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ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 6  |  Page : 1743-1753

Exceptional behavior of breast cancer-associated type 1 gene in breast invasive carcinoma


1 Department of Bioinformatics, Hazara University Mansehra, Mansehra, Pakistan
2 Department of Biochemistry, Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

Date of Submission09-May-2020
Date of Acceptance28-Sep-2021
Date of Web Publication16-Nov-2022

Correspondence Address:
Samina Ejaz
Department of Biochemistry, Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1310_20

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 > Abstract 


Background: Cellular expression level of Breast Cancer-Associated Type 1 (BRCA1) encoded protein is the sign of genome integrity, stability, and surveillance. BRCA1 after sensing DNA damage activates repairing system and if mutated leaves genomic lesions unrepaired and triggers transformation of normal breast cells into cancerous ones.
Aims of study: We conducted in silico study to have a holistic view of BRCA1's correlation with multiple variables of breast invasive carcinoma.
Materials and Methods: We used user-friendly online GeneCardsSuite pathway-level enrichment analysis, UALCAN portal differential expression analysis, cBioPortal cancer genome platform for mutatome map construction, and cancer cell lines encyclopedia genomics of drug sensitivity toolkit to understand correlation of BRCA1 expression with the effectiveness of anti-cancer drugs.
Results: Contrary to general behavior of a tumor suppressor gene our study revealed BRCA1 overexpression under all circumstances. This novel finding needs to be explored further to understand functional impact of BRCA1 overexpression on the expression of many genes which are transcriptionally regulated by BRCA1 and promotion of tumriogenesis.
Conclusion: Our study highlights the potential role of BRCA1-regulated genes in oncogenesis and recommends use of BRCA1-linked genes as future therapeutic targets for effective disease management.

Keywords: Breast cancer-associated type 1, breast invasive ductal carcinoma, DNA damage repair pathways, Double strand brake, genome surveillance


How to cite this article:
Abbas Z, Nouroz F, Ejaz S. Exceptional behavior of breast cancer-associated type 1 gene in breast invasive carcinoma. J Can Res Ther 2022;18:1743-53

How to cite this URL:
Abbas Z, Nouroz F, Ejaz S. Exceptional behavior of breast cancer-associated type 1 gene in breast invasive carcinoma. J Can Res Ther [serial online] 2022 [cited 2022 Dec 2];18:1743-53. Available from: https://www.cancerjournal.net/text.asp?2022/18/6/1743/361201




 > Introduction Top


The most prevalent cancer among women worldwide is the breast cancer which affects women of 154 countries and has the highest mortality rate in 103 countries. In 2018, more than 2 million females were diagnosed with breast carcinoma.[1] Few genes such as breast cancer-associated Type 1 (BRCA1), BRCA2, PTEN, ATM, TP53, HRAS, and CHEK2 and the corresponding proteins are thought to be the significant genetic markers in familial breast carcinoma cases. The BRCA1/2 mutations are the major hallmarks of breast-related malignancies. It is estimated that the carriers of BRCA1 mutation carriers have more than 70% lifetime risk to develop breast cancer other than noncarriers.[2],[3] More than 75% of BRCA1 mutation carriers have a worse prognosis in basal-like (BL1) subtypes of breast cancer.[4],[5] BRCA1 performance during maintenance of genome's integrity depends on the deployment of associated protein complexes for repairing of double-strand breaks (DSB) in DNA.[4] During DSB repairing process, recognition and initiation are mainly triggered through C-terminal domains of BRCA1 which mediates molecular associations with various proteins, including retinoblastoma protein 8, BTB domain, and CNC1 to constitute BRCA1 A complex.[5] The BRCA1-BARD1 complex having E3 ubiquitin ligase activity carries out ubiquitination of a variant histone H2AX which in turn re-localizes TP53 at break sites.[6],[7] The BRCA1 associations with proteins such as MSH2, MSH6, MLH1, ATM, RAD50, MRE11, and NBS1 are necessary for RAD51 recruitment at DSB site. The protein complex containing RAD51 repairs DSBs by identifying homologous sequences.[8],[9] Breast tissue differentiation NOTCH/SLUG regulators have influential interactions with BRCA1. The NOTCH/SLUG regulators elevate the expression of JAG1 which binds with NOTCH receptor, and ultimately components of NOTCH pathway are up-regulated. Higher activity of NOTCH pathway is known to promote progression of cancer.[10],[11],[12] While SLUG protein complex promotes metastasis by conferring stem cell-like features to the tumor cells. BRCA1 negatively regulates SLUG expression.[13]

