BRCA1/2 Serves as a Biomarker for Poor Prognosis in Breast Carcinoma

BRCA1/2 are breast cancer susceptibility genes that are involved in DNA repair and transcriptional control. They are dysregulated in breast cancer, making them attractive therapeutic targets. Here, we performed a systematic multiomics analysis to expound BRCA1/2 functions as prognostic biomarkers in breast cancer. First, using different web-based bioinformatics platforms (Oncomine, TIMER 2.0, UALCAN, and cBioportal), the expression of BRCA1/2 was assessed. Then, the R package was used to analyze the diagnostic value of BRCA1/2 in patients. Next, we determined the relationship between BRCA1/2 mRNA expression and prognosis in patients (PrognoScan Database, R2: Kaplan Meier Scanner and Kaplan–Meier Plotter). Subsequently, the association of BRCA1/2 with mutation frequency alteration and copy number alterations in breast cancer was investigated using the cBioportal platform. After that, we identified known and predicted structural genes and proteins essential for BRCA1/2 functions using GeneMania and STRING db. Finally, GO and KEGG pathway enrichment analyses were performed to elucidate the potential biological functions of the co-expression genes of BRCA1/2. The BRCA1/2 mRNA level in breast cancer tissues was considerably higher than in normal tissues, with AUCs of 0.766 and 0.829, respectively. Overexpression of BRCA1/2 was significantly related to the worse overall survival (p < 0.001) and was correlated to clinicopathological characteristics including lymph nodes, estrogen receptors, and progesterone receptors (p < 0.01). The alteration frequencies of both the gens have been checked, and the results show that BRCA1 and BRCA2 show different alteration frequencies. Their mutation sites differ from each other. GO and KEGG showed that BRCA1/2 was mainly enriched in catalytic activity, acting on DNA, chromosomal region, organelle fission, cell cycle, etc. The 20 most frequently changed genes were closely related to BRCA1/2, including PALB2 and RAD51 relatively. Our study provides suggestive evidence of the prognostic role of BRCA1/2 in breast cancer and the therapeutic target for breast cancer. Furthermore, BRCA1/2 may influence BRCA prognosis through catalytic activity, acting on DNA, chromosomal regions, organelle fission, and the cell cycle. Nevertheless, further validation is warranted.


Introduction
Breast cancer affects a staggering number of 1.7 million women worldwide each year, which is the main leading cause of cancer death in women [1,2]. Five to ten percent of breast cancers are considered to be related to genetic predispositions, and some breast cancers are caused by genetic dominant susceptibility genes, which have a high risk of disease [3].
BRCA1/2 are tumor suppressors that are essential for the faithful repair of doublestrand DNA breaks by homologous recombination. However, BRCA1/2 mutation occurs frequently in breast cancer [4]. The tumor suppressor genes BRCA1/2, which are found on chromosomes 17q and 13q, respectively, and encode factors that restrict cell development, were identified in the early 1990s [5][6][7][8][9]. BRCA1 encodes a nuclear phosphoprotein, which acts as a tumor suppressor gene through maintaining genomic stability [3,10]. BRCA1 is also involved in other cellular functions that are important in maintaining genomic integrity, including mitotic spindle assembly, centrosome replication, cell cycle control, and chromatin remodeling at dsDNA break sites [4]. BRCA2 is a 27-exon gene involved in genome stability, particularly in the homologous recombination (HR) pathway, which repairs double-stranded DNA breaks [3]. BRCA2related cancers, unlike BRCA1, frequently express estrogen and progesterone receptors and have many of the same features as sporadic breast cancer [11]. Women with germline BRCA1 or BRCA2 (BRCA1/2) mutations have a markedly increased risk of breast and ovarian cancer compared with the general population [12][13][14]. PALB2 is a protein-coding gene that is associated with both BRCA1 and BRCA2; it plays a critical role in homologous recombination repair (HRR) through its ability to recruit BRCA2 and RAD51 to DNA breaks [15]. BRCA1 promotes the concentration of PALB2 and BRCA2 at DNA-damage sites, and the interaction between BRCA1 and PALB2 is important for homologous recombination repair [15].
In recent years, BRCA1/2 has attracted considerable attention as a biomarker for predicting cancer prognosis. The identification of biomarkers, such as BRCA1/2, PIK3CA and RSK2, plays an important role in promoting the precise treatment of TNBC [16]. In this study, we assessed the significance of BRCA1/2 expression in breast cancer using multiple online databases and annotation tools. By accessing and analyzing all existing gene expression data, the expression patterns, functions, and prognostic values of BRCA1/2 in breast cancer were comprehensively investigated. These results will facilitate the understanding of the prognostic value of BRCA1/2 in breast cancer in humans.

