Abstract
Therapies targeting estrogen receptor alpha (ERα), including selective ER modulators such as tamoxifen, selective ER downregulators such as fulvestrant (ICI 182 780), and aromatase inhibitors such as letrozole, are successfully used in treating breast cancer patients whose initial tumor expresses ERα. Unfortunately, the effectiveness of endocrine therapies is limited by acquired resistance. The role of microRNAs (miRNAs) in the progression of endocrine-resistant breast cancer is of keen interest in developing biomarkers and therapies to counter metastatic disease. This review focuses on miRNAs implicated as disruptors of antiestrogen therapies, their bona fide gene targets and associated pathways promoting endocrine resistance.
Introduction
The sustained exposure to endogenous estrogens is involved in the initiation and progression of breast cancer (Colditz 1998). The cellular effects of estrogens are mediated by estrogen receptors alpha and beta (ERα and ERβ) and their splice variants (Herynk & Fuqua 2004). Approximately 70% of primary breast tumors express ERα (Clark et al. 1984, Ring & Dowsett 2004), providing the rationale for the successful use of targeted endocrine therapies in breast cancer progression (reviewed in Jordan et al. (2014)).
Endocrine therapies including selective ER modulators (SERMs) such as tamoxifen (TAM); selective ER down regulators (SERDs) such as fulvestrant (ICI 182 780) and aromatase (CYP19A) inhibitors (AIs) such as anastrozole and letrozole, are the frontline adjuvant therapies in treatment of women with ERα+ breast tumors (Regan et al. 2011). These therapies have resulted in substantial improvements in outcomes and quality of life of breast cancer survivors (Jordan et al. 2011). Adjuvant TAM therapy with TAM was the mainstay for ERα+ breast cancer management until clinical trials comparing TAM with AIs that block the conversion of androgens to estrogens (Santen et al. 2009) were proven to provide a significant increase in disease-free survival (Cuzick et al. 2010, Regan et al. 2011). Unfortunately, the effectiveness of TAM and AI therapy is limited, as seen in the relapse of ∼40% of patients (1998). When resistance occurs, it is unclear which subsequent endocrine therapy is most appropriate (Choi et al. 2015). Fulvestrant is used as a second-line therapy for patients with metastatic breast cancer, after developing AI or TAM resistance (Johnston et al. 2005, Perey et al. 2007). Endocrine resistance can be intrinsic (de novo) or acquired (reviewed in Clarke et al. (2003)). In intrinsic resistance, patients are initially unresponsive to endocrine therapies due to lack of ERα, while in acquired resistance, patients become unresponsive after the initial 5-year treatment, even though ERα is still expressed (Dowsett et al. 2010). Biological mechanisms underlying de novo and acquired resistance are therefore of considerable clinical significance. Overall, the mechanisms of endocrine resistance are many and include amplification of multiple growth factor signaling pathways (Ring & Dowsett 2004, Riggins et al. 2005, Bedard et al. 2008, O'Brien et al. 2009, Palmieri et al. 2014). Recently, ERα ligand-binding domain (LBD) mutants that are intrinsically active in the absence of ligand were identified in AI-resistant metastatic disease, but not TAM-resistant (TAM-R) metastases (Li et al. 2013a, Robinson et al. 2013, Toy et al. 2013), providing a new impetus for understanding ERα's role in driving metastatic disease (Jordan et al. 2015). This review will focus on the role of microRNAs (miRNAs) in acquired endocrine-resistant breast cancer.
miRNAs or miRs are small (22 nt), non-protein coding RNAs first identified over a decade ago (Iorio & Croce 2012). Their dysregulation has been implicated in many diseases, including breast cancer (Iorio & Croce 2012). Post-transcriptionally, miRNAs regulate the expression of target genes and are novel candidates for clinical development as therapeutic targets and biomarkers. The role of noncoding RNAs and miRNAs in breast cancer and endocrine-resistant breast cancers have been recently reviewed (Hayes & Lewis-Wambi 2015, van Schooneveld et al. 2015). Here we will review studies demonstrating dysregulation of miRNAs linked to endocrine resistance that result in breast cancer progression. We also describe the bone fide targets of these miRNAs and the molecular pathways dysregulated in conferring resistance. We will summarize miRNAs with predictive and prognostic potential in endocrine-resistant breast cancer.
Overview of ER pathways in breast tumors
The biological effects of estrogens, including estradiol (E2), are mediated by binding to nuclear receptors ERα and ERβ and their splice variants, such as ERα36 and ERα46 and G-protein-coupled ER (GPER). The ERα activation initiated by E2 binding and consequent conformational changes results in ‘nuclear/genomic’ or ‘non-genomic/membrane-initiated’ responses (Watson et al. 2012, Levin 2014). ERα is upregulated in breast tumors (Clark & McGuire 1988). It is the target for therapeutic agents originally termed antiestrogens because they compete with E2 for ERα binding, but now termed SERMs and SERDs because of our greater understanding of their molecular actions (Jordan et al. 2014). Because the role of ERβ in breast cancer remains to be clearly established (reviewed in Thomas & Gustafsson (2011)) and because ERα expression is higher than ERβ in breast tumors and is thus the target of therapeutic intervention, this review will focus primarily on ERα activities related to miRNA expression and activity in endocrine resistance.
In the non-nuclear/membrane-initiated ER pathways, E2 rapidly alters intracellular signaling pathways culminating in changes in gene transcription by processes mediated by plasma membrane (PM)-associated ERα, ERβ, or GPER/GPR30 (reviewed in Riggins et al. (2005), Arpino et al. (2008), Filardo et al. (2008), Watson et al. (2012), and Levin (2014)). These rapid responses include E2 activation of PI3K and Src in the PM, which then activate mTOR through PI3K-mediated AKT phosphorylation. Membrane ER and GPER activate epidermal growth factor receptor (EGFR) with downstream signaling through Ras/Raf and MAPK (Razandi et al. 2003). E2 activation of EGFR can increase ERα phosphorylation (reviewed in Arpino et al. (2008), Johnston (2010), and Renoir et al. (2013)). These signaling pathways subsequently regulate ER transcriptional activity. Despite the numerous studies on ERα, the events and overall processes, including their regulated gene targets, are not completely understood.
Mechanisms of endocrine therapy in breast cancer
Antiestrogen therapies function by two main mechanisms: targeting ERα activity and/or stability. SERMs, e.g., TAM, raloxifene (RAL), and toremifene, compete with E2 for binding the LBD of ERα and inhibit ERα transcriptional activity in a gene- and cell-specific manner. SERMs can be agonists or antagonists depending on the tissue and gene. For example, SERMs are agonists for ERα in the endometrium and increase endometrial tumor incidence (Gottardis et al. 1988, Fornander et al. 1989). As antagonists, SERMs are used in the treatment of breast cancer patients with ERα+ breast tumors (Baum et al. 1983). SERDs, i.e., fulvestrant (ICI 182 780, Faslodex), not only alter the ERα conformation, but stimulate ER protein degradation (Jordan & Brodie 2007, Osborne & Schiff 2011, Zhao & Ramaswamy 2014). AIs inhibit the activity of aromatase (CYP19A1), thus reducing estrogen synthesis in peripheral adipose tissues and within the tumor (Zhao & Ramaswamy 2014). Examples of AIs include letrozole and anastrozole, which are steroidal/irreversible inhibitors, and exemestane, a non-steroidal/reversible inhibitor (Zhao & Ramaswamy 2014).
For postmenopausal women with ERα+ primary tumors, ASCO guidelines recommend AI therapy and for premenopausal women TAM for 10 years (Smith 2014). Adjuvant therapy with TAM for postmenopausal women with endocrine-responsive breast tumors effectively reduced the odds of recurrence by 40% and death from breast cancer after 5 years by 20% (1998). Unfortunately, 40–50% of patients initially responsive to TAM develop TAM resistance (Ring & Dowsett 2004). Likewise, a similar proportion of patients develop AI-resistance (Johnston et al. 2005, Hayashi & Kimura 2015). This indicates that additional mechanisms evolve to promote breast cancer progression in the absence of estrogen signaling.
