Dysregulation of microRNAs in breast cancer and their potential role as prognostic and predictive biomarkers in patient management

The ideal biomarker should be easily accessible, sensitive enough to detect all tumors,
and specific and therefore not detectable in healthy individuals. Because of their
high tissue-specificity, great stability, and aberrant expression in different tumor
types, miRNAs are thought of as specific biomarkers with diagnostic, predictive, and
prognostic potential (Figure 1).

Figure 1. Illustration of the application of microRNAs (miRNAs) as novel diagnostic, predictive,
and prognostic biomarkers in breast cancer management.

MicroRNAs as diagnostic biomarkers for breast cancer heterogeneity

Early diagnosis of breast cancer

Early diagnosis is of utmost importance to reduce the mortality rate in cancer. Therefore,
the search for new diagnostic biomarkers remains a persisting quest. The main examined
diagnostic miRNA signatures will be discussed, with a focus on those miRNAs validated
by more than one study or verified in different cohorts or both. (For a detailed overview
of the individual miRNAs, see Table 4).

Table 4. List of major diagnostic microRNA signatures for the early diagnosis of breast cancer

In a recent study, a signature of nine circulating miRNAs was capable of discriminating
between early-stage breast cancer and healthy controls 62]. A meta-analysis concerning miR-155 showed highly sensitive and specific diagnostic
accuracy 63]. By comparing miRNA profiles between serum samples from breast cancer patients and
healthy volunteers, Chan and colleagues 64] identified four miRNAs as significant diagnostic markers. Cuk and colleagues 65] found another four upregulated miRNAs in plasma of patients with breast cancer, capable
of detecting stage I or II breast cancer, making them attractive candidates for early
breast cancer detection. Another study reported three upregulated miRNAs and one downregulated
miRNA in patients with breast cancer compared with normal controls 66]. A prospective study identified three significantly overexpressed serum miRNAs in
women who eventually developed breast cancer (cases) compared with breast cancer-free
controls 67]. This study introduces a new perspective on the miRNA function by describing their
potential to predict increased risk of developing breast cancer.

Breast cancer molecular subtypes and microRNAs

We have shown that microRNA expression profiles are able to recapitulate the molecular
breast cancer subtypes by using mRNA profiling 60],68]. Molecular miRNA signatures which distinguish between different breast cancer subtypes
were described for the first time (to the best of our knowledge) by Blenkiron and
colleagues 69] and reviewed by Serpico and colleagues 70]. The commonly found molecular subtype-related miRNAs are presented in Table 5.

Table 5. Common subtype-specific microRNAs found by meta-analysis of three independent studies

Blenkiron and colleagues 69] profiled 309 miRNAs in 93 breast tumors with different molecular subtypes. The differential
miRNA expression resulted in a correct classification of basal versus luminal subtypes.
An identified 31-miRNA signature was able to distinguish the different breast cancer
subtypes. de Rinaldis and colleagues 71] found consistent results by revealing a 46-miRNA signature able to differentiate
between breast cancer subtypes (n?=?173). A substantial degree of consistency was
also observed by Dvigne and colleagues 4], and the common miRNA signatures for molecular breast cancer subtypes are listed
in Table 5.

In another study, 453 miRNAs in 29 early-stage breast cancer tumors were profiled,
identifying signatures that accurately predict ER, progesterone receptor (PR), and
HER2 status (Table 6) 72]. miR-342 showed the highest expression in ER-positive and HER2/neu-positive luminal
B tumors, which was verified in recent studies 73],74], and showed a decreased expression in TNBC. MiR-520 g was downregulated in ER- and
PR-positive tumors.

Table 6. MicroRNA signatures for estrogen receptor, progesterone receptor, and HER2/neu receptor
status in breast cancer

Breast cancer histological subtypes and microRNAs

Little is known about the correlation of histological subtypes and specific miRNA
expression patterns and it is definitely worthwhile to further investigate this relation
in the future.

