RESUMEN
Cushing's disease (CD) is caused by pituitary corticotroph adenomas that secrete excess adrenocorticotropic hormone (ACTH). In these tumors, somatic mutations in the gene USP8 have been identified as recurrent and pathogenic and are the sole known molecular driver for CD. Although other somatic mutations were reported in these studies, their contribution to the pathogenesis of CD remains unexplored. No molecular drivers have been established for a large proportion of CD cases and tumor heterogeneity has not yet been investigated using genomics methods. Also, even in USP8-mutant tumors, a possibility may exist of additional contributing mutations, following a paradigm from other neoplasm types where multiple somatic alterations contribute to neoplastic transformation. The current study utilizes whole-exome discovery sequencing on the Illumina platform, followed by targeted amplicon-validation sequencing on the Pacific Biosciences platform, to interrogate the somatic mutation landscape in a corticotroph adenoma resected from a CD patient. In this USP8-mutated tumor, we identified an interesting somatic mutation in the gene RASD1, which is a component of the corticotropin-releasing hormone receptor signaling system. This finding may provide insight into a novel mechanism involving loss of feedback control to the corticotropin-releasing hormone receptor and subsequent deregulation of ACTH production in corticotroph tumors.
Asunto(s)
Adenoma Hipofisario Secretor de ACTH/genética , Proteínas ras/genética , Adenoma/genética , Hormona Adrenocorticotrópica/genética , Adulto , Corticotrofos/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Femenino , Humanos , Mutación , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/genética , Neoplasias Hipofisarias/genética , Receptores de Hormona Liberadora de Corticotropina/genética , Análisis de Secuencia de ADN , Ubiquitina Tiolesterasa/genéticaRESUMEN
BACKGROUND: Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics. METHODS: We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data. RESULTS: We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases. CONCLUSIONS: These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.
Asunto(s)
Variación Genética , Genómica/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Medicina de Precisión/métodos , Adolescente , Adulto , Anciano , Niño , Variaciones en el Número de Copia de ADN , Exoma , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/patología , Polimorfismo de Nucleótido Simple , Pronóstico , Adulto JovenRESUMEN
Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NFκB, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions.
Asunto(s)
Epidermis/metabolismo , Queratinocitos/citología , Psoriasis/inmunología , Psoriasis/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Humanos , Sistema Inmunológico , Inflamación , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Trasplante de Neoplasias , Fenotipo , Piel/patologíaRESUMEN
PURPOSE: To evaluate the potential of targeted photoacoustic imaging as a noninvasive method for detection of follicular thyroid carcinoma. EXPERIMENTAL DESIGN: We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP-activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP-activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual-wavelength imaging method. RESULTS: Active forms of both MMP-2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, nonactivatable, agent. CONCLUSIONS: With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.
Asunto(s)
Adenocarcinoma Folicular/diagnóstico por imagen , Adenocarcinoma Folicular/metabolismo , Técnicas Fotoacústicas , Adenocarcinoma Folicular/patología , Animales , Línea Celular Tumoral , Humanos , Metaloproteinasa 2 de la Matriz/aislamiento & purificación , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/aislamiento & purificación , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Imagen Molecular , RadiografíaRESUMEN
DNA polymerase beta (pol beta) is the key gap-filling polymerase in base excision repair, the DNA repair pathway responsible for repairing up to 20000 endogenous lesions per cell per day. Pol beta is also widely used as a model polymerase for structure and function studies, and several structural regions have been identified as being critical for the fidelity of the enzyme. One of these regions is the hydrophobic hinge, a network of hydrophobic residues located between the palm and fingers subdomains. Previous work by our lab has shown that hinge residues Y265, I260, and F272 are critical for polymerase fidelity by functioning in discrimination of the correct from incorrect dNTP during ground state binding. Our work aimed to elucidate the role of hinge residue I174 in polymerase fidelity. To study this residue, we conducted a genetic screen to identify mutants with a substitution at residue I174 that resulted in a mutator polymerase. We then chose the mutator mutant I174S for further study and found that it follows the same general kinetic pathway as and has an overall protein folding similar to that of wild-type (WT) pol beta. Using single-turnover kinetic analysis, we found that I174S exhibits decreased fidelity when inserting a nucleotide opposite a template base G, and this loss of fidelity is due primarily to a loss of discrimination during ground state dNTP binding. Molecular dynamics simulations show that mutation of residue I174 to serine results in an overall tightening of the hinge region, resulting in aberrant protein dynamics and fidelity. These results point to the hinge region as being critical in the maintenance of the proper geometry of the dNTP binding pocket.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Isoleucina , Nucleótidos/metabolismo , Animales , Disparidad de Par Base , Secuencia de Bases , ADN/genética , ADN/metabolismo , ADN Polimerasa beta/genética , Simulación de Dinámica Molecular , Mutagénesis , Mutación , Conformación Proteica , Pliegue de Proteína , Ratas , Especificidad por SustratoRESUMEN
DNA polymerase beta plays a key role in base excision repair. We have previously shown that the hydrophobic hinge region of polymerase beta, which is distant from its active site, plays a critical role in the fidelity of DNA synthesis by this enzyme. The I260Q hinge variant of polymerase beta misincorporates nucleotides with a significantly higher catalytic efficiency than the wild-type enzyme. In the study described here, we show that I260Q extends mispaired primer termini. The kinetic basis for extension of mispairs is defective discrimination by I260Q at the level of ground-state binding of the dNTP substrate. Our results suggest that the hydrophobic hinge region influences the geometry of the dNTP binding pocket exclusively. Because the DNA forms part of the binding pocket, our data are also consistent with the interpretation that the mispaired primer terminus affects the geometry of the dNTP binding pocket such that the I260Q variant has a higher affinity for the incoming dNTP than wild-type polymerase beta.
