The competency to detect molecular targets concurrently on individual tumor samples could allow association between gene products and proteins in real time. The expression of the targets protein can be monitored before and after treatment and offer a rapid method to measure the efficiency of a targeted therapy of an individual treatment. Some breast cancers put across protein biomarkers i.e. estrogen receptor, progesterone receptor and ErbB on which therapeutic decisions are made^{8, 10}. Treatment options for breast cancer vary depending on the stage at which the cancer is diagnosed. The early stage breast cancer treated by surgery and radiotherapy while more advanced forms of the disease treated with chemotherapy, hormonal and targeted therapies⁷.

To achieve such individualized treatment, appropriate targets must be identified, characterized and validated. Thus, the current work focuses to improve our perceptive of the biology of this disease and to recognize group of biomarkers that may help in early diagnosis and the prediction of treatment response, eventually contributing to more favorable patient response. The objective of this review is to summarize various molecular targets and the clinically approved targeted therapy as well as future agents for the treatment of breast cancer.

TARGETS FOR BREAST CANCER:

A targeted therapy is drug or other substances that block the growth and metastasis of cancer by interfering with specific molecules that are involved in growth, progression and spread of cancer¹¹. Most researchers consider targeted therapy to be a new approach to treat breast cancer that targets the inner workings of cancer cells. Targeted therapies work by going straight to the genes and proteins in cancer cells to stop their growth and spread¹². Genes promoting breast cancer metastasis abound, including ErbB2, CTNNB1, KRAS, PI3KCA, EGFR, MYC, TWIST1, SNAI1, SNAI2, MET, and ID1. Some genes are involved in tumor cell survival and colonization in the metastatic site including PTGS2, EREG, MMP1, LOX, ANGPTL4 and CCL5. Other genes such as PTHLH, IL11, CSF2RB, IL6 and TNF function in a more organ-specific manner¹³.

They are the newest type of treatment for breast and other types of cancers. Various works are carried out to find new ways to target cancer cells as part of treatment and once targets are found, then therapies can then be developed to kill cancer cells. Every cells in the body contains almost 30000 genes, each of these genes makes a different protein¹⁴.

Each protein perform different task for the cell. Many targeted cancer therapies have been approved by the Food and Drug Administration to treat specific types of cancer. Others are being studied in clinical trials and many more are in preclinical testing. Though all these suitability targeted therapy have some limitations. Cancer cells can become resistant to the targeted therapy and this resistance occurs in two ways the targets changes itself by mutation so that the therapy no longer interact with it, and tumors follows a new pathway for the growth that does not depends on targets¹⁵. Various targets for breast cancer management are mainly subdivided into two main topics as

· Clinically approved targeted therapy

· Future targeted therapy

Figure 3: Therapeutic targets for management of breast cancer

Clinically approved therapy:

ErbB Family of receptors:

Around 20% of new breast cancers demonstrated due to the over expression of the human epidermal growth factor receptor-2. The ErbB family of receptor tyrosine kinases consists of four homologous members, as ErbB-1 (EGFR, HER-1), ErbB-2 (HER-2, c-ErbB-2, neu), ErbB-3 (HER-3) and ErbB-4 or HER-4¹⁶. The ErbB receptors regulate organ development, cellular differentiation and morphogenesis in a large variety of tissues. ErbB-1 and ErbB-2 are commonly over expressed leading to deregulated proliferation and development, and contributing to oncogenesis in breast cancer¹⁷. The over expression of ErbB-1leads to about 30% of human breast cancer, and ErbB-2 is amplified and over expressed in up to 20% of primary human breast cancers¹⁸. ErbB-3 involve in breast and ovarian cancers while the ErbB-4 gene is mutated in approximately 20% of melanomas¹⁹.

Anti-ErbB therapies:

A number of new anticancer therapies have been developed to target breast cancers demonstrating ErbB-2 amplification. The most important ErbB-2 targeted therapies include monoclonal antibodies, Antibody-Drug conjugates, Heat-Shock protein-90 inhibitors and Tyrosine kinase inhibitors²⁰.

