In the USA, an estimated 231,840 women were diagnosed with breast cancer in 2015, and there were approximately 40,290 deaths [
1]. Within 3 years of diagnosis, 10–15% of breast cancers develop distant metastases [
2]. Therefore, predictive tests are needed to identify individuals who are at high risk of metastases. To reduce breast cancer metastasis-related morbidity and mortality, the ideal biomarker should be accessible for non-invasive sampling and sensitive enough to detect early onset of tumor metastasis. To date, only a few markers, such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), have been identified as predictors of clinical responses to breast cancer treatments. None of these markers, however, evaluate tumor invasion or provide early detection of tumor metastasis.
MicroRNAs (miRs) are small non-coding RNA molecules that regulate gene expression. In breast cancers, aberrant miR expression influences tumor development and progression [
3]. Some miRs are upregulated in breast cancer, whereas others are downregulated, suggesting that miRs may have tumor-specific profiles. MiRs are remarkably stable in the circulation, and in formalin-fixed, paraffin-embedded tissues [
4]. Thus, they have potential to serve as breast cancer biomarkers. Circulating miRs, which are present in breast cancer patients, have potential as biomarkers that can be obtained by a minimally invasive procedure [
5‐
10]. Members of the human and mouse miR-200 family (miR-200 s), including two clusters (cluster 1: miR-200b, 200a, and 429, on chromosome 1, and cluster 2: miR-200c and miR-141, on chromosome 12), inhibit the epithelial-mesenchymal transition (EMT) [
11,
12] but promote the mesenchymal-epithelial transition (MET) [
13,
14], thereby regulating tumor metastasis by a reversible EMT-MET transition. In cancer cells, miR-200 s induce epithelial differentiation by suppressing ZEB1/2 and subsequently increasing E-cadherin expression [
11,
12]. Apart from the miR-200-ZEB1/2-E-cadherin axis, miR-200 s inhibit Wnt through
CTNNB1, NOTCH through
JAG1, and SNAIL through
SNAI2, and other direct targets,
FN1,
MSN,
NTRK2,
LEPR, and
ARHGAP19, all of which are necessary for tumor metastasis [
15‐
17]. Decreased levels of miR-200 s in tumor cells have been implicated in the invasion and metastasis of breast cancer [
11,
12,
18], but, in preclinical models, restoration of miR-200c reduced metastases [
19], suggesting that the miR-200 s function as tumor suppressors. Conversely, some studies suggest that, in cancer cells, miR-200 s promote tumor metastasis through promotion of tumor colonization at metastatic sites [
13,
14]. In murine cancers and human xenograft models, miR-200-expressing tumor cells and extracellular vesicles from these tumor cells promote breast cancer metastasis and confer the capacity for these cells to colonize distant tissues in an miR-200-dependent manner [
20]. Further, high levels of circulating miR-200 s in breast cancer patients are associated with increased numbers of circulating tumor cells (CTCs) [
21], which are a predictor of metastasis up to 2 years prior to clinical diagnosis [
22] and of shorter brain-metastasis-free survival [
23]. Thus, circulating miR-200 s are promising biomarkers for breast cancer metastasis. However, the cellular origin, mechanism of release, and function of circulating miR-200 s during tumor progression and metastasis remain elusive.
FOXP3 functions as the master regulator in the development and function of regulatory T cells [
24]. Our group found that FOXP3 is also a breast epithelial cell-intrinsic tumor suppressor [
25‐
27]. Unlike normal breast epithelial cells, 60–80% of human breast cancer cells lack nuclear expression of FOXP3 [
25,
26,
28,
29]. Aging female mice with a heterozygous
Scurfy (
sf) mutation of
Foxp3 (
Foxp3
sf/+), which causes a loss of
Foxp3 expression, frequently develop spontaneous breast cancers (ER
+, 14/18; PR
+, 12/18; ErbB2
+, 18/18) after 1 year of age, and 40% of these mice with primary breast tumors also develop lung metastases [
26]. Thus, this mouse model is appropriate for finding and validating breast cancer biomarkers and for evaluating their cellular origin, identifying their mechanism of release, and assessing the function of circulating miRs during tumor progression and metastasis. In the present work, we explored the relevance of FOXP3-mediated transcriptional regulation of miR-200 s in breast cancer cells in mice and humans. We also investigated the cellular origin, mechanism of release, and function of plasma miR-200 s in breast cancer cells and in mouse models.