The Role of MicroRNAs in Estrogen Receptor α-Positive Human Breast Cancer

MicroRNAs (miRNAs) are a class naturally occurring small non-coding RNAs that control gene expression by targeting mRNAs for translational repression or cleavage (Krol et al., 2010). Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers, and that lossor gain-of function of specific miRNAs contributes to breast epithelial cellular transformation and tumorigenesis (Esquela-Kerscher et al., 2006). miRNA expression profiling also revealed that miRNAs are differently expressed among molecular subtypes in breast cancer (Blenkiron et al., 2007; Iorio et al., 2005). There are large-scale molecular differences between estrogen receptor (ER) ┙-positive and ER┙-negative breast cancers (Perou et al., 2000; Sorlie et al., 2003). Endocrine therapy has become the most important treatment option for women with ER┙-positive breast cancer, and approximately 70% of primary breast cancers express ER┙. ER┙ is essential for estrogen-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ER┙-positive breast cancer (Dowsett et al., 2008; Harvey et al., 1999; Yamashita et al., 2006). Multiple mechanisms involved in altering ER┙ gene expression in breast cancer have been proposed, including ER┙ gene amplification (Holst et al., 2007) as well as transcriptional silencing by DNA methylation of CpG islands within the ER┙ promoter (Giacinti et al., 2006) and mutations within the open reading frame of ER┙ (Herynk et al., 2004). However, expression levels of ER┙ in breast cancer tissues differ widely among patients (Yamashita et al., 2011), and frequently change during disease progression and in response to systemic therapies (Yamashita et al., 2009). It was reported that the microRNA miR-206 decreases endogenous ER┙ mRNA and protein levels in human MCF-7 breast cancer cells via two specific target sites within the 3’-untranslated region (UTR) of the human ER┙ transcript (Adams et al., 2007). We found that the expression levels of miR-206 were gradually decreased as ER┙ protein expression increased in breast cancer tissues, suggesting that miR-206 is a key factor for the regulation of ER┙ expression in human breast cancer (Kondo et al., 2008). Moreover, recent studies have shown that ER┙regulating miRNAs, miR-18a, miR-18b, miR-22, miR-193b, miR-302c, and miR-221/222, as well as miR-206, directly targeted ER┙ in 3’UTR reporter assays, and suggested that several miRNAs regulate ER┙ expression.


Introduction
MicroRNAs (miRNAs) are a class naturally occurring small non-coding RNAs that control gene expression by targeting mRNAs for translational repression or cleavage (Krol et al., 2010).Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers, and that loss-or gain-of function of specific miRNAs contributes to breast epithelial cellular transformation and tumorigenesis (Esquela-Kerscher et al., 2006).miRNA expression profiling also revealed that miRNAs are differently expressed among molecular subtypes in breast cancer (Blenkiron et al., 2007;Iorio et al., 2005).There are large-scale molecular differences between estrogen receptor (ER) -positive and ER -negative breast cancers (Perou et al., 2000;Sorlie et al., 2003).Endocrine therapy has become the most important treatment option for women with ER -positive breast cancer, and approximately 70% of primary breast cancers express ER .ER is essential for estrogen-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ER -positive breast cancer (Dowsett et al., 2008;Harvey et al., 1999;Yamashita et al., 2006).Multiple mechanisms involved in altering ER gene expression in breast cancer have been proposed, including ER gene amplification (Holst et al., 2007) as well as transcriptional silencing by DNA methylation of CpG islands within the ER promoter (Giacinti et al., 2006) and mutations within the open reading frame of ER (Herynk et al., 2004).However, expression levels of ER in breast cancer tissues differ widely among patients (Yamashita et al., 2011), and frequently change during disease progression and in response to systemic therapies (Yamashita et al., 2009).It was reported that the microRNA miR-206 decreases endogenous ER mRNA and protein levels in human MCF-7 breast cancer cells via two specific target sites within the 3'-untranslated region (UTR) of the human ER transcript (Adams et al., 2007).We found that the expression levels of miR-206 were gradually decreased as ER protein expression increased in breast cancer tissues, suggesting that miR-206 is a key factor for the regulation of ER expression in human breast cancer (Kondo et al., 2008).Moreover, recent studies have shown that ERregulating miRNAs,, as well as miR-206, directly targeted ER in 3'UTR reporter assays, and suggested that several miRNAs regulate ER expression.