Overview of mechanisms of endocrine resistance
A number of molecular mechanisms have been implicated in promoting endocrine resistance (Fig. 1; Ring & Dowsett 2004, Musgrove & Sutherland 2009, Nagaraj & Ma 2015). For example, ERα expression is silenced by methylation, resulting in reduced ERα (Martinez-Galan et al. 2014). Mutations in ERα (Herynk & Fuqua 2004) or increased expression of truncated forms of ERα, including ERα36 (Deng et al. 2014), are potential mechanisms in acquired resistance. Alterations in ERα coregulators, e.g., increased expression of AP1 and nuclear factor kappa B (NFκB), are associated with endocrine resistance (Johnston et al. 1999, Zhou et al. 2007). Crosstalk between ERα and amplification or activation of receptor tyrosine kinases (RTKs), including EGFR and insulin-like growth factor receptor, have been implicated in endocrine resistance (Arpino et al. 2008). Overexpression of HER2 (ERBB2) can elicit TAM resistance (Arpino et al. 2008), although HER2+ tumors are of a distinct molecular genotype from luminal A/ERα+ breast tumors (Sorlie et al. 2001). Apoptotic and cell survival signals are also dysregulated in TAM-R cells (Riggins et al. 2005). A more extensive review of mechanisms promoting endocrine resistance can be found in several reports (Musgrove & Sutherland 2009, Hasson et al. 2013, Zhao & Ramaswamy 2014). Additional factors are continually identified as playing roles in endocrine resistance.
Summary of the molecular mechanisms promoting acquired endocrine resistance. Activation and/or amplification of receptor RTKs, including insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and HER2 have been detected in tamoxifen-resistant breast cancer cells and endocrine-resistant patient tumors. PM-associated GPER and ERα, including splice variants ERα36 ERα46, are increased in endocrine-resistant breast cancer cells and tumors. Activation of these receptors activate intracellular signaling cascades, including MAPK and PI3K/AKT pathways, that ultimately increase transcription of genes that promote growth and survival and resistance to apoptosis. Additionally, these pathways increase ligand-independent ERα activation by phosphorylation. Alternatively, MAPK and PI3K/AKT can directly promote expression of non-ERE responsive genes by activating other transcription factors, e.g., AP1, not shown here. Promoter methylation of CpG islands and histone deacetylation has been shown to repress ERα expression and promote endocrine resistance. ERα, estrogen receptor α; ERE, estrogen response element; GPER, G protein-coupled estrogen receptor; PM, plasma membrane; RB, retinoblastoma; MYC, v-myc avian myelocytomatosis viral; BCL, B-cell lymphoma; BAK, homologous antagonist killer; BIK, BCL2-interacting killer.
Citation: Endocrine-Related Cancer 22, 5; 10.1530/ERC-15-0355
miRNA biogenesis
miRNAs are evolutionarily conserved, small, non-coding, 22 nt RNAs that post-transcriptionally regulate gene expression by binding to the 3′-UTR of mRNAs to repress transcription or promote degradation (Iorio & Croce 2012). There are an estimated 2588 miRNAs arising from intragenic or intergenic regions of the human genome (June 2014; http://www.mirbase.org/; Kozomara & Griffiths-Jones 2014). Intragenic, i.e., intronic or exonic, miRNAs, which constitute about half of all miRNAs (Berillo et al. 2013), originate from within protein coding genes and can therefore have a shared promoter (and/or transcriptional start site (TSS)) and are expressed simultaneously with their host protein-coding transcript (Rodriguez et al. 2004, Baskerville & Bartel 2005). However, later findings suggest this may not be the case and the regulation of intronic miRNA transcription can occur independent from the host gene (Gennarino et al. 2009, Marsico et al. 2013). Intergenic miRNAs tend to have their own promoters (Gennarino et al. 2009). Determining these TSSs will be essential in understanding the regulation of miRNA expression. Sixty percent of all human protein coding genes are regulated by miRNAs (Friedman et al. 2009), and because miRNAs regulate multiple mRNAs, they are implicated as key regulators in a variety of cellular processes, including cell differentiation, cell death development, proliferation, and metabolism (Bartel 2004).
miRNA biogenesis occurs through canonical and non-canonical pathways. In the canonical pathway of miRNA biogenesis, the primary RNA transcript (pri-miRNA) is transcribed from DNA by RNA polymerase II. Pri-miRNA is further processed to a hairpin precursor transcript (pre-miRNA; ∼70 nt) by a microprocessor complex comprised of Drosha (RNase III enzyme) and associated DiGeorge syndrome critical region gene 8 (DGR8) (Han et al. 2004). DGR8 anchors and recognizes the miRNA region for endonuclease cleavage by Drosha (Kim et al. 2009, Fukunaga et al. 2012). The Drosha microprocessor complex is also implicated in miRNA-independent functions including regulation of heteronuclear ribonucleoproteins and alternative splicing (Macias et al. 2013). Pre-miRNA is exported from the nucleus by Exportin 5 (a RanGTP-dependent dsRNA-binding protein (Bohnsack et al. 2004)) to the cytoplasm where it is further processed by another RNase III enzyme, DICER, in conjugation with trans-activation response RNA-binding protein and protein activator of the interferon-induced protein kinase (PACT; also known as PRKRA) results in a small dsRNA duplex (∼22 nt) (Chendrimada et al. 2005, Kim et al. 2009). One of the duplex strands is included in the RNA-induced silencing complex (RISC), where it recognizes and binds mRNA, resulting in mRNA degradation or translational repression depending on the extent of complementarity (Huntzinger & Izaurralde 2011). The core RISC is composed of four Argonaute (Ago) proteins with AGO2 endonuclease activated upon recruitment of target mRNAs (Meister et al. 2004).
In the non-canonical pathway, miRNAs (miRtrons) are processed by spliceosomes in an RNase III (Drosha)-independent manner (reviewed in Yang & Lai (2011)). The intermediate generated is further processed by lariat debranching enzyme resulting in products that appear as pre-miRNA mimics. These mimics then enter the canonical pathway as Exportin 5 or DICER substrates.
miRNAs are considered to be key players in cellular transformation and in the initiation and progression of cancer (Wiemer 2007). Selected miRNA signatures have been recognized in categorizing developmental lineages and differentiation states of different tumors (Lu et al. 2005, Rosenfeld et al. 2008). In these roles, miRNAs may function as oncogenic miRNAs (oncomiRs) or oncosuppressor miRNAs, although there is an overall downregulation of miRNAs in tumors compared to normal tissues (Lu et al. 2005). Substantial effort is currently underway to understand the molecular mechanisms associated with miRNA dysregulation to assist in early diagnosis and management of breast cancer patients.
miRNA and breast cancer
Since 2005, when miRNA deregulation was first reported in breast cancer (Iorio et al. 2005), over 1000 studies have been published identifying and examining the role of miRNAs in breast cancer. Some of these miRNAs are regulated by E2 and/or influence expression of estrogen-responsive genes (Ferraro et al. 2012, Klinge 2012, 2015). miRNAs have been implicated in regulating hallmarks of breast cancer (reviewed in Goh et al. (2015)), including cell proliferation, cell death, apoptosis, immune response, cell cycle energetics, metabolism, replicative immortality, including senescence, invasion, metastasis (reviewed in Negrini & Calin (2008), O'Day & Lal (2010), Singh & Mo (2013), and McGuire et al. (2015)), and angiogenesis (reviewed in Cortes-Sempere & Ibanez de Caceres (2011) and Goh et al. (2015)).