BRCA1/2 Expression in Human Breast Cancer
The Oncomine database was used to analyze the mRNA levels of BRCA1/2 between tumor tissues and normal tissues. The results showed that BRCA1/2 were overexpressed in breast cancer as compared to their expression levels in normal tissues (Figure 1a). To further evaluate the differential expression of BRCA1/2, we compared its expression levels in the TCGA dataset using the TIMER2.0 database. As shown in Figure 1b, expression of BRCA1/2 in breast cancer tissues is significantly higher than in normal tissue ( Figure 1b). BRCA1 was overexpressed in intraductal cribriform breast adenocarcinoma patients in the TCGA database, with a fold change of 3.454 and a p value of 1.92 × 10 −6 . BRCA2 was also overexpressed in mucinous breast cancer, with a fold change of 3.750 and a p value of 3.22 × 10 −5 . The mRNA levels of BRCA2 were significantly increased in patients with invasive ductal breast carcinoma and invasive lobular breast carcinoma, with fold changes of 3.214 and 2.124, and p values of 1.46 × 10 −25 and 9.36 × 10 −9 , respectively (Table 1). Furthermore, it was discovered that the expression of BRCA1/2 mRNA in breast cancer tissues was significantly higher than that in normal tissues (Figure 2a), and the change rates of BRCA1/2 mRNA in BRCA were 9% and 6%, respectively (Figure 2b), and the AUC of BRCA1/2 was 0.766 and 0.830, respectively (Figure 3), using UALCAN, cBioPortal, and the TCGA database. This graphic generated by Oncomine (https://www.oncomine.org/resource/login.html, accessed 10 February 2022) indicates the numbers of datasets with statistically significant (p < 0.01) mR overexpression (red) or downexpression (blue) of BRCA1/2 (different types of cancer vs. co sponding normal tissue). The threshold was designed with the following parameters: p value of 10 −4 , fold change of 2, and gene ranking of 10%. (b) Human BRCA1/2 expression levels in differ tumor types from TCGA database were determined by TIMER 2.0 (http://timer.cistrome.org, cessed on 10 February 2022) (* p < 0.05; *** p < 0.001).

Clinicopathological Parameters of BRCA1/2 in BRCA Patients
In the TCGA database, overexpression of BRCA1/2 was correlated with clinico logical features and prognosis of BRCA patients ( Table 2). Overexpression of BRC related to lymph node stage, ER status, PR status and PAM 50 subtypes (p < 0.05) expression of BRCA2 was related to age, lymph node stage, ER stage, PR status, status, and PAM 50 subtypes (p < 0.01) ( Table 2).

Clinicopathological Parameters of BRCA1/2 in BRCA Patients
In the TCGA database, overexpression of BRCA1/2 was correlated with clinicopathological features and prognosis of BRCA patients ( Table 2). Overexpression of BRCA1 was related to lymph node stage, ER status, PR status and PAM 50 subtypes (p < 0.05). Overexpression of BRCA2 was related to age, lymph node stage, ER stage, PR status, HER-2 status, and PAM 50 subtypes (p < 0.01) ( Table 2).

Prognostic Value of BRCA1/2 mRNA Expression in BRCA Patients
The prognostic value of BRCA1/2 expression in breast cancer was investigated using the PrognoScan, Kaplan-Meier Plotter and R2 databases to explain how BRCA1/2 affects the prognostic characteristics of breast cancer patients and the associations between variations in the expression of BRCA1/2 genes and clinical outcomes. We observed a positive correlation between BRCA1/2 overexpression and poor patient survival in breast cancer ( Figure 4, p < 0.01). Kaplan-Meier Plotter's survival analysis showed that the overall survival of breast cancer patients was poor in both BRCA1/2 overexpression. In PrognoScan and R2-Kaplan Meier Scanner, overexpression of BRCA1/2 was associated with poor prognosis.

