BIOSCIENCE REPORTS
ACCEPTED MANUSCRIPT Estrogen receptor negativity in breast cancer: A cause or consequence?
Vijaya Narasihma Reddy Gajulapalli, Vijaya Lakshmi Malisetty, Suresh Kumar Chitta and Bramanandam Manavathi Endocrine resistance, which occurs either by de novo or acquired route, is posing a major challenge in treating hormone‐dependent breast cancers by endocrine therapies. The loss of ERα expression is the vital cause of establishing endocrine resistance in this subtype. Understanding the mechanisms that determine the causes of this phenomenon are therefore essential to reduce the disease efficacy. But how we negate estrogen receptor (ER) negativity and endocrine resistance in breast cancer is questionable, to answer that two important approaches are considered: 1) Understanding the cellular origin of heterogeneity and ER negativity in breast cancers, and 2) characterization of molecular regulators of endocrine resistance. Breast tumors are heterogeneous in nature, having distinct molecular, cellular, histological and clinical behaviour. Recent advancements in perception of the heterogeneity of breast cancer revealed that the origin of a particular mammary tumor phenotype depends on the interactions between the cell of origin and driver genetic hits. On the other hand, histone deacetylases (HDACs), DNA methyl transferases (DNMTs), miRNAs and ubiquitin ligases emerged as vital molecular regulators of ER negativity in breast cancers. Restoring response to endocrine therapy through re‐expression of ERα by modulating the expression of these molecular regulators is therefore considered as a relevant concept that can be implemented in treating ER negative breast cancers. In this review, we will thoroughly discuss the underlying mechanisms for the loss of ERα expression and provide the future prospects for implementing the strategies to negate ER negativity in breast cancers.
Cite as Bioscience Reports (2016) DOI: 10.1042/BSR20160228
Copyright 2016 The Author(s). This is an Accepted Manuscript; not the final Version of Record. You are encouraged to use the final Version of Record that, when published, will replace this manuscript and be freely available under a Creative Commons licence. All other rights reserved.
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Estrogen receptor negativity in breast cancer: A cause or consequence?
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Short title: Estrogen receptor negativity in breast cancer
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Vijaya Narasihma Reddy Gajulapalli1#, Vijaya Lakshmi Malisetty2#, Suresh Kumar
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Chitta3 and Bramanandam Manavathi1*
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Dr. Bramanandam Manavathi, PhD Assistant Professor Department of Biochemistry School of Life Sciences University of Hyderabad Hyderabad-500046 India. Email:
[email protected] [email protected]
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#
Molecular and Cellular Oncology Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad-500046, India. 2
Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India. 3
Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515002, Andhra Pradesh, India. *To whom correspondence should be addressed:
These authors contributed equally to this work.
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Abstract
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Endocrine resistance, which occurs either by de novo or acquired route, is posing a
36
major challenge in treating hormone-dependent breast cancers by endocrine therapies. The
37
loss of ERα expression is the vital cause of establishing endocrine resistance in this subtype.
38
Understanding the mechanisms that determine the causes of this phenomenon are therefore
39
essential to reduce the disease efficacy. But how we negate estrogen receptor (ER) negativity
40
and endocrine resistance in breast cancer is questionable, to answer that two important
41
approaches are considered: 1) Understanding the cellular origin of heterogeneity and ER
42
negativity in breast cancers, and 2) characterization of molecular regulators of endocrine
43
resistance. Breast tumors are heterogeneous in nature, having distinct molecular, cellular,
44
histological and clinical behaviour. Recent advancements in perception of the heterogeneity
45
of breast cancer revealed that the origin of a particular mammary tumor phenotype depends
46
on the interactions between the cell of origin and driver genetic hits. On the other hand,
47
histone deacetylases (HDACs), DNA methyl transferases (DNMTs), miRNAs and ubiquitin
48
ligases emerged as vital molecular regulators of ER negativity in breast cancers. Restoring
49
response to endocrine therapy through re-expression of ERα by modulating the expression of
50
these molecular regulators is therefore considered as a relevant concept that can be
51
implemented in treating ER negative breast cancers. In this review, we will thoroughly
52
discuss the underlying mechanisms for the loss of ERα expression and provide the future
53
prospects for implementing the strategies to negate ER negativity in breast cancers.
54 55
Key words:Estrogen receptor alpha/ ER negative breast cancer/ endocrine resistance/
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miRNAs / epigenetic factors /ubiquitin ligases
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Throughout the world, breast cancer remains as one of the prevailing malignancies
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affecting millions of women, although it is scarce in men. Despite of our increased
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understanding of the disease and the improved diagnosis of it, large number of new cases are
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still being registered challenging the current diagnostic measures. For instance, the estimated
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new breast cancer cases and deaths by Sex in United States for the year 2016 is 249,260 and
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40,890, respectively [1]. Breast cancer can originate from different areas of the breast that
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include the ducts, lobules, or in some cases, between the breasts. The majority of breast
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cancers originates from epithelial cells and hence are called ‘carcinomas’ [2]. When left
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untreated, breast cancer can metastasize to other areas of the body, preferably to bone, lung,
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liver or brain and can cause malignancies.
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1. Breast cancer classification
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Breast cancer is heterogeneous in nature as it comprises of various cell types with distinct
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biological features and clinical behaviour. Breast cancers are classified as invasive or non-
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invasive types on the basis of localization and the extent of the tumor spread [3]. On a
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molecular basis (gene expression profile), breast cancers are classified into the following
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major sub-types (Figure 1) [4-12]. Each of these tumors has different risk factors, for
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incidence response to treatment, disease progression, and preferential metastases sites [13,
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14]. Further, the etiology, pathogenesis, and prognosis of breast cancer in patients of various
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races / ethnicities are significantly influenced by intrinsic molecular breast cancer sub-types
79
across the different populations around the globe [15]. PAM50 signature assay is by far the
80
most recent classification of breast cancer by molecular approach techniques, which
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measures 50 genes quantitatively. This assay was developed by Parker et al., for sub-
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classification of breast cancers into the three molecular sub-types (luminal A/B, basal-like,
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and HER2) [16]. The modern classification of breast cancer sub-types based on gene
3
84
expression profiling of the tumors, facilitated the clinical implications and the predictive
85
values of each sub-type. A recent report showed that the St. Gallen surrogate classification of
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breast cancer sub-types can successfully predicts tumor presenting features, nodal
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involvement, recurrence patterns and disease free survival [17]. Further, intrinsic molecular
88
profiling provides clinically relevant information endorsed by St. Gallen Consensus Panel
89
[11]. In view of the heterogenous nature of breast cancer, the optimal classification and sub-
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typing of each tumor will eventually help in the development of a conspicuous therapy.
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1.1 Triple-negative breast cancer (TNBC)
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Based on the immunohistochemical analysis, TNBCs have been identified as breast
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cancers that do not express ERα (estrogen receptor α), PR (progesterone receptor) and Her-2
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(human epidermal growth factor receptor 2) (triple-negative immunophenotype) [18]. Within
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the TNBCs, using gene expression and cluster analysis, Lehmann et al., identified 6 sub-types
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which include two basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal
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(M), a mesenchymal stem-like (MSL), and a luminal androgen receptor (LAR) sub-type [19].