Models for miRNA investigation
The process of investigating miRNAs involved in endocrine resistance usually begins with an initial profiling of miRNA differences between endocrine-sensitive vs -resistant breast cancer cell lines or between breast tumors from patients responsive and non-responsive to endocrine therapies. Methods used in these studies have included microarrays, RNA sequencing, and the relatively recent high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS–CLIP) method with confirmation by quantitative real-time PCR (qPCR). These methods result in the acquisition of huge amounts of data that require integrative analysis and further confirmation. Computational approaches are then utilized to predict possible targets and signaling pathways that are aberrantly regulated by the identified miRNAs. Functional analysis utilizing ectopic expression and forced repression of miRNA expression are performed to validate the role of deregulated miRNAs in tumorigenesis and/or endocrine resistance in vivo and in vitro. The targets of miRNAs are further confirmed by cloning the target 3′-UTR downstream of a luciferase reporter, transfecting a cell line with this reporter plasmid to validate direct inhibition, and western blots and qPCR. Target validation in clinical samples provides human significance to the study. These models have identified miRNAs involved in endocrine resistance that are summarized in Tables 1 and 2.
miRNA associated with antiestrogen resistance in breast cancer. Experimentally proven bona fide targets and method used in confirming target are indicated. Proposed pathways associated with endocrine resistance are included. Method of identification of targets: 3′-UTR luciferase reporter assay (L), downregulation of protein shown by western blot (W), and downregulation of target in quantitative real-time PCR assay (Q)
| miRNA | Treatment/human cell line/tissue | Comments | Targets (method of identification) | Pathway |
|---|---|---|---|---|
| Putative oncomiRs based on experimental data | ||||
| miR-101 | 0, 1, 2, 3, and 4 μM 4-OHT 4 days in MCF7 cells | miR-101 infected cells promote growth stimulatory activity in medium lacking E2 and TAM-R. miR-101 has growth-inhibitory activity in E2-containing medium (Sachdeva et al. 2011) | MAGI2 (L, W) (Sachdeva et al. 2011) | Growth factor receptor (GFR) cytoplasmic signaling |
| miR125b-5p | 0.001–10 μM anastrozole or letrozole in LET-R MCF7 ANA-R MCF7 vs MCF7aro cells Primary breast tumors | Upregulated in LET-R MCF7 cells, ANA-R MCF7 compared to MCF7aro; high miR-125b-5p correlated with earlier relapse in ER+/PR+ patients (Vilquin et al. 2015) | ||
| miR-128a | TAM+LET-R MCF7 cells, MCF7aro cells | Upregulated in TAM+LET-R MCF7 cells vs MCF7aro cells (Masri et al. 2010) | TGFβR1 (L, W) (Masri et al. 2010) | GFR cytoplasmic signaling |
| miR-181b | Human breast samples 1 μM 4-OHT, for 0, 24, 48, and 72 h; 50 nM 4-OHT, 0, 3, and 5 min T47D, TAM-R MCF7 vs TAM-S MCF7 | Enhanced expression in TAM-R MCF7 cells (Lu et al. 2011) Anti-miR-181b suppressed TAM-R xenograft tumor growth in TAM treated mice | TIMP3 (L, W, Q) (Lu et al. 2011) | GFR cytoplasmic signalling |
| miR-205-5p | 1, 10, 100 nM, 1, 10 μM anastrozole; 1, 10, 100 nM, 1, 10 μM letrozole; LET-R MCF7 cells, ANA-R MCF7 vs MCF7aro Primary breast tumors | Upregulated in LET-R MCF7 cells, ANA-R MCF7 compared to MCF7aro cells; high miR-125b-5p correlated with earlier relapse in ER+/PR+ patients (Vilquin et al. 2015) | ||
| miR-210 | Patient samples 100 nM 4-OHT, 0, 1, 2, 3, and 4 days, MCF7, MDA-MB-231 | Increased miR-210 with breast tumor histological grade | ||
| Higher in MDA-MB-231 compared to MCF7 cells (Rothe et al. 2011) | ||||
| miR-222 | Human breast samples 1 μM 4-OHT, for 0, 24, 48, and 72 h; 50 nM 4-OHT, 0, 3, and 5 min T47D, TAM-R MCF7 vs TAM-S MCF7 | Anti-miR-222 suppressed TAM-R xenograft tumor growth in TAM treated mice (Lu et al. 2011) | TIMP3 (L, W, Q) (Lu et al. 2011) | GFR cytoplasmic signaling |
| miR-221/222 | 0-65 μM 4-OHT, 6 days MCF7/TAM-R vs MCF7 measured in conditioned media (Wei et al. 2014) | Sixfold increase in exosomes in MCF7/TAM-S vs MCF7/TAM-R cells (Wei et al. 2014) | P27 and ERα (W, Q) (Wei et al. 2014) | ERα signalling/cell cycle |
| 0, 15, and 20 μM TAM, 16 h TAM-S vs TAM-R MCF7 cells, HER2/neu (+) vs HER2/neu (−) human breast tissue | TAM increased miR-221/222 in TAM-R cells and HER2/neu (+) breast tumors compared to TAM-S MCF7and HER2/neu (−) tumors respectively (Miller et al. 2008) | p27(Kip1) (W, Q) (Miller et al. 2008) | GFR cytoplasmic signaling/cell cycle | |
| 0, 5, 10, and 20 μM 4-OHT, 12, 24, and 48 h, MCF7 and MDA-MB-231 | TIMP3 (W, Q) (Gan et al. 2014) | GFR cytoplasmic signaling | ||
| 10 μM 4-OHT, ±1 nM E2, 48 h MCF7 mammosphere transformed vs MCF7 cells as xenografts in SCID mice | Higher in MCF7 mammospheres compared to MCF7 cells (Guttilla et al. 2012) | ERα (W) | EMT | |
| 100 nM fulvestrant TAM-R MCF7, Ful-R MCF7, MCF7, MCF7–Mek5, MCF7 TNR | Overexpressed in Ful-R MCF7 cells. Indirect activation of β-catenin Repression of TGFβ mediated growth inhibition (Rao et al. 2011) | GFR cytoplasmic signaling | ||
| 0, 5, 10, 20 μM, 10, 20 nM 4-OHT, 72 h MCF7, T47D vs MDA-MB-468 cells Breast tumor tissue | Higher in ERα− MDA-MB-468 breast cancer cells and primary breast tumors vs ERα+ MCF7 and T47D cells (Zhao et al. 2008) | ERα (L, W) (Zhao et al. 2008) | ERα signaling | |