The Relationship of BRCA1/2 Mutations and Copy Number Alterations (CNAs) in Breast Cancer
We individually examined the genetic alterations of BRCA1/2 in breast cancer using cBioPortal. Based on the Breast Invasive Carcinoma (TCGA, Cell 2015) samples and studies, with clinical data from 816 patients, we queried the database for BRCA1/2. The results showed that there was a significant difference in BRCA1/2 alteration frequency in breast cancer. As presented in Figure 5, the alteration frequency of BRCA1/2 was determined. It included mutations (green), amplification (red), deep deletions (blue), and multiple alterations (grey) (Figure 5a). Of the queried samples of BRCA1, 76 (9%) samples were associated with an altered gene set or pathway, resulting in a somatic mutation frequency of 2.2%. There were 18 mutation sites detected located from amino acids 0 to 1863 of the BRCA1 protein, including 12 missenses, 4 truncating, and 1 inframe mutation. Of the queried samples of BRCA2, 76 (9%) samples were associated with an altered gene set or pathway, resulting in a somatic mutation frequency of 2.1%. In BRCA2, a total of 19 mutation sites were detected between amino acids 0 to 3418 of the BRCA2 protein, including 10 missenses, 7 truncating, and 1 inframe mutation (Figure 5b).

The Relationship of BRCA1/2 Mutations and Copy Number Alterations (CNAs) in Breast Cancer
We individually examined the genetic alterations of BRCA1/2 in breast cancer using cBioPortal. Based on the Breast Invasive Carcinoma (TCGA, Cell 2015) samples and studies, with clinical data from 816 patients, we queried the database for BRCA1/2. The results showed that there was a significant difference in BRCA1/2 alteration frequency in breast cancer. As presented in Figure 5, the alteration frequency of BRCA1/2 was determined. It included mutations (green), amplification (red), deep deletions (blue), and multiple alterations (grey) (Figure 5a). Of the queried samples of BRCA1, 76 (9%) samples were associated with an altered gene set or pathway, resulting in a somatic mutation frequency of 2.2%. There were 18 mutation sites detected located from amino acids 0 to 1863 of the BRCA1 protein, including 12 missenses, 4 truncating, and 1 inframe mutation. Of the queried samples of BRCA2, 76 (9%) samples were associated with an altered gene set or pathway, resulting in a somatic mutation frequency of 2.1%. In BRCA2, a total of 19 mutation sites were detected between amino acids 0 to 3418 of the BRCA2 protein, including 10 missenses, 7 truncating, and 1 inframe mutation (Figure 5b).

Co-Expression Analysis of BRCA1/2
There were 3919 positively correlated genes and 37 negatively correlated genes for BRCA1 in the TCGA transcriptome database, and 3720 positively correlated genes and 340 negatively correlated genes for BRCA2. The top 10 co-expressed genes with positive and negative correlations to BRCA1/2 are shown in the form of heat maps (Figure 6a,b). In addition, the Venn diagram indicates that there were 958 intersections of BRCA1/2 coexpressing genes (Figure 6c).

Co-Expression Analysis of BRCA1/2
There were 3919 positively correlated genes and 37 negatively correlated genes for BRCA1 in the TCGA transcriptome database, and 3720 positively correlated genes and 340 negatively correlated genes for BRCA2. The top 10 co-expressed genes with positive and negative correlations to BRCA1/2 are shown in the form of heat maps (Figure 6a,b). In addition, the Venn diagram indicates that there were 958 intersections of BRCA1/2 co-expressing genes (Figure 6c).

Analysis of GO and KEGG Pathway for BRCA1/2
To further understand the potential role of BRCA1/2 in BRCA, GO and KEGG analyses were performed on BRCA1/2 co-expressed genes. The results revealed that BRCA1/2 co-expressed genes were mainly involved in catalytic activity, acting on DNA, chromosomal regions, and organelle fission (Figure 7). KEGG pathway analysis displayed that BRCA1/2 co-expressed genes were enriched in regulating the cell cycle, RNA transport, and the Fanconi anemia pathway (Figure 8).

