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1.
Pathophysiology ; 30(3): 389-399, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37755396

RESUMO

In this article, we discuss a class of MYC-interacting lncRNAs (long non-coding RNAs) that share the following criteria: They are direct transcriptional targets of MYC. Their expression is coordinated with the expression of MYC. They are required for sustained MYC-driven cell proliferation, and they are not essential for cell survival. We refer to these lncRNAs as "MYC facilitators" and discuss two representative members of this class of lncRNAs, SNHG17 (small nuclear RNA host gene) and LNROP (long non-coding regulator of POU2F2). We also present a general hypothesis on the role of lncRNAs in MYC-mediated transcriptional regulation.

2.
Proc Natl Acad Sci U S A ; 120(34): e2304071120, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37585458

RESUMO

Class IA phosphoinositide 3-kinase alpha (PI3Kα) is an important drug target because it is one of the most frequently mutated proteins in human cancers. However, small molecule inhibitors currently on the market or under development have safety concerns due to a lack of selectivity. Therefore, other chemical scaffolds or unique mechanisms of catalytic kinase inhibition are needed. Here, we report the cryo-electron microscopy structures of wild-type PI3Kα, the dimer of p110α and p85α, in complex with three Y-shaped ligands [cpd16 (compound 16), cpd17 (compound 17), and cpd18 (compound 18)] of different affinities and no inhibitory effect on the kinase activity. Unlike ATP-competitive inhibitors, cpd17 adopts a Y-shaped conformation with one arm inserted into a binding pocket formed by R770 and W780 and the other arm lodged in the ATP-binding pocket at an angle that is different from that of the ATP phosphate tail. Such a special interaction induces a conformation of PI3Kα resembling that of the unliganded protein. These observations were confirmed with two isomers (cpd16 and cpd18). Further analysis of these Y-shaped ligands revealed the structural basis of differential binding affinities caused by stereo- or regiochemical modifications. Our results may offer a different direction toward the design of therapeutic agents against PI3Kα.


Assuntos
Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Ligantes , Microscopia Crioeletrônica , Trifosfato de Adenosina/metabolismo
3.
Biochim Biophys Acta Rev Cancer ; 1878(5): 188947, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37394020

RESUMO

Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities.


Assuntos
Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Microscopia Crioeletrônica , Mutação , Domínio Catalítico/genética
4.
Cell Death Dis ; 14(2): 168, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36849510

RESUMO

MYC controls most of the non-coding genome. Several long noncoding transcripts were originally identified in the human B cell line P496-3 and then shown to be required for MYC-driven proliferation of Burkitt lymphoma-derived RAMOS cells. In this study, we used RAMOS cells exclusively as a representative of the human B cell lineage. One of the MYC-controlled lncRNAs required for RAMOS cell proliferation is ENSG00000254887 which we will term LNROP (long non-coding regulator of POU2F2). In the genome, LNROP is located in close proximity of POU2F2, the gene encoding OCT2. OCT2 is a transcription factor with important roles in sustaining the proliferation of human B cells. Here we show that LNROP is a nuclear RNA and a direct target of MYC. Downregulation of LNROP attenuates the expression of OCT2. This effect of LNROP on the expression of OCT2 is unidirectional as downregulation of OCT2 does not alter the expression of LNROP. Our data suggest that LNROP is a cis-acting regulator of OCT2. To illustrate the downstream reach of LNROP, we chose a prominent target of OCT2, the tyrosine phosphatase SHP-1. Downregulation of OCT2 elevates the expression of SHP-1. Our data suggest the following path of interactions: LNROP enables the proliferation of B cells by positively and unidirectionally regulating the growth-stimulatory transcription factor OCT2. In actively proliferating B cells, OCT2 attenuates the expression and anti-proliferative activity of SHP-1.


