Cells for Microscopy using Chamber Slides and Coverslips.


Subject(s)
Cytological Techniques/methods , Microscopy/methods , Cytological Techniques/instrumentation , Fluorescent Antibody Technique/methods , Immunohistochemistry/instrumentation , Immunohistochemistry/methods
14.
Methods Enzymol ; 533: 241-7, 2013.
Article in English | MEDLINE | ID: mdl-24182929

ABSTRACT

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Cells are immobilized on a solid support that is optically suitable for microscopy, and then fixed. When coupled with antibody-based immunofluorescent or immunocytochemical methods, the expression of specific proteins can be quantified and localized. Specialized stains and dyes can also be used to visualize nucleic acids or other cellular structures. See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and Preparation of Cells for Microscopy using 'Cell Blocks'.


Subject(s)
Cytological Techniques/instrumentation , Cytological Techniques/methods , Microscopy/methods , Cells, Cultured , Cells, Immobilized , Fluorescent Antibody Technique/methods , Formaldehyde , Methanol
15.
Methods Enzymol ; 533: 249-55, 2013.
Article in English | MEDLINE | ID: mdl-24182930

ABSTRACT

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Embedding cells in agarose and then in paraffin as 'cell blocks' allows for them to be processed in the same manner that tissue specimens are processed for histology. This method is advantageous because numerous sections can be cut from one cell block. Additionally, sectioning renders antigens within the nucleus and other cell organelles more accessible to antibodies. However, access to specialized equipment for histological tissue processing is required See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and for adherent cells on Preparation of Cells for Microscopy using Chamber Slides and Coverslips.


Subject(s)
Cytological Techniques/methods , Microscopy/methods , Animals , Cells, Immobilized , Cytological Techniques/instrumentation , Paraffin Embedding/instrumentation , Paraffin Embedding/methods , Sepharose
16.
Methods Enzymol ; 529: 161-9, 2013.
Article in English | MEDLINE | ID: mdl-24011044

ABSTRACT

The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses.


Subject(s)
DNA/isolation & purification , Mammals/genetics , Animals , Genome
17.
PLoS One ; 6(10): e26057, 2011.
Article in English | MEDLINE | ID: mdl-22039435

ABSTRACT

The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eµ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.


Subject(s)
Biomass , Genes, myc , Animals , Chromatin Immunoprecipitation , Humans , Lymphoma, B-Cell/genetics , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis
18.
Oncotarget ; 2(9): 669-83, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21941025

ABSTRACT

EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in multiple cancer types and has been implicated in prostate cancer initiation and progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate cancer and show that MYC coordinately regulates EZH2 through transcriptional and post-transcriptional means. Although prior studies in prostate cancer have revealed a number of possible mechanisms of EZH2 upregulation, these changes cannot account for the overexpression EZH2 in many primary prostate cancers, nor in most cases of high grade PIN. We report that upregulation of Myc in the mouse prostate results in overexpression of EZH2 mRNA and protein which coincides with reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate cell types, albeit not in others. Further, in human prostate cancer cells, Myc negatively regulates miR-26a and miR-26b via direct binding to their parental Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in prostate cancer cells results in decreased EZH2 levels and suppressed proliferation. In human clinical samples, miR-26a and miR-26b are downregulated in most primary prostate cancers. As a separate mechanism of EZH2 mRNA upregulation, we find that Myc binds directly to and activates the transcription of the EZH2 promoter. These results link two major pathways in prostate cancer by providing two additional and complementary Myc-regulated mechanisms by which EZH2 upregulation occurs and is enforced during prostatic carcinogenesis. Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate prostate tumor initiation and disease progression.


Subject(s)
DNA-Binding Proteins/biosynthesis , Genes, myc , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Transcription Factors/biosynthesis , Animals , Cell Growth Processes/genetics , Cell Line, Tumor , DNA-Binding Proteins/genetics , Disease Models, Animal , Enhancer of Zeste Homolog 2 Protein , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Immunoprecipitation , Male , Mice , Mice, Transgenic , MicroRNAs/genetics , Polycomb Repressive Complex 2 , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transcription Factors/genetics , Transcriptional Activation , Transfection , Up-Regulation
19.
Am J Pathol ; 178(4): 1824-34, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21435462

ABSTRACT

Increased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia.


Subject(s)
Cell Nucleolus/metabolism , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Adenocarcinoma/metabolism , Animals , Cell Line, Tumor , Epithelial Cells/metabolism , Genes, myc , Humans , Male , Mice , Microscopy, Electron/methods , Oligonucleotide Array Sequence Analysis , Oncogenes , Prostate
20.
PLoS One ; 5(2): e9427, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20195545

ABSTRACT

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina-features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These "Super-Lo-MYC" mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a "paradoxical" increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.


Subject(s)
Epithelial Cells/chemistry , Homeodomain Proteins/metabolism , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/metabolism , Animals , Blotting, Northern , Cell Transformation, Neoplastic , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Homeodomain Proteins/genetics , Immunohistochemistry , Male , Mice , Mice, Transgenic , Prostate/chemistry , Prostate/metabolism , Prostate/pathology , Prostatic Intraepithelial Neoplasia/genetics , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-myc/genetics , Transcription Factors/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...