Analysis of GO and KEGG Pathway for BRCA1/2
To further understand the potential role of BRCA1/2 in BRCA, GO and KEGG analyses were performed on BRCA1/2 co-expressed genes. The results revealed that BRCA1/2 coexpressed genes were mainly involved in catalytic activity, acting on DNA, chromosomal regions, and organelle fission (Figure 7). KEGG pathway analysis displayed that BRCA1/2 co-expressed genes were enriched in regulating the cell cycle, RNA transport, and the Fanconi anemia pathway (Figure 8).

Identification of BRCA1/2 Interacting Genes and Proteins and Genetic Alterations
We constructed the gene-gene interaction networks for BRCA1/2 and the altered neighboring genes by using GeneMania. The results showed that 20 of the most frequently altered genes were closely correlated with BRCA1, including PALB2, RAD51, and PARP1 (Figure 9a). There were 20 most frequently altered genes that were closely correlated with BRCA2, including PALB2, RAD51, and SEM1 (Figure 9b). The A protein-protein interaction (PPI) networks of BRCA1/2 were generated using the STRING database. The result of the PPI network of BRCA1 showed that there were 45 edges and 11 nodes, including PALB2, RAD51, and TP53 (Figure 9c). There were 50 edges and 11 nodes in the result of the PPI network of BRCA2, including RAD51, BARD1 and XRCC3 (Figure 9d).

Identification of BRCA1/2 Interacting Genes and Proteins and Genetic Alterations
We constructed the gene-gene interaction networks for BRCA1/2 and the altered neighboring genes by using GeneMania. The results showed that 20 of the most frequently altered genes were closely correlated with BRCA1, including PALB2, RAD51, and PARP1 (Figure 9a). There were 20 most frequently altered genes that were closely correlated with BRCA2, including PALB2, RAD51, and SEM1 (Figure 9b). The A protein-protein interaction (PPI) networks of BRCA1/2 were generated using the STRING database. The result of the PPI network of BRCA1 showed that there were 45 edges and 11 nodes, including PALB2, RAD51, and TP53 (Figure 9c). There were 50 edges and 11 nodes in the result of the PPI network of BRCA2, including RAD51, BARD1 and XRCC3 (Figure 9d).

Discussion
The prognostic importance of BRCA1/2 in breast cancer was investigated in this research. BRCA1/2 are high-risk breast cancer susceptibility genes with 11-12-fold elevated relative risks in the general population for women [17]. A previous study showed that the probability of breast cancer in BRCA1 mutation carriers is 57-65%, while the probability of breast cancer in BRCA2 mutation carriers is 45-49% [4]. In the past, many studies have explored the relationship between BRCA1/2 mutation and survival. However, due to the relatively low mutation rate, detection rate, and survival bias of BRCA1/2 mutation carriers, the evidence of low survival rate of BRCA1/2 mutation carriers is still inconclusive [18]. With the aim of comprehensively covering the topic of the prognostic role of BRCA1/2 mutations in breast cancer, in this study, we first analyzed the expression of BRCA1/2 in breast cancer tissues and normal tissues. The results showed that BRCA1/2 were overexpressed in breast cancer tissues than in normal tissues, and we obtained the relationship of BRCA1/2 expression, mutation, and survival data in breast cancer from TCGA data analysis, which further verified the above results.
A previous study has reported that BRCA1 and BRCA2 carriers have varied clinical characteristics [19]. Herein, our study found that overexpression of BRCA1 was related to lymph node stage, ER status, PR status and PAM 50 subtypes (p < 0.05), and overexpression of BRCA2 was related to age, lymph node stage, ER stage, PR status, HER-2 status, and PAM 50 subtypes (p < 0.01).