The multivariate interactions of BRCA1 with DNA damage repairing machinery consisting of various tumor suppressor proteins invite us to comprehensively analyze the status of BRCA1 mutations, expression, its promoter methylation pattern, and association with drug sensitivity profile of breast carcinoma tissues. The information obtained will be of clinical significance and can serve as a guide for therapeutic interventions for precision medicines.


 > Materials and Methods Top


Various bioinformatics tools were employed during multi-step experimental strategy used during the study.

Breast cancer-associated type 1 characterization analysis

To characterize BRCA1 data were retrieved from GeneCardsSuite (www.genecards.org) which is a freely available online database containing information of HGNC approved genes (41,846), disease associated genes (13686), protein coding genes (21,137), pseudogenes (22,237), hot genes (500), gene clusters (14), genomic regions (3,780) of biological and clinical relevance, transcriptomics and proteomics data. We queried BRCA1 official gene symbols on homepage through “search box” and pressed “GO” option. The database offered valuable data regarding different aspects of BRCA1, including gene symbols, genomic view summery, protein domains, functions, pathway interactions, druggome information, genomic variants, and associated disorders. We explored information regarding participation of BRCA1 in the pathways which are activated in response to DNA damaging events.

Investigation of breast cancer-associated type 1 expression status

We investigated the status of BRCA1 gene expression in breast invasive carcinoma (BRIC) through UALCAN (www.ualcan.path.uab.edu), an online web portal consisting of The Cancer Genome Atlas (TCGA) ribonucleic acid (RNA)-seq level 3 data of 31 cancer types. The UALCAN portal enabled inquiring relative/differential expression status of BRCA1 across a wide range of normal and cancerous samples in association with multiple parameters including gender, race, age groups, and numerous other pathological features and stage of disease (in case of patients). The information was retrieved in the form of expression data graphs and box plots. We pressed “Analysis” option available on homepage that ultimately opened “Search box” for gene scanning in relevant cancer dataset. We entered BRCA1 gene in “Box,” selected “Beast Invasive Carcinoma” dataset and pressed “Explore” tab which offered whisker box plot representation of differential expression. UALCAN utilized interquartile ranges including minimum, medium, and maximum values. The transcript per million values above/below upper quartile were designated as high and low/medium expression, respectively.

Breast cancer-associated type 1 protein expression analysis in breast cancer

We analyzed BRCA1 protein expression for determining correlation among transcriptomics to proteomics level of gene expression in various breast malignancy-related aspects, including stages, race, age-groups, and cancer subclasses. The UALCAN (www.ualcan.path.uab.edu) online web portal integrates BRCA1 protein expression data from CPTAC consortium and allows retrieval and analysis of protein expression data across the samples of diverse attributes. The web portal presented BRCA1 protein expression data in the form of box plots.

Determination of methylation status of breast cancer-associated type 1 promoter

We analyzed BRCA1 promoter to have an account of its methylation status using UALCAN (www.ualcan.path.uab.edu) an online web portal that allows us to correlate gene expression ratio with DNA methylation rank. UALCAN processes DNA methylation level through beta values from 0 (unmethylated) to 1 (fully methylated) with cut-off values 0.7–0.5 (hyper-methylation) and 0.3–0.2 (hypo-methylation).

