The expression and mutations of BRCA in breast cancer and ovarian cancer: Evidence from bioinformatic analyses

Abstract

BRCA1 and BRCA2 are the best-known genes linked to breast cancer and ovarian cancer, which play crucial roles in DNA repair and transcriptional regulation. Depicting the expression profiles, mutations and interaction networks of BRCA1 and BRCA2 may provide insights to reveal the mechanism of BRCA genes ultimately leading to breast or ovarian tumorigenesis. In this study, BRCA1 and BRCA2 mRNA levels were evaluated using FIREHOSE analysis and Oncomine analysis. cBioPortal analysis and COSMIC analysis were used to explore the BRCA1 and BRCA2 mutations. Kaplan-Meier Plotter was performed to identify the prognostic roles of BRCA1 and BRCA2 in breast cancer and ovarian cancer. Results showed that, first, higher BRCA1 and BRCA2 mRNA expression levels were elevated in breast cancer and ovarian cancer tissues as compare to their matched normal tissues; Second, several common mutations of BRCA1 and BRCA2 genes were identified in both breast cancer and ovarian cancer; Third, NF1, SYCP2 and TP53 were predicted to involve in the interaction network of BRCA1 and BRCA2 in both breast cancer and ovarian cancer. Our results provide a significant insight into that some of the mutations and proteins involved in the interaction network of BRCA1 and BRCA2 that might play common roles in both breast cancer and ovarian cancer. However, the deep mechanism of these observation remains unclear, and further researches need to be performed in the future.

Keywords

Breast cancer ovarian cancer expression profile bioinformatics mutation

Background

Breast cancer is the most frequent cancer which is expected to account for 30% of all new cancer diagnoses in women in the United States.¹ Ovarian cancer is also a significant source of morbidity and mortality, which ranks the seventh-most common cancer and the eighth-most common cause of death from cancer among women.² The vast majority of breast cancer cases are due to gene mutations, particularly mutations in breast cancer 1 (BRCA1) and/or breast cancer 2 (BRCA2), which put them at high risk for developing a second breast cancer and ovarian cancer.³ Moreover, both men and women carrying BRCA1 and/or BRCA2 mutations have a 50% chance to pass the mutations on to their children (hereditary breast and ovarian cancer (HBOC) syndrome), which is characterized by an increased risk of breast cancer and ovarian cancer.⁴ Thus, to understand the expression and mutations of BRCA1/2 in breast cancer and ovarian cancer is an urgent need in clinical practice.

BRCA1 and BRCA2 genes are both tumor suppressors. BRCA1 is a large gene which comprises 24 exons located on chromosome 17 (17q21) and codes for an 1863 amino acid protein with a zinc-binding really interesting new gene (RNG) finger motif at the amino terminus and a conserved acidic carboxyl terminal BRCA1 C terminus (BRCT) motif.^5,6 BRCA2 gene is located on chromosome 13 (13q12), which codes for a 3418 amino acid protein, and shares structural and functional similarities with BRCA1 protein. It is currently quite clear that the normal protein products of BRCA1 and BRCA2 genes play critical roles in double-strand DNA repairs by maintaining genomic integrity through RAD51,⁷ and they also participate in pathways associated with homologous recombination.⁸ However, once either of these genes is mutated or altered, DNA damage may not be repaired properly, likely leading to occurrence of cancers. In patients with BRCA1 and/or BRCA2 mutations, the risk of breast cancer is significantly higher as compared to that in general population, and the histological grade is also more aggressive.^9–11

In this study, we mainly investigated the expression levels and mutations of BRCA1 and BRCA2 in breast cancer and ovarian cancer by Bioinformatic Analyses.

Materials and methods

FIREHOSE analysis for BRCA1 and BRCA2

The expression profile of BRCA1 and BRCA2 across various human cancer types were explored through the Broad Institute FireBrowse portal (http://firebrowse.org/). On the homepage, type “BRCA1” or “BRCA2” in the search box and click on “View Expression Profile.” The boxplots show the expression level of target gene. Red bars are for tumor samples and blue bars are for normal samples.

Oncomine database analysis

BRCA1 and BRCA2 messenger RNA (mRNA) levels in breast cancer and ovarian cancer tissues were compared with their matched normal tissues using The Cancer Genome Atlas (TCGA) datasets in Oncomine database (http://www.oncomine.org). The threshold used to obtain the most significant probes of the queried gene for each microarray data included a two-fold difference in expression between cancers and normal tissues with a p value <1 × 10⁻⁴. For each gene, the mRNA expression level in three independent datasets was analyzed.

