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Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report.

ABSTRACT: Global analysis of gene expression by DNA microarrays is nowadays a widely used tool, especially relevant for cancer research. It helps the understanding of complex biology of cancer tissue, allows identification of novel molecular markers, reveals previously unknown molecular subtypes of cancer that differ by clinical features like drug susceptibility or general prognosis. Our aim was to compare gene expression profiles in breast cancer that develop against a background of inherited predisposing mutations versus sporadic breast cancer. In this preliminary study we analysed seven hereditary, BRCA1 mutation-linked breast cancer tissues and seven sporadic cases that were carefully matched by histopathology and ER status. Additionally, we analysed 6 samples of normal breast tissue. We found that while the difference in gene expression profiles between tumour tissue and normal breast can be easily recognized by unsupervised algorithms, the difference between those two types of tumours is more discrete. However, by supervised methods of data analysis, we were able to select a set of genes that may differentiate between hereditary and sporadic tumours. The most significant difference concerns genes that code for proteins engaged in regulation of transcription, cellular metabolism, signalling, proliferation and cell death. Microarray results for chosen genes (TOB1, SEPHS2) were validated by real-time RT-PCR.

SUBMITTER: Dudaladava V

PROVIDER: S-EPMC3401917 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report.

Dudaladava Volha V Jarzab Michał M Stobiecka Ewa E Chmielik Ewa E Simek Krzysztof K Huzarski Tomasz T Lubiński Jan J Pamuła Jolanta J Pekala Wioletta W Grzybowska Ewa E Lisowska Katarzyna K

Hereditary cancer in clinical practice 20060115 1

Global analysis of gene expression by DNA microarrays is nowadays a widely used tool, especially relevant for cancer research. It helps the understanding of complex biology of cancer tissue, allows identification of novel molecular markers, reveals previously unknown molecular subtypes of cancer that differ by clinical features like drug susceptibility or general prognosis. Our aim was to compare gene expression profiles in breast cancer that develop against a background of inherited predisposin ...[more]

PMID: 20223001

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Project description:Background: Breast cancer is a complex disease, encompassed by different clinically and molecularly stratified entities. In 2000, Perou and colleagues demonstrated that tumor phenotypic diversity correlates with differences in global gene expression patterns, which in turn reflect aspects of the biological behavior of the tumors. Gene expression profiling has distinguished sporadic breast tumor sub-classes with genetic, histopathological and clinical differences, however, little is known about the molecular classification of hereditary breast tumors. It has been recently suggested that most tumors arising in BRCA1 mutation carriers display a basal-like phenotype, with the percentages reported in different studies ranging from 44 to almost 100%. In the present study we used expression profiling to produce a molecular classification of BRCA1-associated breast tumors and applied an integrative approach to examine biological dependencies or differences. Methods and Findings: We used frozen tumor tissue from fourteen patients all of which harbored germline pathological mutations in BRCA1. Total RNA extraction, amplification and labeling with Cy5 were performed using standard protocols. Universal Human Reference RNA (Stratagene) was used as a reference and labeled with Cy3. Each pair of Cy3/Cy5 samples was hybridized onto the CNIO human OncoChip V2, which consists of a spotted microarray with 11,675 cDNA clones from the I.M.A.G.E. Consortium. Two channel ratios (Cy5/Cy3) for each spot were generated and quantified using GenePix Pro 5.1 (Axon Instruments, Inc., Union City, CA, USA). Data were normalized and filtered and multiple statistical analyses were performed. The data are deposited in the GEO database under the accession number GSE12350. A tissue microarray (TMA) containing an independent series of 15 BRCA1 tumors was used to validate some of the results obtained. We have described molecular signatures that define BRCA1 subclasses depending on the expression of the gene encoding for Estrogen Receptor, ESR1. Signatures were found to be molecularly associated with different biological processes and transcriptional regulatory programs. The signature of ESR1-positive tumors was mainly linked to cell proliferation-related processes and, therefore, regulated by the estrogen receptor, while the signature of ESR1-negative tumors was mainly linked to the immune response and possibly regulated by transcription factors of the REL/NFκB family. These signatures were then verified in an independent series of hereditary and sporadic breast tumors, which revealed a possible prognostic value for each subclass. Over-expression of immune response genes appears to be a common feature of ER-negative sporadic and hereditary breast cancer and is associated with good prognosis. Interestingly, the ESR1-negative tumors were sub-stratified into two groups presenting light differences in the magnitude of the expression of immune response transcripts and REL/NFκB transcription factors, and this could be dependent on the type of germline alteration. In addition, analysis of the human protein-protein interaction network provides the wiring diagram of critical molecular associations in BRCA1 tumorigenesis and identifies close relationships between the different signatures. Conclusions: In summary, in the present study we have established the gene expression profiling of a series of BRCA1 tumors and found that there is a further degree of heterogeneity beyond the main classification by the expression of ESR1 and the presence or absence of a basal-like phenotype. We have identified specific signatures for ESR1-positive and ESR1-negative BRCA1 tumors, the latter characterized by the enrichment of immune response and cell cycle genes, and have found that slight differences in the level of expression of the immune response stratify the ESR1-negative BRCA1 tumors into two additional sub-groups (A and B) with possible prognostic differences. NFkB could be a major driver responsible for the levels of both immune response and apoptotic genes in this group of tumors, which could in turn be related to the type of germline mutation in BRCA1. This study reveals the molecular complexity of BRCA1 breast tumors, which are found to display similarities to sporadic tumors, and suggests possible prognostic implications. 14 samples (primary breast tumors containing a BRCA1 mutation) were hybridized to a cDNA microarray in order to investigate the possible heterogeneity within the BRCA1 group.

