RESUMEN

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

Asunto(s)

Proteínas de Unión al Calcio/genética , Predisposición Genética a la Enfermedad/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Haploinsuficiencia , Heterocigoto , Humanos , Immunoblotting , Neoplasias Renales/genética , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma

RESUMEN

Heritable germline mutations in major cancer genes generally lead to a restricted pattern of tissue-specific malignancies, yet many of the same mutations frequently occur somatically in a broad range of spontaneous neoplasms affecting different organs. Might this reflect a difference in tumorigenesis in children and adults?

Asunto(s)

Transformación Celular Neoplásica/genética , Genes del Tumor de Wilms , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Proteínas Tirosina Quinasas Receptoras/genética , Adulto , Quinasa de Linfoma Anaplásico , Niño , Humanos , Neuroblastoma/genética , Especificidad de Órganos/genética , Retinoblastoma/genética , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/genética

Asunto(s)

Personajes , Historia del Siglo XX , Italia , Estados Unidos

RESUMEN

OBJECTIVE: To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes. METHODS: Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC. RESULTS: A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an 'interferon signature' may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis. CONCLUSIONS: These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently-incurable malignancy.

Asunto(s)

Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , FN-kappa B/genética , Carcinoma de Células Renales/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Redes Reguladoras de Genes , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunohistoquímica , Interferones/genética , Interferones/metabolismo , Neoplasias Renales/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Mutación , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Transducción de Señal/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Análisis de Supervivencia , Transcriptoma , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo

Asunto(s)

Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Proteínas F-Box/genética , Modelos Genéticos , Mutación , Neoplasias/genética , Ubiquitina-Proteína Ligasas/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Humanos

RESUMEN

This year, 2011, marks the forty-year anniversary of the statistical analysis of retinoblastoma that provided the first evidence that tumorigenesis can be initiated by as few as two mutations. This work provided the foundation for the two-hit hypothesis that explained the role of recessive tumour suppressor genes (TSGs) in dominantly inherited cancer susceptibility syndromes. However, four decades later, it is now known that even partial inactivation of tumour suppressors can critically contribute to tumorigenesis. Here we analyse this evidence and propose a continuum model of TSG function to explain the full range of TSG mutations found in cancer.

Asunto(s)

Genes Supresores de Tumor/fisiología , Modelos Genéticos , Neoplasias/genética , Neoplasias/patología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Predisposición Genética a la Enfermedad/genética , Haploinsuficiencia/genética , Humanos , Neoplasias/metabolismo , Neoplasias/terapia

RESUMEN

We hypothesized that cells bearing a single inherited "hit" in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of "one hit" represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention.

Asunto(s)

Biomarcadores de Tumor/genética , Células Epiteliales/fisiología , Perfilación de la Expresión Génica , Genes BRCA1 , Genes BRCA2 , Predisposición Genética a la Enfermedad , Mama , Minería de Datos , Femenino , Expresión Génica , Heterocigoto , Humanos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovario , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

We previously concluded, from our analysis of the published data of other investigators, that the yield of germ-line and somatic mutations after exposure to ionizing radiation is parabolically related to the logarithm of the dose-rate at which a given dose is administered. Here we show that other data reveal a similarly parabolic relationship for other ionizing radiation-associated phenomena, namely, genetic recombination, chromosomal translocation, cell inactivation and lethality, and human leukemogenesis. Furthermore, the minima for all effects fall in a relatively narrow range of the dose-rate logarithms. Because the only mechanism common to all of these phenomena is the double-strand break (DSB) in DNA, we refer to our previous analysis of the endogenous production of DSBs, from which we concluded that approximately 50 endogenous DSBs occur per cell cycle, although most are repaired without error. Comparison then reveals that their rate of production falls within the range of minima for the several end points pursuant to radiation-induced DSBs. We conclude that the results reflect a physiological principle whereby signals originating from induced DSBs elicit responses of maximal effectiveness when they are produced at a rate near that of the production of endogenous DSBs. We refer to this principle as "signaling resonance."

Asunto(s)

Daño del ADN , ADN/efectos de la radiación , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Leucemia/etiología , Mitosis/fisiología , Mutación/efectos de la radiación , Recombinación Genética/efectos de la radiación , Translocación Genética/efectos de la radiación

RESUMEN

Twenty years ago, antiestrogen therapy with tamoxifen played only a secondary role in breast cancer care. All hopes to cure metastatic breast cancer were still pinned on either the discovery of new cytotoxic drugs or a dose-dense combination of available cytotoxic drugs with bone marrow transplantation. A similar strategy with combination chemotherapy was employed as an adjuvant for primary breast cancer. Simply stated, the goal was to kill the cancer with nonspecific cytotoxic drugs while keeping the patient alive with supportive care. However, medical research does not travel in straight lines, and an alternative approach emerged to solve the problem of controlling tumor growth with minimal side effects: targeted therapy. The approach of using long-term antihormone therapy to control early-stage breast cancer growth would revolutionize cancer care by targeting the tumor estrogen receptor (ER). The success of the strategy would contribute to a decrease in the national mortality figures for breast cancer. More importantly, translational research that targeted the tumor ER with a range of new antiestrogenic drugs would presage the current fashion of blocking survival pathways for the tumor by developing novel targeted treatments. But a surprise was in store when the pharmacology of "antiestrogens" was studied in detail: The nonsteroidal "antiestrogens" are selective ER modulators--ie, they are antiestrogens in the breast, estrogens in the bone--and they lower circulating cholesterol levels. This knowledge would establish a practical approach to breast cancer chemoprevention for women at high risk (tamoxifen) and low risk (raloxifene).