Kaplan–Meier plotter analysis

The prognostic value of BRCA1 and BRCA2 gene in breast cancer and ovarian cancer was analyzed using Kaplan–Meier Plotter (http://kmplot.com/analysis/). Overall survival of the patient with high and low levels of BRCA1 or BRCA2 was shown by using a Kaplan–Meier survival plot.

COSMIC analysis for BRCA1 and BRCA2 mutations

The Catalogue of Somatic Mutations in Cancer (COSMIC) database (http://cancer.sanger.ac.uk/cosmic) was used for analysis of BRCA1 and BRCA2 mutations. Pie charts were generated for a distribution overview and substitutions on the coding strand in breast cancer and ovarian cancer.

cBioPortal analysis for alteration frequency and interaction network of BRCA1 and BRCA2

Alteration frequency of BRCA1 and BRCA2 mRNA in breast cancer and ovarian cancer was performed using cBioPortal for Cancer Genomics (http://www.cbioportal.org/). All searches and analyses were performed according to the online instructions of cBioPortal.^12,13

Results

BRCA1 and BRCA2 were upregulated in breast cancer and ovarian cancer

We surveyed the level of BRCA1 and BRCA2 gene expression in 38 human cancers by using the TCGA database. The columns in the Figure 2 represented the accurate quantification of gene and isoform expression from RNA-Seq data. The results showed that higher levels of BRCA1 (Figure 1(a)) and BRCA2 (Figure 1(b)) transcripts were observed in almost all cancer tissues compared with their matched normal tissues. Interestingly, the BRCA1 and BRCA2 gene exhibited the similar expression pattern in various cancer types, including breast cancer and ovarian cancer (Figure 1).

Figure 1.

FIREHOSE analysis for BRCA1 and BRCA2 expression profile. The (a) BRCA1 and (b) BRCA2 genes show higher mRNA expression level in multiple human cancers as compared to their matched normal tissues. The boxplots show the expression level, red bars are for tumor samples and blue bars are for normal samples. Results showed that the expression of both BRCA1 and BRCA2 is the highest in acute myeloid leukemia (LAML) and lowest in kidney chromophobe (KICH).

Figure 2.

BRCA1 and BRCA2 mRNAs were evaluated in breast cancer and ovarian cancer using Oncomine analysis. (a) BRCA1 and (b) BRCA2 mRNA levels in breast cancer samples. (c) BRCA1 and (d) BRCA2 mRNA levels in ovarian cancer samples. For each gene, the mRNA expression levels were analyzed in two independent datasets (*p < 0.05 vs normal tissues).

Oncomine analysis of cancer versus normal tissue also showed that the BRCA1 and BRCA2 were significantly upregulated in breast cancer and ovarian cancer (Figure 2) in three independent studies as compared to corresponding normal tissue.^14,15 Kaplan–Meier analysis revealed that high BRCA1 expression level was correlated with a poor survival rate in breast cancers (p = 1.51E−11; Figure 3(a)), while there was no significant correlation between BRCA1 expression level and the survival rate in ovarian cancers (p = 0.13; Figure 3(c)). In addition, we found that high BRCA2 expression level was correlated with poor survival rate in both breast cancers (p = 0.00093; Figure 3(b)) and ovarian cancers (p = 1.0E−9; Figure 3(d)).

Figure 3.

Kaplan–Meier plotter analysis of BRCA1 and BRCA2 in breast cancer and ovarian cancer. (a) BRCA1 and (b) BRCA2 expression levels were negatively related to overall survival of breast cancer. (c) BRCA1 expression level exhibited no significant correlation with the overall survival of ovarian cancer. (d) BRCA2 expression level showed negative correlation with the overall survival of ovarian cancer. Red line shows the overall survival with high levels of BRCA1 or BRCA2, and black line shows the low levels of BRCA1 or BRCA2 in breast cancer and ovarian cancer.

BRCA1 and BRCA2 mutation in breast cancer and ovarian cancer

We studied the mutation of BRCA1/2 using COSMIC, and the information of mutations of substitution missense, nonsense, synonymous, and insertion frame-shift is shown in pie chart (Figure 4). In both breast cancer and ovarian cancer, the most frequent mutation is substitution missense in BRCA1/2 genes. In the mutation samples of breast cancer, 50% of BRCA1 and 54.9% of BRCA2 were substitution missense (Figure 4(a) and (c)). In ovarian cancers, 31.87% of BRCA1 and 44.62% of BRCA2 were substitution missense (Figure 4(b) and (d)). Mutation samples of breast cancer have 23.33% G > A, 20.00% G > C and 20.00% G > T substitution in BRCA1 coding strand (Figure 4(a)), and 22.58% G > C and 19.35% G > A substitution in BRCA2 coding strand (Figure 4(c)). In ovarian cancer mutation samples, there were 32.61% G > T and 21.71% G > A substitution in BRCA1 coding strand (Figure 4(b)) and 23.68% G > T and 21.05 G > A substitution in BRCA2 coding strand (Figure 4(d)).