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Recently, Prat et al., sub-classified TNBCs into basal-like (BL) (70%) or non basal-like
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breast cancers (NBL) (~25%) based on gene expression profiling data [20]. Irrespective of
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these different classifications, basically all TNBCs are aggressive in nature and are associated
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with more proliferation and metastasis than other sub-types. TNBCs account for up to 20% of
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all breast cancers. These types of tumors are associated with BRCA1 and BRCA2 mutations
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[21]. With respect to treatment, basal-like breast cancer patients within TNBC, but not in
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non-basal type, appear to benefit with either carboplatin or bevacizumab, an anti-VEGF
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monoclonal antibody therapy in neoadjuvent setting [22]. On the other hand, the non-Basal-
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like (i.e. Luminal A, Luminal B and HER2-enriched) or AR-positive, estrogen receptor (ER),
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and progesterone receptor (PgR)-negative metastatic breast cancers might benefit from anti4
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androgens [23]. However, in many cases the option for treatment is chemotherapy only, as
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the TNBC tumors are not amenable to conventional targeted therapies [24].
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1.2 Her-2 positive breast cancers:
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Her-2 positive breast tumors are characterized by the lack of expression of
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luminal/ER-related genes and over-expression or augmentation of Her-2 gene associated with
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aggressive phenotypes. ERBB2 gene encodes for a trans-membrane tyrosine kinase receptor
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(Her-2) which belongs to the epidermal growth factor (EGFR) family. These tumors are
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frequently high-grade and 50% of them exhibit p53 mutations and are associated with poor
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prognosis [16, 25]. This sub-types comprise approximately of about 14% of all the breast
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tumors and can be effectively treated by various anti-Her-2 therapies such as Transzumab or
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Lapatinib [25].
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1.3 Luminal breast cancer:
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About 2/3rd of breast cancers are ER-positive [26-28] that are specified by the expression
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of estrogen receptor α (ERα) and progesterone receptor (PR) in breast tumors. Because these
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tumors depend on estrogen for their growth, treatment with selective ER modulators (SERM)
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such as tamoxifen or raloxifene; or aromatase, which are crucial for estrogen biosynthesis,
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inhibitors like anastrozole or letrozole have better outcome in these patients. However, many
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patients with ER-positive breast tumors fail to respond to endocrine therapy with tamoxifen,
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an anti-estrogen, and most tumors that are initially responsive acquiring resistance by various
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mechanisms [29-31]. In recent years, high-throughput gene expression screening studies
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identify specific gene expression signatures that predict response to endocrine therapy and
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directs breast cancer patients for more appropriate therapeutic options [32, 33]. In other
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studies while using gene expression screening in mammary tumors, it was indicated that ER-
5
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positive breast tumors with poor response to endocrine therapy tend to have lower ERα
135
expression and high levels of proliferation-associated genes [32, 34-36]. Based on the
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proliferative index, luminal or ER-positive tumors were further classified into two intrinsic
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subtypes, Luminal A, and Luminal B [37]. Luminal A breast cancers express high levels of
138
ERα, lack of HER-2 expression, low expression of proliferative genes such as Ki67 and low-
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grade (1 or 2). These tumors grow very slowly and have the better prognosis than luminal B
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type [38]. These tumors (luminal A) are successfully treated with endocrine therapy and have
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the best prognosis with high survival rates with low recurrence. On the other hand, low levels
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of ERα are expressed by Luminal B tumors, which constitute about 10-20%, whereas Her-2
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positive are often high grade (2 or 3). Expression of proliferative markers like Ki67 and
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Cyclin B1 is higher in Luminal B tumors than in luminal A. Tumors of this sub-group are
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associated with a unfavourable prognosis than in Luminal A type and may benefit from the
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chemotherapy [39]. They can be treated with targeted therapies, for e.g., selective estrogen
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receptor modulators (SERMs), such as tamoxifen or, in postmenopausal women aromatase
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inhibitors such as anastrozole [40].
149 150
2. ER negativity and endocrine resistance in breast cancer
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Anti-estrogen resistance is likely to develop over time because of the highly pliable
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and adaptive nature of breast cancers to various selective pressures [41, 42]. Anti-estrogen
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resistance is of two types: de novo and acquired. The absence of both ERα and progesterone
154
receptor (PR) expressions represents the prevailing mechanisms of de novo resistance.
155
However, approximately 25% of ER+/PR+, 66% of ER+/PR-, and 55% of ER-/PR+ breast
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tumors do not respond to anti-estrogens [42]. Several experimental studies suggest that loss
157
of ERα can be due to long-term activation of growth factor signaling pathways.
158
Approximately 30% of the patients display loss of ERα where EGFR/HER2 activity is high 6
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[43, 44], where the acquired resistance is defined by loss of anti-estrogen responsiveness by
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initially responsive tumors. Most of the initial responsive breast tumors to anti-estrogens
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confer acquired resistance [29], which express ERα at recurrence on anti-estrogen therapy
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and are considered as ER+ tumors [45]. Although, tamoxifen has been shown to diminish
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the relapse and mortality rates of ER-positive breast cancers, a significant number of ER-
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positive tumors develop resistance to tamoxifen and become ER-negative [41]. It appears
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that a loss of ERα expression does not represent the major mechanism, driving acquired anti-
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estrogen resistance. Furthermore, it is very difficult to attribute any single mechanism that
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confers anti-estrogen resistance. Accumulating evidence suggests that several mechanisms
168
acting at cellular or molecular levels are likely to be responsible for the endocrine resistance
169
as discussed below.
170
Endocrine resistance is posing a major challenge today in treating significant
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percentage of breast cancers by hormone therapy. Understanding the mechanisms that
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underlie the causes of this phenomenon is therefore essential to reduce the burden of this
173
disease. But how we negate ER negativity and endocrine resistance in breast cancers is
174
questionable, to answer that two important approaches are considered: 1) Understanding the
175
origin of heterogeneity and ER negativity, and 2) characterization of molecular regulators of
176
endocrine resistance.
177 178
2.1 Understanding the origin of heterogeneity and ER negativity
179
Breast cancers are heterogeneous anomalies, having distinct molecular, cellular,
180
histological and clinical behavior [13]. Tumor heterogeneity is of two types: intra-tumor
181
(within the tumor) and inter-tumor (inter tumor). Breast cancers exhibit both intra-tumor as
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well as inter-tumor heterogeneity. But the underlying biology, causing tumor heterogeneity is
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yet to be fully understood. Due to the intra-tumor heterogeneity, breast cancer treatment has 7
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become more challenging today in clinical oncology studies [46]. To understand the tumor
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heterogeneity, it is essential to define the origin of each tumor cell type. Recent evidence
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suggests that the genetic lesions determine the tumor phenotype and cancers of distinct sub-
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types within a tissue, which may be derived from different 'cells of origin'. Defined genetic
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alterations/changes may lead to the initiation of respective breast cancer cell type [47].
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Although, identification of cell-of-origin of each sub-type of breast cancer is challenging, it
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would provide the identity and degree of transformation, which eventually enables us in
191
better understanding of the breast tumor sub-types as well as it would help in predicting the
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tumor behaviour and early detection of malignancies. In normal breast cells where ER-
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positive cells rarely proliferate, whereas in breast tumors ER drives cell proliferation [48].
