Therapeutic Drug Targets and Phytomedicine For Triple Negative Breast Cancer

By Acharya Balkrishna

Triple negative breast cancers (TNBC) are a biologically aggressiveform of breast cancer and constitute approximately 10-15% of all breast cancerpatients. Distant metastasis, lack of clinically targeted therapies andprognostic markers, makes the disease difficult to treat. Till now not muchwork has been carried out on this deadly disease. This book provides an overview of TNBC etiology, its treatmentstrategies and prognostic markers to identify the outcome of standard therapies.Signalling pathways namely cell proliferation, angiogenesis, invasion andmetastasis, apoptosis, autophagy and others involved in complicating thedisease have been described in the chapters to convey an understanding aboutthe disease mechanisms. All the possible drugs either in pre-clinical orclinical stages have also been mentioned with data that depicts their efficiencyin targeting altered genes. The book also introduces the reader to herbalmedicine exhibiting high potency to target TNBC, their synthetic analogs usedduring chemotherapy and their ability to fight against chemoresistance. Theconcept of phytonanotechnology has also been discussed. The book helps createawareness among a broad range of readers about TNBC. It points to prioritizingthe upgradation of health care facilities and re-designing future treatmentstrategies to provide maximum benefit to breast cancer patients.

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Jan 13, 2023
• ISBN: 9789815079784

Book preview

Etiological Insights into TNBC and their Related Catastrophic Risks

Haritha H Nair¹, Ruby John Anto¹, *

¹ Cancer Research Program, Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India

Abstract

Triple-negative breast cancer is the most aggressive form of breast cancer that lacks expression of estrogen, progesterone, and human epidermal growth factor receptor 2. TNBC is characterized by poor clinic-pathological attributes, prognostic markers, unavailability of efficient therapeutic approaches, and higher chances of disease relapse along with metastasis to distant sites. Dysregulated epigenetic and transcriptional profiling was involved in cancer progression including histone modification, altered miRNA, DNA methylation, and long non-coding RNA signatures. This chapter will provide an insight into the molecular biology of TNBC including gene expression patterns and their subtypes. TNBC molecular spectrum was extensively studied to depict the distant metastasis-free survival and overall survival rate in affected individuals. Prevalence and epidemiology trends of TNBC patients across the globe were also studied to determine the impact of genetic predisposition and socioeconomic factors behind its aggressive behavior.

Keywords: Basal like, Epidemiology, Epigenetic profiling, Immune-modulatory, Intratumor heterogeneity, LncRNAs, Luminal androgen receptor, Mesenchymal stem-like, Metastatic, MiRNA, Molecular classification.

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* Corresponding author Ruby John Anto: Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India, Tel: [phone]; Fax: [phone]; E-mail: [e-mail]

INTRODUCTION

Breast cancer is one of the most common malignancies among women globally [1]. In 2018, approximately 2.1 million cases of breast cancers were diagnosed globally which account for 11.6% of women’s population, and a mortality rate of 6.6 has been reported across the world [2]. The heterogeneous nature of breast cancer might be responsible for such aggressive behavior. Identification of diverse cell phenotypes, their localization, and cell density may help in predicting the heterogeneous nature of the disease. A substantial difference in incidence and mortality rate has been observed so far depending upon the geographical location.

According to GLOBOCAN reports, incidences of breast cancer are more prevalent in Australia, Northern Western Europe, and North America. Whereas the incidences of the disease are slightly lower in Africa, South America, and Asia. However, a sudden increase in the number of cases in the latter regions has also been observed in the past few years (Fig. 1) [1, 3].

Fig. (1))

Incidences and mortality rate amongst females due to different types of cancers according to GLOBOCAN reports.