Figure 4.

COSMIC analysis for BRCA1 and BRCA2 mutations. Pie-chart showed the percentage of the mutation type of BRCA1 and BRCA2 in breast cancer and ovarian cancer according to COSMIC database. (a) BRCA1 and (c) BRCA2 mutations in breast cancer. (b) BRCA1 and (d) BRCA2 mutations in ovarian cancers.

Furthermore, alteration frequency of BRCA1 and BRCA2 in breast cancer and ovarian cancer was analyzed using BioPortal. Eight studies of breast cancer and two studies of ovarian cancer were included in the database. Results showed that about 2%–8% of breast cancer and 6%–22% of ovarian cancer clinical samples contained BRCA1 and/or BRCA2 mutation (Figure 5(a) and (b)). In breast cancer clinical samples, there were 81 mutations observed in BRCA1, 43 of which were missense mutations and 38 were in-frame mutation and truncating; there were 88 mutations in BRCA2, 42 of which were missense mutations and 46 were in-frame mutations and truncating. In ovarian cancer, there were 48 mutations of BRCA1 gene, only 2 of which were missense mutations and 46 were in-frame mutations and truncating; there were 46 mutations in BRCA2, 5 of which were missense mutations and 41 were in-frame mutations and truncating. Several common mutations were observed in BRCA1 (E1346Kfs*20, E23Vfs*17, and Q1756Pfs*74) and BRCA2 (V220Ifs*4, N1784Hfs*2, and S1982Rfs*22) in both breast cancer and ovarian cancer by pairwise analysis (Tables 1 and 2).

Figure 5.

Alteration frequency and interaction networks of BRCA1 and BRCA2 in breast cancer and ovarian cancer analyzed using cBioPortal. (a and b) The mutation studies of BRCA1 and BRCA2 in breast cancer and ovarian cancer. Result showed that about 2%–8% of breast cancer and 6%–22% of ovarian cancer clinical samples contained BRCA1 and/or BRCA2 mutation. (c and d) The interaction network analysis using cBioPortal.

Table 1.

Common mutations in BRCA1 observed in both breast cancer and ovarian cancer.

Cancer study	AA change	Type	Copy number	Number of mutations in sample
Breast (TCGA pub)	E1346Kfs*20	FS del	ShallowDel	138
Breast (TCGA pub)	E23Vfs*17	FS del	Gain	118
Breast (TCGA pub)	Q1756Pfs*74	FS ins	ShallowDel	65

TCGA: The Cancer Genome Atlas.

Table 2.

Common mutations in BRCA2 observed in both breast cancer and ovarian cancer.

Cancer study	AA change	Type	Copy number	Number of mutations in sample
Breast (TCGA pub)	V220Ifs*4	FS del	Diploid	69
Breast (TCGA pub)	N1784Hfs*2	FS del	ShallowDel	49
Breast (TCGA pub)	S1982Rfs*22	FS del	Diploid	48

TCGA: The Cancer Genome Atlas.

Interaction networks of BRCA1 and BRCA2 in breast cancer and ovarian cancer

The interaction networks of BRCA1 and BRCA2 were analyzed using BioPortal (Figure 5(c) and (d)). Among the genes involved in the interaction network, over 30% of them could form complexes with BRCA1 and BRCA2 in both breast cancer (30.6%) and ovarian cancer (36.5%). In breast cancer, BRCA1 could control the expression of cyclin-dependent kinase inhibitor 1B (CDKN1B, p27^Kip1). TEX15 could control the state of BRCA1 and BRCA2. Moreover, we found that three genes (NF1, SYCP2, and TP53) were involved in both breast cancer and ovarian cancer. BRCA1 could form complex with the protein of these three genes and also control the state change of SYCP2 in both breast cancer and ovarian cancer, which was related to cell cycle, mitosis, and meiosis.