Discussion
The prognostic importance of BRCA1/2 in breast cancer was investigated in this research. BRCA1/2 are high-risk breast cancer susceptibility genes with 11-12-fold elevated relative risks in the general population for women [17]. A previous study showed that the probability of breast cancer in BRCA1 mutation carriers is 57-65%, while the probability of breast cancer in BRCA2 mutation carriers is 45-49% [4]. In the past, many studies have explored the relationship between BRCA1/2 mutation and survival. However, due to the relatively low mutation rate, detection rate, and survival bias of BRCA1/2 mutation carriers, the evidence of low survival rate of BRCA1/2 mutation carriers is still inconclusive [18]. With the aim of comprehensively covering the topic of the prognostic role of BRCA1/2 mutations in breast cancer, in this study, we first analyzed the expression of BRCA1/2 in breast cancer tissues and normal tissues. The results showed that BRCA1/2 were overexpressed in breast cancer tissues than in normal tissues, and we obtained the relationship of BRCA1/2 expression, mutation, and survival data in breast cancer from TCGA data analysis, which further verified the above results.
A previous study has reported that BRCA1 and BRCA2 carriers have varied clinical characteristics [19]. Herein, our study found that overexpression of BRCA1 was related to lymph node stage, ER status, PR status and PAM 50 subtypes (p < 0.05), and overexpression of BRCA2 was related to age, lymph node stage, ER stage, PR status, HER-2 status, and PAM 50 subtypes (p < 0.01).
In this study, we found that overexpression of BRCA1/2 was significantly related to worse overall survival (p < 0.001). As a result, these genes might be used as novel anticancer targets. GO analysis showed that BRCA1/2 co-expressed genes were mainly involved in catalytic activity, acting on DNA, chromosomal regions, and organelle fission. KEGG pathway analysis displayed that BRCA1/2 co-expressed genes were enriched in regulating the cell cycle, RNA transport, and the Fanconi anemia pathway. In gene-gene interaction networks, 20 of the most frequently altered genes were closely correlated with BRCA1, including PALB2, RAD51, and PARP1. There were 20 most frequently altered genes that were closely correlated with BRCA2, including PALB2, RAD51, and SEM1. In the PPI network, there were 45 edges and 11 nodes in BRCA1, including PALB2, RAD51, and TP53. There were also 50 edges and 11 nodes in BRCA2, including RAD51, BARD1 and XRCC3. PALB2 was initially identified as a protein that interacts with BRCA2 and plays a crucial role in the BRCA2 genome caretaker function and was subsequently shown to also interact with BRCA1 [15,[36][37][38]. Biallelic germline loss-of-function mutations in PALB2 cause Fanconi's anemia, whereas monoallelic loss-of-function mutations are associated with an increased risk of breast cancer [39]. RAD51 is a strand transferase that helps DNA strands exchange with undamaged homologous chromatids by polymerizing nucleoprotein filaments on single-stranded DNA [40,41]. Overexpression of RAD51 is associated with tumor aggressiveness and is known to confer treatment resistance in a variety of tumors, including breast cancer [42]. PARP1 has been shown to interact with DNA polymerase α and multiprotein DNA replication complexes [43][44][45]. BRCA1 and BARD1 both bind DNA and interact with RAD51, and BRCA1-BARD1 enhances the recombinase activity of RAD51 [35].
The present study used a systematic multiomics approach to investigate BRCA1/2 functions as prognostic biomarkers in breast cancer. Although we investigated the correlation between BRCA1/2 and breast cancer patients, some limitations remain. Nevertheless, there is a lack of in-depth insight into the relevant mechanisms involved, and further research is required. Overall, our study provides suggestive evidence of the prognostic role of BRCA1/2 in breast cancer and the therapeutic target for breast cancer. Furthermore, BRCA1/2 may influence BRCA prognosis through catalytic activity, acting on DNA, chromosomal regions, organelle fission, and the cell cycle. Nevertheless, further validation is warranted.

Patient Datasets
The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/about-nci/ organization/ccg/research/structural-genomics/tcga, accessed on 10 February 2022) is a publicly funded project initiated by the United States government with the goal of classifying and discovering the major genomic alterations that cause cancer to create a comprehensive The Cancer Genome Atlas database that includes more than 20,000 molecular characterizations of primary cancers and matched normal samples for 33 cancer types [46]. It was used to download all the raw data for BRCA1/2, including transcriptome RNA-seq data and clinical information.