Intertumor or intratumor heterogeneity is majorly responsible for the evolution of different subtypes within the same tumor. These subtypes can be further characterized by their morphology, molecular profiling, or hormone receptors which can be used as a specific biomarker of the disease, for example, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). Such a huge variation in individual tumors suggested that tumor cells may exhibit different phenotypes with diversified functions and multiple expression markers of the disease. Furthermore, this intratumor heterogeneity enhances the ability of tumor cells to adapt to near micro-environmental conditions. A tumor sample dissected from the patient's body during biopsy cannot be considered as a true representative of the real tumor. A single tumor may comprise different cancer cell populations with diverse phenotypes, and properties and may show resistance to drugs. Collectively all these factors contribute to the complication of the disease and thereby making it more cumbersome to treat [4, 5]. Triple-negative breast cancer (TNBC) is another subtype of breast cancer that was deprived of the expression of ER, PR, and HER2. TNBCs are highly heterogeneous in the name of morphology, presentation, and genetic aberration. TNBC is characterized by high tumor grade, early relapse of the disease, high proliferation rate, and decreased overall survival rate [6, 7]. Processes like angiogenesis, uncontrolled proliferation, invasion and metastasis, and inhibition of apoptosis were the predominant pathways involved in complicating TNBC (Fig. 2).

Fig. (2))

Figure representing uncontrolled proliferation, angiogenesis, apoptosis, and invasion & metastasis in TNBC.

So far on the basis of transcriptional profiles, TNBC has been classified into 6 major molecular subtypes exhibiting differential responses to diverse chemotherapeutic regimens (Fig. 3) [8, 9]. According to Lehmann et al, these 6 subtypes include; mesenchymal (M), basal-like (BL-1 & 2), mesenchymal stem-like (MSL1), immune-modulatory (IM), and claudin low.

Fig. (3))

Figure representing different subtypes of TNBC including their percentage and genes involved.

Previously there was a certain confusion amongst TNBC and basal-like tumors as the latter ones are also high grade, aggressive with a high TP53 mutation rate and often occur in younger patients in comparison to the older population. Several studies clarified the fact that tumors expressing basal-like gene expression patterns could be considered TNBC but all the TNBC could not be classified as basal like [10]. Claudin low subtypes were further classified through hierarchical clustering which lies in proximity to basal-like tumors. Both of these subtypes exhibited similar gene expression signatures, for example, reduced expression of HER2 and genes lying in the luminal cluster of patients. There are several factors named as constraints in health care facilities, epidemiology, and cultural factors which pose a burden on TNBC patients and further reduce the life span of the diseased individual. The lack of treatment strategies, prognostic markers, and detection biomarkers has made the disease more complicated to treat. In this chapter, we will discuss the etiological insights into TNBC, its incidence, and the mortality rate caused due to the disease across the world [11-13].

Epigenetic Profiling of Triple Negative Breast Cancer

Altered epigenetic profiles were predominantly tangled in breast cancer pathogenesis. Molecules involved in dysregulated epigenesis such as chromatin regulators were considered emerging therapeutic drug targets. Transcriptional and epigenetic profiling of HCC1806 were extensively studied to depict molecular mechanisms involved in epigenetic theories [14]. These analyses also help in determining the functional genomic framework and outline chromatin and transcriptional regulators involved in interaction with the genome. Protein-DNA contact profiling and nascent transcriptional profiling are the functional tools that enable the investigation of protein-DNA interaction and RNA synthesis respectively. In this section, we will briefly discuss the role of DNA methylation, histone modification, miRNA profile, and long non-coding RNA signatures in altering TNBC expression [15, 16].

DNA Methylation Altering Triple Negative Breast Cancer Expression

DNA methylation constitutes a major epigenetic event involved in enhancing complications associated with TNBC. GC-rich, CpG island accounts for a short DNA sequence and represents a non-methylated state. DNA methyl transferase introduced certain modifications in DNA methylation that disrupted the structure of heterochromatin and caused an alteration in gene expression. Around 10% of gene promoters were associated with CpG island whose methylation status is dependent upon the transcriptional activity. In the case of TNBC, the DNA methylation patterns were observed to be similar to that of other breast cancer subtypes [17-19]. 5 genes named as CD44, RB, p73, CDKN2B, and MGMT are observed to be methylated along with other 11 non-methylated genes such as PMS2, MLH1, GSTP1, MSH2, MSH6, DLC1, MSH3, CACNAIA, CACNAIG, ID4 and Twist1 in TNBC. Genome-based applications to assess DNA methylation and miRNA expression in primary TNBC cells, its adjacent tissues, and lymph node metastasis have emerged as a promising technique to identify the new biomarker and other signaling pathways involved in the survival and proliferation of TNBC [20, 21].