| 100 nM 4-OHT and 100 nM fulvestrant, 6 h, 2 days, TAM-S MCF7 vs TAM-R LY2 cells | 4-OHT upregulates miR-221/222 in LY2 TAM-R cells and downregulates miR-221/222 in MCF7 cells (Manavalan et al. 2011) | ERBB3, ERα (Q) (Manavalan et al. 2011) | ERα signaling/GFR cytoplasmic signaling | |
| miR-301 | 300 nM 4-OHT, 24, 48, and 72 h MCF7, MDA-MB-231 | Higher in MCF7, T47D, MDA-MB-231, and MDA-MB-231 compared to MCF7 10A. Higher expression in lymph node negative (LNN) invasive ductal breast cancer (Shi et al. 2011) | FOXF2, BBC3, PTEN (L, W, Q), and COL2A1 (L, Q) (Shi et al. 2011) | GFR cytoplasmic signaling |
| miR-519a | 0, 5, and 10 μM 4-OHT, 3 and 72 h TAM-R MCF7 vs MCF7, HEK 293FT, breast cancer patient datasets | Upregulated in TAM-R MCF7 cells compared to TAM-S MCF7 cells (Ward et al. 2014) | CDKNIA, RB1, and PTEN (L, W, Q) (Ward et al. 2014) | Cell cycle |
| miR-1280 | Patient blood samples | Higher in blood from patients with metastatic breast cancer after cytotoxic chemotherapy or undefined endocrine therapy (Park et al. 2014) | ||
| Putative oncosuppressor miRNAs based on experimental data | ||||
| Let7b/Let7i | TAM-R MCF7, MCF7, MDA-MB-231 Breast cancer tissues | Overexpression of Let7b/Let7i enhanced sensitivity of TAM-R MCF7 cells to TAM only in hormonal withdrawal medium and not in normal growth medium (Zhao et al. 2011) | ERα36 (L, W, Q) (Zhao et al. 2011) | ERα signaling |
| Let7i | 5, 10, 15, and 20 μM 4-OHT, 48 h, ZR-75-1 cells | Overexpression of Let71 increased TAM-S in ZR-75-1 cells | Apoptosis/cell survival signaling | |
| Inverse correlation of Let7i and TNF receptor associated factor 1 (TRAF1; Weng et al. 2014) | ||||
| miR-10a | Primary breast tumors | Higher expression in patient tumors was associated with longer relapse-free time. Increased expression predicted tumor relapse in TAM-treated ER+ postmenopausal breast cancer patients (Hoppe et al. 2013) | ||
| miR-15a/16 | 100 pM E2, 1 μM 4-OHT, and 100 nM fulvestrant, E2+4-OHT, E2+ICI, for 24, 24, and 72 h TAM-R MCF7/HER2Δ16 vs MCF7/HER2 cells (Cittelly et al. 2010a) | Suppressed expression of miR-15a and miR-16 in HER2Δ16 mutant cells associated with inverse expression of BCL2 protein and mRNA and decreased sensitivity to TAM and ICI (Cittelly et al. 2010a) | Apoptosis/cell survival signaling | |
| miR-30a-3p | Patient samples (Rodriguez-Gonzalez et al. 2011) | Increased expression in ER+ primary breast tumors of patients who received TAM and showed longer progression-free survival; inverse correlation with HER2 and RAC1 cell motility signaling pathways (Rodriguez-Gonzalez et al. 2011) | ||
| miR-126 | Primary breast tumors | Higher expression in patient tumors was associated with longer relapse-free time. Increased expression predicted tumor relapse in TAM-treated ER+ postmenopausal breast cancer patients (Hoppe et al. 2013) | ||
| miR-200b/200c | 100 nM 4-OHT and 100 nM fulvestrant, 6 h, 2 days, TAM-S MCF7 vs TAM-R LY2 (Manavalan et al. 2013) | Decreases in TAM-R LCC1, LCC2, LCC9, and LY2 cells vs MCF7 cells (Manavalan et al. 2013) | ZEB1/2 (W, Q) (Manavalan et al. 2013) | EMT |
| 100 nM 4-OHT and 100 nM ICI, 6 h, 2 days, TAM-S MCF7 vs TAM-R LY2 | Increased in TAM-S MCF7 and decreased in TAM-R LY2 cells (Manavalan et al. 2011) | CYP1B1 (Q) (Manavalan et al. 2011) | ||
| miR-342-3p | 24 h 100 pM E2, 1 μM 4-OHT, E2+4-OHT in TAM-R MCF7/HER2Δ16, MCF7/HER2, TAM-R1, LCC2 cells, breast tumors (Cittelly et al. 2010b) | Downregulated in TAM-R MCF7/HER2Δ16 cell, TAM-R1, LCC2 cells, and TAM refractory human breast tumors vs MCF7 cells and TAM-S tumors. TXNIP is an indirect target of miR-342 (Cittelly et al. 2010b) | BMP7, GEMIN4 (microarray, L, Q), and SEMAD (microarray, Q) (Cittelly et al. 2010b) | GFR cytoplasmic signalling |
| Primary breast tumors 10 nM E2, 20 μM 4-OHT, 72 h, MCF7 vs SKBR3 and MDA-MB-231 cells | Decreased in ERα− SKBR3 and MDA-MB-231 cells vs MCF7 cells | ERα signaling | ||
| Direct correlation between miRNA-342 expression and ERα expression (He et al. 2013) | ||||
| miR-375 | 5 μM 4-OHT TAM-R MCF7 cells vs TAM-S MCF7 cells | Lower in TAM-R MCF7 vs MCF7 cells (Ward et al. 2013) | MTDH (L, W, Q) (Ward et al. 2013) | EMT |
| miR-424-3p | 0.001–10 μM anastrozole or letrozole; LET-R MCF7 cells, ANA-R MCF7 vs MCF7aro Primary breast tumors | Downregulated in LET–RMCF7 cells, ANA-R MCF7 compared to MCF7aro (Vilquin et al. 2015) | ||
| miR-451 | 1 μM 4-OHT for 0, 4, 8, and 24 h, TAM-R MCF7 vs TAM-S MCF7 cells | Reduced levels in TAM-R vs TAM-S MCF7 cells (Bergamaschi & Katzenellenbogen 2012) | 14-3-3ς (W, Q) (Bergamaschi & Katzenellenbogen 2012) | GFR cytoplasmic signaling |
| miR-574-3p | 1 μM 4-OHT, MCF7 cells/tissue sample | Lower in TAM-R MCF7 cells and clinical breast cancer tissues compared to TAM-S MCF7 cells and adjacent normal control respectively (Ujihira et al. 2015) | Clathrin heavy chain (CLTC) (L, W, Q) (Ujihira et al. 2015) | GFR cytoplasmic signaling |
| miR-873 | 1, 10, 100 nM, 1, 5 μM 4-OHT, 7 days, MCF7/TAM-R vs MCF7 cells and xenograft tumors | Downregulated in TAM-R MCF7 and breast tumors compared to TAM-S and normal tissues respectively | Cyclin-dependent kinase 3 (CDK3) (L, W, Q) (Cui et al. 2014) | Repressed ERα transcriptional activity (Cui et al. 2014) |
4-OHT, 4-hydroxytamoxifen; TAM-S, TAM-sensitive; TAM-R, TAM-resistant; LET-R, letrozole-resistant; ANA-R, anastrozole-resistant; Ful-R, fulvestrant-resistant. MCF7aro cells are MCF7 cells stably overexpressing aromatase.