Monoclonal antibodies function by arresting cell cycle in G1 phase and work by binding to the extracellular domain of the receptor, recruiting cytotoxic lymphocytes and perturbing ErbB-2 mediated signaling events²¹. Trastuzumab is a humanized monoclonal antibody that was approved for metastatic and adjuvant therapy, and is considered the backbone treatment for ErbB-2 positive breast cancer. The mechanisms of action of trastuzumab are not fully understood, but studies have shown that trastuzumab functions by antibody-dependent cellular cytotoxicity, inhibition of the cleavage of the HER2 extracellular domain, inhibition of signaling mediated by HER2 through PI3K, and MAPK cascades^22,
23. Subsequent data from the North Central Cancer Treatment Group (NCCTG)

N9831 study demonstrated that administration of trastuzumab concurrent with chemotherapy is preferred²⁴. Regrettably not all patients with HER2 breast cancer respond to trastuzumab therapy. The proposed mechanisms of resistance include inability for trastuzumab to bind to its target due to a truncated HER2 receptor that lacks the extracellular domain or due to steric hindrance by over expression of the membrane-associated glycoprotein²⁵.

Pertuzumab a monoclonal anti-ErbB2 antibody binds to subdomain II intended for an epitope distinct from that targeted by trastuzumab, which hamper with receptor homo and heterodimerization. It demonstrate significant efficacy in preclinical studies and good tolerability, bioavailability, and clinical activity upon three week intravenous administrations²⁶. The combination therapy Pertuzumab and Trastuzumab were generally preferred and well tolerated with common adverse effect diarrhea in randomized patients with metastatic HER2 breast cancer²⁷.

Trastuzumab- emtansine (T-DM1) is an antibody drug conjugate in which trastuzumab is linked to the microtubule-inhibitory agent mertansine (DM1) consists of trastuzumab conjugated to DM1 and is a novel category of antibody-drug conjugate therapeutics²⁸. Based on the results of the EMILIA trial, T-DM1 was approved by the FDA in February 2013 for the treatment of patients with HER2 metastatic breast cancer who had previously received trastuzumab and a taxane, separately or in combination²⁹.

The proper folding of the ErbB-2 protein following its biosynthesis in the ribosome is performed by heat-shock protein-90. By inhibiting this chaperone function of heat shock protein-90 with inhibitors such as tanespimycin (also known as 17-AAG), the stability of ErbB2 is undermined³⁰.

Tyrosine kinase inhibitors a small molecular compound have been developed that block the nucleotide binding site within the intracellular domain of ErbB proteins. Lapatinib is a derivative of 4-anilinoquinoline and inhibits the phosphorylation and subsequent activation of the PI3K-Akt and Ras-Raf-mitogen activated protein kinase signaling cascades, increasing apoptotic activity and decreasing cellular proliferation. It targets both ErbB-1 and ErbB-2 by reversibly attaching to the intracellular adenosine trisphosphate binding sites of kinases³¹.

The deep access in to the cleft of ErbB-1 has been permitted by large aniline quinazoline head group of Lapatinib. The FDA have been approved only tanstuzumab and Lapatinib for clinical use in ErbB-2 positive breast cancer patients³². Combined HER2 targeting with lapatinib and trastuzumab has been shown to be effective in the treatment of trastuzumab-resistant metastatic HER2 breast cancer³³.

PARP receptors:

PARP1 is a multifunctional enzyme; best known for its role in the repair of single strand DNA breaks³⁴. Germline BRCA1/2 mutations are responsible for only a marginal of all breast and ovarian cancers comprised less than 10% of these malignancies³⁵. Several drugs targeting poly ADP-ribose polymerase enzymes are under development. The results observed in patients with germline mutations in BRCA1 and BRCA2, with further data supporting antitumour activity of PARP inhibitors in sporadic ovarian cancer³⁶. Iniparib was supposed to be a PARP inhibitor that showed promising results in randomized phase II trials in patients with triple-negative breast cancer but showed negative results in phase III trial³⁷.

Olaparib was a more potent inhibitor of cell growth than iniparib. The inhibition mechanism of both drugs was cell line-dependent and independent of the molecular subtype status of the cells³⁸. Olaparib and iniparib are among two of the PARP inhibitors most extensively investigated in clinical trials in patients with breast cancer^{39, 40}.