Transcript Expression Analysis Using Oncomine Platform
The Oncomine database (https://www.oncomine.org/resource/login.html, accessed on 10 February 2022) is a web-based data mining platform that includes 264 separate datasets and aims to collect, standardize, evaluate, and distribute transcriptomic cancer data for biomedical research [47]. The expression levels of BRCA1/2 in breast cancer were retrieved from the Oncomine platform. The p value 1 × 10 −4 , fold-change 2, and gene ranking in the top 10% were used to calculate the fold change in the expression of BRCA1/2 in clinical cancer specimens compared to normal controls. The co-expression profile of the BRCA1/2 in breast cancer was shown as a heat map.

TIMER 2.0 Analysis
Tumor IMmune Estimation Resource 2.0 (http://timer.cistrome.org, accessed on 10 February 2022) is a comprehensive resource for systematical analysis of immune infiltrates across diverse cancer types [48]. In this study, TIMER2.0 was used to determine the expression of BRCA1/2 in breast cancer and normal tissues across all TCGA cohorts.

Analysis of Gene Expression and Mutation Alterations Using cBioPortal
The cBioPortal for Cancer Genomics (http://www.cbioportal.org, accessed on 10 February 2022) is a well-known and frequently used online website that allows large-scale cancer genomics datasets to be visualized, analyzed, and downloaded [50]. In our study, cBioPortal was used to analyze the frequency of mutations and copy number alterations of BRCA1/2 with appropriate parameter settings.

Correlation Analysis of BRCA1/2 and Breast Cancer Stage
The ggplot2 program was used to perform a correlation analysis of BRCA1/2 expression and cancer stage. The ROC curve for diagnosis analysis was created using R packages. This section's clinical data were obtained from the TCGA database.

Prognosis Analysis Using PrognoScan
PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/, accessed on 10 February 2022) is a widely used online database for meta-analysis of the prognostic significance of genes [51]. In this study, PrognoScan was used to analyze the association of BRCA1/2 expression with the survival in breast cancer patients.

Survival Analysis Using Kaplan-Meier Plotter
The Kaplan-Meier plotter (https://kmplot.com/analysis/, accessed on 10 February 2022) is an online application tool that can evaluate the impact of the expression of more than 54,000 genes on the survival of 21 different cancer types [52]. It was used to analyze the relationship between BRCA1/2 expression and the survival of patients with breast cancer in this study.

Survival and Correlation Analysis Using R2
R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi, accessed on 10 February 2022) is an online tool that can be used for a variety of genomics research and visualization tasks. In this study, we used R2 platform to analyze the correlations between BRCA1/2 expression and the survival of patients with breast cancer.

GO and KEGG Enrichment Analysis
To investigate probable biological activities and signaling pathways influenced by BRCA1/2, we used the package R "cluster profile" to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on co-expressed genes. GO analysis is a commonly used method for large-scale functional enrichment research; gene functions can be classified into biological process (BP), cellular component (CC), and molecular function (MF).

Analysis of BRCA1/2 Interacting Genes and Proteins
The GeneMANIA database (http://www.genemania.org, accessed on 10 February 2022) was applied to construct the gene-gene interaction network of BRCA1/2. The STRINGdb analysis tool (https://string-db.org/cgi/input.pl, accessed on 10 February 2022) is a database of known and predicted protein interactions and was used to construct a protein-protein interaction (PPI) network of BRCA1/2.

Statistical Analysis
R (v.3.6.3) was used to perform the statistical analysis. Differences between groups were compared using the Wilcoxon rank-sum test or Student's t test, as appropriate. Pearson or Spearman correlation tests, as appropriate, were used to determine correlations. Survival curves were generated using PrognoScan, the Kaplan-Meier Plotter, and the R2. All results were shown as p values of the log-rank test. They were created, and log-rank tests were performed to identify the significance of the difference between the survival curves, and differences with a p < 0.05 were considered statistically significant.

Data Availability Statement:
The datasets created and/or analyzed during this investigation are available to the public. In the Materials and Methods section, each portal's persistent web link has been supplied.