RESUMEN
Uterine leiomyomata (fibroids) are common and clinically important tumors, but little is known about their etiology and pathogenesis. We previously mapped a gene that predisposes to multiple fibroids, cutaneous leiomyomata and renal cell carcinoma to chromosome 1q42.3-q43 (refs 4-6). Here we show, through a combination of mapping critical recombinants, identifying individuals with germline mutations and screening known and predicted transcripts, that this gene encodes fumarate hydratase, an enzyme of the tricarboxylic acid cycle. Leiomyomatosis-associated mutations are predicted to result in absent or truncated protein, or substitutions or deletions of highly conserved amino acids. Activity of fumarate hydratase is reduced in lymphoblastoid cells from individuals with leiomyomatosis. This enzyme acts as a tumor suppressor in familial leiomyomata, and its measured activity is very low or absent in tumors from individuals with leiomyomatosis. Mutations in FH also occur in the recessive condition fumarate hydratase deficiency, and some parents of people with this condition are susceptible to leiomyomata. Thus, heterozygous and homozygous or compound heterozygous mutants have very different clinical phenotypes. Our results provide clues to the pathogenesis of fibroids and emphasize the importance of mutations of housekeeping and mitochondrial proteins in the pathogenesis of common types of tumor.
Asunto(s)
Carcinoma Papilar/genética , Carcinoma de Células Renales/genética , Fumarato Hidratasa/genética , Mutación de Línea Germinal , Neoplasias Renales/genética , Leiomioma Epitelioide/genética , Leiomioma/genética , Neoplasias Uterinas/genética , Alelos , Cromosomas Humanos Par 1 , Exones , Femenino , Fumarato Hidratasa/metabolismo , Eliminación de Gen , Genes Dominantes , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Masculino , Mutación , Linaje , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
DNA mismatch repair (MMR)-deficient cells typically accumulate mutations in short repetitive DNA tracts. This microsatellite instability (MSI) facilitates malignant transformation when affecting genes with growth-related and caretaker functions. To date, several putative MSI target genes have been proposed mainly based on high mutation frequency within their coding regions. However, some intronic repeat mutations have also been suggested to associate with MSI tumorigenesis, indicating the need for additional analyses on noncoding repeats. Here we have analyzed an intronic T9 repeat of semenogelin I (SEMG1) and report mutation frequencies of 51% (75 of 146) and 62% (8 of 13) in MMR-deficient primary colorectal cancers and cell lines, respectively. The putative effect of the SEMG1 mutations was assessed by RNA and protein level analyses, but no differences were detected between colorectal cancer cell lines with different SEMG1 status. Subsequently, the general background mutation frequency of MSI colorectal cancers was assessed by screening for intergenic T9 repeat alterations. One of 10 examined repeats was mutated in 70% (102 of 145) of the colorectal cancers evaluated. The frequencies observed here are notably higher than previously published in noncoding repeats shorter than 10 bp in MMR-deficient primary tumors. Our results indicate that high mutation frequencies, similar or higher than those observed in proposed and approved target genes, can be detected in repeat tracts of MSI tumors without any apparent selection pressure. These data call for urgent and thorough large-scale evaluation of mutation frequencies in neutral short repetitive sequences in MMR-deficient tumors.
