RESUMO
Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, but account for only a small fraction of breast cancer susceptibility. To find additional genes conferring susceptibility to breast cancer, we analyzed CHEK2 (also known as CHK2), which encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53 (refs 3,4,5). We show that CHEK2(*)1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2 (P = 0.00000003), including 13.5% of individuals from families with male breast cancer (P = 0.00015). We estimate that the CHEK2(*)1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway.
Assuntos
Neoplasias da Mama/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Deleção de Sequência , Estudos de Casos e Controles , Quinase do Ponto de Checagem 2 , Feminino , Genes BRCA1 , Genes BRCA2 , Testes Genéticos , Heterozigoto , Humanos , Masculino , Repetições de Microssatélites , Mutação , Linhagem , Fatores de RiscoRESUMO
Mutations of E-cadherin have been identified in half of lobular breast cancers and diffuse-type gastric cancers, two tumor subtypes with remarkably similar pathological appearances including small rounded cells with scant cytoplasm and a diffuse growth pattern. A causal role for E-cadherin gene mutations in the lobular breast cancer phenotype was recently demonstrated in E-cadherin knock-out mice. These observations suggested that another gene in the E-cadherin tumor suppressor pathway might be mutated in lobular breast cancers with wild-type E-cadherin genes. Here, we identified E-cadherin gene mutations exclusively in human breast cancer cell lines that grow with a rounded cell morphology. Using expression analyses and gene mutation analyses, we have identified four biallelic inactivating alpha-catenin mutations among 55 human breast cancer cell lines. All four alpha-catenin mutations predicted premature termination of the encoded proteins, and concordantly, none of the four mutant cell lines expressed alpha-catenin proteins. Importantly, three of the alpha-catenin mutant cell lines had the rounded cell morphology and all 14 cell lines with the rounded cell morphology had mutations of either E-cadherin or alpha-catenin. As anticipated, loss of alpha-catenin protein expression was associated with the lobular subtype in primary breast cancers. Together, our observations suggest that alpha-catenin may be a new tumor suppressor gene that operates in the E-cadherin tumor suppressor pathway.
Assuntos
Neoplasias da Mama/patologia , Carcinoma/patologia , Códon sem Sentido , Genes Supressores de Tumor , Proteínas de Neoplasias/genética , alfa Catenina/genética , Alelos , Neoplasias da Mama/genética , Caderinas/genética , Caderinas/fisiologia , Carcinoma/genética , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Adesão Celular/genética , Linhagem Celular Tumoral/metabolismo , Forma Celular/genética , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/deficiência , Análise de Sequência com Séries de Oligonucleotídeos , alfa Catenina/biossíntese , alfa Catenina/deficiênciaRESUMO
Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Análise Mutacional de DNA , Feminino , Expressão Gênica , Humanos , MutaçãoRESUMO
BACKGROUND: Low-risk breast cancer susceptibility alleles or SNPs confer only modest breast cancer risks ranging from just over 1.0 to 1.3 fold. Yet, they are common among most populations and therefore are involved in the development of essentially all breast cancers. The mechanism by which the low-risk SNPs confer breast cancer risks is currently unclear. The breast cancer association consortium BCAC has hypothesized that the low-risk SNPs modulate expression levels of nearby located genes. METHODS: Genotypes of five low-risk SNPs were determined for 40 human breast cancer cell lines, by direct sequencing of PCR-amplified genomic templates. We have analyzed expression of the four genes that are located nearby the low-risk SNPs, by using real-time RT-PCR and Human Exon microarrays. RESULTS: The SNP genotypes and additional phenotypic data on the breast cancer cell lines are presented. We did not detect any effect of the SNP genotypes on expression levels of the nearby-located genes MAP3K1, FGFR2, TNRC9 and LSP1. CONCLUSION: The SNP genotypes provide a base line for functional studies in a well-characterized cohort of 40 human breast cancer cell lines. Our expression analyses suggest that a putative disease mechanism through gene expression modulation is not operative in breast cancer cell lines.
Assuntos
Alelos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Éxons , Perfilação da Expressão Gênica , Genes Neoplásicos , Predisposição Genética para Doença , Genótipo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RiscoRESUMO
Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.