Future targated thearapy:

Proliferative index (Ki-67):

Ki-67 is the proliferation marker and one of the most controversially discussed parameter for treatment choice in breast cancer patients. Gerdes firstly identified a nuclear protein being related with cellular proliferation⁴¹. Ki-67 is present in all proliferating cells, and there is great interest in its role as a proliferation marker⁴².

The Ki-67 antibody reacts with nuclear non-histone protein that is present in all active phases of cell cycle, except the G₀phase. The Oncotype dx^TM assay used to predict the risk of occurrence and the degree of chemotherapy benefits in women with node-negative, ER-positive breast cancer in which the one of the 21 potential favored genes is Ki-67⁴³. The immunohistochemical evaluation is the most widespread analysis method for detection of Ki-67 antigen. It was shown that Ki-67 nuclear antigen is expressed in certain phases of the cell cycle namely S, G_1,G₂, and M phases, but is non-existing in G₀phase⁴⁴. Ki-67 has also been appreciated as a prognostic factor being associated with breast cancer outcomes^{45, 46}. The study demonstrated that Ki-67 is widely applied in routine clinical work and associated with common histopathological parameters, but was shown to be an independent prognostic parameter for disease free survival in breast cancer patients⁴².

Micro-RNA:

The mature miRNAs are small, around 18-25 nucleotides long and non-protein coding RNAs. The studies in 2005 involving cell lines, in vivo models and clinical specimens have concerned several functions for miRNAs and recognized association of miRNAs with breast cancer. The functions including hold down oncogenesis and tumors, promoting or restraining metastasis, and rising sensitivity or resistance to chemotherapy and targeted agents in breast cancer⁴⁷. The miRNAs have been recently more widely investigated due to their potential role as target for cancer therapy.

Micro-RNAs are known to regulate cell cycle and development that’s why now widely believed to play a vital role in many malignancies and act as tumor suppressors or oncogenes^{48, 49}. The majority of miRNA functions are locate on suppression of their target genes, which mean that a certain miRNAs will be tumor oppressive if its target gene is an oncogene⁵⁰. Gene regulation by miRNAs is important for the commencement and progression of several human cancers. The progression and development of breast cancer might manipulate due to various miRNAs shown by recent studies on breast cancer^{51,
52, 53}. The miRNAs can either promote or suppress tumor genesis and metastasis as shown in Table 2. An ability to target gene networks at multiple levels is an attractive characteristic of miRNAs.

The reduced tumor development, metastasis, improve response to treatment, and prevent resistance to therapy has carried out by modulating miRNA expression⁶³. Targeting the precursors of mature miRNAs can inhibit production and antisense oligonucleotides or their chemically tailored analogs may be used to inactivate an oncogenic miRNA⁶⁴.

The miR-497 functions mainly as a tumor suppressor gene. However it also acts as an oncogene in different cancers by directly targeting various downstream genes and multiple signaling pathways. The study shown that miRNA-497 can serve as a diagnostic and prognostic biomarker and as a promising therapeutic target in future clinical application⁶⁵.

Table 2: Micro-RNAs involved in breast cancer migration, invasion and metastasis

Sr. no.	miRNAs	Potential target	References
	Tumor suppressor miRNAs
1	miR-125a and miR-125b	HER2, HER3	54, 55
2	miR-205	HER3	56
3	miR-7	Pak1	57
4	miR-342	HER2	58
5	miR-34a	Bcl-2	59
	Oncogenic miRNAs
1	miR-21	BCL-2	60
2	miR-373 and miR520c	CD44	61
3	miR-375	No target determined	62

However, in the last few years there has been increasing interest in circulating miRNAs as cancer biomarkers, due to their high stability and the noninvasiveness of their detection. There are many pre-analytical (whole blood collection to plasma/serum preparation) and analytical (handling, extraction method and banking to RNA extraction and miRNA quantification) aspects as well as donor related factors can interfere with accurate quantification of circulating miRNA. If methodological parameters could be correctly considered in study design, some individual and environmental factors could not be properly assessed and taken into account during circulating miRNA analysis, thus probably influencing circulating miRNA application in clinical practice⁶⁶.