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The lack of proliferation in the ER-positive ductal epithelium indicates a positive link
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between ERα expression and terminal differentiation in the normal breast cells and it further
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implies that ER-positive and -negative tumors arise from distinct cell types. Recent studies in
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model systems reported that luminal progenitors will serve as precursors for basal-like tumors
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if they receive a genetic or epigenetic event(s) that could change the phenotypes [49-53]. For
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instance, deletion of BRCA1 or PTEN in luminal epithelial cells results in loss of luminal
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differentiation, and then oncogenic insults in these cells, leading to the formation of basal-
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like tumors [54].
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Mouse models were used to address if the origin of a particular mammary tumor
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phenotype depends on the interactions between the cell of origin and driver genetic hits.
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Melchor et al., generated mice deleted of Pten, p53, and Brca2 in mammary basal epithelial
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cells or luminal ER-negative cells. Conditional deletion of Brca2 and p53 in either basal or
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luminal ER-negative cells resulted in tumors with different latencies and histopathological
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features. For example, tumors in mice derived upon of p53, Pten or Brca2 depletion in basal
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epithelial tumor cells displayed features of basal-like cells, whereas luminal ER-negative cell-
8
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origin tumors mimicked molecular sub-types of breast cancer, including basal-like and
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luminal B [55]. Transcriptome analysis from these tumors further provided the molecular link
211
between the genetic lesion and tumor type. Consistent with the phenotypic data, gene
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expression signature of Brca1:p53 mouse correlated with the human basal-like sub-type and
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with human BRCA1 breast cancers. The tumors of Pten deleted mice matched with the
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molecular features of Luminal A and non-BRCA1/2 cancers, whereas Brca2:p53/Pten:p53
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gene signature had been seen across the range of human breast cancer molecular sub-types.
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Based on these observations, it has been concluded that the initiating genetic lesion is the
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primary determinant of the molecular expression pattern of the resulting tumors.
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Furthermore, the genetic lesions together with a cell of origin serve as strict drivers of tumor
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phenotype but not the cell of origin alone, reiterating the fact that mammary tumor
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heterogeneity is a result of interactions between the cell of origin and early genetic events.
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The breast cancer can be initiated in a single cell by a combined effect of genetic and
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epigenetic events, suggesting that breast cancer is a monoclonal disease. Subsequent tumor
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progression is driven by the accumulation of additional genetic changes combined with clonal
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expansion and selection. The two models such as the cancer stem cell (CSC) and the clonal
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evolution and selection hypotheses agree that tumors originate from a single cell. However,
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controversies prevail regarding the tumor heterogeneity, progression, and development of
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drug resistance. The differences between two models depict how a transformed cell acquires
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multiple mutations and unlimited proliferative potential. In particular, these two models
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explain tumor heterogeneity with different mechanisms: CSC suggests tumor heterogeneity
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as a program of aberrant differentiation, whereas clonal evolution supports that it is a result of
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competition among tumor cells with different phenotypes [56, 57].
232
Tamoxifen treatment and heterogeneity have an intimate association in the
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development of endocrine resistance in breast cancer. Many breast cancers that arise after 9
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tamoxifen treatment are typically ER-negative, although premalignant lesions such as
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atypical ductal hyperplasia are highly ER-positive. The p53 null mouse mammary epithelial
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transplant model is characterized by ER-positive premalignant lesions that give rise to both
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ER-positive and -negative tumors. Given this progression from ER-positive to ER-negative
238
lesions, Medina et al tested the ability of tamoxifen to block or delay mammary
239
tumorigenesis in several versions of this model. Tamoxifen blocked estrogen signalling in
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these mice as evidenced by a decrease in progesterone-induced lateral branching and
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epithelial proliferation in the mammary epithelium. Tamoxifen also significantly delayed
242
tumorigenesis in ER-positive high premalignant line PN8a from 100% to 75%. From this
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study the authors derive that tamoxifen delays the emergence of ER-negative tumors if given
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in early stages of premalignant progression [58].
245
Recently, attempts were made to generate a novel heterogeneous, spontaneous
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mammary tumor animal model of Kunming mice (Mus musculus Km) which is ER-negative
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that have developed invasive ductal tumors which spread through the blood vessel into the
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liver and lungs. The mammary tumors are either ER or PR negative, while human epidermal
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growth factor receptor-2 (HER-2) protein is weakly positive. In addition, these tumors also
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had high expression of vascular endothelial growth factor (VEGF), moderate or high
251
expression of c-Myc and cyclin D1 which elucidates that this is one of the first spontaneous
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mammary model displaying colony-strain of outbred mice and could serve as a pivotal tool in
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understanding the biology of anti-hormonal breast cancer in women [59]. These mouse
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models can be further explored to study the origin of ER negativity and to further understand
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the endocrine resistance.
256 257
2.2 Characterisation of molecular regulators of endocrine resistance in breast cancer
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Because ERα is responsible for the development and progression of majority of breast
259
cancers, current therapies target ERα functions where tamoxifen, an anti-estrogen, has been
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the principal front-line therapy for breast cancers for the last three decades [60, 61]. But a
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large number of patients displayed tamoxifen resistance posing a major challenge in treating
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these patients [36, 62]. Although reduced expression of ERα is one of the major contributing
263
factors to the endocrine resistance [63, 64], the mechanism of ERα down regulation in
264
endocrine resistance is not fully understood. Recent advancements in the field suggest that
265
epigenetic modifications, miRNA-mediated gene silencing and proteasomal degradation,
266
either of which can cause loss of ERα expression resulting in ER negativity of breast cancers
267
(Figure 2).
268 269
2.2.1 Epigenetic regulation of ERα and development of ER negativity in breast cancer
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Mammalian genomes contain a high degree of punctuated DNA sequences of CpG
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called CpG islands [65]. Methylation of DNA at these CpG sites in the proximal regions of
272
gene promoters is quite often linked to suppression of the respective gene expression [66],
273
which is an epigenetic mechanism in which methyl groups are covalently attached to the
274
5´carbon of a cytosine ring in a CpG dinucleotide. Although CpG island methylation occurs
275
in normal developmental processes such as X chromosome inactivation and genomic
276
imprinting, these CpG islands are usually not methylated in normal cells [67].
277
Methylation of the ERα gene promoter is intimately linked to loss of ERα expression
278
in breast cancers [68]. Re-expression of ERα upon treatment of MDA-MB231 cells, an ER
279
negative breast cancer cell line, with 5-azacytidine, a DNA methyltransferase (DNMT)
280
inhibitor, provided initial clues about the role of DNA methylation on ERα expression [69].