Asunto(s)
Neoplasias Colorrectales/genética , Mutación del Sistema de Lectura , Repeticiones de Microsatélite/genética , Proteínas de Secreción de la Vesícula Seminal/genética , Alelos , Disparidad de Par Base/genética , Secuencia de Bases , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Reparación del ADN/genética , ADN de Neoplasias/genética , Inestabilidad Genómica , Humanos , Intrones/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Secreción de la Vesícula Seminal/biosíntesis , Proteínas de Secreción de la Vesícula Seminal/metabolismoRESUMEN
We have recently reported that low tumor levels of SMAD4, a key mediator of transforming growth factor-beta superfamily signaling, can predict the probability of recurrence in patients with Dukes C colorectal cancer who had surgery as the only form of treatment. However, standard treatment for Dukes C colorectal cancer patients currently involves the administration of 5-fluorouracil (5-FU)-based adjuvant chemotherapy after surgery. Approximately 30% to 40% of these patients present with recurrence and die within 5 years, and there is great need for markers capable of predicting poor prognosis after the combined surgery/adjuvant treatment. In this study, we evaluate the prognostic value of SMAD4 in patients treated with surgery and 5-FU-based adjuvant therapy. We used immunohistochemistry and quantitative real-time reverse transcription-PCR to measure the levels of SMAD4 protein and mRNA expression in the primary tumors and a number of lymph node metastases from a series of 75 Dukes C colorectal cancer patients with at least 6 years of follow-up. Patients with tumors expressing low levels of SMAD4 protein or mRNA showed significantly shorted disease-free and overall survival than patients with high tumor levels of SMAD4. The median survival of patients with low SMAD4 protein or mRNA tumor levels was 1.4 and 1.2 years, respectively, whereas patients with high SMAD4 tumor level had a median survival of >9.3 years. In addition, the protein and mRNA levels of SMAD4 in lymph node metastases was significantly lower than in primary tumors (P = 0.006). In contrast, allelic imbalance in chromosome 18q21 was of no prognostic significance in these patients. In conclusion, low SMAD4 tumor levels identified a subset of patients with poor prognosis following surgery and 5-FU-based adjuvant therapy; therefore, these patients could be good candidates to receive combined treatment with additional chemotherapeutic agents such as CPT-11 and/or oxaliplatin.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas de Unión al ADN/metabolismo , Fluorouracilo/uso terapéutico , Transactivadores/metabolismo , Desequilibrio Alélico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Quimioterapia Adyuvante , Cromosomas Humanos Par 18/genética , Neoplasias Colorrectales/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Humanos , Técnicas para Inmunoenzimas , Metástasis Linfática , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , ARN Mensajero/metabolismo , Proteína Smad4 , Tasa de Supervivencia , Transactivadores/genéticaRESUMEN
More than 50% of patients with Dukes C colorectal cancer have disease recurrence and die within 5 years after surgical removal of their primary tumor. It is currently not possible to distinguish patients with good and bad prognosis. SMAD4 is an important tumor suppressor gene that mediates transforming growth factor-beta superfamily signaling and is located in chromosome 18q21, a region with frequent genetic losses in these tumors. Allelic imbalance in 18q has been linked to poor prognosis in a subset of colorectal cancer patients. Therefore, we generated a tissue microarray containing triplicate tumor samples from 86 Dukes C patients and used immunohistochemistry to assess the relative expression level of SMAD4 and its value as a prognostic marker. In addition, SMAD4 was screened for mutations and two polymorphic microsatellite markers were used to assess the presence of allelic imbalance in these tumors. Patients with tumors expressing high SMAD4 levels had significantly better overall (P < 0.025) and disease-free (P < 0.013) survival than patients with low levels. This identifies SMAD4 as a prognostic marker for Dukes C colorectal cancer. Although all tumors with absent SMAD4 staining showed allelic imbalance in 18q21, tumors with 18q21 allelic imbalance as a group showed no difference in SMAD4 levels compared with tumors without allelic imbalance, suggesting that additional mechanisms of SMAD4 down-regulation exist. In addition, although SMAD4 mutations were found in five tumors, they were not associated with shorter survival. In conclusion, the level of expression of SMAD4 was found to be a more sensitive marker than 18q21 allelic imbalance and SMAD4 mutations, which were of no prognostic significance for these patients.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/genética , Transactivadores/genética , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Cromosomas Humanos Par 18/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Análisis Mutacional de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Proteínas de Unión al ADN/análisis , Femenino , Humanos , Inmunohistoquímica , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Proteína Smad4 , Análisis de Supervivencia , Transactivadores/análisisRESUMEN
Loss of function mutations in the fumarate hydratase (fumarase, FH) gene were recently identified as the cause for dominantly inherited uterine and cutaneous leiomyomas and renal cell cancer. To further evaluate the role of FH in tumorigenesis, we screened FH mutations from tumor types seen in hereditary leiomyomatosis and renal cell cancer mutation carriers-41 uterine and 10 cutaneous leiomyomas, 52 renal cell carcinomas, 53 sarcomas, 29 prostate carcinomas, and 15 lobular breast carcinomas. Few mutations were detected. Biallelic inactivation of FH was found in one uterine leiomyosarcoma, one cutaneous leiomyoma, and one soft tissue sarcoma. Whereas the two former lesions were shown to originate from a germ-line mutation, the soft tissue sarcoma is to our knowledge the first example of purely somatic inactivation of FH in tumors.