TP-53:

The p53 protein discovered by Levine et al. in 1979. TP-53 is encoded by the TP53 gene positioned on the short arm of chromosome 17 and contains 11 exons but the first exon does not encode and is located about 10Kb from other exons; a protein that is highly preserved across animal species⁶⁷. Vogelstein’s team discovered that the TP53 gene is inactivated in human cancers in 1989⁶⁸. The p53 protein enclose 393 amino acids is divided in to the regions highly sealed during evolution, and its function in numerous regulatory mechanisms has been well recognized⁶⁹.

The function of p53 is altered due to mutation in the DNA binding domain or deletion of the carboxy-terminal domain in nearly 50% of cancers and 30% of breast cancers⁷⁰. The Triple negative breast cancers, defined by lack of expression of estrogen receptor, progesterone receptor, and HER2, probably include both basal like breast cancer and some poorly differentiated luminal breast cancers. TNBCs also have an increased frequency of TP53 mutations. An increased expression of genes associated with ErbB-2 amplicon and TP53 mutation shown in HER-2 like tumors^{71, 72, 73}. The study revealed that alteration in TP53 were identified in 72/81 (89%), which is similar to other studies of basal-like or TNBC including The Cancer Genome Atlas dataset⁷⁴. TP53 status illustrates a strong prognosis impact and this could be useful in choosing best treatment for breast cancer.

Androgen Receptors:

The hormones play an essential role in breast carcinogenesis and therefore this provides a foundation for their use as anticancer therapies⁷⁵. The male sex hormones play an important role in normal female breast physiology and hence research has also been conducted in the use of androgens for breast cancer therapy. The hormonal imbalance may be results of breast cancer this ratio of estrogens to androgens is vastly different between women and men. AR is the most commonly expressed nuclear hormone receptor in the breast cancer^76,
77. The assays for ER and progesterone receptor (PR) are performed routinely and have been shown to predict for response to currently approved endocrine therapies.

In addition, AR targeted therapies may also be important for breast cancers that have developed resistance to current hormone and HER2 directed therapies⁷⁸. The inverse correlation establishes between histopathological grade and the expression of all sex hormone receptors in breast tumors. AR expression diminished from 88% to 47% in invasive carcinoma as tumor grade progressed from 1 to 3⁷⁹. The probable extrapolative importance of AR may useful to identify more assessable surrogate biomarkers for AR expression in breast cancer. AR is a marvelous target for therapy because it has been shown historically to be an efficient target for prostate cancer treatment and is commonly expressed in the majority of breast cancers⁸⁰.

The first clinical trial to report anti-androgen activity of bicalutamide was carried out in advanced breast cancer and establishes the potential of targeting AR in AR-dependent, ER-independent disease. The possibility of dual pathway inhibition of androgen and HER2, MEK, or PI3K/AKT as suggested by preclinical trials is unexplored clinically⁸¹.

3-Phosphoinositide-dependent protein kinase-1 (PDK1):

3-phosphoinisitide dependent protein kinase-1 is a protein encoded by the PDK-1 gene, which plays a vital role in the signaling pathways elicited by several growth factors such as protein kinase B, protein kinase C (PKC), p70 ribosomal protein S6 kinases, and serum glucocorticoid dependent kinases by phosphorylating serine or threonine residues in the activation loop. The regulations of physiological processes related to metabolism, growth, proliferation, and survival carryout by AGC kinases^82,
83. The changes in action and expression of PDK1 and several AGC kinases have been connected to human diseases including cancer. The critical component of oncogenic phosphoinositide 3-kinase signaling is alteration of PDK1 and suggesting that inhibition of PDK1 can inhibit breast cancer progression⁸⁴.

PDK1 protein and messenger ribonucleic acid are over expressed in a majority of human breast cancers. PDK1 is highly expressed in a bulk of human breast cancer cell lines. The PI3K pathway is one of the most frequently deregulated pathways in human malignancies, including activating and deactivating mutations, copy number changes, and post transcriptional epigenetic irregularities. The migration and experimental metastasis of human breast cancer cells have inhibited by down regulation of PDK1 level^{85, 86}. Thus targeting PDK1 may be a valuable anticancer strategy that may advance the worth of chemotherapeutic strategies in breast cancer patients.