281
Indeed, this was further supported by the observation that ER-negative tumors maintained the
282
methylation status of ESR1 gene (encodes ERα) promoter, but not in ER-positive tumors
11
283
implying that DNA methylation is the potential contributing factor for ER negativity in breast
284
cancers [70]. Yan et al showed that DNMT1 is responsible for ESR1 promoter methylation in
285
ER-negative breast cancer cell lines, MDA-MB-231. When DNMT1 expression was silenced
286
by antisense oligonucleotides, the expression of ERα was retained in MDA-MB-231 cells
287
[71]. Increased total DNMT activity and elevated levels of DNMT3B in a set of ER-negative
288
cell lines as compared to ER positive cell lines further attributed to higher rates of
289
methylation on promoters of ESR1 in ER-negative cells [72]. In other studies, methyl-CpG-
290
binding protein 2 (MeCP2) was shown to stabilize the methylation status of the ESR1 gene
291
promoter [73]. The MeCP2 is a component of NuRD complex, nuclear remodeling and
292
deacetylation complex is a large protein complex containing the dual core histone
293
deacetylases 1 and 2 (HDAC1 and 2), the metastasis-associated proteins MTA1 (or
294
MTA2/MTA3), the methyl-CpG-binding domain protein MBD3 or MeCP2, the
295
chromodomain-helicase-DNA-binding protein CHD3 (Mi-2α) or CHD4 (Mi-2β) and the
296
histone binding proteins RbAp46 and RbAp48. As the Mi-2/nucleosome remodeling and
297
deacetylase (NuRD) complex contains deacetylase activity, MeCP2-NuRD complex
298
represses ER expression by a dual mechanism involving methylation and deacetylation of
299
ESR1 promoter. Similarly, silencing of MTA1, another component of NuRD complex, is also
300
shown to reduce the ERα expression in ER-positive breast cancer cells [74]. Binding of the
301
NuRD complex to the ERα-target gene promoters has also been observed in ER-negative
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breast cancer cells re-expressing functional ERα in response to tamoxifen [75]. In contrast to
303
these observations, a recent study postulated that an increased ERα expression in ERα-
304
negative cells also increased its expression in ER-positive cells upon MTA1
305
silencing,differential recruitment of MTA1 transcriptional complex bound to ER promoter
306
has been identified as the underlying mechanism causing it. The transcriptional factors AP-2γ
307
(TFAP2C) and the IFN-γ-inducible protein 16 (IFI16) were associated with MTA1 complex
12
308
in MCF7 cells, in which TFAP2C activated ESR1 gene transcription in contrast to MDA-
309
MB231 cells where MTA1 complexed with IFI16 repressed the promoter activity and
310
silenced the MTA1 which increased the expression of ERα [76]. In another study, a different
311
model of epigenetic regulation of the ESR1 promoter was proposed based on the
312
experimental evidence obtained from ER–postive and –negative cell lines. In this model, an
313
activator complex composed of pRb2/E2F4/5/HDAC1/SUV39H1/p300 binds to E2F boxes in
314
the promoter region of ESR1 gene. However the presence of p300, a HAT, overcomes the
315
repressor activity imposed by both HDAC1 and the HMT SUV39H1 on ESR1 promoter.
316
Whereas in MDA-MB231 cells, methylation of CpG by Dnmt3a/3b on this promoter induces
317
the recruitment of ICBP90 (inverted CCAAT box binding protein of 90 kDa) and
318
consequently facilitate the replacement of p300 by Dnmt1 in the repressor complex
319
pRb2/E2F4/5/HDAC1/SUV39H1/Dnmt1 to silence the ESR1 gene expression [77].
320
Subsequently MeCP2 is recruited to the methylated ESR1 promoter to ensure its complete
321
repression [78] which infers that distinct protein complexes with opposing transcriptional
322
activities contribute to the epigenetic regulation of ESR1 gene expression in different breast
323
cancer cells. Similarly, inhibition of EZH2, a histone H3 Lys 27 (H2K27) methyltransferase
324
and Polycomb group protein, is associated with upregulation of ERα in breast cancer cells,
325
suggesting that targeting of EZH2 provides an option for restoring response to tamoxifen in
326
endocrine-resistant breast cancers [79]. In addition to these intrinsic regulators, arsenic also
327
has been shown to induce re-expression of functional ERα in MDA-MB231 cells [80]. The
328
re-expression of ERα by arsenic involves repression of DNMT1 and DNMT3a expression
329
along with partial dissociation of DNMT1 protein from the ESR1 promoter in these cells.
330
Thus, it can be concluded that ESR1 promoter is under constant threat from the protein
331
complexes that contain methylation and deacetylation enzymes and, provides an option to
13
332
target these mechanisms to re-express ERα which eventually restores the hormone sensitivity
333
and response to endocrine therapy in ER-negative breast cancers.
334
Attempts were made to test the therapeutic effects of methylation and deacetylation
335
inhibitors both in vitro and in vivo. Zhou et al showed that treatment with HDAC inhibitor
336
suberoylanilide hydroxamic acid (SAHA) resulted in the re-expression of ERα coupled with
337
the loss of EGFR in ER-negative MDA-MB231 cells and restored tamoxifen sensitivity in
338
these cells [81]. Down-regulation of EGFR by SAHA is due to the attenuation of its mRNA
339
stability. In contrary, Yi et al reports that SAHA enhances ER degradation through CHIP-
340
mediated proteasomal pathway in MCF7 cells, an ER-positive breast cancer cell line [82] and
341
thus can be postulated that opposing effects of SAHA in different breast cancer cells could be
342
due to the cell lines used, however precise mechanisms are yet to be identified. The combined
343
therapy using both DNMT and HDAC inhibitors displays better assurance to treat ER-
344
negative breast cancers [83]. Valproic acid (VPA), a HDAC inhibitor, is also shown to
345
restore estrogen sensitivity in MDA-MB231 cells by inducing the re-expression of ERα and
346
FoxA1, a coactivator of ERα [84]. Another study showed that letrozole treatment in
347
combination with Entinostatin, a HDAC inhibitor, increased the sensitivity in xenografts
348
where Letrozole alone had significant reduction in the expression of ERα but there was a
349
marked increase in the expression of Her-2 also [85]. As growth factor signalling antagonizes
350
ERα expression, treating it with trastuzumab (anti-Her-2 antibody) ablates Her-2 action,
351
leading in increased expression of ERα and enhances its sensitivity to endocrine therapy [86,
352
87]. However, the exact mechanism of trastuzumab blocking Her-2 leading to up regulation
353
of ERα remains elusive. A recent study shows that trastuzumab treatment enhances Myc–
354
SMRT interactions in Her-2 over expressing breast cancer cells and inhibits expression of the
355
Myc target gene, survivin. Further trastuzumab treatment induces the interaction between
356
CAAT box binding protein (CBP) and ERα which in turn enhances ERα transcriptional
14
357
activity and expression of the ERα target gene, pS2. Furthermore, metastatic tissues from
358
patients who had failed for trastuzumab therapy were pS2-positive providing the proof that
359
trastuzumab treatment can benefit endocrine-resistant breast cancer patients with hormone
360
therapy [88]. Recent studies also showed that FTY720 and avermectin, inhibitors of histone
361
deacetylase and SIN3 corepressor, as a novel strategy to restore tamoxifen sensitivity in ER-
362
negative and TNBC tumors [89, 90]. Overall, these studies showed the combination therapy
363
using various inhibitors of epigenetic modulators providing a new arsenal to the limited list of
364
therapies to endocrine-resistant breast cancer treatments.