Asunto(s)
Sustitución de Aminoácidos , ADN Polimerasa beta/química , Cartilla de ADN/química , Desoxirribonucleótidos/química , Mutación Missense , Disparidad de Par Base , Emparejamiento Base/fisiología , Dominio Catalítico/fisiología , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Desoxirribonucleótidos/genética , Desoxirribonucleótidos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.
Asunto(s)
Transformación Celular Neoplásica/genética , ADN Polimerasa beta/genética , Reparación del ADN/fisiología , Mutación/genética , Neoplasias de la Próstata/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dicroismo Circular , ADN Polimerasa beta/metabolismo , ADN Circular , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Neoplasias de la Próstata/enzimología , Pliegue de Proteína , Moldes GenéticosRESUMEN
Thirty percent of the 189 tumors studied to date express DNA polymerase beta variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein. Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorage-independent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase beta variant proteins, implying that it has a mutational basis. Because DNA polymerase beta functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Reparación del ADN/genética , Mutación/genética , Fenotipo , Animales , Western Blotting , Línea Celular , Transformación Celular Neoplásica/genética , Vectores Genéticos/genética , Ratones , RetroviridaeRESUMEN
The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , ADN/química , ADN/metabolismo , ADN Polimerasa beta/genética , Glutamina , Isoleucina , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
DNA polymerases ensure efficient insertion of the correct dNTP into the DNA substrate. They have evolved mechanisms for discriminating among very similar dNTP substrates. DNA polymerase beta is a repair polymerase that provides a model system for a direct study of insertion fidelity. In this study, we examined the role of hinge residue Ile260 of the rat Polbeta on enzyme activity and accuracy. We changed residue I260 to every other amino acid residue and used genetic screens to assess the activity and fidelity of the resulting mutants. The I260D, -E, -K, -N, and -R mutants are significantly less active than wild-type Polbeta. Interestingly, I260H and I260Q are active but exhibit mutator activity. This suggests that the nonpolar nature of residue 260 is important for maintaining the activity and fidelity of Polbeta. We employ molecular modeling as an aid in explaining the observed phenotypes and propose a mechanism whereby the positioning of the DNA substrate in the enzyme and within the surface of the hinge may be a key player in forming an optimal active site for phosphodiester bond formation between Watson-Crick base pairs.
Asunto(s)
ADN Polimerasa beta/metabolismo , Isoleucina/metabolismo , Fragmentos de Péptidos/metabolismo , Sustitución de Aminoácidos/genética , Animales , Arginina/genética , Asparagina/genética , Ácido Aspártico/genética , ADN Polimerasa beta/antagonistas & inhibidores , ADN Polimerasa beta/genética , Activación Enzimática/genética , Estabilidad de Enzimas/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Prueba de Complementación Genética , Variación Genética , Ácido Glutámico/genética , Glutamina/genética , Histidina/genética , Isoleucina/genética , Lisina/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fenotipo , Pliegue de Proteína , Estructura Terciaria de Proteína/genética , Ratas , Especificidad por Sustrato/genéticaRESUMEN
Recent small-scale studies have shown that 30% of human tumors examined to date express DNA polymerase beta variant proteins. One of the DNA polymerase beta colon cancer-associated mutants, K289M, has been shown to synthesize DNA with a lower fidelity than wild-type Pol beta. Thus, the K289M protein could confer a mutator phenotype to the cell, resulting in genomic instability. Another DNA polymerase beta variant identified in colon carcinoma interferes with base excision repair in cells. This may result in unfilled gaps which can serve as substrates for recombination and result in genomic instability. DNA polymerase beta has also been shown to be overexpressed in a variety of tumors. In some cases, overexpression of polymerase beta in cells confers a transformed phenotype to the cells. In other cases, overexpression results in telomere fusions. Thus, mutant forms or aberrant quantities of polymerase beta confer a mutator phenotype to cells. Combined with the small-scale tumor studies, these mechanistic studies implicate variant forms of DNA polymerase beta in the etiology of human cancer.
Asunto(s)
ADN Polimerasa beta/genética , Variación Genética/genética , Neoplasias/enzimología , Neoplasias/genética , Sustitución de Aminoácidos/genética , Animales , HumanosRESUMEN
Previous investigations have shown that approximately 35% of the 90 tumors analyzed to date contain mutations within the DNA polymerasebeta (pol beta) gene. The existence of pol beta mutations in a substantial fraction of human tumors studied suggests a link between DNA pol beta and cancer. A DNA pol beta variant, in which Lys-289 has been altered to Met, was identified previously in a colorectal carcinoma. The K289M protein was expressed in mouse L cells containing the lambda cII mutational target. The lambda DNA was packaged and used to infect bacterial cells to obtain the spontaneous mutation frequency. We found that expression of K289M in the mouse cells resulted in a 2.5-fold increase in the mutation frequency. What was most interesting was that expression of K289M in these cells resulted in a 16-fold increase in the frequency of C to G or G to C base substitutions at a specific site within the cII target. By using this cII target sequence, kinetic analysis of the purified K289M protein revealed that it was able to misincorporate dCTP opposite template C and dGTP opposite template G with significantly higher efficiency than the wild-type pol beta protein. We provide evidence that misincorporation of nucleotides by K289M results from altered positioning of the DNA within the active site of the enzyme. Our data are consistent with the interpretation that misincorporation of nucleotides resulting from altered DNA positioning by the K289M protein has the potential to result in tumorigenesis or neoplastic progression.