Asunto(s)
Carcinoma de Células Renales/genética , Fumarato Hidratasa/genética , Mutación de Línea Germinal , Neoplasias Renales/genética , Leiomioma/genética , Neoplasias Cutáneas/genética , Neoplasias Uterinas/genética , Secuencia de Bases , Carcinoma de Células Renales/enzimología , Femenino , Humanos , Neoplasias Renales/enzimología , Leiomioma/enzimología , Leiomiosarcoma/enzimología , Leiomiosarcoma/genética , Masculino , Datos de Secuencia Molecular , Mutación Missense , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias Cutáneas/enzimología , Neoplasias Uterinas/enzimologíaRESUMEN
We have allelotyped a series of 104 Finnish colorectal cancers (CRCs) using 372 polymorphic markers spaced, on average, at 10 cM intervals, and have made a comparison of the differences in the frequency of allelic imbalance (AI) between familial and sporadic cases. Differences in the frequency of allelic imbalance (loss of heterozygosity or amplification) at a number of loci were detected and these were evaluated through analysis of additional series of cancers using specific markers. The most consistent difference was observed at chromosome 20q13.1-13.3 characterized by a two fold difference between familial and nonfamilial disease in a total of 99 familial and 186 sporadic Finnish cases. This difference was not observed in a UK set of 67 familial and 96 sporadic CRCs. The genome-wide effort resulted in a large data set giving clues to the location of putative CRC predisposition genes in the genome. The approach provides an alternative strategy for detecting cancer predisposition genes solely reliant on the molecular analysis of single cases obviating the requirement to collect multiple samples from families.
Asunto(s)
Cromosomas Humanos Par 20 , Neoplasias Colorrectales/genética , Adulto , Anciano , Desequilibrio Alélico , Femenino , Finlandia , Predisposición Genética a la Enfermedad , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana EdadRESUMEN
PURPOSE: A considerable fraction (30% to 70%) of families with verified or putative hereditary nonpolyposis colorectal cancer fails to show mutations in DNA mismatch repair (MMR) genes. Our purpose was to address the genetic etiology of such families. MATERIALS AND METHODS: We scrutinized a population-based cohort of 26 families from Finland that had screened mutation-negative by previous techniques. Blood was tested for allelic messenger RNA (mRNA) expression of MLH1, MSH2, and MSH6 by single nucleotide primer extension (SNuPE), and tumor tissue for MMR protein expression by immunohistochemistry (IHC) as well as for microsatellite instability (MSI). Full-length cDNAs of genes implicated by SNuPE or IHC were cloned and sequenced. RESULTS: Unbalanced mRNA expression of MLH1 alleles was evident in two families. An inherited nonsense mutation was subsequently identified in one family, and complete silencing of the mutated allele was identified in the other family. Extinct protein expression by IHC implicated MLH1 in these two and in four other families, MSH2 in four families, and MSH6 in one family. Although no unequivocal genomic mutations were detected in the latter families, haplotype and other findings provided support for heritable defects. With one exception, all tumors with IHC alterations showed MSI, in contrast to the remaining families, which showed neither IHC changes nor MSI. CONCLUSION: Our expression-based strategy stratified the present "mutation-negative" cohort into two discrete categories: families linked to the major MMR genes MLH1, MSH2, and MSH6 (11 [42%] of 26) and those likely to be associated with other, as yet unknown susceptibility genes (15 [58%] of 26).
