|Year : 2015 | Volume
| Issue : 3 | Page : 508-513
Current and emerging breast cancer biomarkers
Maryam Sana, Hassan Jameel Malik
Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad 38000, Pakistan
|Date of Web Publication||9-Oct-2015|
National Institute for Biotechnology and Genetic Engineering, Faisalabad 38000
Source of Support: None, Conflict of Interest: None
Breast cancer treatment has experienced several advancements in the past few decades with the discovery of specific predictive and prognostic biomarkers that make possible the application of individualized therapies. In addition to traditional prognostic factors of breast carcinoma, molecular biomarkers have played a significant role in tumor prediction and treatment. The most frequent genetic alterations of breast cancer are gained along chromosome 1q, 8q, 17q, 20q, and 11q and losses along 8p, 13q, 16q, 18q, and 11q. Interestingly, many of these chromosomal fragments harbor known proto oncogenes or tumor suppressor genes such as BRCA1, BRCA2, p53, HER2-neu, cyclin D1, and cyclin E, which are briefly described in this review.
Keywords: Biomarkers, breast cancer, individualized therapy, mutation, prognostic and predictive factors
|How to cite this article:|
Sana M, Malik HJ. Current and emerging breast cancer biomarkers. J Can Res Ther 2015;11:508-13
| > Introduction|| |
Breast carcinoma is highly heterogeneous disorder both etiologically and genetically. In 2008, approximately 1.38 million new breast cancer cases were diagnosed that commonly occurring cancer worldwide. In Eastern Africa, the incidence rate of breast cancer varies from 19.3/100,000 women and in the developed regions of the world (except in Japan), the incidence rate is very high that is 80/100,000 women. 
Incidence rates of breast cancer in most regions of the world especially in the developing nations are increasing, like incidence rate of breast cancer in Pakistan is 50.1/100,000 women per year. ,
Although still in the early stages of research, molecular breast cancer subtypes may become useful in planning treatment and developing new therapies. Most of the studies divide breast cancer into four major molecular subtypes as shown in [Table 1]. 
In the last 50 years, despite efforts to limit the occurrence of cancer globally, breast cancer has become the leading cause of death in women and rank second in cancer-related mortality. Early stage diagnosis is necessary for the management of this high-risk cancer, which requires sensitive and specific biomarkers as a marker for current or future disease state. 
A biomarker is a feature that is objectively evaluated and measured for the indication of pathogenic processes, normal biological processes, and pharmacologic responses to treatment. It is a biological indicator that detects molecular changes in the tissue and fluids of the body that associated with the disease. 
These biomarkers provide the biological material to estimate the risk of having and getting cancer, classify cancer and provide insights into prognosis and prediction, so have a therapeutic advantage. Cancer biomarkers may be changed in gene expression or mutation in some genes or methylation of promoter region of cancer-specific genes which caused a change in expression of proteins. 
Many potential prognostic and predictive biomarkers for breast cancer have been investigated, in the past few decades. With the progression of new techniques for gene expression profiling, it revealed that in comparison to traditional prognostic factors, novel biological factors and molecular markers have more important roles. Some established and emerging biomarkers are discussed here.
p53 is one of the mostly studied biomarkers for breast cancer. It is a transcription factor that regulates the cell cycle, so it acts as tumor suppressor. The gene is named Tp53 after the protein which it codes. It is present on the short arm of chromosome 17 (17p 31.1 ). The name p53 is due to its molecular mass. Mutation in the p53 genes causes the formation of proteins that could not quickly degraded as wild type protein, so it accumulates and could be analyzed by immunohistochemistry. , The human p53 gene is a nuclear phosphoprotein composed of 393 amino acids that causes both repression and activation of gene expression by binding at many sites of chromatin. , It has short half-life so maintained at low level in unarrested cells, but in stress conditions such as in case of DNA damage, it is stabilized by posttranslational modifications. It also plays an important role in stem cell biology. 