Expression of ERα in human breast cancer tissues 2.1.1 ERα expression in normal and malignant breast epithelial cells
ER expression in normal and premalignant breast epithelial cells has been assessed by immunohistochemistry.It was reported that on average, normal premenopausal terminal duct lobular units contain about 30% ER -positive cells (Allred et al., 2004).In contrast, nearly all cells express very high levels of ER in the majority of premalignant breast lesions, including atypical ductal hyperplasia, atypical lobular hyperplasia and lobular carcinoma in situ.DCIS (Ductal carcinoma in situ) shows various levels of ER expression ranging from high levels of lower-grade lesions to low levels of higher-grade lesions, including entirely ER -negative.Furthermore, the wide range of ER expression is observed in invasive ductal carcinoma.There is a substantial (~25%) subset of invasive breast cancers that does not contain any ER -expressing cells.The majority (~75%) of invasive breast cancers, however, does contain ER -expressing cells, but the proportion varies ranging from very low, to intermediate, to very high.We examined ER expression in breast cancer tissues by immunohistochemistry using Allred score (Allred et al., 1998), and demonstrated that breast tumors show a wide range of ER expression levels (Yamashita et al., 2011).Moreover, intensity of ER expression is not equal among breast cancer cells in an individual tumor.Eastern cooperative oncology group compared the distribution of ER protein expression by immunohistochemistry and ER mRNA expression by quantitative reverse transcription (RT)-PCR in ECOG 2197 study (Badve et al., 2008).They reported that ER mRNA expression in breast cancer tissues also showed continuous and the wide range.

Role of expression levels of ERα in human breast cancer
It is well established that expression levels of ER govern response to endocrine therapy and prognosis in ER -positive human breast cancer.Allred and colleagues first reported that expression levels of ER assessed by immunohistochemistry affected prognosis (Harvey et al., 1999).ER status using Allred score was a highly significant predictor of disease-free survival for patients who received adjuvant endocrine therapy.Recently, relationship between expression levels of ER and prognosis was analyzed in adjuvant endocrine therapy trials for postmenopausal breast cancer, and demonstrated that diseasefree survival or time to recurrence were significantly different according to expression levels of ER (Dowsett et al., 2008;Viale et al., 2007).It was reported that the use of a cutoff of 1% staining cells for ER indicated a better prognosis and at least some degree of endocrine responsiveness (Hammond et al., 2010).In the neoadjuvant endocrine therapy trial for postmenopausal women, correlation between expression levels of ER assessed by Allred score and response to neoadjuvant endocrine therapy was analyzed (Ellis et al., 2001).It showed that letrozole response rates were superior to tamoxifen response rates in every ER Allred score from 3 to 8, indicating that letrozole is more effective than tamoxifen regardless of the level of ER expression.We also studied expression levels of ER on pretreatment biopsies and post-treatment surgical specimens in postmenopausal patients with ERpositive primary breast cancer who were treated with aromatase inhibitors for 6 months (Yamashita et al., 2009).We showed that ER expression was decreased in post-treatment tumors compared to pretreatment specimens.On the other hand, we analyzed expression levels of ER in primary breast cancer specimens from 75 metastatic breast cancer patients who received endocrine therapy on relapse, and analyzed the correlation between expression levels of ER and response to endocrine therapy and post-relapse survival (Yamashita et al., 2006).Our results indicated that patients with higher ER expression responded significantly to endocrine therapy, and that patients with higher ER expression had better survival after relapse.Moreover, we recently reported that patients whose breast tumors contained high ER expression effectively responded to aromatase inhibitors and displayed longer time to progression during first-line endocrine therapy with aromatase inhibitors and time to endocrine therapy failure (Endo et al., 2011).