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN/biosíntesis , Predisposición Genética a la Enfermedad , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Adaptadoras Transductoras de Señales , Adulto , Edad de Inicio , Anciano , Disparidad de Par Base , Proteínas Portadoras , Análisis Mutacional de ADN , Reparación del ADN , ADN Complementario/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Linaje , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Factores de RiesgoRESUMEN
The expression patterns of cancer-related genes in 13 cases of squamous cell lung cancer (SCC) were characterized and compared with those in normal lung tissue and 13 adenocarcinomas (AC), the other major type of nonsmall cell lung cancer (NSCLC). cDNA array was used to screen the gene expression levels and the array results were verified using a real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). Thirty-nine percent of the 25 most upregulated and the 25 most downregulated genes were common to SCC and AC. Of these genes, DSP, HMGA1 (alias HMGIY), TIMP1, MIF, CCNB1, TN, MMP11, and MMP12 were upregulated and COPEB (alias CPBP), TYROBP, BENE, BMPR2, SOCS3, TIMP3, CAV1, and CAV2 were downregulated. The expression levels of several genes from distinct protein families (cytokeratins and hemidesmosomal proteins) were markedly increased in SCC compared with AC and normal lung. In addition, several genes, overexpressed in SCC, such as HMGA1, CDK4, IGFBP3, MMP9, MMP11, MMP12, and MMP14, fell into distinct chromosomal loci, which we have detected as gained regions on the basis of comparative genomic hybridization data. Our study revealed new candidate genes involved in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , Neoplasias de Células Escamosas/genética , Adulto , Anciano , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana EdadRESUMEN
In a previous genome-wide effort we identified a novel candidate colorectal cancer (CRC) susceptibility locus by allelotyping tumors from familial and sporadic CRC patients. Familial cases harbored amplifications significantly more frequently in 20q13, indicating the presence of a putative oncogene within the amplicon. A kinase-encoding AURKA is located in 20q13 and recently a Phe31Ile (91T > A) change of AURKA was suggested to function as a low penetrance tumor-susceptibility factor. Association analysis suggested an increased cancer risk in 91A homozygous individuals. In addition, tumors from heterozygous individuals showed preferential amplification of 91A allele and were more aneuploid, important findings that have not been confirmed to date. To evaluate whether AURKA is a target for the observed 20q13 amplifications, we assessed the frequency of the 91T > A change and possible preferential amplification of either allele in 125 familial and 110 sporadic Finnish CRC cases. We observed a preferential amplification of 91A with a significant difference between the alleles in the familial group (p = 0.03). Furthermore, a trend between younger age at diagnosis and genotype in the familial group was observed (p = 0.06). Other possible AURKA germline variants were screened by sequencing 10 of the familial cases. The frequency and amplification patterns of the observed variants were assessed in a larger set of familial and sporadic CRC but no evidence on tumorigenic role of the other sequence alterations was obtained. Thus our results support the importance of AURKA 91A as a low penetrance CRC susceptibility factor.
Asunto(s)
Neoplasias Colorrectales/genética , Amplificación de Genes , Predisposición Genética a la Enfermedad , Proteínas Serina-Treonina Quinasas/genética , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Aurora Quinasa A , Aurora Quinasas , Estudios de Casos y Controles , Femenino , Finlandia , Genotipo , Humanos , Masculino , Persona de Mediana Edad , LinajeRESUMEN
BACKGROUND & AIMS: Although approximately 50% of Dukes' C colorectal cancer patients are surgically cured, it is currently not possible to distinguish these patients from those at high risk of recurrence. The recent advent of routine adjuvant chemotherapy for these patients has greatly complicated the identification of new markers predicting the response to surgery, which is now reliant on archived materials. Microarray analysis allows fine tumor classification but cannot be used with paraffin-embedded archival samples. METHODS: We used microarray analysis of a unique set of fresh-frozen tumor samples from Dukes' C patients who had surgery as the only form of treatment to identify molecular signatures that characterize tumors from patients with good and bad prognosis. RESULTS: Unsupervised hierarchical clustering and a K-nearest neighbors-based classifier identified groups of patients with significantly different survival (P = .019 and P = .0001). Expression profiling outperformed previously reported genetic markers of prognosis such as TP53 and K-RAS mutational status and allelic imbalance in chromosome 18q, which were of limited prognostic power in this study. Functional categories significantly enriched in gene-expression differences included protein transport and folding. The prognostic potential of the RAS homologue RHOA, one of the most differentially expressed genes, was further investigated using immunohistochemistry and a tissue microarray containing 137 independent Dukes' C tumor samples. Reduced RHOA expression was associated with significantly shorter survival (P = .01). CONCLUSIONS: This study shows that gene-expression profiling of surgical tumor samples can predict recurrence in Dukes' C patients. Therefore, this approach could be used to guide decisions concerning the clinical management of these patients.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Perfilación de la Expresión Génica , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/mortalidad , Supervivencia sin Enfermedad , Genes p53 , Genes ras , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , Pronóstico , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Recurrencia , Análisis de Supervivencia , Factores de Tiempo , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Microsatellite instability (MSI) secondary to loss of DNA mismatch repair (MMR) is present in adenomas and colorectal carcinomas from individuals with hereditary nonpolyposis colorectal cancer (HNPCC). To better characterize when MMR loss occurs during HNPCC progression, the extent of deletions in noncoding polyA sequences were compared between 6 adenomas (all < or = 1.0 cm in size) and 10 cancers. Numbers of deleted bases reflect time since loss of MMR because polyA deletions are stepwise. Adenoma deletions were nearly the same (85%) as the cancers with sum total deletions at four different polyA loci of -32.7 bases in adenomas and -38.4 bases in cancers. Intervals between negative clinical examinations and tumor removal (average of 2.1 years) were known for six tumors. There were no significant differences in the extent of deletions in tumors removed under clinical surveillance (-34.8 bases) versus tumors removed without prior negative examinations (-36.5 bases). These findings illustrate that MSI is extensive in both small adenomas, and tumors which appear after negative clinical examinations, consistent with an early loss of MMR in HNPCC, even before a gatekeeper mutation.