p53 contains two transactivation domains on its N-terminus that is TAD1 and TAD2, in which mutation causes inactivation of p53 completely as tumor suppressor. At C terminal, there is a tetramerization domain that directly interacts with single-stranded DNA.  Many different p53 protein isoforms are encoded by human p53 gene that is p53α, p53β, p53γ, ∆133p53, ∆133p53β, ∆133p53ɤ, and ∆ 40p53. These isoforms are due to alternative splicing of intron 9 and 2, using alternative promoter in intron 4 and alternative initiation of translation. 
p53 controls cellular functions such as cell cycle control, DNA repair, apoptosis, angiogenesis, and cellular stress response through its target genes such as Mdm2, WAFI/CIPI, WIPI, BAX, PIG3, FASL, CSR, P21, etc.  Regulation of p53 occurs at different levels that are by postraslational modifications, by increasing the protein concentration and by cellular localization. 
After 15 years of discovery of p53, two genes were identified which were related to p53 that is p63 and p73. These genes have structurally and functionally similarity with p53 and work in a signaling network for controlling different cellular functions such as cell death, differentiation, and proliferation.  The Tp53 gene is found altered in breast carcinomas in approximately 20-40% of all cases depending upon tumor size and stage of the disease. It seems to be an early event in breast tumorigenesis.  About 75% of Tp53 mutations are missense, 9% are deletions and frame shift insertions, 7% are nonsense mutations, and 5% are silent mutations. Different mutant forms of p53 protein have different biological and functional effects. These mutations result in significant loss in transactivation capacity as well as in DNA binding activity. ,
The most heterogeneous group of posttranslational modifications known in proteins is glycoproteins. Many prognostic and predictive biomarkers in cancer are glycoproteins in nature, for example, HER2-neu in case of breast cancer. The human epidermal growth factor receptor 2 gene is present on the long arm of chromosome 17 (17q 21.1 ). It encodes a receptor of molecular mass 185 kDa. These receptors are single pass transmembrane proteins and consist of intracellular tyrosine binding domain with various tyrosine phosphorylation sites and an extracellular domain for ligand binding and a cytoplasmic tail.  HER2 is a member of HER family, the other structurally related members of this family are HER1 (ErbB1 or epidermal growth factor receptor), HER3 (ErbB3), and HER4 (ErbB4). HER2-neu form heterodimers with other HER members preferably although it has no identified ligand. Major ligands of the other HER family receptors are transforming growth factor, epidermal growth factor (EGF), amphiregulin, heregulin, EGF, EPI, and NRG. ,
HER2 form homodimers and heterodimers and after dimerization, it induces different cellular functions such as cell growth, differentiation, and survival through different signaling cascade for example PLC gamma, MAPK, and PI3K. These pathways are influenced by pattern of dimerization. To prevent apoptosis and to promote cell proliferation, MAPK and PI3K are key signaling pathways. 
In normal cells, the number of HER2 receptor is 20,000 but in case of tumor, this number is increased up to 200,000 receptors. It will lead to aggressive form of breast cancer if left untreated. Twenty-five percent of all breast cancer cases are due to overexpression of HER2.  The HER2 positive breast cancer patients have more survival rate than patients without HER2 overexpression and significant apoptosis is induced in the breast cancer cells by the inhibition of HER2 overexpression. Both predictive and prognostic values of HER2 have been established. 
HER2 and Tp53 genes both are present on chromosome 17, HER2 on 17q 21.1 and Tp53 on 17p 13.1 . It is still unknown that how this proximity affects the interaction between these genes. There is a strong association between HER2 positive breast cancer and p53 mutations. Approximately, 71% of HER2 positive breast cancer patients contain mutated p53. If the patients have elevated level of HER2, indication for germline Tp53 mutation increased by almost seven-fold. 