Exosomal miRNAs described in breast cancer
| EV miRNA composition | Direction of miRNA expression | Sample source | Time in culture prior to harvesting exosome | Comments | Targets analyzed in study | References | ||
|---|---|---|---|---|---|---|---|---|
| Cell line | Patient | Xenograft | ||||||
| miR-21, let7a, miR-100, miR-125b, miR-720, miR-1274a, and miR-1274b miR-205 | ↑ ↓ | MCF7 MCF10A | No | No | 72 h | Guzman et al. (2015) | ||
| miR-10a, miR155, miR-373, miR-10b, miR-21, and miR-27a | ↑ | MCF7 MCF10A NmuMG MDA-MB-231 | Yes | Yes | 24 h 72 h | OncomiRs miR-10b and miR-21 confirmed by northern blot | Melo et al. (2014) | |
| miR-10b and miR-10a, miR-218, miR10a, miR-99a, miR-142-3p; miR-32, miR-138, miR-7e, miR-106b | ↑ | MCF7 MDA-MB-231 MCF10A HMLE HEK-293T | No | No | 48 h | OncomiR | HOXD10 and KLF4 (L, W) (targets for miR-10b) | Singh et al. (2014) |
| miR-140 | ↓ | MCF10DCIS MCF7 | No | No | 3 days | Li et al. (2014) | ||
| miR-29a and miR-21 | ↑ | MDA-MB-231 HEK-293T | ||||||
| miR-373, miR-101, and miR-372 | ↑ | MCF7 | Yes (serum) | No | Serum used | High in TNBC; over-expression of miR-373 promotes loss of ER and resistance to camptothecin | Eichelser et al. (2014) | |
| miR-221/222 | ↑ | MCF7 TAM-R/MCF7 | No | No | 72 h | Exosomal transport may be an additional mechanism by which miR-221/222 promote TAM-R | P27 (Q, W) and ERα (Q, W) | Wei et al. (2014) |
| miR-23a and miR1246 | ↑ | MCF7 MCF7/Doc | No | No | 12 h 24 h | May contribute to cisplatin resistance | Chen et al. (2014b) | |
| miR-100, miR-17, miR-222, miR-342-3p, miR-451, and miR-30a | ↑ | MCF7 MCF7/Adr MCF7/Doc | No | No | 72 h | PTEN (Q; target for miR-222) | Chen et al. (2014c) | |
| Let7a, miR-328, miR-130a, miR-149, miR-602, and miR-92b | ↑ | MCF7 and MDA-MB-231 | No | No | Kruger et al. (2014) | |||
| miR-198 | ↓ | |||||||
| miR-16 | ↑ | MSCs 4T1 SVEC | No | No | 48 h | VEGF (Q) | Lee et al. (2013) | |
| miR-16, miR-720, miR-451, and miR-1246 | ↑ | MCF7 MDA-MB-231 | No | No | 5 days | Palma et al. (2012) | ||
| miR-451 and miR-1246 | ↑ | MCF7 MDA-MB-231 SKBR3 BT-20 | No | No | 5 days | Pigati et al. (2010) | ||
| miR-223 | ↑ | Macrophages SKBR3 MDA-MB-231 | No | No | 24–48 h | Released by macrophages and promotes breast cancer cell invasion | Mef2c (L, W) | Yang et al. (2011) |
| miR-210 | ↑ | MCF7 | No | No | 48 h | Released in response to hypoxia | King et al. (2012) | |
↑, Increased expression; ↓, reduced expression. EV, extravessicular; Adr, adriamycin; Dox, doxorubicin; MSCs, mesenchymal stem cells; 4T1, mouse breast cancer cell line; SVEC, mouse endothelial cell line; NMuMG, nontumorigenic mouse mammary epithelial cells; HMLE, human mammary epithelial cell line; MCF10DCIS, MCF10 ductal carcinoma in situ cells.
There are limitations to these approaches. For example, integrative analysis for identifying aberrantly expressed miRNAs is limited by the set of computational parameters used in the study and rarely are these parameters applied to other studies. Functional investigations need to be performed for each miRNA and its target. The acquisition of massive amounts of information, which, though relevant, do not often translate to physiological significance, necessitates further studies within clinical settings. HITS–CLIP is technically challenging and complex, requiring great skill, but has the advantage of capturing interactions ‘frozen’ by u.v.-crosslinking under physiological conditions without the use of exogenous crosslinking agents, which can lead to artificial interactions (Moore et al. 2014).
miRNAs regulating ERα protein and signaling
The role of miRNAs in promoting endocrine resistance is exemplified by, but not limited to, their involvement in regulating ERα (Fig. 2). Decreased ERα expression is involved in endocrine-resistant breast cancer progression. miRNAs, including miR-221/222 (Zhao et al. 2008), miR-342-3p (He et al. 2013), miR-873 (Rothe et al. 2011), and Let7b/Let-7i (Zhao et al. 2011), downregulate ERα protein expression (Table 1). miR-221 and miR-222 are overexpressed in TAM-R and ERα− breast cancer cell lines and tumors (Miller et al. 2008, Zhao et al. 2008, Manavalan et al. 2011). The 3′-UTR of ERα is a direct target of miR-221/222 decreasing ERα protein but not mRNA (Zhao et al. 2008). Transient overexpression of miR-221/222 in TAM-sensitive (TAM-S) MCF7 and T47D cells resulted in TAM resistance, whereas the downregulation of miR-221/222 in ERα−/TAM-R MDA-MB-468 cells restored ERα expression and sensitized cells to TAM-induced cell cycle arrest and apoptosis (Zhao et al. 2008). ERα is not a direct target of miR-342-3p, but loss of miR-342-3p was associated with a concomitant loss in ERα expression and resulted in TAM resistance (He et al. 2013). Conversely, forced overexpression of miR-342-3p sensitized MCF7 cells to TAM-induced apoptosis (He et al. 2013). The exact mechanism promoting loss of ERα expression upon downregulation of miR-342-3p is yet to be determined (van Schooneveld et al. 2015).
Established targets of miRNAs in endocrine-resistant breast cancer. miRNAs associated with ERα signaling, growth factor/RTK signaling, EMT, dysregulation of cell cycle kinetics, and apoptosis and their targets in these pathways involved in endocrine resistance are shown. The miRNAs and their targets are described in the text and summarized in Tables 1 and 2. The established, bona fide targets are indicated with solid black lines. The dotted arrows indicate observed correlations with unknown mechanisms.
Citation: Endocrine-Related Cancer 22, 5; 10.1530/ERC-15-0355
Increased expression of ERα splice variants has also been reported to be associated with poor prognosis and contribute to endocrine resistance (Shi et al. 2009, Li et al. 2013b). ERα36 is an N-terminal truncated 36 kDa variant of full-length ERα (ERα66; Wang et al. 2006). ERα36 lacks AF1 and AF2 of ERα66, retains DNA binding and dimerization domains and binds E2, but is not inhibited by TAM or fulvestrant (Wang et al. 2006). ERα36 is expressed in ERα− breast cancer cells and tumors (Zhang et al. 2012) and overexpressed in TAM-R breast cancer cells (Li et al. 2013b). Increased ERα36 protein expression is proposed to be mediated by decreased let7, since transfection of let7b and let7i mimics repressed ERα36 expression and sensitized TAM-R MCF7 cells to TAM growth inhibition (Zhao et al. 2011). Let7 family members are downregulated in breast cancer tissues and TAM-R MCF7 cells (Vadlamudi et al. 2001, Zhao et al. 2011).
ERα is regulated by post-translational modifications including phosphorylation, methylation, sumoylation, and palmitoylation (Li et al. 2003, Acconcia et al. 2004, Sentis et al. 2005, Zhang et al. 2013, Cui et al. 2014). These modifications influence ERα interaction with other molecules, including transcriptional coregulators, hence regulating gene transcription (Fig. 1). These post-translational events also contribute to endocrine resistance (Anbalagan & Rowan 2015). miRNAs are implicated in altering post-translational ERα modifications to promote TAM resistance (Cui et al. 2014). For example, miR-873 targets CDK3, which phosphorylates ERα at Ser104/116 and Ser118 (Cui et al. 2014). miR-873 expression was downregulated in TAM-R/MCF7 breast cancer cells and forced overexpression of miR-873 in these cells reversed TAM resistance and decreased xenograft tumor growth. The authors postulated that the decrease in miR-873 resulted in enhanced ERα phosphorylation and ligand-independent activity in TAM-R/MCF7 cells (Cui et al. 2014).
miRNA regulation of ERα protein interactors in breast cancer
ERα interacts with other transcription factors, e.g., AP1, Sp1, NFκB, and the forkhead transcription factor (FOXM1), to regulate gene expression (Petz et al. 2002, Pradhan et al. 2010, Sanders et al. 2013). Increased activity of these transcription factors is associated with endocrine resistance (Johnston et al. 1999, Schiff et al. 2000, Zhou et al. 2007, Bergamaschi et al. 2014). FOXM1 is overexpressed in many cancers, including breast cancer, and its ectopic expression promotes cell invasiveness (Bergamaschi et al. 2014). Repression of FOXM1 was associated with increased miR-211 (Song & Zhao 2015) and miR-23a (Eissa et al. 2015), and repressed breast cancer cell growth, migration, and invasion in animal models.