Breast cancer-associated type 1 mutatome map mining

We explored BRCA1 mutatome map from cBioPortal for Cancer Genomics (www.cbioportal.org), an online database, by querying BRCA1 gene symbol with the selection of “BRIC TCGA, Cell 2015” and pressed “Submit” query option. The portal presented “Mutation” tab provided graphical summery and table of all BRCA1 gene attributes, i.e., nonsynonymous mutations, positions and frequency in BRIC samples. Moreover, the cBioPortal revealed comprehensive information regarding BRCA1 genomic lesions including missense, nonsense, nonstart, and frameshift insertions/deletions.

Correlational analysis of breast cancer-associated type 1 expression and drug resistance

We investigated relationship of BRCA1 expression status with the response of tumor to anti-cancer drugs used for breast cancer treatment. The information was obtained through cancer cell lines encyclopedia and the genomics of drug sensitivity (CCLE GDSC) gene expression-drug sensitivity correlations toolkit (www.public.tableau.com/CCLE_GDSC_Correlations). We selected BRCA1 gene from CCLE GDSC “gene list” and adjusted “EC50/IC50” coefficient concentrations. The CCLE GDSC toolkit revealed green (negative nonresistant) or red (positive resistant) colored representations. Data were tabulated to reflect the association of BRCA1 expression with drug effectiveness.

Association of breast cancer-associated type 1 expression and survival of patients

The impact of BRCA1 expression on survival of patients was accessed through information available on UALCAN (www.ualcan.path.uab.edu), an online web portal, in the form of Kaplan–Meier (KM) survival plot. The web portal provided KM plots to indicate the relation of BRCA1 expression with three parameters, i.e., time from days of diagnosis to death or days to last fellow-up, status of alive or dead, and finally expression of high, medium, and low. The KM survival curves of high, medium, and low expression were compared through log-rank test analysis.


 > Results Top


Breast cancer-associated type 1 enrichment analysis

The information extracted through GeneCardsSuite v4.12 revealed BRCA1 as a nuclear 1863 amino acid containing phosphoprotein encoded by a gene located on chromosome 17q21.31 (125,951 bases from 43,044,295 to 43,170,245) which participates in maintenance of genome stability and prevention of tumriogenesis.[14] The BRCA1 protein forms complex with RNA Polymerase II, and histone deacetylases using its BRACT domain and mediates transcription of a set of genes.[15] Among the BRCA1-regulated genes, many participate in the homologous recombination-based repair mechanism of double-strand brakes.[16] The maintenance of genome integrity is a vital phenomenon to ensure survival and development of eukaryotic multicellular organisms. The base modifications, interstrand cross-links, and strand breaks are characterized as DNA damages. The natural cellular signaling system senses and responds to any DNA damage based upon the location, chemical nature, cell cycle timing, and type of damage events. BRCA1 induces BRCA1-associated genome surveillance complex consisting of multiple proteins such as MSH2, MSH6, MLH1, ATM, BLM, and RAD50-MRE11-NBS1 protein complexes for the detection and repairing of DNA lesions. In response to any DNA damage BRCA1 and RAD51 co-localize to subnuclear region by FANCD2 ubiquitylation due to the activation of nuclear complex comprised of proteins such as FANCA, FANCC, FANCE, FANCF, and FANCG. The homologous recombination repairing process is initiated and progresses through BRCA1, BRCA2, RAD51, and FANCD2 in DNA repairing mechanism. The nuclear phosphoprotein BRCA1 establishes interactions with RAD50-RAD51-MRE11-NBS1 complex. The CHK2 phosphorylation is triggered through ATM/ATR activation which further phosphorylates BRCA1. The phosphorylated BRCA1 influences extent of ubiquitin ligation by associating with BARD1 to ensure the role of BRCA1 in the maintenance of genome stability. The BRCA1 is known to activate GADD45 which has a significant role in DNA damage repairing via regulating G2/M and G1/S checkpoints through CHK1/PLK1 complex. BRCA1-STAT1 complex inhibits growth by activating RB and IFN-gamma genes. The chromatin remodeling and homologous recombination repair process are initiated through BRCA1-BACH1-SWI/SNF complex that regulates HDACs.[17],[18],[19] Information regarding BRCA1 interaction profile is summarized in [Figure 1].
Figure 1: Breast cancer associated type 1 DNA damage repairing pathway (Figure is retrieved from genecards database)