Assuntos
Neoplasias da Mama/genética , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas ras/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Análise Mutacional de DNA , Humanos , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase , Proteínas ras/metabolismoRESUMO
Germ line mutations of the BRCA1 gene confer a high risk of breast cancer and ovarian cancer to female mutation carriers. The BRCA1 protein is involved in the regulation of DNA repair. How specific tumor-associated mutations affect the molecular function of BRCA1, however, awaits further elucidation. Cell lines that harbor BRCA1 gene mutations are invaluable tools for such functional studies. Up to now, the HCC1937 cell line was the only human breast cancer cell line with an identified BRCA1 mutation. In this study, we identified three other BRCA1 mutants from among 41 human breast cancer cell lines by sequencing of the complete coding sequence of BRCA1. Cell line MDA-MB-436 had the 5396 + 1G>A mutation in the splice donor site of exon 20. Cell line SUM149PT carried the 2288delT mutation and SUM1315MO2 carried the 185delAG mutation. All three mutations were accompanied by loss of the other BRCA1 allele. The 185delAG and 5396 + 1G>A mutations are both classified as pathogenic mutations. In contrast with wild-type cell lines, none of the BRCA1 mutants expressed nuclear BRCA1 proteins as detected with Ab-1 and Ab-2 anti-BRCA1 monoclonal antibodies. These three new human BRCA1 mutant cell lines thus seem to be representative breast cancer models that could aid in further unraveling of the function of BRCA1.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Mutação , Adenocarcinoma/genética , Adulto , Sequência de Bases , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
Seminomas and nonseminomas represent the invasive stages of testicular (TGCTs) of adolescents and adults. Although TGCTs are characterized by extra copies of the short arm of chromosome 12, the genetic basis for gain of 12p in the pathogenesis of this cancer is not yet understood. We have demonstrated that gain of 12p is related to invasive growth and that amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates with reduced apoptosis in the tumors. Here we show that three known genes map within the newly determined shortest region of overlap of amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to the telomeric region of the SROA, DAD-R is the first gene at the centromeric region within the 12p amplicon. Although all three genes are amplified to the same level within the SROA, expression of DAD-R is significantly up-regulated in seminomas with the restricted 12p amplification compared with seminomas without this amplicon. DAD-R is also highly expressed in nonseminomas of various histologies and derived cell lines, both lacking such amplification. This finding is of particular interest because seminomas with the restricted 12p amplification and nonseminomas are manifested clinically in the third decade of life and show a low degree of apoptosis. In contrast, seminomas lacking a restricted 12p amplification, showing significantly lower levels of DAD-R with pronounced apoptosis, manifest clinically in the fourth decade of life. A low level of DAD-R expression is also observed in normal testicular parenchyma and in parenchyma containing the precursor cells of this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression in seminomas and nonseminomas correlates with invasive growth and a reduced level of apoptosis associated with an earlier clinical presentation. These data implicate DAD-R as a candidate gene responsible in part for the pathological effects resulting from gain of 12p sequences in TGCTs. In addition, our results also imply differences in expression regulation of DAD-R between seminomas and nonseminomas.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Seminoma/genética , Adulto , Fatores Etários , Cromossomos Humanos Par 12/genética , Proteínas de Ligação a DNA/biossíntese , Amplificação de Genes , Humanos , Masculino , Proteínas Nucleares/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Pseudogenes , Proteínas Repressoras/biossíntese , Fatores de Transcrição SOXD , Seminoma/metabolismo , Seminoma/patologia , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling-normal-like, basal, HER2-positive, and luminal A and B-were identified by CellSearch, a US Food and Drug Administration-approved test that uses antibodies against the cell surface-expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed.
Assuntos
Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/sangue , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/imunologia , Células Neoplásicas Circulantes , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Feminino , Perfilação da Expressão Gênica , Humanos , Células Neoplásicas Circulantes/patologia , Valor Preditivo dos Testes , Projetos de PesquisaRESUMO
BACKGROUND: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples. PRINCIPAL FINDINGS: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number of candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs). CONCLUSIONS: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes.
Assuntos
Neoplasias da Mama/genética , Éxons/genética , Genes Neoplásicos , Sequência de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Genética Populacional , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , Polimorfismo de Nucleotídeo Único , Transcrição GênicaRESUMO
The p53 tumor suppressor gene is frequently mutated in breast cancer. Here, we used direct sequencing to screen the complete coding sequence of the p53 gene from 41 human breast cancer cell lines. We identified 32 cell lines (78%) with a p53 gene alteration that predicted a change in the encoded protein. Thirty-one of these mutations were accompanied by loss of the other p53 allele. All mutations but one were unique and 27 mutations had previously been identified in uncultured human cancers. Ten mutations were predicted to encode a truncated p53 protein and 22 missense mutations were identified. p53 transcript expression was analyzed by semi-quantitative RT-PCR and p53 protein expression was determined by Western blotting. Our analyses revealed three p53 expression patterns: wild-type p53 cell lines had normal transcript levels and low or no detectable protein expression; cell lines with a p53 truncating mutation had low transcript levels and low or no detectable protein expression; and cell lines with a p53 missense mutation had highly variable transcript and protein expression levels. As a whole, our data represent a p53 mutation profile in breast cancer cell lines, providing a model for structural, functional and pharmacological studies on p53 in human cancer.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Genes p53 , Mutação , Proteína Supressora de Tumor p53/biossíntese , Alelos , Linhagem Celular Tumoral , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Reação em Cadeia da PolimeraseRESUMO
Because of genetic heterogeneity, the identification of breast cancer-susceptibility genes has proven to be exceedingly difficult. Here, we define a new subset of families with breast cancer characterized by the presence of colorectal cancer cases. The 1100delC variant of the cell cycle checkpoint kinase CHEK2 gene was present in 18% of 55 families with hereditary breast and colorectal cancer (HBCC) as compared with 4% of 380 families with non-HBCC (P<.001), thus providing genetic evidence for the HBCC phenotype. The CHEK2 1100delC mutation was, however, not the major predisposing factor for the HBCC phenotype but appeared to act in synergy with another, as-yet-unknown susceptibility gene(s). The unequivocal definition of the HBCC phenotype opens new avenues to search for this putative HBCC-susceptibility gene.