Activation of NFκB contributes to endocrine resistance in breast cancer (Keklikoglou et al. 2011). A genome-wide miRNA screen in HEK-293T cells identified 13 miRNAs regulating NFκB transcriptional activity (Keklikoglou et al. 2011). Subsequent studies in MDA-MB-231 TNBC cells demonstrated that miR-570 and miR-373 inhibited TGFα-activation of NFκB-induced transcription of pro-inflammatory cytokines, e.g., IL6, IL8, CXCL1, and ICAM1, and TGFβ signaling by direct targeting of RELA and TGFBR2. The authors reported that transient overexpression of miR-520 or miR-373 inhibited TGFβ-induced MDA-MB-231 cell invasion. While they did not detect miR-373 in human breast tumors, a correlation of higher miR-520c expression in ERα tumors and lower TGFBR2 transcript expression was observed, allowing the authors to suggest loss of miR-520 expression may play a role in ERα-tumor progression via altered NFκB signaling (Keklikoglou et al. 2011).
The nuclear receptor co-activator proline glutamic acid leucine rich protein (PELP1) interacts with ERα (Vadlamudi et al. 2001) to modulate genomic (Nair et al. 2004) and nongenomic functions of ERα (Barletta et al. 2004, Vadlamudi et al. 2005). As a proto-oncogene, PLEP1 is upregulated during breast cancer metastasis and promotes human breast tumor xenograft growth in nude mice (Vadlamudi et al. 2005, Rajhans et al. 2007, Roy et al. 2012). In MCF7 cells, cytoplasmic localization of PELP1 conferred resistance to TAM (Vadlamudi et al. 2005). Although the mechanisms of PELP1 promotion of TAM resistance is not fully known, the binding of PELP to the proximal promoters of the oncosuppressors miR-200a and miR-141 recruited histone-deacetylase 2 (HDAC2) and repressed their transcription (Roy et al. 2014). The attendant decrease in miR-200a and miR-141 was suggested to stimulate metastatic growth (Becker et al. 2015).
ERα coactivator nuclear receptor co-activator 3 (NCOA3, also known as SRC3 (Liao et al. 2002) and AIB1 (Anzick et al. 1997)), is overexpressed in 50% of breast tumors (Anzick et al. 1997). Targeting SRC3 is of clear clinical interest (Tien & Xu 2012). SRC3 overexpression results in constitutive activation of ERα-mediated transcription, breast tumor growth and resistance to TAM in vivo and in xenograft models (List et al. 2001, Ring & Dowsett 2004). SRC3 translation is repressed by miR-17-5p. Overexpression of miR-17-5p in MCF7 cells repressed E2-induced proliferation and endogenous cyclin D1 transcription (Hossain et al. 2006). miR-195 negatively regulates SRC3 in human hepatoma cells (Jiang et al. 2014), but whether it does so in breast cancer is unknown.
ERα corepressors including nuclear receptor co-repressor 1 (NCoR1) influence gene transcription by recruiting HDAC complexes to promote chromatin condensation and repression of gene transcription (Lavinsky et al. 1998, Ring & Dowsett 2004). NCoR1 is reduced in TAM-R MCF7 xenograft tumors grown in nude mice (Lavinsky et al. 1998). However, both NCoR and the corepressor SMRT stimulated 4-hydroxytamoxifen (4-OHT)–ERα agonist activity on an estrogen response element-driven luciferase reporter in transiently transfected Rat-1 cells. Blockage of NCoR1 promoted the agonistic activity of TAM (Lavinsky et al. 1998). To our knowledge, there are no reports of miRNA regulation of NCoR1. However, inhibition of miRNA synthesis by knocking down DICER in LNCaP prostate cancer cells increased NCoR1 transcription and likewise, NCoR1 increased in the prostate of DICER−/− mice (Narayanan et al. 2010). Conversely, ectopic expression of DICER mediates metastasis and TAM resistance in breast cancer cells (Selever et al. 2011).
miRNA activation of growth factor receptor signaling in endocrine resistance cancer
Endocrine-resistant breast cancer cells and tumors show increased EGFR signaling (Aiyer et al. 2012). Although trastuzumab is a targeted therapy widely used in patients whose breast tumors overexpress HER2, these patients also benefit from TAM (Huynh & Jones 2014). Unfortunately, overexpression of an isoform of HER2, HER2Δ16, which is associated with metastasis (Mitra et al. 2009), also promotes TAM resistance (Cittelly et al. 2010a,b). Decreased expression of miR-15a, miR-16, and miR-342-3p contribute to endocrine resistance in TAM-R MCF7/HER2Δ16 breast cancer cells (Cittelly et al. 2010a,b). miR-342 was also downregulated in TAM-non-responsive breast tumors and HER2-negative, TAM-R TAM-R1 and LCC2 cells (Cittelly et al. 2010b). Transient overexpression of miR-342 resensitized TAM-R MCF7/HER2Δ16 and TAM-R1 cells to TAM-induced apoptosis; decreased BMP7, GEMIN4, and SEMAD are proposed as direct miR-342 targets mediating this response. However, the role of these targets in directly promoting TAM resistance was not examined.
Decreased miR-451 also regulates mitogenic signaling to promote TAM resistance (Bergamaschi & Katzenellenbogen 2012). TAM, but not RAL or fulvestrant, downregulates miR-451 in TAM-R cells (Bergamaschi & Katzenellenbogen 2012). Downregulation of miR-451 was associated with upregulation of its target protein 14-3-3ζ, a scaffolding protein whose high expression is correlated with early time to disease recurrence in patients treated with TAM. Overexpression of miR-451 in MCF7 cells decreased 14-3-3ζ and reduced activation of HER2, EFGR, and MAPK signaling, resulting in decreased cell proliferation and migration and increased apoptosis. In addition, overexpression of miR-451 restored the inhibitory effectiveness of SERMs in TAM-R cells.
A recent paper used a miRNA library screen to identify miRNAs associated with TAM-S in MCF7 cells (Ujihira et al. 2015). The authors identified miR-105-2, miR-877, let7f, miR-125a, and miR-574-3p as ‘dropout’ miRNAs that were downregulated in 4-OHT-treated compared to vehicle control-treated MCF7 cells. Of these miRNAs, miR-574-3p, was found to be downregulated in breast cancer tissue samples compared to adjacent normal tissue samples. Luciferase reporter assays and knockdown or overexpression of miR-574-3p identified clathrin heavy chain (CLTC) as a bona fide miR-574-3p target. Low CLTC transcript levels were correlated with better survival in breast cancer patients. This study outlines a new role of miR-574-3p in mediating TAM responses; however, whether upregulation of miR-574-3p will sensitize TAM-R cells to TAM remains to be determined.
Earlier, we discussed downregulation of ERα protein by increased miR-221/222 in endocrine-resistant breast cancer. Dysregulation of miR-221/222 was also reported to regulate multiple stages of RTK pathways to promote anti-endocrine resistance. In vitro analysis confirmed that miR-221/222 was increased in endocrine-resistant HER2-positive primary human breast cancer tissues compared to HER2-negative tissue samples (Miller et al. 2008). Overexpression of miR-221/222 made TAM-S MCF7 cells resistant to TAM and decreased protein expression of its known target p27. Overexpression of p27 enhanced TAM-induced cell death in TAM-R MCF7 cells (Miller et al. 2008). The same lab reported that repression of miR-222 and miR-181b suppressed growth of TAM-R MCF7 tumor xenografts in mice (Lu et al. 2011). Reduced expression of tissue metalloproteinase inhibitor 3 (TIMP3), a common target of miR-221/222/181b, in primary breast carcinomas was also reported to mediate TAM resistance by relieving repression of ADAM10 and AMAM17. ADAM10 and AMAM17 are critical for growth of TAM-R cells (Lu et al. 2011). Ectopic expression of TIMP3 repressed growth of TAM-R cells and reduced phosphoMAPK- and EGF-induced phosphoAKT levels. Conversely, repression of TIMP3 in TAM-S MCF7 promoted phosphorylation of MAPK and AKT and desensitized the cells to growth inhibition by TAM in vitro and in vivo. In another study, the same group showed that sensitivity to TAM upon inhibition of miR-221/222 was unique to ERα+ MCF7 cells and not ERα− MDA-MB-231 cells, although TIMP3 was a miR-221/222 target in both cells (Gan et al. 2014).