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Breast cancer-associated type 1 differential expression analysis

Breast cancer has various types under TNM staging system based on tumor size (T), tumor-lymph node interaction (N), and tumor-derived metastasis (M). When breast carcinoma is diagnosed at higher stages, it reduces survival chances, and lowered stages are closer to normal cellular physiology with less aggressive tumorigenic signatures.[20] Stage 1–3 are invasive in nature, but existing cancerous cells remain confined within in the breast tissue but in Stage 4 tumor cells spread into regional lymph nodes and distant body organs with worse prognosis.[21] Our results showed that BRCA1 is significantly overexpressed (P < 0.005) in Stage 1 (10.55), Stage 2 (11.8), Stage 3 (10.48), and Stage 4 (8.65) of the disease as compared to the normal expression (4.95). Moreover, Stage 1 also differed significantly (P < 0.005) than Stage 2 and Stage 3 [Figure 2]. Breast cancer showed interesting prevalence record in less or more-developed populations with high incidence rates in European and African countries. BRCA1 was observed to have a higher expression (P < 0.005) in Caucasian (10.86), African-American (11.23), and Asian (11.36) races as compared to the normal expression (4.95). Breast cancer reported in above 50-year-old females is mostly aggressive due to several factors, including abnormal menopause setup, poorer medical conditions, and late diagnosis. BRCA1 has shown up-regulation (P < 0.005) in 21–40 years (10.69), 41–60 years (11.06), 61–80 years (11.44), and 81–100 years (9.79) as compared to normal expression (4.95). While the difference between patients of various population- and age-based groups was found insignificant (P > 0.005). Majority of the published breast cancer-related work revolves around females rather than males due to rare chances of this disease in males. However, BRCA1 is known to have higher expression (P < 0.005) level in both male (27.31) and female (11.16) patients as compared to the expression level documented in healthy cancer-free cells (4.95). Moreover, BRCA1 expression level documented in male patients differed significantly (P < 0.005) than females. BRCA1 expression in association with different factors is shown in [Figure 2].
Figure 2: Association of breast cancer associated type 1 expression with various stages of disease, and race, gender and age of breast cancer patients. The number of samples in a particular dataset is indicated by n. Statistical analysis revealed significant variations (P < 0.005) among following comparisons: Normal-versus-Stage1, Normal-versus-Stage2, Normal-versus-Stage3, Normal-versus-Stage4, Stage1-versus-Stage2, Stage1-versus-Stage3, normal-versus-caucasian, normal-versus-African American, normal-versus-Asian, normal-versus-male, normal-versus-female, male-versus-female, normal-versus-age (21–40 years), normal-versus-age (41–60 Years), normal-versus-age (61–80 Years) and normal-versus-age (81–100 years). However, variations among various patients groups (based upon individual cancer stages and patient's race) and patients-versus-normal group were found to be insignificant (P > 0.05). Detail of statistical significance level is given in supplementary data