CXCL12/ CXCR4 axis:

The cell surface receptor CXCR4 were first recognized as regulators of lymphocytes trafficking to the bone marrow and there after proposed to control the trafficking of breast cancer cells to sites of metastasis^{87, 88, 89}. The fundamental role in cancer cell proliferation, invasion and dissemination in the mainstream of malignant diseases including breast, ovarian, lung, colon, prostate, kidney, melanoma, brain, esophageal, pancreatic and many forms of leukemia was performed by CXCR4^{90, 91}. The common biological activity in stimulating the migration of different types of cells including monocytes, neutrophils, endothelial cells, mesenchymal stem cells and malignant epithelial cells carryout by a class of small (8-10 kDa) inflammatory or homeostatic cytokines CXCL12^{92, 93}. Chemokines are classified in to four groups based on the number and spacing of their N-terminal cysteine residues as CXC, CC, C and CX3C⁹⁴.

Chemokine receptors belong to a family of G protein coupled receptors containing seven transmembrane spanning α-helix domains. One of the intracellular loops of the chemokine receptors combines with heterotrimeric G proteins that mediate a cascade of intracellular signaling following ligand binding⁹⁵. It has been stated that several chemokine receptors including CCR2, CCR5, CXCR1, CXCR2, CXCR4 and CXCR7 can undergo homo and heterodimerization upon ligand binding; a method that was projected to regulate distinct intracellular pathways^{96, 97}. The upregulated CXCR7 is found to be involved in tumor cell growth, survival and metastasis in many malignant cells, including breast, lung, cervical, pancreatic and prostate cancer cells^{98, 99, 100}.

The CXCR4/CXCL12 axis also indirectly promotes tumor metastasis by arbitrate invasion and proliferation of tumor cells, and enhancing tumor associated neoangiogenesis. The CXCR4/CXCL12 signaling in tumor cells is regulated at several levels^{101, 102, 103}. Multiple agents currently developed to target CXCR4/CXCL12 signaling in breast cancer. The anti-CXCR4 drug AMD-3100 also known as plerixafor approved for stem cell mobilization in patient with non-Hodgkin’s lymphoma and multiple myloma. CTCE-9908 is approved for clinical use in patients with osteosarcoma which is a CXCL12 analog¹⁰⁴.

CONCLUSION:

Breast cancer is the leading disease in all over the world, and the various new drugs and targets are significant to lower the mortality rate of breast cancer. The biological markers or the therapeutic targets are the new technique to treat the breast cancer and other malignancies. The biomarkers are involved in the oncogenesis and development of cancer cells and hence inhibition of this pathway is very imperative for the management of breast cancer. Targeted therapy works by inhibiting the proteins and stop working of various proteins for the development of tumors and metastasis. The utilization of chemotherapy and radiotherapy is also beneficial for the treatment of breast cancers but they have various side effects and alter the quality of patient’s life. The use of targeted therapy which inhibits all these targets and their pathways utilized for the carcinogenesis and development of metastasis.

All above mentioned biomarkers are helpful to search for the new targeted drug therapy for the treatment of breast cancer. This review compiles the novel therapeutic targets for the management of breast cancer and the novel therapeutic drugs to minimize the risk of breast cancer. It also affords information to researchers, scientists and organizations to search for the various therapeutic drugs for the mitigation, prognosis and treatment of breast cancer.

CONFLICT OF INTREST:

The authors declare no conflict of interest.

ABBREVIATION:

EGFR (ErbB): epidermal growth factor receptor

HER: human epidermal growth factor receptor

PARP: poly ADP-ribose polymerase

PDK1: 3-phosphoinositide-dependent protein kinase-1

AR: androgen receptor

PR: progesterone receptor

TNBC: Triple negative breast cancer

miR: micro-RNA

ER: estrogen receptor

PKC: protein kinase C

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Received on 28.12.2017 Modified on 16.01.2018

Res. J. Pharmacology and Pharmacodynamics.2018; 10(1): 29-37.

DOI: 10.5958/2321-5836.2018.00006.X

Stage	Description
Stage I	The tumour is no larger than two centimeters, and has not spread to the lymph nodes
Stage II	The tumour is around five centimeters in size and may have spread to the lymph nodes under the arm
Stage III	The tumour may have spread to lymph nodes, be clumped together or sticking to other structures. The tumour may have also spread to surrounding breast tissue.
Stage IV	Tumour that have spread to other organs in the body e.g. lungs, liver or bone. This is sometimes referred to as ‘invasive cancer