Asunto(s)

Antineoplásicos Hormonales/administración & dosificación , Neoplasias de la Mama/prevención & control , Quimioprevención/tendencias , Sistemas de Liberación de Medicamentos/tendencias , Oncología Médica/tendencias , Moduladores Selectivos de los Receptores de Estrógeno/administración & dosificación , Tamoxifeno/administración & dosificación , Investigación Biomédica/tendencias , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Industria Farmacéutica , Femenino , Humanos , Sobrevivientes

RESUMEN

The past 60 years surely constitute a Golden Age for biomedical science, and for medical genetics in particular. A personal experience began with an encounter with inborn errors of metabolism, selection, and the incidences of hereditary diseases, and peaked with molecular biology, virology, and cytogenetics, finally focusing all three on the problem of cancer.

Asunto(s)

Genética Médica , Animales , Niño , Genética Médica/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Pediatría/historia , Retinoblastoma/historia , Estados Unidos

RESUMEN

Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/análisis , ARN/biosíntesis , Humanos , ARN/genética , Reproducibilidad de los Resultados

RESUMEN

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Terapia por Láser , Microdisección , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Línea Celular Tumoral , ADN Complementario/análisis , ADN Complementario/biosíntesis , ADN Complementario/genética , Perfilación de la Expresión Génica/instrumentación , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

BACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identify early molecular changes associated with dominantly inherited predisposition to "two-hit" renal tumors, the expression profiles of primary cultures of phenotypically normal renal epithelial cells from individuals bearing a germline mutation in either the von Hippel-Lindau (VHL) or the tuberous sclerosis complex (TSC) gene were compared to that of renal epithelial cells from control nonmutation carriers by microarray analysis. RESULTS: Reliability of the microarray data from pooled samples was confirmed by real-time RT-PCR. Principal Component Analysis revealed substantial differences in the gene expression profiles of the renal epithelial cells from VHL and TSC mutation carriers. In several instances, the microarray data confirm our present knowledge of the cellular pathways affected by biallelic VHL and TSC mutations. CONCLUSIONS: These findings demonstrate that heterozygosity for a mutant tumor suppressor gene may alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner. Detectable effects of "one-hit" represent early molecular changes in tumorigenesis that may serve as targets for chemopreventive intervention.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Proteínas de Unión al Calcio/genética , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Mutación de Línea Germinal/genética , Riñón/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Biomarcadores de Tumor/genética , Células Epiteliales/citología , Células Epiteliales/patología , Heterocigoto , Humanos , Riñón/citología , Riñón/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Enfermedad de von Hippel-Lindau/genética

RESUMEN

This article extends our previous quantitative analysis of the relationship between the dynamics of the primary structure of DNA and mutagenesis associated with single-strand lesions to an analysis of the production and processing of endogenous double-strand breaks (EDSBs) and to their implications for oncogenesis. We estimate that in normal human cells approximately 1% of single-strand lesions are converted to approximately 50 EDSBs per cell per cell cycle. This number is similar to that for EDSBs produced by 1.5-2.0 Gy of sparsely ionizing radiation. Although EDSBs are usually repaired with high fidelity, errors in their repair contribute significantly to the rate of cancer in humans. The doubling dose for induced DSBs is similar to doubling doses for mutation and for the induction of carcinomas by ionizing radiation. We conclude that rates of production of EDSBs and of ensuing spontaneous mitotic recombination events can account for a substantial fraction of the earliest oncogenic events in human carcinomas.

Asunto(s)

Daño del ADN , Reparación del ADN/genética , Replicación del ADN/genética , Mutación , Neoplasias/genética , Animales , Transformación Celular Neoplásica/genética , Escherichia coli/genética , Rayos gamma , Humanos , Incidencia , Modelos Animales , Neoplasias/epidemiología , Oncogenes , Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Xenopus laevis

Asunto(s)

Biología Molecular/historia , Neoplasias/genética , Neoplasias/historia , Historia del Siglo XVIII , Historia del Siglo XIX , Humanos

RESUMEN

Cancer is a genetic disease of somatic cells. Tumor karyotypes are rarely normal, and most show multiple abnormalities of both number and structure. The first direct evidence for this concept of cancer came from studies of tumor-specific translocations in leukemias and lymphomas, revealing the importance of oncogenes and the regulation of gene transcription in cancer. A second major source of information about human cancer genes is hereditary cancer. Genetic predisposition of the autosomal dominant type imposes a high relative risk for one or more kinds of cancer. In the past decade or so, more than 30 mutant genes for such hereditary cancers have been cloned. Penetrance depends upon additional, somatic, mutations. A few of the genes are oncogenes or DNA repair genes, but most are tumor suppressor genes. Some tumor suppressors regulate transcription, while others operate in signal transduction pathways that are involved in regulating processes of cell birth, differentiation, and death. The knowledge gained is stimulating new approaches to the treatment and prevention of cancer.

Asunto(s)

Neoplasias/genética , Adolescente , Adulto , Apoptosis/genética , Ciclo Celular/genética , Clonación Molecular , Reparación del ADN/genética , Femenino , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Humanos , Masculino , Mutación , Neoplasias/epidemiología , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neurocutáneos/genética , Oncogenes , Proteína de Retinoblastoma/fisiología , Selección Genética , Proteína p53 Supresora de Tumor/fisiología