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Breast cancer is categorized into luminal, human epidermal growth factor receptor 2 (HER2)+ and triple-negative breast cancer (TNBC) based on the expression status of cellular estrogen receptors, progesterone receptors, and HER2. In TNBC, the expression all three receptors is lost but prolactin and androgen receptors are expressed frequently.[22] BRCA1 was examined to have higher expression (P < 0.005) in Luminal (10.46), HER2+ (8.16), and TNBC (13.68) as compared to the one observed in normal cellular expression (4.95). Breast cancer has a diverse association with menstrual cycle and considerably varies from Pre-Peri-Postmenopausal situations due to production of estrogen. BRCA1 was examined to have overexpression (P < 0.005) in premenopause (10.51), peri-menopause (13.53), and postmenopause (11.21) with reference to normal expression (4.95). Breast cancer is histologically categorized mainly into three types, i.e., invasive ductal carcinoma (IDC) cancer of milk transporting duct, and invasive lobular carcinoma (ILC). The cancer of milk-producing glands and metaplastic type of carcinoma spreads into distant parts of body.[23] IDC has various types such as mucinous, medullary, and papillary which have specified morphological features. However, few not otherwise specified forms of IDC also exists which are termed as INOS.[24] BRCA1 was documented to have up-regulation (P < 0.005) in IDC (12.04), ILC (9.36), Mucinous (8.94), Medullary,[15] Mixed (11.27), and others (10.12) as compared with normal expression (4.95). The metaplastic (8.73) group had significantly higher expression of BRCA1 than IDC and other types. Moreover, IDC exhibited higher expression (P < 0.0005) than ILC. TNBC has various forms based on genomic mutations which are BL1, BL2, immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptors (LAR). The analysis showed significant overexpression (P < 0.005) of BRCA1 in BL1 (22.54), BL2 (9.37), IM (11.75), M (14.19), MSL (7.33), and LAR (6.54) as compared to normal expression (4.95). Information is summarized in [Figure 3].
Figure 3: Status of breast cancer associated type 1 expression in different subclasses, triple negative breast cancer, Menopause states and histological forms of breast cancer. The number of samples in a particular dataset is indicated by n. Statistical analysis revealed significant variations (P < 0.05) among following comparisons: Normal-versus-luminal, normal-versus-human epidermal growth factor receptor 2 positive, normal-versus-triple negative breast cancer, normal-versus-triple negative breast cancer- basal-like 1, normal-versus- triple negative breast cancer-Immunomodulatory, normal-versus-triple negative breast cancer-Mesenchymal, normal-versus- triple negative breast cancer-universitas sebelas maret, luminal-versus-triple negative breast cancer-mesenchymal stem-like, triple negative breast cancer- basal-like 1-versus-triple negative breast cancer- basal-like 2, triple negative breast cancer- basal-like 1-versus-triple negative breast cancer-mesenchymal stem-like, normal-versus-premenopause, normal-versus-peri-menopause, normal-versus-postmenopause, normal-versus-invasive ductal carcinoma, normal-versus-invasive lobular carcinoma, normal-versus-mixed, normal-versus-Other, normal-versus-mucinous, normal-versus-medullary, invasive ductal carcinoma-versus-invasive lobular carcinoma, invasive ductal carcinoma-versus-metaplastic and other-versus-metaplastic. However, variations among various patients groups (based upon breast cancer subclasses, major subclasses (with triple negative breast cancer types), menopause Status and histologic subtypes) and patients-versus-normal group were found to be insignificant (P > 0.05). Detail of statistical significance level is given in supplementary data

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Breast cancer-associated type 1 protein expression analysis

The BRCA1 protein expression was higher in Stage II (2.03 Z-value), Stage III (1.62 Z-value), Caucasian (2.02 Z-value), Asian (2.01 Z-value), African-American (1.79 Z-value), 41-60 (2.03 Z-value), 21–40 (1.79 Z-value), 61–80 (1.52 Z-value), Luminal (2.02 Z-value), and TNBC (1.86 Z-value) than Stage I (0.37 Z-value), 81–100 (0.66 Z-value), and HER2+ (0.03 Z-value). Information has been summarized in [Figure 4].
Figure 4: Breast cancer associated type 1 protein expression analysis in breast cancer stages, race, age-groups and cancer subtypes. All variations were statistically insignificant (P > 0.05) except caucasian-versus-Asian

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Methylation status of breast cancer-associated type 1 promoter