Volinia and colleagues 75] found a nine-miR invasiveness signature by profiling miRNAs in ductal carcinoma in situ and invasive ductal carcinoma (IDC). Giricz and colleagues 76] revealed six differentially expressed miRNAs during progression from lobular carcinoma
in situ to invasive lobular carcinoma. Van der Auwera and colleagues 77] showed a differential expression of 13 miRNAs in inflammatory breast cancer (IBC)
versus non-IBC. Another study examined the miRNA expression profile in IBC and found
five miRNAs able to accurately classify between IBC and non-IBC 78].

Predictive microRNAs

Predictors help to individualize therapy and diagnosis of breast cancer, correlate
with response to a given treatment, and determine the treatment benefit. Recently,
several miRNAs have been described to serve as putative therapeutic targets. (See
Table 7 for described miRNAs and Additional file 1: Table SI 1 for a more extensive overview of predictive miRNAs).

Table 7. Predictive microRNAs – microRNAs involved in response (sensitivity/resistance) to
conventional breast cancer therapeutic strategies

MicroRNAs associated with hormone therapies

MiRNAs appear to be involved in the process of endocrine resistance 79], and research has been conducted to identify miRNAs associated with clinical benefit
of hormone therapies.

miR-375

miR-375 demonstrated its sensitizing effect on tamoxifen response via direct targeting
of metadherin (MTDH). Loss of MTDH restored sensitivity to tamoxifen and correlated
with disease-free survival (DFS) in tamoxifen-treated patients 80].

miR-342

He and colleagues 74] found that miR-342 expression positively correlates with ER? expression and that
introducing miR-342 into estrogen-dependent breast cancer cell lines enhanced sensitivity
to tamoxifen-induced apoptosis. Cittelly and colleagues 81] verified these findings and reported miR-342 downregulation to be associated with
tamoxifen resistance.

miR-221/222

The miR-221/222 cluster is an inhibitor of ER? and is being associated with tamoxifen
resistance in breast cancer cells 82]-84]. This miRNA cluster has also been involved in resistance to fulvestrant, a selective
ER downregulator 85].

Let-7f

Aromatase inhibitors are used in endocrine therapy since they decrease estrogen production
by blocking the aromatase gene CYP19A1, a direct target of let-7 f. MiRNA expression profiling before and after treatment
with letrozole showed an increase in let-7f in preclinical as well as clinical settings
86].

MicroRNAs associated with targeted therapies

miR-210

Jung and colleagues 87] examined the plasma miR-210 expression level in patients with HER2⁺ breast cancer before (that is, baseline expression) and after neoadjuvant chemotherapy
(NCT) that included trastuzumab. MiR-210 was the only miRNA with a significant upregulated
baseline expression in the residual disease group before treatment. Therefore, high
miR-210 baseline expression was associated with resistance to trastuzumab-included
chemotherapy. Results were validated by comparing miR-210 expression in trastuzumab-sensitive
and -resistant breast cancer cells and in an independent set of pre- and post-operative
plasma samples. A significantly higher miR-210 level was found in the trastuzumab-resistant
cells and in the independent patient cohort before surgery.

MicroRNAs associated with response to chemotherapeutic agents

Drug sensitivity varies with each patient, making predictors of benefit or resistance
toward proposed chemotherapeutic agents essential. In this way, the proportion of
patients with a beneficial treatment would increase and toxicity of ineffective treatments
would be avoided.

miR-125b

Zhou and colleagues 88] reported resistance to various chemotherapies induced by miR-125b targeted repression
of Bak1 (BCL2 antagonist killer 1), which was verified by the clinical findings of
Wang and colleagues 89], who reported higher miR-125b expression in non-responsive patients after admission
of 5-fluorouracil. Climent and colleagues 90] reported miR-125b deletion on chromosome 11q to be correlated with benefit of anthracycline-based
chemotherapy and low recurrence rate in patients with lymph node-negative breast cancer.

miR-30c

Bockhorn and colleagues 91] described how increased levels of miR-30c sensitized the drug response of breast
cancer cell lines to paclitaxel and doxorubicin, and this was also seen in the preclinical
model by Fang and colleagues 92].

miR-21

Mei and colleagues 93] reported miR-21 upregulation to be associated with taxol resistance in breast cancer
cells. These results were validated in a recent study, in which miR-21 upregulation
resulted in an increase of the anti-apoptosis protein BCL-2 and chemoresistance in
breast cancer cells 9]. The increase of BCL-2 expression was induced by direct targeting by miR-21, which
was able to unconventionally upregulate the expression of its direct target by binding
to its 3? UTR 94].