365 366
2.2.2 Role of miRNAs in the development of ER negativity in breast cancer
367
MicroRNAs (miRNAs) are small non-coding RNA molecules with a length of 18–22
368
nucleotides, miRNAs are naturally synthesized by mammalian cells which mostly are
369
evolutionary conserved. These small RNAs modulate post-transcriptional expression of
370
protein-coding genes in diverse biological processes including cell cycle, survival,
371
differentiation, autophagy and senescence [91, 92]. miRNAs bind to 3’untranslated region
372
(UTR) of mRNA transcripts and inhibit their translation either by degradation or
373
destabilization of target mRNA [93]. Large data suggest that dysregulated expression of
374
miRNAs is found in many cancers, including breast cancer [94-97].
375
The connection between miRNAs and breast cancers was derived from studies
376
investigating the expression of miRNAs in breast cancer cell lines and tumor samples. As 3’
377
UTR of ERα mRNA, which is about ~4.3 kb long, contains several putative binding sites for
378
various miRNAs created curiosity to investigate the role of miRNAs on ERα functions and its
379
functional relevance to breast cancer development. miR-206 was the first miRNA reported to
380
regulate ERα expression in breast cancer cells,miR-206 has two binding sites within the 1200
381
bp region in the 3’UTR of ERα. Overexpression of miR-206 in MCF7 cells led to the 15
382
decrease in ERα levels, but has no effect on ERβ and the expression levels of ERα target
383
genes such as PR, CCDN1, and pS2 [98]. Similar to miR-206, miR-221 and miR-222 levels
384
that are elevated in ER-negative breast cancers could decrease ERα protein levels by binding
385
to 3’UTR of ERα. miR-221/222 expression confers tamoxifen and fulvestrant resistance in
386
ER-positive breast cancer cells indirectly contributing to ER negativity [99, 100]. It appears
387
that miR-221/222 expression confers fulvestrant resistance by activating β-catenin and
388
modulating TGF-β and p53 signalling [101]. Further, elevated levels of miR-221/222 were
389
found in ER-negative and Her-2-positive breast cancer cells. Silencing of these two miRNAs
390
partially restores ERα protein expression, tamoxifen-induced cell growth arrest and
391
apoptosis. In contrast, ectopic expression of miR-221/222 in ER-positive cells reduced levels
392
of ERα and conferred resistance to tamoxifen [63, 102]. In another study, miR-22 was
393
identified as a potential ERα-targeting miRNAs [103]. Ectopic expression of miR-22 caused
394
degradation of ERα mRNA and inhibition of ERα-dependent proliferation of breast cancer
395
cells. Further, miR-22 expression was found to be down regulated in ER positive human
396
breast cancer cell lines and tumor specimens [103, 104]. High level expression of miR-22 in
397
MDA-MB231 decreased ERα levels and subsequently induced apoptosis. let-7 is an ERα
398
targeting miRNA whose expression is low in ER-positive breast cancer cell lines. Studies by
399
Zhao et al., revealed that ectopic expression of let-7 miRNA in MCF7 cells decreases ERα
400
activity and cell proliferation, and subsequently induces apoptosis in MCF7 cells [105].
401
Furthermore, let-7 expression was inversely correlated with invasion and metastasis, which
402
indicates that loss of ER expression by let-7 may result in poor clinical outcomes and
403
resistance to endocrine therapy [106]. Since the activity of co-regulators is crucial for ERα
404
functioning, miRNAs that target co-regulators could also indirectly influence the
405
functionality of ERα in breast cancer cells. Consistent with this notion, miR-17-5p, represses
406
the AIB1/SRC-3, a coactivator of ERα, thereby attenuating ERα-mediated cell proliferation
16
407
[107]. Expression of miR-17-5p was low in breast cancer cell lines. Hossain et al found that
408
down regulation of AIB1 by miR-17-5p results in decreased ERα target gene expression and
409
proliferation of breast cancer cells [107].
410
In addition, high throughput analysis of miRNAs expression in breast cancers brings
411
about the prognostic value of breast cancer status irrespective of the influence of estrogen on
412
their expression and whether these miRNAs target ERα or not. For example, a microarray-
413
based study identified ERα is a target of miRNAs, miR-18a/b, miR-193b, miR-206 and miR-
414
302c [108]. Furthermore, high expression levels of miR-18a and miR-18b were correlated
415
with ER-negative status in breast tumors [109]. Another recent study found that 20 miRNAs
416
were significantly dysregulated in ER-positive compared to ER-negative breast cancers. Of
417
which, 12 miRNAs are up-regulated and eight are down-regulated. In particular, a miR-190b
418
expression is found to be 23 folds higher in ER-positive as compared to ER-negative breast
419
tumors [109]. Although the miR-190b expression is high in ER-positive breast tumors, its
420
expression is not directly influenced by estrogen and does not affect breast cancer cell
421
proliferation.
422
In order to identify the miRNA-mediated tamoxifen resistance in breast cancers,
423
Miller et al performed microarray studies comparing the miRNA profiles in tamoxifen-
424
resistant vs. tamoxifen-sensitive MCF7 breast cancer cell lines [102] which revealed that
425
eight miRNAs were significantly up-regulated while seven miRNAs were markedly down-
426
regulated in tamoxifen-resistant MCF7 breast cancer cells as compared to tamoxifen-sensitive
427
cells. Reintroduction of low expressing miRNAs in tamoxifen-resistant breast cancer cell
428
lines could restore tamoxifen sensitivity. For instance, down regulation of miR-342 in Her-2-
429
positive and negative cell lines as well as in tamoxifen refractory breast tumors were found
430
To be sensitive to tamoxifen when the expression of miR-342 was restored. Hence, restoring
431
miR-342 expression could be a novel approach to sensitize refractory breast tumors to 17
432
endocrine therapy [110, 111]. Together, these studies imply that miRNAs those target ERα,
433
contributes to the ER negativity in breast cancers and therefore, serves as potent therapeutic
434
markers as well as targets in endocrine-resistant breast cancers (Table 1). Additional studies
435
are required to confirm the roles of miRNAs in a clinical setting to get clear results. For,
436
clinical applications, miRNA expressions should be carefully validated prior to being
437
adopted.
438 439
2.2.3 Role of ubiquitination on ERα stability and breast cancer phenotype
440
The cellular levels of crucial regulators like kinases, receptors, phosphatases,
441
transcription factors, etc. are tightly regulated as their persistent high expression may have
442
undesirable effects on the cell. Ciechanover et al., first reported the selective degradation of
443
protein through the conjugation of ubiquitin molecules in an ATP-dependent manner [112].
444
Ubiquitinated proteins are recognized and degraded by the multi-subunit complex called the
445
26S proteasome [113], This ubiquitin–proteasome pathway has a role in diverse cellular
446
processing such as cell-cycle regulation, cell proliferation differentiation, apoptosis, etc. in
447
higher eukaryotes. Depending on the number of ubiquitins added to the target protein,
448
ubiquitination is two types: monoubiquitination and polyubiquitination. Although
449
monoubiquitination is associated with diverse processes ranging from membrane transport to
450
transcriptional regulation, polyubiquitination is mainly known to regulate protein turnover
451
through proteasome-mediated degradation [114].