Project description:BACKGROUND:MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene expression regulation and have been described as key regulators of carcinogenesis. Aberrant miRNA expression has been frequently reported in sporadic breast cancers, but few studies have focused on profiling hereditary breast cancers. In this study, we aimed to identify specific miRNA signatures in hereditary breast tumors and to compare with sporadic breast cancer and normal breast tissues. METHODS:Global miRNA expression profiling using NanoString technology was performed on 43 hereditary breast tumors (15 BRCA1, 14 BRCA2, and 14 BRCAX), 23 sporadic breast tumors and 8 normal breast tissues. These normal breast tissues derived from BRCA1- and BRCA2- mutation carriers (n =?5) and non-mutation carriers (n =?3). Subsequently, we performed receiver operating characteristic (ROC) curve analyses to evaluate the diagnostic performance of differentially expressed miRNAs. Putative target genes of each miRNAs considered as potential biomarkers were identified using miRDIP platform and used for pathway enrichment analysis. RESULTS:miRNA expression analyses identified several profiles that were specific to hereditary breast cancers. A total of 25 miRNAs were found to be differentially expressed (fold change: >?2.0 and p <?0.05) and considered as potential biomarkers (area under the curve >?0.75) in hereditary breast tumors compared to normal breast tissues, with an expressive upregulation among BRCAX cases. Furthermore, bioinformatic analysis revealed that these miRNAs shared target genes involved in ErbB, FoxO, and PI3K-Akt signaling pathways. CONCLUSIONS:Our results showed that miRNA expression profiling can differentiate hereditary from sporadic breast tumors and normal breast tissues. These miRNAs were remarkably deregulated in BRCAX hereditary breast cancers. Therefore, miRNA signatures can be used as potential novel diagnostic biomarkers for the prediction of BRCA1/2- germline mutations and may be useful for future clinical management.