MicroRNAs associated with radiotherapy

miR-34a

Low levels of miR-34a rendered breast cancer cells more resistant to radiotherapy
95]. These findings were verified by Stankevicins and colleagues 96], who postulated that miR-34a was involved in breast cell responses to low-dose X
radiation. Moreover, miR-34a was found to be upregulated by p53 in response to radiation
in normal and breast cancer cell lines 96].

Prognostic microRNAs

Since patients at higher risk might acquire differential therapeutic interventions,
the search for prognostic biomarkers remains a continuous work in progress. Several
gene-expression studies have identified new or improved miRNA prognostic markers,
giving information on the course and outcome of disease in different subgroups of
patients. To narrow this extensive area, we will emphasize those prognostic miRNAs
that are being validated (that is, examined in more than one study or tested in the
same study) but verified in more than one cohort or dataset. An overview of the miRNAs
described in this review can be found in Table 8 and Table 9; for a more extensive list, see Additional file 1: Tables S2A and S2B.

Table 8. List of positive prognostic microRNA signatures in breast cancer

Table 9. List of negative prognostic microRNA signatures in breast cancer

MicroRNAs associated with positive prognosis

Let-7b and miR-205

Quesne and colleagues 97] revealed association of let-7b and miR-205 with prognosis in breast cancer. They
applied in situ hybridization to study miRNA expression in a population-based breast tumor cohort
and validated their findings by use of qRT-PCR. Deregulation of let-7b, which targets
the oncogenes H-RAS and HMGA2, often occurs early in breast cancer progression and its expression is known to be
downregulated during EMT and associated with less aggressive tumors 69],98]-100]. Within luminal breast cancer, increased let-7b expression was positively associated
with survival.

Additionally, the authors reported miR-205 as another miRNA with positive prognostic
value 97]. MiR-205 regulates EMT by inhibiting E-cadherin suppression. MiR-205 expression is
associated with tumors of ductal morphology and independently predicts survival within
this tumor subtype.

Two independent studies verified these results. Ma and colleagues 101] showed how low let-7b expression levels in a heterogeneous breast cancer cohort associated
with poor prognosis reflected in lower overall survival (OS) and relapse-free survival
(RFS) times. Markou and colleagues 102] detected miRNA-205 levels in a heterogeneous breast cancer cohort and demonstrated
how downregulation was associated with longer DFS.

miR-375

Wu and colleagues 57] applied deep sequencing methods on blood of patients with primary stage II or III
breast cancer to indicate the miR-375 prognostic value and validated these findings
in an independent cohort by using qRT-PCR. miR-375 is known to inhibit migration and
invasion which is partially carried out by targeting JAK2 103]. A differential expression exists between patients with and without metastatic relapse.
Higher circulating levels reflect more favorable clinical outcome in terms of pathologic
complete response to NCT and absence of relapse 57].

miR-30a

The prognostic features of miR-30a were investigated in 221 patients with IDC 104], showing that miR-30a, which has been implicated in regulation of several biological
processes 105]-107], negatively regulates vimentin expression and overexpression suppresses migration
and invasiveness 104]. Reduced expression is associated with unfavorable outcome (decreased RFS and DFS).
The prognostic value of miR-30a was also examined by Zhang and colleagues 108] on a heterogeneous set of 96 patients with breast cancer. The authors attributed
the tumor-suppressive nature of this miRNA to its ability to target MTDH, thereby
suppressing breast tumor growth and metastasis. Decreased expression of miR-30a was
associated with an unfavorable outcome in terms of metastasis development 108].

miR-342-5p

miR-342-5p has a role in cell cycle progression and cell growth regulation. The latest
research by Leivonen and colleagues 109] demonstrated how higher expression of miR-342-5p, which is an efficient negative
regulator of the HER2 pathway, was significantly associated with better survival in
two heterogeneous breast cancer cohorts.

miR-497

miR-497 is a tumor-suppressive miRNA and its biological role is found in the regulation
of the nervous system. Several studies examined miR-497 and found its expression to
be significantly decreased in breast cancer compared with normal breast and negatively
correlated with adverse clinicopathological characteristics. Shen and colleagues 110] reported how elevated miR-497 expression rendered IDC patients with better prognosis.
An independent study verified its prognostic role in a population-based breast cancer
cohort, in which higher expression was correlated with a better 5-year DFS and OS
111].