452
The first report about ER ubiquitination was investigated by Nirmala and Thampan
453
[115]. They identified that the ERα in the uterus is ubiquitinated and this ubiquitination is
454
enhanced by estradiol treatment. The half-life of ERα in the presence of estrogen is about 3-4
455
hours [115] which was further supported by Nawaz et al,depicted that ubiquitin activating
456
enzyme, UBA, and ubiquitin conjugating enzymes, UBCs, can degrade ER protein in vitro. 18
457
Treatment of cells with the proteasome inhibitor MG132 or lactacystin could significantly
458
enhance the stability of ERα [116]. Subsequent studies clearly established that ERα
459
undergoes ubiquitination upon ligand binding and this modification is important for efficient
460
transactivation by the receptor [117]. Other than natural ligand, anti-estrogen ICI-182,780 can
461
induce proteasome-dependent proteolysis of ERα and therefore considered as a therapeutic
462
drug for treating ER-positive breast cancers [118].
463
Many ubiquitin ligases are known to directly interact with ERα and stimulate its
464
degradation and associate with breast cancer phenotype [119]. Fan and his colleagues
465
identified that the C-terminus of Hsp70-interacting protein (CHIP), a chaperone-dependent
466
E3 ligase, interacts directly with ERα and promote ERα degradation through ubiquitination-
467
proteasomal degradation pathway [120]. The U-box (containing ubiquitin ligase activity) and
468
the tetratricopeptide repeat (TPR, essential for chaperone binding) domains of CHIP are
469
necessary for CHIP-mediated ERα degradation. Ectopic expression of the CHIP, resulted in
470
decreased levels of endogenous ERα protein and impairment of ERα-mediated gene
471
expression, and hormone responsiveness in ER-positive cells. Notably, PES1, an estrogen-
472
inducible gene, inhibits CHIP-mediated ERα degradation mediated by CHIP. In contrast,
473
PES1 promotes CHIP-mediated ERβ ubiquitination and degradation. This differential
474
regulation of ER protein stability lies in the interaction of PES1 with AF1 domain of ERα but
475
not with ERβ. PES1 expression displayed good clinical outcome in breast cancers [121].
476
Whereas, suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was
477
reported to enhance ERα degradation through a CHIP-mediated proteasomal pathway in
478
breast cancer MCF7 cells, suggesting the positive crosstalk between CHIP and SAHA in ER-
479
positive breast cancers [82]. von Hippel-Lindau (VHL), another E3 Ub ligase and a tumor
480
suppressor, also regulates ERα stability. Ectopic expression of pVHL suppresses endogenous
481
ERα levels and also promotes ubiquitinylation-mediated degradation of ERα [122]. pVHL19
482
mediated ERα suppression is critical for the maintenance of microtubule organizing center
483
(MTOC) as elevated ERα promotes MTOC amplification through disruption of BRCA1-
484
Rad51 interaction and induces γ-tubulin expression [123]. Furthermore, activation of ERα
485
signalling can increase γ-tubulin, a core factor of TuRC which render resistance to Taxol in
486
breast tumors. Together, these findings suggest that pVHL-mediated ERα suppression is
487
important for regulation of MTOC as well as drug resistance in breast tumors [123]. The
488
speckle-type POZ protein (SPOP), an adaptor of Cullin3 based E3 ubiquitin ligase, also binds
489
to ERα and targets ERα for ubiquitination-dependent degradation [124].
490
Neural precursor cell developmentally expressed down-regulation of 8 (NEDD8) -
491
Uba3 pathway which is shown to mediate ERα proteolysis [125]. Uba3 interacts with ligand-
492
bound ERα through NR boxes that are important for the interaction between co-regulators
493
and steroid hormone receptors. Uba3 has neddylation activity, which is required for inhibition
494
of steroid receptor transactivation [126]. Duong et al., reported that Mdm2, an oncogenic E3
495
ubiquitin-ligase, directly interacts with ERα in a ternary complex involving p53. This
496
complex regulates both ligand-dependent and independent reduction of ERα stability in
497
human breast cancer cell lines, MCF7 [127]. Recent findings by Pan and colleagues showed
498
that CUE domain containing protein CUEDC2 could promote ERα degradation through the
499
ubiquitin-proteasome pathway. By studying specimens from a large cohort of subjects with
500
breast cancers, the authors found a strong inverse correlation between CUEDC2 and ERα
501
expression. Notably, patients with high levels of CUEDC2 expression had poor
502
responsiveness to tamoxifen treatment and high potential for relapse. Further, ectopic
503
CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen,
504
implying that CUEDC2 can contribute to resistance in breast cancer.
20
505
Not only the polyubiquitination but monoubiquitination of ERα has been associated
506
with its functional activity. For instance, lysine 302 of ERα is subjected to mono-
507
ubiquitination by BRCA1/BARD1E3 Ub ligase [128]. Down-regulation of BRCA1 leads to
508
activation of ERα, conversely ectopic expression of BRCA1 down regulates ERα activity
509
[129]. In contrary, monoubiquitination at lysine 302 and 303 is shown to be important for
510
ERα transcriptional activity and estrogen-induced cell proliferation [130]. RNF31, an atypical
511
E3 ubiquitin ligase, is also shown to monoubiquitinate ERα and increases ERα stability. This
512
is consistent with the previous reports supporting the stabilization of ERα by its
513
monoubiquitination. RNF31 and ERα association mainly occurs in the cytosol and activates
514
the non-genomic mechanism, by which RNF31 via stabilizing ERα levels, controls the
515
transcription of estrogen-dependent genes linked to breast cancer cell proliferation [131].
516
Other than ubiquitination, ERα phosphorylation is also prone to proteasomal degradation and
517
breast cancer phenotype. For instance, mitogen-activated protein kinase (p38MAPK)
518
mediated phosphorylation of ERα at Ser-294 is prone to its turnover via the SCF (Skp2)
519
proteasome-mediated pathway. Surprisingly inhibition of p38MAPK or Skp2 knockdown
520
restored functional ERα protein levels in ERα-negative breast cancer cells which, suggests
521
that p38MAPK or Skp2 are responsible for the loss of ER protein expression in ER-negative
522
breast cancer cells [132].
523
Over a decade of research on these aspects revealed that ERα regulators such as
524
epigenetic factors and ubiquitin ligases emerged as vital contributors of ER negativity in
525
breast cancers. The optimal balance between the expression of these regulators may predicts
526
the outcome of the endocrine response in breast cancer (Figure 3). With this data, we propose
527
a model wherein various epigenetic factors and ubiquitin ligases directly or indirectly
528
contribute to ER negativity and endocrine resistance in breast cancers by inhibiting ER
529
expression/functionality. The ER negativity along with PR (progesterone receptor) and Her-2 21
530
negativity together contributes to TNBC phenotype. As estrogen signalling via the ERα has
531
been shown to up-regulate the expression of the PR gene and thus the majority of ER-positive
532
tumors are also PR-positive. Therefore, loss of ERα expression could lead to PR negativity.
533
Since Her-2 over-expression or amplification is associated with loss of ERα expression and
534
vice versa, Her-2 overexpression is also a potential mechanism for ER negativity in breast
535
cancer (Figure 4).