Assuntos
Linfoma de Burkitt , RNA Longo não Codificante , Humanos , Linfócitos B , Proliferação de Células/genética , RNA Longo não Codificante/genética , Fatores de Transcrição
5.
Proc Natl Acad Sci U S A ; 119(46): e2215621119, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36343266

RESUMO

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.


Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Microscopia Crioeletrônica , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Domínio Catalítico/genética , Neoplasias/genética
6.
Proc Natl Acad Sci U S A ; 119(38): e2210769119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095215

RESUMO

Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.


Assuntos
Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase , Classe I de Fosfatidilinositol 3-Quinases/química , Classe Ia de Fosfatidilinositol 3-Quinase/química , Humanos , Espectrometria de Massas/métodos , Anticorpos de Domínio Único
7.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34725156

RESUMO

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/química , Tiazóis/química , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/ultraestrutura , Microscopia Crioeletrônica , Conformação Proteica , Células Sf9 , Spodoptera
9.
Bioorg Med Chem ; 42: 116246, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34130216

RESUMO

We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.


Assuntos
Desenvolvimento de Medicamentos , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade
10.
Nat Chem ; 13(6): 540-548, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33833446

RESUMO

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.


Assuntos
Sistemas de Liberação de Medicamentos , Região Variável de Imunoglobulina/farmacologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , DNA/química , Descoberta de Drogas , Fluorescência , Biblioteca Gênica , Humanos , Região Variável de Imunoglobulina/química , Camundongos , Microscopia de Fluorescência , Neoplasias , Neoplasias Experimentais
11.
Adv Sci (Weinh) ; 7(22): 2001970, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33240760

RESUMO

A versatile and Lipinski-compliant DNA-encoded library (DEL), comprising 366 600 glutamic acid derivatives coupled to oligonucleotides serving as amplifiable identification barcodes is designed, constructed, and characterized. The GB-DEL library, constructed in single-stranded DNA format, allows de novo identification of specific binders against several pharmaceutically relevant proteins. Moreover, hybridization of the single-stranded DEL with a set of known protein ligands of low to medium affinity coupled to a complementary DNA strand results in self-assembled selectable chemical structures, leading to the identification of affinity-matured compounds.

12.
Proc Natl Acad Sci U S A ; 117(12): 6571-6579, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156728

RESUMO

MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras/metabolismo , Aptâmeros de Nucleotídeos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteína 9 Associada à CRISPR/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Regiões Promotoras Genéticas , Domínios Proteicos , RNA Guia de Cinetoplastídeos , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transcrição Gênica
13.
Annu Rev Virol ; 6(1): 31-47, 2019 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-31567064

RESUMO

I always loved biology and to do experiments. This passion and a great deal of good fortune and serendipity landed me in the field of retrovirology at the time when it opened to experimental analysis. I became involved in viral replication, genetics, and viral oncogenes. In more recent years, I have applied what I learned in tumor virology to human cancer. The early years of my personal life were marked by displacements and migration: deportation into East Germany, escape to the West, and emigration to the United States. As a young man I faced heartbreaking personal tragedies but attained a peaceful and steady course in the second half of my life. I am fortunate to have found my home in Southern California and to continue in cancer research.


Assuntos
Oncogenes , Retroviridae , Vírus/genética , Alemanha , História do Século XX , História do Século XXI , Humanos , Vírus Oncogênicos/genética , Estados Unidos
14.
Cancers (Basel) ; 11(11)2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31652979

RESUMO

Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.

15.
Viruses ; 11(1)2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669277

RESUMO

Jan Svoboda triggered investigations on non-defective avian sarcoma viruses. These viruses were a critical factor in the genetic understanding of retroviruses. They provided the single and unique access to the field and facilitated the discovery of the first oncogene src and of the cellular origin of retroviral oncogenes. They continue to be of importance as singularly effective expression vectors that have provided insights into the molecular functions of numerous oncogenes. Combined with the contributions to the validation of the provirus hypothesis, Jan Svoboda's investigations of non-defective avian sarcoma viruses have shaped a large and important part of retrovirology.