DNA methylation involves the addition of methyl group to CpG islands present in promoter or other regions of any gene. The promoter methylation promotes transcriptional silencing and it is the most prevalent gene silencing mechanism, specifically in cases of tumor suppressor genes.[25] In cancer, usually, cell proliferation-associated genes are hypomethylation while DNA repairing genes are hypermethylated.[26] In our study, BRCA1 appeared to have very lower degree of hyper-methylation in majority of the samples and normal subjects as indicated by the lower beta value (i.e., ranging from 0.33 to 0.35). Statistical analysis was carried out to compare normal subjects' methylation status (0.36) with the patients. Results revealed significant variations (P < 0.005) in Stage 1 (0.35), Stage 2 (0.35), Stage 3 (0.35), Caucasian (0.35), African-American (0.34), Asian (0.35), female (0.35), 41–60 years (0.35) and 61–80 years (0.35). Data are summarized in the form of box and whisker plot [Figure 5].
Figure 5: Breast cancer associated type 1 promoter methylation status in in breast cancer patients of different stages, gender, race and age. The number of samples in a particular dataset is indicated by n. Beta value is used to reflect the extent of DNA methylation and it ranged from 0 (unmethylated) to 1 (fully methylated). The lower cut-off value (0.3–0.25) indicates hypo-methylation while hyper-methylation is considered to have higher beta value (0.7 – 0.5) as per defined criteria. The number of samples in a particular dataset is indicated by n. Statistical analysis revealed significant variations (P < 0.005) among following comparisons: Normal-versus-Stage1, normal-versus-Stage2, normal-versus-Stage3, normal-versus-caucasian, normal-versus-African American, normal-versus-Asian, normal-versus-female, normal-versus-age (41–60 Years) and normal-versus-age (61–80 Years). However, variations among methylation status of various patients groups and patients-versus-normal group were found to be insignificant (P > 0.05). Detail of statistical significance level is given in supplementary data

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Extraction of breast cancer-associated type 1 mutatome map

We determined BRCA1 differential expression in BRIC-TCGA Cell 2015 dataset of 816 samples in which 40 samples (4.9%) showed altered expression. While 16 samples had missense mutation unknown significance and 1 sample was observed to have inframe mutation of unknown significance. Overall, BRCA1 showed 2.0% amplification, 0.9% homozygous deletion, and 2.2% mutation in BRIC dataset [Figure 6].
Figure 6: Mutational spectrum of breast cancer associated type 1 in breast invasive carcinoma

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BRCA1 has 5 significant domains, i.e., zf_C3HC4 zinger finger type (Ring finger 24–64 AA), serine-rich domain associated with BRCT (344–507 AA), ethylene insensative 3 (EIN3 648–978 AA), and BRCA1 C Terminus domain (1662-1723/1757-1842 AA). In the present study, among 1863 amino acids of BRCA1 18 BRIC-associated mutations were detected through in silico analysis. The detected mutations included 12 missense mutations documented in IDC of breast, i.e., G1788V, Q1811 L, P1238H, D1344H, E9Q, K50T, S1027I, D366N, D96H, D2E, S915C, and P1126Q. While 5 truncating mutations were recorded in IDC (X1559_Splice, E720 and P1614Qfs19) and mixed both invasive ductal/lobular carcinoma cases (Q934 and T688Vfs12). A single in-frame mutation C644del has been noticed in IDC [Figure 7].
Figure 7: Breast cancer associated type 1 mutatome map in breast invasive carcinoma

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Correlation of breast cancer-associated type 1 and drug sensitivity profile of breast invasive carcinoma

BRCA1 expression enhances the effectiveness of many drugs which either inhibit tumor growth preventing (Obatoclax Mesylate), cancer cells migration (AG-014699/Rucaparib), and tumor angiogenesis (BX-912) or act as antineoplastic agent (BMS-754807), apoptosis inducer (CAL-101 or Idelalisib), cell growth regulatory BRAF inhibitor (Debrafenib), hedgehog signaling mediated oncogenes suppressor (GDC0449 or Vismodegib), antitumor metabolic activist (GSK1070916), angiogenesis-based metastatic inhibitor (KIN001-236), growth-mediated signaling transduction pathway inhibitor (LFM-A13), S-phase inhibitor (MLN4924 or Pevonedistat), cell cycle regulation inhibitor (NPK76-II-72-1), cellular adhesion inhibitor (PF-562271), anti-proliferatory agent (Ruxolitinib), stress response inducer (Salubrinal), tumor cell growth inhibitor (SNX-2112), and anti-metastatic (XMD14-99). Information regarding drug sensitivity profile is summarized in [Table 1].
Table 1: Breast cancer associated type 1 association with drug sensitivity status of breast invasive carcinoma