Regulation of expression levels of ERα
Multiple mechanisms involved in the regulation of ER expression in breast cancer have been identified, including mutations of ER gene (Herynk et al., 2004) and transcriptional silencing by DNA methylation within the ER promoter (Gaudet et al., 2009;Giacinti et al., 2006;Iwase et al., 1999).It was recently reported ER gene amplification in breast cancer (Holst et al., 2007;Tomita et al., 2009).Holst and colleagues demonstrated that ER gene amplification was frequent and more than 20% of breast cancers harbored genomic amplification by FISH analysis.They reported that ESR1 amplification was tightly linked to ER protein expression, and that ESR1 amplification was also found in benign and precancerous breast diseases, such as atypical ductal hyperplasia, ductal carcinoma in situ and lobular carcinoma in situ.However, breast cancer patients show a wide range of ER expression levels (Yamashita et al., 2011), and the levels of expression in individual patients change during disease progression and in response to systemic therapies (Yamashita et al., 2009).Therefore, other mechanisms may also regulate ER expression in breast cancer.

miRNA biogenesis and function
MicroRNAs (miRNAs) comprise a large family of ~21-nucleotide-long RNAs that have emerged as key post-transcriptional regulators of gene expression (Krol et al., 2010).In mammals, miRNAs are predicted to control the activity of ~50% of all protein-coding genes.Functional studies indicate that miRNAs participate in the regulation of almost every cellular process investigated so far and that changes in their expression are associated with many human pathologies.Primary miRNA transcripts are cleaved into 70-to 80-nucleotide precursor miRNA (pre-miRNA) hairpins by RNase III Drosha in the cell nucleus and transported to the cytoplasm, where pre-miRNAs are processed by RNA Dicer into 19-to 25-nucleotide miRNA duplexes.One strand of each duplex is degraded, and the other strands become mature miRNAs, which, incorporated into the RNA-induced silencing complex, recognize sites in the 3'-UTR of the target mRNAs and cause translational repression or mRNA cleavage.miRNAs are a new player among gene regulation mechanisms, and their functions have not been fully explored but are known to include the regulation of cellular differentiation, proliferation and apoptosis.Recent evidence has shown that miRNA mutations or mis-expression are associated with various human cancers (Esquela-Kerscher et al., 2006).

Regulation of expression levels of ERα by miRNAs
Adams and colleagues first identified two potential miRNA miR-206 target sites within the 3'-UTR of ER mRNA via in Silico analysis (Adams et al., 2007).The validation assay revealed that both miR-206 target sites specifically interacted with miR-206, which in turn repressed the corresponding ER mRNA and protein expression.They also demonstrated that expression levels of endogenous miR-206 were significantly higher in ER -negative MDA-MB-231 cells than in ER -positive MCF-7 cells.Moreover, they showed that miR-206 repressed ER mRNA and protein expression in MCF-7 and T47D cells.We showed that miR-206 expression assayed by quantitative RT-PCR analysis was inversely correlated with ER but not ER mRNA expression in human breast cancer tissues (Kondo et al., 2008).Moreover, miR-206 expression levels were gradually decreased as ER protein expression increased in human breast cancer.Transfection experiments revealed that introduction of miR-206 in estrogen dependent MCF-7 cells inhibited cell growth.Transfection of miR-206 into MCF-7 cells suppressed ER expression and inhibited cell growth in a dose-dependent manner.Furthermore, introduction of miR-206 produced a dose-dependent decrease of mRNA expression of ER -target genes, such as progesterone receptor, cyclin D1 and pS2.Adams and colleagues recently reported that miR-206 coordinately targeted mRNAs encoding the coactivator proteins SRC-1 and SRC-3, and the transcription factor GATA-3, all of which contribute to estrogenic signaling and a luminal A phenotype (Adams et al., 2009).Furthermore, they identified that miR-206 contributed to the epidermal growth factor (EGF) induced repression of ER signaling in MCF-7 cells.