Assuntos
Vírus do Sarcoma Aviário/genética , Genes Virais , Oncogenes , Animais , Humanos , Provírus/genética
16.
Expert Opin Ther Targets ; 22(10): 869-877, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30205700

RESUMO

INTRODUCTION: The main regulatory subunits of Class IA phosphatidylinositol 3-kinase (PI3K), p85α and p85ß, initiate diverse cellular activities independent of binding to the catalytic subunit p110. Several of these signaling processes directly or indirectly contribute to a regulation of PI3K and could become targets for therapeutic efforts. Areas covered: This review will highlight two general areas of p85 activity: (1) direct interaction with regulatory proteins and with determinants of the cytoskeleton, and (2) a genetic analysis by deletion and domain switches identifying new functions for p85 domains. Expert Opinion: Isoform-specific activities of regulatory subunits have long been at the periphery of the PI3K field. Our understanding of these unique functions of the regulatory subunits is fragmentary and raises many important questions. At this time, there is insufficient information to translate this knowledge into the clinic, but some tempting targets have emerged that could move the field forward with the help of novel technologies in drug design and identification.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Desenho de Fármacos , Terapia de Alvo Molecular , Animais , Humanos , Isoenzimas , Fosfatidilinositol 3-Quinases/metabolismo , Subunidades Proteicas , Transdução de Sinais/fisiologia
17.
Bioorg Med Chem ; 26(14): 4234-4239, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30037753

RESUMO

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology.


Assuntos
Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Solubilidade , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
18.
Oncotarget ; 8(34): 55863-55876, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28915558

RESUMO

Our understanding of isoform-specific activities of phosphatidylinositol 3-kinase (PI3K) is still rudimentary, and yet, deep knowledge of these non-redundant functions in the PI3K family is essential for effective and safe control of PI3K in disease. The two major isoforms of the regulatory subunits of PI3K are p85α and p85ß, encoded by the genes PIK3R1 and PIK3R2, respectively. These isoforms show distinct functional differences that affect and control cellular PI3K activity and signaling [1-4]. In this study, we have further explored the differences between p85α and p85ß by genetic truncations and substitutions. We have discovered unexpected activities of the mutant proteins that reflect regulatory functions of distinct p85 domains. These results can be summarized as follows: Deletion of the SH3 domain increases oncogenic and PI3K signaling activity. Deletion of the combined SH3-RhoGAP domains abolishes these activities. In p85ß, deletion of the cSH2 domain reduces oncogenic and signaling activities. In p85α, such a deletion has an activating effect. The deletions of the combined cSH2 and iSH2 domains and also the deletion of the cSH2, iSH2 and nSH2 domains yield results that go in the same direction, generally activating in p85α and reducing activity in p85ß. The contrasting functions of the cSH2 domains are verified by domain exchanges with the cSH2 domain of p85ß exerting an activating effect and the cSH2 domain of p85α an inactivating effect, even in the heterologous isoform. In the cell systems studied, protein stability was not correlated with oncogenic and signaling activity. These observations significantly expand our knowledge of the isoform-specific activities of p85α and p85ß and of the functional significance of specific domains for regulating the catalytic subunits of class IA PI3K.

19.
Proc Natl Acad Sci U S A ; 114(30): 8071-8076, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28696308

RESUMO

Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter "tsRNAs") is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called "ts-101" and "ts-53," respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.


Assuntos
Neoplasias/metabolismo , Pequeno RNA não Traduzido/metabolismo , Células A549 , Estudos de Casos e Controles , Células HEK293 , Humanos , Oncogenes
20.
Mol Cell Oncol ; 3(1): e1029063, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27308543

RESUMO

A point mutation in PIK3CA, the gene encoding the α isoform of class I phosphatidylinositol 3-kinase, induces extensive remodeling of the transcriptome and proteome, resulting in a gene signature that specifically resembles that of the basal subtype, but not other types, of breast cancer.

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