536 537
3. ERα rescue therapy
538
The percentages of breast cancer cells, which become ER-negative that are initially ER-
539
positive are not very high (10%) [133]. Due to acquired resistance, initially sensitive ERα+
540
breast cancers response to a second and even third line therapies falls with increasing lines of
541
treatment [134]. It implies that the selective growth of ER-negative populations is not a
542
common contributor to acquired resistance. However, it is difficult to assess whether ERα+
543
breast cancers that do not respond will become ER-negative with treatment or not. But this
544
could be due to either the loss of ERα functionality or cells might have lost their dependence
545
on ERα to drive proliferation, and so the presence of functional ERα is no longer a
546
requirement for cell survival and proliferation [41]. On the other hand, tumors that exhibit de
547
novo resistance had an association between lower ERα expression for lesser extent and lower
548
rate of response to endocrine therapy [135]. This raised the possibility that re-expression of
549
ERα may benefit the endocrine therapy in these patients, but not in those who had tumors
550
with acquired resistance.
551
Rescue therapy, also known as salvage therapy, is a form of therapy given to the
552
patients who does not respond to the standard therapy. As the effects of anti-estrogens such as
553
tamoxifen are primarily mediated through the ERα, breast tumors expressing the receptor
554
respond well to SERM (selective estrogen receptor modulators) therapy. However, about 22
555
30% of invasive breast cancers are hormone-independent because they lack ERα expression
556
due to inactive ESR1 promoter [136]. Many of the tumors that initially respond to tamoxifen
557
can acquire resistance during and after tamoxifen therapy [30]. Therefore, ER negativity in
558
breast carcinomas confronts to treat with anti-estrogens. A hypothesis was emerged where re-
559
expression of the ERα could restore the endocrine response in ER-negative cells. When ERα
560
was ectopically expressed in an ER-negative breast cancer cell line (MDA-MB-231), 17-β-
561
estradiol inhibited the proliferation of these cells, while the anti-estrogens ICI182780, and
562
tamoxifen blocked this effect indicating that ERα reexpression restores tamoxifen sensitivity
563
in ER-negative cells [137]. Later on, several investigations led to provide the crosstalk
564
between ERα expression and growth factor signalling [138, 139]. Analysis of breast tumors
565
using phosho-specific growth factor receptor antibodies revealed that erbB-2/Her-2 over-
566
expressing tumors are ER-/PR-negative [140], indicating that increased Her-2 receptor is
567
associated with the ER-negative phenotype. Because ER-negative tumors often display over-
568
expression or amplification of growth factor receptors of the erbB family, particularly EGFR
569
and erbB-2, and consequently, elevated growth factor signaling and resultant MAP kinase
570
(ERK) activity, EGFR or Her2 over-expression in ER-positive breast cancer cells was
571
investigated. Accordingly, over expression of either EGFR or Her-2 in MCF7 cells results in
572
acquisition of estrogen-independence due to loss of ERα expression further supporting the
573
fact that growth factor signaling and ERα expression have mutual inhibitory action in breast
574
cancer cells [141, 142]. Since MAPK is the downstream molecule of these growth factor
575
signaling pathways, inhibition of this hyperactive MAPK restores ERα and acquired anti-
576
estrogen response [143, 144]. An exception to this relationship is that hyperactivation of
577
MAPK does not lead to re-expression of ERα in SUM-102 and SUM159, two ER-negative
578
basal type breast cancer cell lines which are found to exhibit hypermethylation of the ESR1
579
promoter suggesting that additional mechanisms may operate to repress ERα expression in
23
580
these cell lines [44]. Summing these studies, it can be concluded that the re-expression of
581
ERα in ER-negative breast cancer cells by inhibiting EGFR or Her-2 signaling restores, at
582
least in part, a hormone-responsiveness and could be useful as a potential therapeutic
583
approach to endocrine-resistant breast cancer.
584
Initial studies on ER-negative breast cancer cells by treating with demethylating
585
agents and histone deacetylase inhibitors led to the expression of ER mRNA and functional
586
protein. Fan et al reported that ERα can be re-expressed in ER-negative breast cancer cells by
587
both DNA methyltransferase-1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (AZA) and
588
histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic
589
acid (SAHA)1 [145]. Another study by Zhou et al., showed that ERα reactivation can be
590
achieved using clinically relevant HDAC inhibitor LBH589 without demethylation of the
591
CpG island within the ESR1 promoter [146]. These studies provide evidence that ER-
592
negative breast cancer cells can be sensitized with anti-tumor effects of tamoxifen by
593
combining treatment with 5-aza-dC/TSA. As indicated earlier, inhibition of growth factor
594
signaling by trastuzumab that blocks Her-2/MAPK activation renders ERα re-expression and
595
acquire the tamoxifen sensitivity. These studies provide new treatment options for patients
596
with de novo resistance to endocrine therapies.
597
ERα re-expression is a win-win strategy to combat ER-negative breast cancer
598
(personal opinion). Because the application of HDAC, DNMT or MEK inhibitors restores
599
ERα expression in ER-negative breast cancer cells, these cells have responded to selective
600
ERα antagonists [143, 145]. However, studies by Al-Eshry’s group demonstrated that ERα
601
re-expression does not always result in effective responses to SERM therapy [44], which is
602
because certain cancer cells fail to re-express ERα upon inhibition of the growth factor
603
pathway. Over a period of time, the heterogeneity of a tumor might have changed due to
604
which these tumor cells did not re-express ERα. Moreover, the systemic factors that 24
605
accounts for establishing the local ecosystem within the tumor had opposed the re-
606
expression of ERα. It implies that although combined therapy using these inhibitors along
607
with Tamoxifen has shown promising results in vitro and in vivo models, the following
608
concerns need to be fully addressed before implementation of re-expression of ER therapy
609
in clinics: 1) does all tumor cells respond to anti-estrogens? In case of tumors that exhibit
610
acquired resistance has developed more heterogeneity and may respond poorly to anti-
611
estrogens, 2) re-expression of ERα in those tumors with the application of HDAC, DNMT
612
or MEK inhibitors may develop resistance to these inhibitors, 3) since ER-positive breast
613
cancer cells die without ERα and ER-negative breast cancer copes without the receptor, why
614
does one want to give another selective advantage to these tumor cells? and 4) because breast
615
cancer cells will gain also the proliferative advantage given by the endogenous circulating
616
estrogen, will that not affect the quality of the life of the patient? Therefore, the ERα re-
617
expression in ER-negative breast cancer cells for restoring response to endocrine therapy
618
need to be thoroughly investigated using large cohorts of clinical trials.