Histone Modification Altering Triple Negative Breast Cancer

Histone modification is also referred to as post-translational alterations of the N-terminal domain which may hinder the chromatin structure and abrupt the process of transcription. Certain principal genes were also affected due to alteration in the N-terminal of histone proteins linked with TNBC which may induce more complications. Different cases of histone methylation and acetylation have been observed in different breast carcinoma subtypes so far. Analysis of histone modification could further support the process of diagnosis and prognosis in TNBC cells associated with epithelial-mesenchymal transition. Histone modification may help in differentiating breast cancer subtypes for example, the expression level of H3K4ac and H3K4me helps in differentiating between MCF-7 and MDA-MD-231. H3K4ac was involved in ER signalling pathways in MCF-7 whereas H3k4me and H3k4ac were chiefly involved in EMT mediated metastatic cascade in the MDA-MB-231 cell line of TNBC [22, 23].

Histone methyl transferase (HMT) catalyzes the methylation process of amino acid residues such as arginine and lysine. 4 HMTs named SETDB1, SMYD2, SMYD3, and ASHIL exhibited miRNA amplification in basal-like breast cancer on the other hand miRNA expression levels of 8 HMTs such as SETDT, E2H1, EHMT1, SMYD3, SETD3, WHSC1, SETD8, and SETDB were found to be reduced. Moreover, 12 other HMTs were also observed to be upregulated in BL in comparison to other breast cancer subtypes. Therefore, it can be clearly said that histone modifications may help in identifying and differentiating different subtypes and could significantly alter several signaling pathways to abrupt normal functioning thereby complicating TNBC [24].

miRNA Altering Triple Negative Breast Cancer

mRNA patterns that can be predicted through the transcriptomic analysis may help in understanding cell genetic code. In case of TNBC, miRNA may play a dual role both as a tumor suppressor or oncomiR. miRNA signature pattern plays a pivotal role in the prognosis and stratification of disease into its sub-classes [25-27]. Rinaldis et al. in their study identified 14 miRNAs associated with cell motility mechanism and thereby may act as an optimal prognostic marker of the disease. TNBC subtypes can further be classified on the basis of miRNA expression for example 13 miRNA were identified to be over-expressed in BLBC [28]. Gasparini et al. carried out IHC to divide TNBC Cohort into CK5/6-positive, core basal with EGFR, and into 5NP including 5 negative markers. They also identified four miRNA signatures named miR-493, miR-30c, miR-27a, and miR-155 to be dysregulated in TNBC. Hence on the basis of miRNA expression, TNBC patient population could be stratified into high-risk and low-risk IHC subgroups. The core basal subgroup depicted the worst prognosis. Therefore, miRNA could be potentially considered as a prognostic marker to predict the likely outcome of different chemotherapeutic approaches [29]. OncomiRs could also function as a negative prognostic marker as their elevated expression is directly linked with the enhanced proliferation index of TNBC. Kalecky et al. revealed that miR-17-92 overexpression could distinguish between BL1 and BL2 subtypes of TNBC [30]. To identify the miRNA signatures involved in several pro-survivals, proliferation, and metastatic pathways and their possible role in enhancing disease-free survival and overall survival rate, different studies were carried out. Several researchers opted for an integrated analytical approach to identify miRNA/mRNA/lncRNA expression in TNBC patients [31-33]. The expression level of miR-1305, miR-221, miR-4708, and RMDN2 mRNA could help in segregating lower or high-risk groups of TNBC, and subsequently, appropriate treatment could be assigned to benefit the patients [34].