Asunto(s)
Adenoma/genética , Carcinoma/genética , Neoplasias Colorrectales/genética , Eliminación de Gen , Mutación , Poli A/genética , Adulto , Disparidad de Par Base , Humanos , Persona de Mediana Edad , Vigilancia de la Población , Factores de TiempoRESUMEN
A pretumor progression model predicts many oncogenic cancer mutations may first accumulate in normal appearing colon. Although direct observations of early pretumor mutations are impractical, it may be possible to retrospectively reconstruct tumor histories from contemporary cancer mutations. To infer when and in what order mutations occur during occult pretumor progression, we examined 14 cancers from individuals with heterozygous germline mutations in DNA mismatch repair (MMR) genes or hereditary nonpolyposis colorectal cancer (HNPCC). Somatic inactivation of the normal allele occurs sometime during a lifetime and results in loss of MMR, elevated mutation rates, and subsequent widespread somatic microsatellite mutations in HNPCC cancers. Patient ages at MMR loss can be estimated because intervals between MMR loss and cancer removal can be inferred from numbers of microsatellite tumor mutations. The relative order of MMR loss during pretumor progression may also be inferred from its collective ages of occurrence. Somatic MMR loss preceded cancer removal by an average of 6.1 years, occurred relatively late in life (average of 41.6 versus 47.7 years at cancer removal), and was a surprisingly late (fifth or sixth) step. Calculations indicate five or six oncogenic mutations could accumulate with relatively normal replication fidelity in normal appearing colon. HNPCC pretumor progression essentially begins from birth and ends with MMR loss, implying elevated mutation rates and tumorigenesis may be unnecessary for most progression.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN/genética , Proteínas de Neoplasias/genética , Lesiones Precancerosas/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Adaptadoras Transductoras de Señales , Adulto , Factores de Edad , Disparidad de Par Base/genética , Proteínas Portadoras , Transformación Celular Neoplásica , Análisis Mutacional de ADN , Reparación del ADN/genética , Progresión de la Enfermedad , Mutación de Línea Germinal , Humanos , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Modelos Biológicos , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Estudios Retrospectivos , Factores de TiempoRESUMEN
The colorectum and uterine endometrium are the two most commonly affected organs in hereditary nonpolyposis colon cancer (HNPCC), but the genetic basis of organ selection is poorly understood. As tumorigenesis in HNPCC is driven by deficient DNA mismatch repair (MMR), we compared its typical consequence, instability at microsatellite sequences, in colorectal and endometrial cancers from patients with identical predisposing mutations in the MMR genes MLH1 or MSH2. Analysis of non-coding (BAT25, BAT26, and BAT40) and coding mononucleotide repeats (MSH6, MSH3, MLH3, BAX, IGF2R, TGF beta RII, and PTEN), as well as MLH1- and MSH2-linked dinucleotide repeats (D3S1611 and CA7) revealed significant differences, both quantitative and qualitative, between the two tumor types. Whereas colorectal cancers displayed a predominant pattern consisting of instability at the BAT loci (in 89% of tumors), TGF beta RII (73%), dinucleotide repeats (70%), MSH3 (43%), and BAX (30%), no such single pattern was discernible in endometrial cancers. Instead, the pattern was more heterogeneous and involved a lower proportion of unstable markers per tumor (mean 0.27 for endometrial cancers versus 0.45 for colorectal cancers, P < 0.001) and shorter allelic shifts for BAT markers (average 5.1 bp for unstable endometrial cancers versus 9.3 bp for colorectal cancers, P < 0.001). Among the individual putative "target" loci, PTEN instability was associated with endometrial cancers and TGF beta RII instability with colon cancers. The different instability profiles in endometrial and colorectal cancers despite identical genetic predisposition underlines organ-specific differences that may be important determinants of the HNPCC tumor spectrum.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/complicaciones , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN , Neoplasias Endometriales/genética , Repeticiones de Microsatélite , Proteínas Adaptadoras Transductoras de Señales , Alelos , Proteínas Portadoras , Repeticiones de Dinucleótido/genética , Femenino , Mutación de Línea Germinal , Humanos , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/genética , Proteínas Nucleares , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Proteínas Proto-Oncogénicas/genética , Expansión de Repetición de Trinucleótido , Proteínas Supresoras de Tumor/genéticaRESUMEN