MiRNAs that directly target ERα in human breast cancer
Zhao and colleagues demonstrated that miR-221 and miR-222 were highly expressed in ER -negative breast cancer cells, and that miRNA in situ hybridization analyses also showed overexpression of miR-221 and miR-222 in ER -negative breast tumors (Zhao et al., 2008).Recently, two groups reported several miRNAs that down-regulated ER in breast cancer cells.They identified the target sites of miRNAs, such as miR-18a and b, miR-302, miR-193b, miR-22, and miR221/222 as well as miR-206, in the ER 3'-UTR, and showed that these miRNAs inhibited estrogen signaling by directly targeting ER mRNA (Leivonen et al., 2009;Pandey et al., 2009) (Fig. 1).We recently analyzed expression levels of miRNAs that directly target ER , in human breast cancer samples by quantitative RT-PCR analysis (Yoshimoto et al., 2011).Our results showed that miR-18a expression was much higher in ER -negative than ER -positive tumors, with the expression levels of miR-18a not differing in ER -positive breast cancer as a function of ER protein level.Surprisingly, expression levels of miR-193b and miR-221 were significantly lower in ER -negative than ER -positive tumors, and the levels of these miRNAs gradually increased as ER protein expression increased.There was no statistically significant association between miR-22 and ER expression, and miR-302c expression was minimal in human breast cancer samples.Prognostic analysis showed that low miR-18b expression was significantly associated with improved survival in HER2negative breast cancer.Our results suggest that miRNAs that directly target ER have distinct roles in not only regulating ER but also regulating other target genes in human breast cancer, and that some miRNAs might be associated with characteristics of ERpositive breast cancer.

Role of miRNAs in breast cancer
miRNAs can function as tumor suppressors and oncogenes (Esquela-Kerscher et al., 2006).The reduction or deletion of a miRNA that functions as a tumor suppressor leads to tumor formation.On the other hand, the amplification or overexpression of a miRNA that function as an oncogene results in tumor formation.Lu and colleagues first analyzed miRNA expression profiling in normal and tumor samples, and revealed global changes in miRNA expression (Lu et al., 2005).It was found that miRNA expression seems g l o b a l l y h i g h e r i n n o r m a l t i s s u e s c o m p a r e d w i t h t u m o r s .M o r e o v e r , m i R N A s a r e differentially expressed in various cancers.miRNA expression profiling also revealed that miRNAs are differently expressed among molecular subtypes in breast cancer (Blenkiron et al., 2007;Iorio et al., 2005).Iorio and colleagues first reported the miRNA gene expression profile in human breast cancer (Iorio et al., 2005).Compared with normal breast tissue, miRNAs are aberrantly expressed in human breast cancer.They also reported differentially expressed miRNAs associated with ER expression, including miR-206.Blenkiron and colleagues analyzed miRNA expression in human breast cancer, and found that many miRNAs were differently expressed between breast cancer subtypes, such as luminal A, luminal B, HER2-positive, basal-like and normal-like (Blenkiron et al., 2007).They also found significant association between miRNA expression profiling and clinicopathological factors such as ER status and tumor grade.Furthermore, recent studies have demonstrated that loss-or gain-of function of specific miRNAs contributes to breast epithelial cellular transformation and tumorigenesis.The interconnections between miRNAs and tumor suppressor genes and oncogenes in breast cancer were summarized by Zoon and colleagues (Zoon et al., 2009).

Conclusion
It is well recognized that individual miRNA sequences can suppress the production of hundreds of proteins (Krol et al., 2010).Reduction of protein levels in this way is often modest, however, and many such RNAs probably collectively fine-tune gene expression.Accumulating evidence supports the hypothesis that the ability of miRNAs to simultaneously regulate many target genes makes them attractive candidates for regulating normal and cancer cells.miRNAs are potential therapeutic targets for more tailored treatment strategies for breast cancer.

Fig. 1 .
Fig. 1.Schematic representation of the human ER mRNA and the predicted miRNA target sites.