Long Non-coding RNA Signature Altering Triple Negative Breast Cancer Expression

Long non-coding RNAs (LncRNAs) were also observed to play a pivotal role in carcinogenesis. LncRNAs could function as important diagnostic markers and promising therapeutic agents but were observed to be dysregulated in the case of TNBC [35, 36]. With the help of the bioinformatics tool, different TNBC-linked lncRNA genes have been identified and can be considered potential therapeutic targets. For example, BCAR4, a highly dysregulated lncRNA/gene was observed to be overexpressed in breast tumors. Elevated expression of BCAR4 was linked with a poor overall survival rate in TNBC patients. BCAR4 is also involved in the hedgehog signaling pathway and contributes to events of invasion and migration [37, 38]. MEG3 is another tumor suppressor lncRNA gene that can potentially retard cell invasion, proliferation, angiogenesis, pro-survival pathways such as PI3K, Akt, and TGF-β, and several other pathways [39]. Whereas the role of H19 has remained to be controversial as it can exhibit both oncogenic and tumor-suppressive properties [40, 41]. Expression levels of lncRNA could further be influenced by inducing epigenetic modifications; for example, CpG island linked with promoter region named LOC554202 was found to be hypermethylated which ultimately may downregulate the expression of Loc55420 in TNBC cells. Further, the transcription rate also played an important role in regulating the expression of the lncRNA gene (Fig. 4) [42, 43].

Fig. (4))

Figure depicted the impact of DNA methylation, long non-coding RNAs, miRNA alteration on TNBC.

An Insight of Molecular Characterization of Triple Negative Breast Cancer

To unveil an efficient therapeutic approach to target TNBC, it is highly needed to understand the biology of the disease. A comprehensive evaluation of genetic portraits involved in complicating the disease would further help in predicting targeted mutations and the possible drug candidates to target them. Based on disease progression, TNBC can be classified in different stages:

Stage 0: Stage 0 indicated that TNBC cells were still intact inside the duct and have not yet invaded deeper inside the nearby breast fatty tissue. In this stage, cancer is not yet spread to lymph nodes or any distant sites, and this stage is referred to as ductal carcinoma in situ or called a non-invasive TNBC subtype.

Stage IA: In this particular stage, the size of the tumor lesion was around 2 cm or even lesser and has not yet spread to distant sites or lymph nodes.

Stage IB: This stage refers to the tumor of around 2 cm or even slightly lesser than that but has micro-metastasize to 1-3 axillary lymph nodes. In lymph nodes, the size of cancer cells was around 0.2 mm and included around 200 cells. In stage 1B, cancer cells did not metastasize to distant locations.

Stage IIA: Tumor lesion was of around 2 cm size and has entered into 1-3 axillary lymph nodes and cancerous cells inside these lymph nodes were more than 2 mm in size. These cells were generally found in mammary lymph nodes as evident from lymph node biopsy results.

Stage II B: In this stage, the tumor lesion was between 2-5 cm in size and has spread to 1-3 axillary lymph nodes which can be found in internal mammary lymph nodes in the biopsy. This stage also included those tumor cells which were larger than 5 cm but have not yet grown into the chest wall or skin and have not yet spread to lymph nodes.

Stage III A: This stage includes those tumor cells which were not more than 5cm in size and have spread to 4-9 axillary lymph nodes but do not grow into the chest wall or skin but might be found in internal mammary nodes too.

Stage III B: During this stage, tumor cells have grown into the chest wall or skin but have spread to either 1-3 or 4-9 axillary lymph nodes, or may have enlarged internal mammary lymph nodes.

Stage III C: This stage tumor belonged to any recognizable size but has spread to either:

• 10 or more axillary lymph nodes

• Lymph nodes lying under or above clavicle region

• Tiny amount of cancer cells has reached internal mammary lymph nodes.

Stage IV: During this advanced stage cancer cells have spread to nearby lymph nodes, and to other organs including liver, brain, lung, or bone [32, 44].