PTEN on 10q23.3 encodes a dual-specificity phosphatase that negatively regulates the phosphoinositol-3-kinase/Akt pathway and mediates cell-cycle arrest and apoptosis. Germline PTEN mutations cause Cowden syndrome and a range of several different hamartoma-tumor syndromes. Hereditary nonpolyposis colon cancer (HNPCC) syndrome is characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. Although both colorectal carcinoma and endometrial carcinoma are the most frequent component cancers in HNPCC, only endometrial cancer has been shown to be a minor component of Cowden syndrome. We have demonstrated that somatic inactivation of PTEN is involved in both sporadic endometrial cancers and HNPCC-related endometrial cancers but with different mutational spectra and different relationships to MSI. In the current study, we sought to determine the relationship of PTEN mutation, 10q23 loss of heterozygosity, PTEN expression, and MSI status in colorectal cancers (CRCs). Among 11 HNPCC CRCs, 32 MSI+ sporadic cancers, and 39 MSI- tumors, loss of heterozygosity at 10q23.3 was found in 0%, 8%, and 19%, respectively. Somatic mutations were found in 18% (2 of 11) of the HNPCC CRCs and 13% (4 of 32) of the MSI+ sporadic tumors, but not in MSI- cancers (P = 0.015). All somatic mutations occurred in the two 6(A) coding mononucleotide tracts in PTEN, suggestive of the etiological role of the deficient MMR. Immunohistochemical analysis revealed 31% (14 of 45) of the HNPCC CRCs and 41% (9 of 22) of the MSI+ sporadic tumors with absent or depressed PTEN expression. Approximately 17% (4 of 23) of the MSI- CRCs had decreased PTEN expression, and no MSI- tumor had complete loss of PTEN expression. Among the five HNPCC or MSI+ sporadic CRCs carrying frameshift somatic mutations with immunohistochemistry data, three had lost all PTEN expression, one showed weak PTEN expression levels, and one had mixed tumor cell populations with weak and moderate expression levels. These results suggest that PTEN frameshift mutations in HNPCC and sporadic MSI+ tumors are a consequence of mismatch repair deficiency. Further, hemizygous deletions in MSI- CRCs lead to loss or reduction of PTEN protein levels and contribute to tumor progression. Finally, our data also suggest that epigenetic inactivation of PTEN, including differential subcellular compartmentalization, occurs in CRCs.
Asunto(s)
Cromosomas Humanos Par 10 , Neoplasias Colorrectales/genética , Repeticiones de Microsatélite/genética , Mutación , Monoéster Fosfórico Hidrolasas/genética , Proteínas Supresoras de Tumor/genética , Disparidad de Par Base , Neoplasias Colorrectales/metabolismo , Reparación del ADN , Humanos , Pérdida de Heterocigocidad , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Inactivating germ-line mutations of LKB1 lead to Peutz-Jeghers syndrome (PJS). We have generated mice heterozygous for a targeted inactivating allele of Lkb1 and found that they develop severe gastrointestinal polyposis. In all cases, the polyps arising in the Lkb1+/- mice were found to be hamartomas that were histologically indistinguishable from polyps resected from PJS patients, indicating that Lkb1+/- mice model human PJS polyposis. No evidence for inactivation of the remaining wild-type Lkb1 allele in Lkb1+/- -associated polyps was observed. Moreover, polyps and other tissues in heterozygote animals exhibited reduced Lkb1 levels and activity, indicating that Lkb1 was haploinsufficient for tumor suppression. Analysis of the molecular mechanisms characterizing Lkb1+/- polyposis revealed that cyclooxygenase-2 (COX-2) was highly up-regulated in murine polyps concomitantly with activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). Subsequent examination of a large series of human PJS polyps revealed that COX-2 was also highly up-regulated in the majority of these polyps. These findings thereby identify COX-2 as a potential target for chemoprevention in PJS patients.