By promoting cell growth, miR-221/222 also promotes resistance to fulvestrant (Rao et al. 2011). Ectopic expression of miR-221/222 in TAM-R MCF7 and TAM-R BT474 cells increased β-catenin and relieved TGFβ-mediated growth inhibition. Inhibition of β-catenin decreased estrogen-independent growth in pre-miR-221/222-transfected MCF7 cells (Rao et al. 2011). The TGFβ signaling pathway was inhibited in letrozole-resistant, aromatase-stably transfected MCF7 (T+LET-R) breast cancer cells, and miR-128a was upregulated in these cells (Masri et al. 2010). miR-128a targeted and repressed TGFβ receptor 1 (TGFβR1) protein expression in T+LET-R cells compared to the parental MCF7 cells stably transfected with aromatase (MCF7aro). Repression of miR-128a re-sensitized T+LET-R cells to TGFβ growth inhibition.
Loss of PTEN is associated with poor outcome in HER2+ breast tumors (Stern et al. 2015). PTEN is downregulated by miR-101 (Sachdeva et al. 2011). Overexpression of miR-101 promotes MCF7 cell growth and TAM resistance in estrogen-free growth medium but suppressed cell growth in E2-containing medium (Sachdeva et al. 2011). TAM resistance was mediated by Akt activation and was independent of ERα expression. miR-101 repressed its target membrane-associated guanylate kinase inverted 2 (MAGI2), a scaffolding protein required for PTEN activity, thus reducing PTEN activity leading to activation of Akt. PTEN is also a bona fide target of the oncomiR miR-301 (Shi et al. 2011). Transient repression of miR-301 in MCF7 cells decreased cell viability and sensitized cells to TAM (Shi et al. 2011).
miRNAs as cell cycle regulators in endocrine-resistance
SERMs can be cytostatic and cytotoxic by promoting G1-phase cell cycle arrest (Subramani et al. 2015). miR-221/222 (Miller et al. 2008) and miR-519a (Ward et al. 2014) have been implicated in altering expression of molecular regulators of the cell cycle to promote endocrine resistance. miR-221/222 represses p27 to promote TAM resistance in breast cancer cells (Miller et al. 2008, Wei et al. 2014).
Recently, miR-519a was reported as a novel oncomiR by increasing cell viability and cell cycle progression (Ward et al. 2014). miR-519a was upregulated in TAM-R MCF7 cells compared with TAM-S MCF7 cells. Elevated levels of miR-519a in primary breast tumors were associated with reduced disease-free survival in ERα+ breast cancer patients and miR-519a was suggested to contribute to TAM resistance. Knockdown of miR-519a in TAM-R MCF7 cells sensitized the cells to TAM growth inhibition. Concordantly, overexpression of miR-519a in TAM-S MCF7 cells desensitized the cells to TAM by preventing growth inhibition while promoting caspase activity and apoptosis. Tumor suppressor genes involved in PI3K signaling CDKNIA (which encodes p21), RB1, and PTEN, were reported to be bona fide targets of miR-519a, although the role of these targets in mediating TAM-R have not been explored.
miRNAs in epithelial-to-mesenchymal transition
Changes involved in tumor progression include acquisition of migration/invasion, gain of front-rear polarity, resistance to anoikis, and mesenchymal transition (Howe et al. 2012). Genetic changes that occur during epithelial-to-mesenchymal transition (EMT) include but are not limited to activation of SNAIL, increased zinc-finger E-box-binding 1 (ZEB1), reduced E-cadherin and increased vimentin and N-cadherin (Lamouille et al. 2014). EMT is also implicated as a mechanism by which tumors enact resistance to TAM (Steinestel et al. 2014).
To identify miRNAs that mediate TAM resistance, we used a microarray to identify miRNAs differentially regulated between endocrine-sensitive MCF7 cells and an endocrine-resistant MCF7 variant LY2 cells, with selected results confirmed by qPCR (Manavalan et al. 2011). Among these, miR-200a, miR-200b, and miR-200c were found to be downregulated in LY2 cells and other TAM-R breast cancer cell lines (LCC9) compared with parental TAM-S MCF7 cells (Manavalan et al. 2011, 2013). The decrease in miR-200 family expression was associated with increase in ZEB1. ZEB1 is an EMT-inducing transcription factor that represses E-cadherin (Hurteau et al. 2007). Ectopic expression of miR-200b and/or miR-200c altered LY2 morphology to a more epithelial-like phenotype and inhibited cell migration. These phenotypic changes were associated with repression of the mesenchymal markers N-cadherin, vimentin, and ZEB1 and an increase in the epithelial marker E-cadherin. Further, upregulation of miR-200b/200c or ZEB1 knockdown sensitized LY2 cells to TAM- and fulvestrant-induced growth inhibition. However, overexpression of miR-200b/200c in MCF7 cells did not promote resistance to TAM or fulvestrant, indicating that cellular changes in addition to downregulation of miR-200 family members are involved in TAM resistance in LY2 cells.
In another miRNA microarray study, miR-375 was found to be downregulated in a mesenchymal TAM-R MCF7 cell line model (Ward et al. 2013). Re-expression of this miR-375 sensitized TAM-R cells to TAM and reduced invasiveness by decreasing expression of mesenchymal markers fibronectin, ZEB1, and SNAI2 while increasing the epithelial markers E-cadherin and ZO1. This resulted in partial reversal of EMT called mesenchymal-to-epithelial transformation. Metadherin (MTDH), a cell surface protein upregulated in breast tumors that mediates metastasis (Brown & Ruoslahti 2004), was identified as a direct, bona fide miR-375 target mediating this response. TAM-treated patients whose primary tumor showed high MTDH showed shorter disease-free survival and a higher risk of relapse. This study exemplifies the role of miRNAs in mediating cellular transformations that foster tumor progression and TAM resistance.
EMT allows the emergence of cancer stem cells (CSCs) that have properties including self-renewal potential and tumorigenicity (Singh & Settleman 2010, Ward et al. 2013). Mammosphere culture is widely utilized to enrich the population of mammary epithelial stem cells and breast CSCs in vitro (Dontu et al. 2003, Charafe-Jauffret et al. 2009). Mammosphere culture of MCF7 cells (MCF7M cells) resulted in permanent EMT with increased miR-221/222 and loss of their target ERα mRNA expression (Guttilla et al. 2012). MCF7M cells were also characterized by downregulation of epithelial-associated tumor suppressor miRNAs including miR-200c, miR-203, and miR-205. MCF7M cells were resistant to TAM-induced cell death. These data reinforce other studies discussed above demonstrating a role for increased expression of miR-221/222 in driving TAM-R.
miRNAs as regulators of apoptosis/cell survival signaling
Tumor growth reflects a balance between cell growth and cell death. Endocrine inhibitors activate apoptotic and stress signals to inhibit breast cancer cell growth (Mandlekar & Kong 2001, Riggins et al. 2005, Musgrove & Sutherland 2009). However, the molecular mechanisms behind these observations are yet to be fully defined. Activation of antiapoptotic proteins such as BCL2, crosstalk between apoptotic effects of antiestrogens and the TNFα pathway and promotion of survival signals including PI3k/Akt and NFκB have been documented to promote resistance endocrine therapy (Riggins et al. 2005). miRNAs can directly target antiapoptotic transcripts or regulate mediators of the survival signaling pathways. For example, low miR-15a/16 expression correlated with upregulation of BCL2 in TAM- and fulvestrant-treated TAM-R MCF7/HER2Δ16 cells and xenograft tumor promotion in vivo (Cittelly et al. 2010a). RNAi targeting of BCL2 or reintroduction of miR-15a/16 decreased TAM-induced BCL2 expression in TAM-R MCF7/HER2Δ16 cells resulting in TAM-induced decrease in cell growth and promotion of apoptosis. Conversely, repression of miR-15a/16 in TAM-S MCF7/vector or MCF7/HER2 increased BCL2 expression and promoted resistance to TAM by inhibiting apoptosis and preventing growth inhibition.