619
As the mechanisms underlying endocrine resistance is very complex, for the benefit
620
of these patients, exploring combination therapies are extremely important for improving
621
the overall survival. Indeed, endocrine therapy combined with Gefitinib, lapatinib or
622
everolimus is currently under investigation in clinical trials. The study results have
623
provided the evidence that combination therapy may improve the progression free survival
624
in treated patients [147, 148]. A recent study also showed that Gefitinib could reverse TAM
625
resistance in breast cancer cells by inducing ERα re-expression [149]. The same group also
626
previously showed that elemene (ELE), a traditional Chinese medicine, could reverse the
627
TAM resistance of breast cancer cells and that ERα loss was the primary cause for the
628
development of TAM resistance in these cells [150]. ELE appears to induce ERα re-
629
expression by increasing the ERα transcript level to sensitize the cells to anti-estrogens. It 25
630
implies that re-exposure of ER-negative breast cancer patients to either drugs such as
631
Gefitnib, decitabine, elemene (ELE) or LBH589 followed by endocrine therapy may benefit
632
these patients and provide a novel therapeutic strategy for endocrine therapy.Although one
633
such attempt was made, unfortunately, the clinical trial of combination therapy using
634
tamoxifen in combination with decitabine, demethylating agents and LBH589, deacetylation
635
inhibitor was discontinued. The reason being for early termination of the study was due to
636
small numbers of participant’s analyzed and technical problems.
637
4.
Concluding remarks and future prospects
638
Because endocrine resistance possesses a major challenge in treating the significant
639
number of breast cancer cases, understanding the mechanisms that underlie the causes of this
640
phenomenon is essential to reduce the burden of this disease. Although significant
641
advancements are being made in the identification and characterization of several factors that
642
contribute to the endocrine resistance, but our present understanding of this phenomenon is
643
still at premature stage. Lack of ERα expression due to hypermethylation of ESR1 promoter
644
made researchers in this field to draw new strategies to re-express ERα in ER-negative breast
645
cancers. Indeed, such strategies were successful in pre-clinical trials, but yet to reach the
646
clinics. Of note, drugs such as SAHA in combination with Herceptin perceived greater
647
attention to show the promise in endocrine therapy [151]. Several miRNAs have been
648
differentially expressed in endocrine cancers and emerged as new prognostic markers of the
649
disease. More importantly, expression profiling studies showed over-expression of several
650
ERα targeting miRNAs in ER-negative breast cancers suggesting that they can be served as
651
bio-markers in the diagnosis and also in the management of breast cancer. Furthermore,
652
developing the miRNA mimics as therapeutic drugs targeting these miRNAs will have the
653
greater clinical value, but future awaits improving our technological advances in delivering
26
654
these agents in the form of drugs into the sites of tumor. The other contributing factor for
655
endocrine resistance is ERα-specific ubiquitin ligases. Because several lines of evidence
656
suggest that re-expression of ERα in ER-negative breast cancer cells can restore sensitivity to
657
tamoxifen, restoring the ERα expression by inhibiting ERα-specific Ub ligases provide
658
potential novel strategies for restoring tamoxifen sensitivity. Therefore, small molecule
659
inhibitors specific to these Ub ligases may overcome tamoxifen resistance in breast cancers.
660
In particular, whether ER negativity is a cause or a consequence of the disease progression is
661
a million dollar question in this field. Therefore the debate continues until to unravel the
662
precise mechanism/s that explains the origin of ER negativity in breast cancer. Besides this,
663
understanding tumor heterogeneity and real time monitoring of early resistance to targeted
664
therapies by analyzing the resistant tumors through integrated approach is needed. We
665
envisage more intensive research and debates with a resurgence of interest to better
666
understand the ER negativity in breast cancer.
667 668
Acknowledgments
669
This work is supported by the Department of Biotechnology (DBT), India grants No-
670
BT/MED/30/SP11273/2015 and BT/PR8764/MED/97/104/2013; and Department of Science
671
and Technology (DST) grant No- SB/SO/BB/013/2013, (to BM) India, DST-PURSE and
672
UGC-UPE2, UGC-DRS grant. No conflict of interest exist to disclose.
673 674 675 676 677 678 679 680 681 682
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Figure Captions/Legends
1025
Table 1. The expression of miRNAs, their function and the breast cancer phenotype.
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Figure 1. The pie diagram represents percentage of different molecular subtypes of breast
1027
cancers.
34
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Figure 2. Schematic representation of role of various regulatory mechanisms in loss of ERα
1029
expression and function in ER-negative breast cancer. Epigenetic regulators such as DNA
1030
methyl transferases (DNMT), histone deacetylases (HDACs) and ER-specific miRNAs
1031
negatively regulate ERα expression. The ERα expression is also lost by hyperactive MAPK
1032
pathway. ER-specific Ubiquitin ligases promote ERα degradation through ubiquination
1033
mechanism. These three types of molecular regulators ensure endocrine resistance in ER-
1034
negative breast cancer. SERMs, specific estrogen receptor modulators; Me, DNA
1035
methylation; Ac, histone acetylation.
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Figure 3. Schematic representation of a model depicting the subtle balance between ER
1037
regulators (+ ve / - ve) dictate ER negativity and therefore, endocrine resistance in breast
1038
cancer.
1039
Figure 4. Schematic representation of a model depicting the role of miRNAs, epigenetic
1040
factors and ubiquitin ligases which directly or indirectly regulate ER expression and causes
1041
ER negativity and endocrine resistance in breast cancer. The ER negativity along with PR
1042
and Her-2 negativity together contribute to TNBC phenotype. As PR expression is dependent
1043
on ERα, loss of ERα expression led to PR negativity. Because growth factor signalling
1044
antagonises ERα expression, Her-2 negativity may lead to re-expression of ERα. But whether
1045
Her-2 negativity opposes ER negativity in breast cancer is unknown.
1046
35
Table 1. The effect of various miRNAs on ERα expression and the breast cancer phenotype. Name of miRNA miR-22 miR-206 miR-221 miR-222 Let-7 miR-193b miR-190b miR-302c miR-342 miR-17-5p
miRNA function ERα levels decreased ERα levels decreased ERα levels decreased ERα levels decreased ERα levels decreased ERα levels decreased ERα levels high ERα levels decreased Sensitive to tamoxifen Represses AIB1/SRC-3 (ERα coactivator)
Phenotype (Breast cancer) ERα-negative
References
ERα-negative
[98]
ERα-negative
[99,100]
ERα-negative
[99,100]
ERα-negative
[105]
ERα-negative
[108]
ERα-positive ERα-negative
[109] [108]
ERα-positive ERα-negative
[103,104]
[110, 111] [107]
Figure 1
Her2
14
[ER+|PR+] HER2-Ki67-
11.2
[ER+|PR+] HER2-Ki67+ Luminal 7.8
Luminal 20.1
38.8
TNBC
[ER+|PR+] HER2+Ki67+ Her2 overexpression
12.3
Basal Normal-Like
23.7
Her2 TNBC
Figure 2
E2 DNMT
Me
HDAC MAPK
Me
Ac
Ac
ERα mRNA
Ub Ligases
miRNAs
ERα degradation ERα
ESR1 promoter SERMs
Loss of ERα Target gene expression
Figure 3
ER +ve regulators ER -ve regulators Endocrine response
Endocrine resistance
Figure 4
TNBC ?
EREpigenetic factors DNMT1, DNMT3B MTA1/IFI16, MeCP2, HDACs, EZH2
miRNAs miR-22 miR-221 miR-342 miR-18a/b miR-206 Let-7
miR-206 miR-222 miRNA-449 miR-193b miR-302c
Ub Ligases CHIP SPOP MDM2 SKP2
VHL NEDD4 CUEDC2