MicroRNAs associated with negative prognosis

miR-122

Wu and colleagues 57] identified circulating miR-122 in a heterogeneous breast cancer exploration and validation
cohort, by sequencing and RT-PCR, respectively. They found miR-122 to be associated
with outcome in terms of relapse and identified a distinct expression in patients
undergoing metastatic relapse and those who remained relapse-free.

miR-27b-3p

The miR-27 family is known to regulate cell cycle progression and survival by targeting
the tumor-suppressive FOXO1 gene and is highly expressed in breast cancer 112]. In a recent study, Shen and colleagues 113] aimed to identify and validate a prognostic signature for patients with TNBC (n?=?58)
and found that lymph node status and miR-27b-3p were independent predictors of poor
prognosis in terms of distant metastasis-free survival. This result was validated
in a TNBC cohort (n?=?41) 113].

miR-21

miR-21, which has its biological role in development, morphogenesis, and differentiation,
is known to be overexpressed in breast cancer 114]. The significance of this miRNA as a prognostic factor was examined in two independent
studies. Lee and colleagues 115] found significantly higher expressions in IDC compared with normal breast tissue,
which positively associated with tumor size, stage, grade, and Ki-67 expression. A
higher miR-21 expression also correlated with ER negativity and HER2 positivity. A
lower OS could be noticed in patients with higher miR-21 expression levels 115]. In a more recent study, miR-21 was found to inversely correlate with DFS 102].

miR-210

Volinia and colleagues 75] used deep sequencing to determine miR-210 in patients with IDC. miR-210 showed association
with time to metastasis and OS by uni- and multivariate analyses. Various breast cancer
genes—such as BRCA1, E-cadherin, PARP1, and RB1—with an antagonistic behavior to miR-210
were identified. More recently, systematic reviews and meta-analyses of previous clinical
research were performed to recapitulate the significance of increased miR-210 in the
prognosis of cancer. Summarizing nine published studies, the authors found that miR-210
could predict outcome, especially in patients with breast cancer, and that miR-210
overexpression predicts DFS and RFS 116],117].

miR-9

The functional significance of miR-9 is evidenced by its regulative role in neurogenesis,
development, and apoptosis. Dysregulation of miR-9 influences proliferation or metastasis
formation and has been reported in cancer 118]. In breast cancer, upregulation of miR-9 suppresses E-cadherin, leading to increased
cell motility and invasiveness 119]. Zhou and colleagues 120] found miR-9 to discriminate between cases with and without local recurrence (LR),
in which the latter group showed a significantly lower expression. In ER⁺ cases, a lower 10-year LR-free survival was seen in patients with high miR-9 expression
by using miRNA arrays 120].

miR-187

Using an in silico method in two independent breast cancer cohorts, Mulrane and colleagues 121] identified and validated miRNA-187 as being involved in breast cancer progression.
miR-187 is independently associated with poor outcome in breast cancer (particularly,
lymph node-positive samples) in terms of reduced breast cancer-specific survival.

miR-155

Several studies demonstrated the involvement of miR-155 in biological processes, such
as cell survival, growth, migration, and invasion 23],25]. Song and colleagues 122] found a significantly higher miR-155 expression in formalin-fixed paraffin-embedded
tumor compared with normal tissues. Multivariate analysis showed an inverse (yet insignificant)
correlation with breast cancer outcome in terms of OS. Recently, Kong and colleagues
123] verified these findings and reported miR-155 to be frequently upregulated in various
types of cancer where it plays pro-angiogenic, proliferative, and migratory roles.
Moreover, they noticed miR-155 expression levels to be associated with poor prognosis
in terms of OS 123].