Asunto(s)
Proteínas Portadoras , Isoenzimas/biosíntesis , Síndrome de Peutz-Jeghers/enzimología , Síndrome de Peutz-Jeghers/genética , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas Quinasas Activadas por AMP , Proteínas Adaptadoras Transductoras de Señales , Animales , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/metabolismo , Inducción Enzimática , Genes Supresores de Tumor , Heterocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular , Linfocinas/metabolismo , Proteínas de la Membrana , Ratones , Ratones Noqueados , Síndrome de Peutz-Jeghers/tratamiento farmacológico , Síndrome de Peutz-Jeghers/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Mutations in the DNA MMR genes MSH2, MLH1, MSH6 and PMS2 underlie a large subset of HNPCC cases, and a hallmark of the tumors is MSI. In many HNPCC families, however, a causative mutation has not been found. Therefore, the involvement of additional, thus far unknown, genes in MSI as well as MSS colorectal tumor predisposition is possible. The role of a relatively recently cloned MMR gene, MLH3, in familial CRC has been studied; but the results appear somewhat conflicting. To further evaluate the role of MLH3 in CRC predisposition, we analyzed 30 Finnish CRC cases for germline mutations by sequencing. These cases were selected from a large series of Finnish CRC patients, to match features previously proposed to associate with MLH3 germline defects. We found 5 missense variants, 4 of which were also found in Finnish cancer-free controls. The only remaining variant does not appear to be an attractive candidate for a disease-associated mutation because the amino acid change is located outside the conserved residues. We also screened for the previously reported variants, including a frameshift change, the most likely pathogenic MLH3 mutation observed so far. The frameshift was not present in the 30 CRC cases or in 700 cancer-free controls. While it is a difficult task to exclude a role of MLH3 in HNPCC, our study could not confirm a role for MLH3 in CRC predisposition.
Asunto(s)
Proteínas Portadoras/genética , Neoplasias Colorrectales/genética , Reparación del ADN/genética , Predisposición Genética a la Enfermedad/genética , Mutación/genética , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Cartilla de ADN/química , ADN de Neoplasias/análisis , Finlandia , Frecuencia de los Genes/genética , Pruebas Genéticas , Humanos , Proteínas MutL , Reacción en Cadena de la PolimerasaRESUMEN
Germline mutations in the fumarate hydratase (FH) gene at 1q43 predispose to dominantly inherited cutaneous and uterine leiomyomas, uterine leiomyosarcoma, and papillary renal cell cancer (HLRCC syndrome). To evaluate the role of FH inactivation in sporadic tumorigenesis, we analyzed a series of 299 malignant tumors representing 10 different malignant tumor types for FH mutations. Additionally, 153 uterine leiomyomas from 46 unselected individuals were subjected to and informative in loss of heterozygosity analysis at the FH locus, and the five (3.3%) tumors displaying loss of heterozygosity were subjected to FH mutation analysis. Although mutation search in the 299 malignant tumors was negative, somatic FH mutations were found in two nonsyndromic leiomyomas; a splice site change IVS4 + 3A>G, leading to deletion of exon four, and a missense mutation Ala196Thr. The occurrence of somatic mutations strongly suggests that FH is a true target of the 1q43 deletions. Although uterine leiomyomas are the most common tumors of women, specific inactivating somatic mutations contributing to the formation of nonsyndromic leiomyomas have not been reported previously. Taking into account the apparent risk of uterine leiomyosarcoma associated with FH germline mutations, the finding raises the possibility that also some nonsyndromic leiomyomas may have a genetic profile that is more prone to malignant degeneration. Our data also indicate that somatic FH mutations appear to be limited to tumor types observed in hereditary leiomyomatosis and renal cell cancer.