Molecular Classification of Triple Negative Breast Cancer

Sorlie et al. in their study identified five molecular subtypes of TNBC based on its intrinsic gene expression profiling. Analysis of around 500 genes was carried out using hierarchical clustering with help of a cDNA microarray study of 65 breast tumor samples procured from 42 diverse diseased individuals [45]. Luminal A and luminal B subtypes were named on the basis of their possible luminal epithelial cell origin. These subtypes exhibit significant similarity in gene expression profiling and expression of ER whereas the HER-2 enriched subtype often depicted elevated expression of HER-2 gene and other closely associated genes for example GRB7. On the other hand, HER-2 enriched subtype possessed a reduced expression of luminal and hormone receptor-related genes. Few researchers revealed that HER-2-positive cancer cannot be referred to as HER-2 enriched subtype as they are often found to be belonging to luminal subtype as they co-express ER. These tumor subtypes were observed to be biologically different from HER-2 enriched subtype. Contrarily, normal-like breast cancer exhibited a similar expression pattern to that of normal breast tissue. Although, the biological significance of this subtype is yet to be elucidated. HER2 enriched subtype is of vital significance for prognosis whereas basal-like subtypes were often linked with the worst clinical outcomes. A novel gold standard intrinsic subtype test has been developed which may be of great clinical utility, as it involves 50 gene subtype predictors (PAM 50) and an RT-PCR assay to determine the intrinsic subtype of tumor samples using RNA of the tumor sample. Almost all the TNBC intrinsic subtypes have been identified so far and amongst all, basal-like subtype is found to be the most common subtype followed by the recently unveiled claudin-low subtype. In this sub-section of the chapter, we will briefly discuss the molecular subtype of TNBC based on their gene expression profiling [46, 47].

Basal Like (BL1 & BL2) Subtype of Triple Negative Breast Cancer

In the BL1 subtype of TNBC, components and pathways of the cell cycle and cell division were found to be actively involved. In this subtype, cell proliferation pathways were found to be linked with an over-expressed DNA damage response pathway named ATR/BRCA. Elevated Ki-67 mRNA expression level indicated that this subtype showed a high proliferation rate in comparison to other TNBC subtypes. In BL2 subtype several cells signaling growth factors such as MET, NGF, EGF, IGFIR, and Wnt/B-Catenin along with glycolysis and gluconeogenesis were observed to be involved. The most prominent growth factor receptors found in the BL2 subtype are EPHA2, MET, and EGFR [48]. Burstein et al in their study also confirmed the existence of 2 basal-like subtypes in basal-like clusters. B, T, NK cells, immune-regulating pathways, and cytokine signaling pathways were found to be down-regulated in the case of the BLIS subtype. This subtype showed reduced expression of molecules involved in innate and adaptive immune cell communication, antigen presentation, and differentiation of immunological cells, furthermore showing the worst prognosis. The expression of the Sox family transcription factor is another major characteristic feature of this subtype. According to Lehmann et al., the BL2 subtype is similar to the immune-modulatory subtype [10, 49]. The gene expression pattern of cell lines possessing BRCA1 and BRCA2 mutation coincides with a basal-like subtype of TNBC henceforth, it will not be untrue to say that BRCA mutant tumors are often basal-like TNBC [50].

Immunomodulatory Subtype of Triple Negative Breast Cancer

The immunomodulatory (IM) subtype of TNBC was observed to be extensively rich in factors linked with immunological processes. Immune cell signaling was commonly observed in the IM subtype. Genes involved in cell signaling substantially coincide with the genes involved in medullary breast cancer which is another rare form of TNBC. In comparison to BLIS, the IM subtype expressed an elevated level of genes involved in the control of the immunological response, for example, B, T, and NK cells. This subtype of TNBC showed better prognostic outcomes. Elevated expression of the STAT gene and its transcription factor-mediated cell signaling pathways leads to a better prognosis of this subtype of TNBC. Active involvement of STAT genes in IM type also widened the possibility to search for an efficient therapeutic approach against this particular subtype [49].

Mesenchymal and Mesenchymal Stem Like Subtype of Triple Negative Breast Cancer

Mesenchymal stem like (MSL) subtype of TNBC comprises of components and pathways associated with the cell differentiation pathway, cell motility, extracellular receptor interaction, and several others. The mesenchymal (M) subtype of TNBC also exhibited genes similar to MSL for common biological processes. However, certain genes are uniquely present in the MSL TNBC subtype which were predominantly linked to growth factor signaling pathways such as EGFR, calcium signalling, ERK1/2, PDGF, ABC transporters, G protein-coupled receptor, and adipocytokine signaling. Burstein et al in their study said that this subtype of TNBC exclusively represents genes related to adipocytes (ADIPOQ, PLIN1) and osteocytes (OGN). Important growth factor-1 was also observed to be over-expressed in this subtype of TNBC [49].