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Association of breast cancer-associated type 1 expression and survival of patients

There is no effect of BRCA1 expression level on the survival of BRCA patients as it is reflected by the KM survival curve analysis [Figure 7]. About 70% of the BRCA patients having high and low/medium expression level exhibited the same survival pattern. While 25% of the patients having higher expression of BRCA1 had slightly higher survival rate than patients having low/medium expression level, but the difference is statistically insignificant (P = 0.15). The same was true for the effect of BRCA1 expression in combination with race (P = 0.57), gender (P = 0.15) and cancer type (P = 0.089) on the survival of BRCA patients [Figure 8].
Figure 8: Kaplan–Meier plots to reflect the association of breast cancer associated type 1 expression status alone and in correlation with race, gender and cancer type on survival of breast cancer associated type 1 patients. Statistical analysis revealed that all variations were insignificant

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 > Discussion Top


Among women, the prevalence ratio of breast-related malignancy is 30% of the total known cancer reports. While TNBC accounts for 12%–17% of the breast carcinoma cases.[27],[28] BRCA1 is a shuttle protein that is translocated from cytoplasam to nucleus as part of TP53 signaling pathway in response to the DNA damage.[29],[30] The 1863 amino acids encoding BRCA1 gene is positioned at chromosome 17q21 and consists of 24 exons. The BRCA1 protein is composed of many functional domains, including RING finger N-terminal domain, BRCT domains, coiled-coiled domain, SQ cluster, and two nuclear localization signals. BRCA1 mutations are associated with both heritable and sporadic types of breast cancer. Various studies have reported the role of BRCA1 mutations in breast, prostate, peritoneal, ovarian, fallopian tube, and pancreatic cancers in either women or men. Multiple evidences are available regarding numerous mutations in RING finger and BRCT domains. Both domains mediate influential protein-protein interactions essential for nucleotide repairing processes.[31],[32],[33],[34],[35],[36],[37],[38],[39],[40] Initial studies mentioned significance of BRCA1 in tumorigenesis, hyperplasia, and abnormal ductal development. Several high-grade carcinomas exhibit loss of BRCA1 function or lowest level of expression with higher proliferation speed.[40],[41],[42],[43] BRCA1 is influential member of BRCAness phenomenon and undergoes mutations, copy number alterations, aberrant expression, and promoter methylation. The dysfunctional BRCA1 is the indictor of less effective nucleotide repairing mechanism which ultimately leads to the generation of genomic lesions.[44] The impact of BRCA1 gene expression on diverse breast cancer-associated attributes, i.e., TNBC subtypes, stages, age-groups, race, menopause status, and histological forms has not been extensively investigated [Figure 4].

Due to the lack of confirmed drug targets in TNBC, it behaves clinically aggressively that leads to worse or poor prognosis as compared with other molecular subtypes of breast cancer. It is known that chance of TNBC patients to have mutated BRCA1 is 75% higher than patients of other molecular subtypes.[45],[46] In our study, all molecular subtypes of TNBC have elevated BRCA1 expression which shows a worse prognosis with lethal invasive nature of proliferation due to diverse set of molecular alterations. Several studies reported worse prognosis in TNBC patients having BRCA1 mutations in their genome compared with BRCA1 nonmutated carriers.[47],[48],[49],[50],[51],[52] Here, the availability of normal BRCA1 protein could protect the error-prone genomic lesions which may ultimately lead to the activation of oncogenes. These in silico findings invite researchers to conduct more rational experimental studies to elucidate the impact of BRCA1 expression in TNBC patients.