Due to poor clinical outcome, HER2 overexpression represents an attractive target for therapeutic intervention. Trastuzumab is a monoclonal antibody, which targets the extracellular domain of the receptor and stops intracellular signaling thus prevent proliferation. It is approved as anti-HER2 antibody for the treatment of breast cancer patients with over-expression of HER2.  Among HER2 over-expressing tumor cells, HER2 protein levels are relatively homogenous, so in patients, anti-HER2 therapy targets most cancer cells. Nearly, all patients treated with trastuzumab ultimately experienced progression and disease recurrence. As trastuzumab binds with the extracellular domain of HER2, so physically it's binding with the receptor is prevented by shedding the extracellular domain which contains the trastuzumab binding site. 
BRCA1 and BRCA2
Approximately, 5-10% of breast cancers are hereditary, most of which are due to the mutations in tumor suppressor gene BRCA1 and BRCA2. BRCA1 is identified in 1994, located on chromosome 17q 21.31 which encodes a 220 kDa nuclear phosphoprotein. Much has been learned about the function of the BRCA1 in the past 10 years. BRCA1 spans about 110 kb of DNA by its 22 exons and contains tandem duplication of approximately 30 kb.  At its N-terminus, BRCA1 has a ring domain (amino acid 20-64). This domain mediates interaction of BRCA1 with other proteins and exhibit E3 ubiquitin ligase activity. At its C-terminus domain (BRCT), it is shared with other proteins which play an important role in maintaining genome integrity.  DNA repair and DNA response signaling functions of BRCA1 are associated with these domains. BRCA1 is a part of many super complexes which play an important role in different cellular functions like double strand break repair, activation of cell cycle checkpoints and activation of DNA damage response. Normally, BRCA1 form heterodimer with BARD1, which is also a RING/BRCT containing protein. BRCA1 is unstable in the absence of BRAD1 and rapidly degraded. 
BRCA2 is present on chromosome 13q 12.3 and contains 27 exons. In BRCA2, translation start sites present in exon 2 and in AT rich coding sequences. It encodes a 3418 residue protein and contains eight BRCT repeats. By the age of 70, high lifetime risk of breast cancer associated with BRCA1 and BRCA2 is 80% and 50% respectively.  In the subnuclear clusters, BRCA2 directly interact with RAD51 which is a key component of DNA damage repair via its C-terminus domain or BRC repeats, while in case of BRCA1 during this process, its activity is modulated by phosphorylation. If BRCA2 is defective, cells become hypersensitive to agents which break double stranded DNA or cross-link DNA strand. If cells contain nonfunctional BRCA2, repair of double strand breaks occur in error prone fashion. The errors result in chromosomal instability which is a feature of carcinogenesis. 
Up to 30% of the breast cancers are attributed to mutations in BRCA1 and BRCA2. In these mutations, three founder mutations are commonly encountered that is, c.5266dup (5382insC), c.68_69del (185delAG) in BRCA1, and c.5946del (6174delT) in BRCA2. It is highly unlikely that a different mutation will be found, in case if one of these mutations is not present. ,
Progesterone receptor encoded by gene PGR and consists of 933 amino acids. It is located on chromosome 11q 21.1 . It is composed of three domains: A DNA binding domain, a modulating N-terminal domain and a ligand binding C-terminal domain. It is involved in cell-cell signaling, negative regulation of gene expression and transcription initiation from RNA polymerase II promoter. 
PR sequestered in a nonproductive form associated with cellular chaperones and other heat shock proteins, in the cells which lack the progesterone. In the absence of progesterone, the receptor is not able to control the transcription rate of its cognate promoters. In the presence of progesterone, PR undergoes many changes which involve dissociation from the heat shock protein complexes, conformational changes, phosphorylation, sumoylation, ubiquitination, dimerization, and nuclear translocation. These changes allow progesterone receptor to bind with progesterone response elements, which are present in the regulatory regions of target genes. This binding causes the upregulation of target gene transcription by recruitment of basal transcriptional machinery and co-activators. 