To identify potentially ethnic group-specific TAM-S biomarkers, Weng et al. performed an integrative genomic analysis on 58 African-derived HapMap YRI lymphoblastoid cell lines (YRI LCLs; breast cancer cells; Bradley & Pober 2001). Genetic variants (including 50 SNPs with effects on 34 genes and 30 miRNAs) were identified to be sensitive to endoxifen, an active metabolite of TAM. Among the genes identified, increased TNF receptor-associated factor 1 (TRAF1) and decreased let7i expression correlated with endoxifen resistance in 44 YRI LCLs. TRAFs are intracellular signal transducers for death receptor superfamily TNF receptor (TNFR; Bradley & Pober 2001). TRAF1 associates with TRAF2 to form a protein complex that interacts with inhibitor-of-apoptosis protein (IAP) to mediate anti-apoptotic signals (MAPK8/JNK and NFκB) from the TNFR (Wang et al. 1998, Wajant et al. 2003). Repression of TRAF1 or overexpression of let7i in ZR-75-1 luminal breast cancer cells enhanced sensitivity to TAM and decreased the number of viable cells. These data show that by regulating death signals, miRNAs can also mediate response to antiestrogens.
Extracellular vesicular (exosomes) transport of miRNAs in endocrine resistance
Extracellular vesicles (EVs), produced by outward budding of the PM, contain proteins and nucleic acids that can be transported in blood between tissues and cells (Chiba et al. 2012, Gong et al. 2012). The contents of EVs can facilitate tumor growth including angiogenesis, invasion, metastasis, and immune suppression. They also play a role in reducing effectiveness of drugs (Chen et al. 2014a). Exosomes are smaller EVs of endosomal origin and formed by the fusion of multivesicular bodies with PMs (Johnstone et al. 1987, Jung et al. 2012). Exosomes transport miRNAs in circulation. Mechanisms of exosomal formation and delivery are cell-specific and display proteins from their tissue of origin and are specific to the target cells (Clayton et al. 2001, Simpson et al. 2009, Braicu et al. 2015). It was initially reported that miRNAs were randomly packaged into exosomes with no specific miRNA preferentially incorporated (Valadi et al. 2007, Skog et al. 2008). Studies now indicate different miRNAs are associated with specific customized exosomes (Palma et al. 2012). However, the selection mechanism for incorporating miRNAs into exosomes is yet to be elucidated.
EVs or exosomal delivery of miRNAs is thought to play roles in breast tumorigenesis and metastasis (Table 2). Cell culture studies showed that exosomes secreted by TAM-S MCF7 cells were larger in size and number compared to TAM-R MCF7 cells and were taken up by the TAM-S cells (Wei et al. 2014). This study showed that miR-221/222 were released from exosomes into TAM-S MCF7 cells, resulting in the reduction of their target genes p27 and ERα and enhanced TAM resistance. Transfection of a miR-221/222 inhibitor in MCF7 cells treated with TAM-R MCF7 exosomes reduced TAM resistance. These data again support the importance of miR-221/222 in TAM resistance in MCF7 cells.
The potential use of exosomal miRNAs as candidate biomarkers for cancer diagnosis and prognosis remains to be definitively proven. The manipulation of exosomal miRNAs suggests a new therapeutic approach for drug delivery, but requires further research.
miRNAs of unknown function in endocrine resistance
Other miRNAs identified by microarray or library screen and confirmed by qPCR to promote antiestrogen resistance in breast cancers, but having undetermined functional roles include: miR-10a, miR-21, miR-22, miR-29a, miR-181a, miR-125b, miR-205, which mediate resistance to TAM (Manavalan et al. 2011), and miR-125a and miR-877, which also mediate TAM resistance (Ujihira et al. 2015) Other miRNAs identified by integrative analysis to make up network clusters that contribute to antiestrogen resistance include: miR-146a, miR-27a, miR-145, miR-21, miR-155, miR-125b, and let7s (Xin et al. 2009).
Roles of Drosha, DICER, and AGO2 in endocrine resistance
As described earlier in this review, Drosha and DICER function in miRNA processing. The role of Drosha in endocrine resistance has not been ascertained, despite the observation that reduced cytoplasmic Drosha is predictive of better endocrine therapy response (Khoshnaw et al. 2013).
Loss of DICER is predictive of a better response to endocrine therapy (Khoshnaw et al. 2012). Elevated DICER was associated with TAM resistance in metastatic breast tumors and tumor xenografts (Selever et al. 2011). DICER-overexpressing cells were enriched with the breast cancer resistance protein (BCRP), a member of the ATP-binding cassette transporter superfamily that causes resistance to several chemotherapeutic agents (Doyle & Ross 2003). Increased BCRP resulted in a more efficient efflux of TAM in DICER-overexpressing cells compared to control cells. Inhibition of BCRP inhibited TAM efflux and restored TAM-S in DICER-overexpressing cells. In ERα− breast cancer cells, DICER is targeted by oncomiRs including miR-103/107 (Martello et al. 2010), let7, miR-222/221, and miR-29a (Cochrane et al. 2010). Whether repression of DICER by these miRNAs mediates endocrine resistance is yet to be determined.
AGO2 recruits mRNA and miRNA into the RISC and is the catalytic component of the RISC. AGO2 is elevated in ERα− compared to ERα+ breast cancer cell lines and tumors (Adams et al. 2009). Expression of AGO2 is mediated by ERα/estrogen signaling and EGFR/MAPK signaling pathways (Adams et al. 2009). Ectopic expression of full length AGO2 in MCF7 cells promoted cell proliferation, reduced cell–cell adhesion, and increased cell migration (Adams et al. 2009). Whether AGO2 plays a role in endocrine resistance is yet to be determined.
We examined the association of AGO2 expression and overall survival rate in breast cancer patients using the online survival analysis tool, Kaplan–Meier plotter (http://kmplot.com/backup/breast; Gyorffy et al. 2013). It assesses the association gene expression on breast cancer prognosis using microarray data from 3554 patients. The patient data are from GEO. Higher AGO2 expression correlates with reduced relapse-free survival in all breast cancer patients and those whose primary tumors are ER+/PR+ (Fig. 3).
Higher expression of AGO2 is statistically associated with decreased relapse-free survival in all breast cancer cases and in patients whose primary tumors are ERα+/PR+. The Kaplan–Meier plots of AGO2 expression in breast tumors with breast cancer survival were generated using http://kmplot.com/analysis/index.php?p=service (Gyorffy et al. 2010). (A) All breast tumors, n=3557 and (B) ERα+/PR+, n=701.
Citation: Endocrine-Related Cancer 22, 5; 10.1530/ERC-15-0355
Conclusion
miRNAs are dysregulated in endocrine-resistant breast cancer and these miRNAs regulate specific genes in growth-promoting, apoptosis-resistant, and EMT pathways that result in TAM and AI resistance (Tables 1 and 2). The involvement of exosomes containing miRNAs in mediating endocrine resistance provides a new target for biomarker identification and therapeutic intervention to block metastatic spread. Although identifying new miRNAs mediating endocrine resistance is important, research effort is needed to determine the mechanisms and functional roles of already identified miRNAs with unknown roles in endocrine resistance and to develop targeted therapeutics to counter miRNA dysregulation and enhance hormonal sensitivity.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was supported by National Institutes of Health R01 CA138410 and in part by a grant from the University of Louisville School of Medicine to C M Klinge.
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(P Muluhngwi and C M Klinge contributed equally to this work)