Project description:ObjectiveBreast cancer susceptibility gene 1/2 (BRCA1/2) is the most important susceptibility gene associated with hereditary ovarian cancer (HOC). We aimed to screen BRAC1 and BRAC2 gene mutations in a member of a hereditary ovarian cancer family in China, and to analyze the structure and function of the mutant protein.MethodsA typical HOC family was selected. Blood samples and pathological tissue samples were taken from the female members of the family. Blood samples from two patients with sporadic ovaries of the same pathological type were taken as a control group. After RNA extraction, PCR amplification was applied and the PCR products were directly sequenced and aligned, prediction and analysis of protein structure and molecular conformation that may be caused by BRCA1/2 mutation.ResultsThe whole gene analysis of BRCA1 and BRCA2 in ovarian cancer patients in the family showed that there were 8 mutations in BRCA1 whole gene sequencing, including 3 nonsense mutations (2314C>T, 2543T>C, 4540T>C); two mutations have been recorded, which are associated with cervical cancer (2844C>T) and endometriosis (3345A>G); three newly discovered mutations (3780A>G, 5069A>G, 3326A>T). Among them, 3780A>G and 5069A>G caused amino acid changes, while 3326A>T mutation caused Arg mutation to stop codon. A total of 7 mutations were detected in BRCA2 whole-genome sequencing, including 5 non-significant mutations (3623A>G, 4034T>C, 4790A>G, 6740G>C, 7469A>G); one no-record mutation (1716T>A), and 1 recorded mutation (1342A>C), which was associated with breast cancer and ovarian cancer. BRCA1 (3326A>T) and BRCA2 (1342A>C) mutations were co-existing in patients (II1, II3, and II5) identified as serous adenocarcinoma grade II. Two cases of ovarian serous cystadenocarcinoma with no history of family tumors were normalized for BRCA1/2 gene sequencing. In the gene detection of III generation female, four females with BRCA2 (1342A>C) mutation were found, and one of them also carried the BRCA1 (3326A>T) mutation, who can be considered a high-risk group of HOC in this family. Online protein structure predictions revealed that BRCA1 (3326A>T) mutations mutated AGA at this site to TGA resulting in a translated Arg (arginine) mutation as a stop codon, while BRCA2 (1342A>C) mutated AAT at this site to CAT resulting in a translated Asn mutation to His.ConclusionThe BRCA1 (3326A>T) and BRCA2 (1342A>C) were detected in the HOC family, which may be the susceptibility gene of the family's HOC. The BRCA1/2 gene screening may be possible to obtain high-risk populations in this family.

Project description:Array CGH analysis of breast tumors has contributed to the identification of different genomic profiles in these tumors. Loss of DNA repair by BRCA1 functional deficiency in breast cancer has been proposed as a relevant contribution to breast cancer progression for tumors with no germline mutation. Identifying the genomic alterations taking place in BRCA1 not expressing tumors will lead us to a better understanding of the cellular functions affected in this heterogeneous disease. Moreover, specific genomic alterations may contribute to the identification of potential therapeutic targets and offer a more personalized treatment to breast cancer patients.Forty seven tumors from hereditary breast cancer cases, previously analyzed for BRCA1 expression, and screened for germline BRCA1 and 2 mutations, were analyzed by Array based Comparative Genomic Hybridization (aCGH) using Agilent 4x44K arrays. Overall survival was established for tumors in different clusters using Log-rank (Mantel-Cox) Test. Gene lists obtained from aCGH analysis were analyzed for Gene Ontology enrichment using GOrilla and DAVID tools.Genomic profiling of the tumors showed specific alterations associated to BRCA1 or 2 mutation status, and BRCA1 expression in the tumors, affecting relevant cellular processes. Similar cellular functions were found affected in BRCA1 not expressing and BRCA1 or 2 mutated tumors. Hierarchical clustering classified hereditary breast tumors in four major, groups according to the type and amount of genomic alterations, showing one group with a significantly poor overall survival (p = 0.0221). Within this cluster, deletion of PLEKHO1, GDF11, DARC, DAG1 and CD63 may be associated to the worse outcome of the patients.These results support the fact that BRCA1 lack of expression in tumors should be used as a marker for BRCAness and to select these patients for synthetic lethality approaches such as treatment with PARP inhibitors. In addition, the identification of specific alterations in breast tumors associated with poor survival, immune response or with a BRCAness phenotype will allow the use of a more personalized treatment in these patients.

Dataset Information

Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report.

Publications

Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report.

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