Progesterone has two receptors that are PRA and PRB, which are transcribed by the same gene using alternative promoter. Both these proteins are identical, but PRB is longer than PRA because it contains extra 164 amino acids on its N-terminal. PRA is a repressor of activity of PRB which is a major activator of transcription factors.  During tumorigenesis, there is a change in the ratio between PRA and PRB that is more PRA than PRB. PR expression is a marker for normal ER functions, and approximately 60% of breast cancer expresses PRA or PRB. Breast cancer patients contain steroid receptors show better prognosis and response to endocrine therapy than patients that lack these receptors. 
| > Emerging biomarkers|| |
MKi-67 is a nuclear protein of molecular mass 359 kDa and commonly used for the detection and quantification of proliferating cells. Its expression is increased, associated with cell growth. It is commonly used as a diagnostic marker in various cancers because its expression reflects the cellular proliferation rate. 
Ki-67 gene is present on the long arm of chromosome 10 (10q 26.2 ). The complete sequence of cDNA encoding for the protein was published by Schluter in 1993. Alternative splicing results in two alternative mRNA species, encodes two isoforms of the protein. The small isoform has a calculated molecular mass of 320 kDa and large isoform, a mass of 359 kDa. A sequence of 22 amino acids can be found called Ki-67 motif, which includes the epitope (FKEL) targeted by original Ki-67 antibody and is highly conserved between species. In the carboxy terminal region of the molecule, a potential ATP/GTP site was predicted called P-loop. The antigen is exclusively detectable in the nuclei of the cells during interphase. 
It plays a critical role in cell division and thought to be required for maintaining cell proliferation, DNA metabolic process, cellular response to heat, meiosis, and organ regeneration. The difficulties in determining the functional role of Ki-67 is due to the absence of similarity with other proteins. Until recently, no protein except some Ki-67 equivalents in other mammals exhibits obvious homology with the human Ki-67, although the presence of conserved domains shared with proteins of characterized functions. 
Throughout the cell cycle, the expression of Ki-67 varies in intensity. This shows that it could lead to a misclassification of cycling cells as resting ones. During G1 and early S-phase, the levels of Ki-67 are low, during mitosis progressively increase to maximum level while, during anaphase and telophase, a rapid decrease in expression starts. The degree of expression can be used as a marker of different conditions of growth. Ki-67 has a half-life cycle of around 60-90 minutes. During cell cycle, the differences in expression are due to variable de-novo synthesis not due to the accumulation of nondegraded protein. Throughout the cell cycle, the location and cellular appearance is not homogenous. 
Within the growth fraction of given population, the expression of the Ki-67 is thought to be an indicator that the fraction of cells born into the proliferative category. A proliferation marker cannot predict the actual division of the cell, but can be used only to indicate the potential of certain cell to divide.  Methods that are mostly used in experimental research are for ethical and practical reasons difficult to apply to human. In contrast, Ki-67 immunostaining can be performed on various types of histological and cytological preperations and is more sensitive and rapid. One drawback is that formalin-fixed paraffin sections, which are routinely used in histopathology, could not be used for the original Ki-67 antibody. 
G1/S-specific cyclin D1 also called Bcl-1 and PRAD1 oncogene is a protein in human that is encoded by the CCND1 gene. It consists of 295 amino acids with molecular weight of about 34 kDa and is located on chromosome 11q 13.3 . CCND1 contains 5 exons and spans approximately 15 kb. Cyclin D1 belongs to cyclin family, whose members are characterized by periodicity in protein abundance during the cell cycle. 
After the discovery of CDK in yeast, now in human genome, more than 13 CDKs and 25 proteins have been identified with homology in the cyclin box, a region of 100 amino acids near the amino terminal. The CDKs form dimmers with different regulatory subunits called cyclins. Among these cyclins, cyclin D1 is the most important for its role in tumorigenesis. 