Discussion

In women, BRCA1 or BRCA2 mutation results in about 40%–80% risk of developing breast cancer and about 11%–40% risk of developing ovarian cancer, respectively.^16–19 Recently, genetic testing for BRCA1 and BRCA2 mutations was used for identifying individuals at high risk of developing breast cancer and ovarian cancer.^20,21 In this study, we surveyed the latest evidence of the expression profiles and mutations of BRCA1 and BRCA2 using Bioinformatic Analyses/TCGA data portal. Higher mRNA level of BRCA1 and BRCA2 was identified in both breast cancer and ovarian cancer in three independent RNA-seq datasets, and there is a positive correlation between the mRNA expression level of BRCA genes and poor survival in breast cancer and ovarian cancer.

BRCA1 and BRCA2 gene expression is potentially an important tool for use in cancer management. A study in lung cancer showed that BRCA1 gene could serve as an indicator of chemo-resistance, and reconstitution of wild-type BRCA1 function into lung cancer cells results in thousand-fold increase in sensitivity to paclitaxel and vinorelbine.²² Another preclinical study in breast cancer has emphasized the potential of using BRCA1 and BRCA2 dysfunction to predict response to clinical treatment.²³ However, previous study showed that BRCA1 protein level exhibited a significant reduction in sporadic breast and ovarian cancers,^24,25 probably partially due to the different splice variants or localization of BRCA1 protein. Actually, the BRCA1 and BRCA2 expression level in human mammary epithelial and cancer cells varies with the cell cycle, which expressed in a cell-cycle-dependent manner, peaking at the G1/S boundary.

In this study, about 81 mutations in BRCA1 gene and 88 mutations in BRCA2 gene in breast cancer samples and 48 mutations in BRCA1 and 46 mutations in BRCA2 in ovarian cancer samples were observed. One striking point in this study is that three mutations in BRCA1 and three in BRCA2 are observed in both breast cancer and ovarian cancer, respectively, which indicated that these mutations may play common roles in both breast cancer and ovarian cancer. Moreover, the common mutations in BRCA1 and BRCA2 are frame-shift deletion and known to be oncogenic.²⁶

It is well known that BRCA1 and BRCA2 are involved in DNA repair, cell-cycle checkpoint regulation, and transcription,²⁷ and these processes are dictated through its crosstalk with a network of proteins. It is now clear that BRCA1 and BRCA2 protein co-localizes with RAD51 complexes on mitotic and meiotic chromosomes after exposure to ionizing radiation (IR) or hydroxyurea.^8,28 There are some other proteins reported to interact with BRCA1, such as ATM/ATR, CHK2, and Aurora A protein kinase, and regulate cell-cycle progression.²⁹ In this study, we identified three genes (NF1, SYCP2, and TP53) associated with BRCA1 in both breast cancer and ovarian cancer. Neurofibromin 1 (NF1) is a tumor suppressor gene that comprises 60 exons coding a protein called neurofibromin, which is related to neurofibromatosis–Noonan syndrome and neurofibromatosis, type 1. Among its related pathways are mitogen-activated protein kinase (MAPK) signaling pathway and Ras signaling pathway.³⁰ Both NF1 and BRCA1 gene are located in the long arm of the chromosome 17, while the role of NF1 in breast cancer has been suggested in some publications.^31,32 Synaptonemal complex protein 2 (SYCP2) is a testis-specific human gene and aberrantly expressed in human papillomavirus (HPV)+ cancers³³ and head and neck squamous cell carcinoma.³⁴ In our study, we found that BRCA1 could form complex with NF1 and SYCP2 in both breast cancer and ovarian cancer, respectively, which suggested that BRCA1 gene could interact with NF1 and SYCP2 directly or indirectly in cell-cycle regulation. However, further research will be required to discern the deep mechanisms of these observations in the future.

In summary, our findings revealed not only the increased expression pattern of BRCA1 and BRCA2 genes in breast cancer and ovarian cancer but also provided an understanding on the mutations and interaction networks of these two genes in the cancers mentioned above. Our results also provide a significant insight into some of the mutations and proteins involved in the interaction network that might play common roles in both breast cancer and ovarian cancer.

Footnotes

Acknowledgements

Z.W. and Y.W. have contributed equally to this study.

Contributorship

ZW performed the experiments and drafted the manuscript. YW performed the Oncomine analysis and cBioportal analysis. ZX performed the Kaplan-Meier plotter analysis. JZ and HL conceived of the study and its design, obtained fundings for the study, drafted and revised the manuscript. All authors read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by Shenzhen Science and Technology Program (Basic Research Project, No. JCYJ20150402144905865).

Guarantor

Not available

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