In our study, BRIC datasets showed BRCA1 overexpression in all BRIC-associated variables including stage of disease, race, age, gender, menopause status, molecular subtypes, histological subtypes, and TNBC types. Higher BRCA1 expression observed in Stage 2 than Stage 1 and Stage 3 reflects insignificant impact of BRCA1 elevated expression on initiation step of breast tissues transformation. In Stage 1–3, breast tumor cells are ready to invade nearby lymph node tissues by replicating supportive genomic alterations.[53] The status of BRCA1 expression is more dysregulated in the Asian race comparative to African, American, and Caucasian populations. From 41 to 81-year-old age, cases showed elevated expression of BRCA1 than younger patients. This finding indicates regulatory impact of BRCA1 in age-based carcinoma and suggests its role as invasive carcinoma marker in old age patients. Our study validates previous reports regarding higher breast cancer prevalence in old-aged patients and has been linked with the lack of awareness and delayed diagnosis among the Asian community.[54],[55],[56],[57] BRCA1 deregulation in Asian race with elder ages confirmed. Low prevalence male BRIC patients have been shown to exhibit higher expression of BRCA1 that attracts researchers to investigate the factors responsible for cancer progression in male breast carcinomas. During pre- and postmenopausal conditions of BRIC, patients BRCA1 overexpression indicates its associations with multi-faceted hormonal pathways. In breast malignancy, pre-postmenopausal status is very crucial factor that plays a role in receptor-mediated carcinogenesis triggered by altered regulation of estrogen and follicular stimulating hormone.[58],[59],[60] In breast ductal carcinoma (medullary form), patients BRCA1 overexpression highlights its clinical significance. In all TNBC types, elevated BRCA1 expression, specifically BL1/M/IM suggests its contribution in stem cell-like behavior.

In our study, the BRCA1 promoter methylation status insignificantly varied than normal subjects. However, previous studies have reported hypermethylation and silencing of BRCA1 promoters in breast cancer patients.[61]

BRCA1 mutatome map showed significant mutations in BRIC dataset. Majority of the missense mutations observed in IDC are localized in a variety of domains, including BRCT domain, SQ cluster, RING finger motif, and nonhomologous end-joining region. These missense mutations make BRCA1 protein nonfunctional in the context of protein binding sites for phosphorylation and ubiquitination which are essential for normal repairing processes. IDC patients 3 truncating mutations were detected in BRCT, a coiled-coil region, and RING finger domains. These domains are essential for DSBs repairing process.[62] There is a single inframe mutation in homologous recombination RAD50/RAD51 binding region which is important for nuclear localization, DNA binding, and transcriptional regulation. Our results revealed that BRCA1 enhances effectiveness of many therapeutic anti-cancer drugs. Our findings validate BRCA1 interaction with moesin/radixin/ezrin drugs, reduced motility, and number of malignant cells.[63] We further observed that variations in the BRCA1 expression level have no influence on the survival rate of BRCA patients.

Our study raises several serious concerns regarding BRCA1 overexpression in breast cancer. BRCA1 is a tumor suppressor that performs key role in repairing DSBs, but why it is overexpressed in carcinoma? Which molecular factors are responsible for its upregulation in tumorigenic conditions? Why promoter methylation mediating pathways are deactivated in BRIC patients? Why BRCA1 overexpression is not sufficient to repair genomic lesions? Which are the molecular determents that manipulate BRCA1 overexpression to promote tumriogenesis? What is the role of endocrinological factors to determine BRCA1 fate under diverse molecular microenvironment? How BRCA1 mutations dysregulate expression rate or lead to a nonfunctional protein?


 > Conclusion Top


BRCA1 is an essential factor which is known to regulate normal development and physiology of breast tissues. While its aberrant expression promotes breast cancer progression due to lack of genome surveillance machinery continuously recognizing and fixing the damages.[64] Researchers have previously suggested that tumor biology can be better understood by focusing on the role of BRCA1 in maintaining genome integrity and mediating normal cell cycle.[65] Our study proposes that BRCA1 upregulation may facilitate tumor progression and demands extensive molecular analysis of regulatory factors to understand role of BRCA1 in carcinogenesis. This in silico study provided enough data which after experimental validation can be employed in future to develop versatile therapeutic strategies based on BRCA1-mediated oncogenesis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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