Luminal Androgen Receptor Subtype of Triple Negative Breast Cancer

Luminal androgen receptor (LAR) was the highly differential subtype of TNBC in comparison to other subtypes. They exhibited ER-negative status, but certain genes were mostly enriched in pathways that were hormonally regulated such as androgen/estrogen metabolism, porphyrin metabolism, and steroid synthesis. From gene expression profiling, it was observed that the LAR subtype involved ESR1 (gene encoding for ERα). Several other estrogen-regulated genes such as GATA3, FOXA, PGR, and XBP1 were involved in the LAR subtype referred to as ER-negative tumor and provided molecular evidence of activation of other estrogen receptors [49]. In LAR, AR mRNA was found to be over-expressed, and a 9-fold higher expression was observed in comparison to other subtypes. The immunohistochemistry analysis demonstrated that AR expression is 10 times higher in the LAR subtype when compared with other TNBC subtypes. From these observations, it can be concluded that LAR subtypes are androgen receptor-driven tumors thus AR could be considered a potential therapeutic target [50].

Claudin Low Subtype of Triple Negative Breast Cancer

Recently, another subtype of claudin-low has been identified through gene expression profiling [51]. Claudin low which constitutes an important part of tight junction that was observed to cover the available space between coinciding epithelial cells, EPCAM, mucin-1, and E-cadherins. Approximately 61-71% of claudin-low tumors were triple negative whereas 25-39% of TNBC belongs to claudin low subtype hence it could be predicted that neither all the claudin low subtypes were TNBC nor vice versa. Due to downregulated proliferation genes and uneven expression of basal keratin proteins, claudin low subtype greatly differs from basal-like tumors. Luminal markers were downregulated in this subtype and epithelial to mesenchymal transition markers, cancer stem-like features were observed to be overexpressed. Elevated expression of EMT marker was associated with resistance to chemotherapy and enhanced metastatic potential and played a prominent role in enhancing the complexity of disease in this subtype. In response to neoadjuvant chemotherapy, a declined pathological complete remission rate was observed in the LAR subtype which was lower than the basal-like subtype but was significantly higher than the LAR subtype. Depiction of LAR has further provided evidence for the etiology of TNBC and subsequently has enhanced our understanding of its biology and ultimately would help in the discovery of potential therapeutic implications to target the disease [50-53].

Heterogeneous Nature of Triple Negative Breast Cancer: A Stumbling Block

Different histological variants exist in TNBCs, for example, existence of metaplastic elements, stromal lymphocytic response, medullary elements, and poor tumor differentiation. TNBC spectrum was generally considered as a lower graded neoplasm with no or very less possibility of metastatic potential to invasive carcinomas. So far, different researchers could only identify two subsets of lower graded TNBC and named them as [54-57]:

(A) Low-grade TN breast neoplasia family

(B) Salivary gland-like tumors

High genetic instability with complex genomes has been observed in TNBC. Somatic mutations were highly prevalent in tumor suppressor genes such as TP53 and PTEN (phosphatase and tensin homolog). In 10% of TNBC cases, mutations in PI3K/Akt and PIK3CA pathways have also been observed [28]. Bareche et al., in their study, revealed genomic alterations in TNBC molecular subtypes. BL1 tumors were associated with chromosomal instability to a great extent along with approximately 92% of TP53 mutation rate, amplified PI3KCA, and AKT2, high copy number with several genomic deletions were involved in DNA repair mechanism. Whereas in the LAR subtype of TNBC, high mutation rates have been observed especially in CDH1, AKT1, and PI3KCA. Genes associated with angiogenesis pathways were observed to be altered in M and MSL subtypes of TBNC. In the IM subgroup, genes related to immunological response and checkpoint inhibitors were observed to be overexpressed. IM tumors were concluded to be contaminated with immune infiltrates [58-61]. The prognosis rate was linked with the worst prognosis conversely, IM subtype exhibited a better prognosis.