By alternative splicing two transcripts are formed, transcript a and b. Cyclin D1b encoded by transcript b contains 274 amino acids and different C-terminus domain from cyclin D1a.  Transcript b expressed in breast cancer cell lines and primary tumors, but at very low levels than transcript a. In breast cancer samples, overexpression of cyclin D1 is primarily accounted for by transcript a. In primary breast cancers, the relative levels of cyclin D1a and cyclin D1b proteins isoforms have not been reported so far. 
Cyclin D1 is very important in the development of neoplasia. It is an established oncogene for which the amplification and genomic rearrangement leading to overexpression is commonly found in multiple types of human cancer. In the cyclin D1 gene, these tumor-specific alterations reflect the critical selective advantage conferred by it's over expressed gene product and shows the importance of cyclin D1 in neoplastic process. There is uncertainty about the precise downstream cellular mechanisms that lead to neoplasia by dysregulated cyclin D1.  Strong evidence emphasizes that in human breast cancer cyclin D1 overexpression and amplification act as a driving force. Three- to ten-fold amplification of DNA on 11q 13.14 to 11q 13.5 is present in approximately 13% to 20% of the breast cancers. 
Cyclin E is a G1 cyclin which was first identified in 1991. It is located on chromosome 19q 13.1 containing 395 amino acids and molecular weight of 45 kDa. During the cell cycle, the cyclin E levels are periodic and its level is maximum during G1 phase with maximum enzymatic function of cyclin E-cdk2 complex. 
It regulates the cell transit from G1 phase to the S-phase of the cell cycle. In the nucleus, the cyclin E accumulates at the G1/S phase boundary and as cell progress through the S-phase it is degeraded. In cancer this firm regulation of cell growth by cyclin is lost.  In many breast cancers, the cyclin E is constitutively present in complex with cyclin-dependent kinase 2. This can lead to elimination of important checkpoint control and phosphorylation of substrates at inappropriate times during the whole cell cycle phases. Deregulation of cyclin E causes accelerated S-phase entry, genetic instability and tumorigenesis. It has been shown that in human mammary epithelial cells overexpression of cyclin E induces chromosomal aneuploidy. 
In the mid-1990s, the critical role of cyclin E in regulating G1 to S-phase was confirmed by two studies. In one study, constitutive overexpression of cyclin E resulted in diminished requirement for growth factors, decrease in cell size and shortening of the G1 phase.  In the other study, it was found that during G1 phase the microinjection of anti-cyclin E antibody into fibroblast resulted in cell cycle arrest.  In some breast cancer cell lines, the cyclin E gene is amplified. This amplification can results in constitutively overexpression of cyclin E mRNA throughout the cell cycle up to 64-fold. This overexpression results in chromosomal instability which provides strong support for the role of cyclin E in breast cancer. 
Posttranslational cleavage of full-length cyclin E results into low molecular weight forms that are more hyperactive than full-length protein. In addition to overexpressing the 50 kDa full-length cyclin E protein, some breast cancer cell lines express up to five low molecular weight isoforms of cyclin E which are unique to tumor cells. These are related to the increasing grade and stage of breast cancer. 
| > Future prospective|| |
As breast cancer is a heterogeneous disease with different risk profiles, biological behavior, molecular profiles, and several subtypes, it poses a challenge for its management. Prognostic and predictive biomarkers provide an important tool for individualized therapies and efficient treatment. Some biomarkers such as p53, BRCA1, and HER2 have proved clinical applicability, but there is still need to overcome the deficiencies in the methodologies of their assessment. More tools are required to facilitate clinical decision-making processes in the early stages. There is a need to pay attention on the design and conduct of clinical trials and for the validation of emerging biomarkers for clinical use and reterospective evaluation. With the effort being exploited in characterizing the molecular features of individual cancer and discover of new biomarkers with promising use in clinics, we would able to find unique case-specific patterns of biomarkers, to help in optimization of tailored therapies and breast cancer patient care at individualized level.
We are thankful to the National Institute for Biotechnology and Genetic Engineering for the technical support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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