MicroRNAs in the development and neoplasia of the mammary gland

Introduction

MicroRNAs (miRs) are small, endogenous noncoding RNAs that regulate gene expression post-transcription, by degrading target mRNA or repressing translation. They are hardy in nature, being resistant to RNAse degradation, acidic pH, and repeated freezing and thawing, and are stable at room temperature¹. Matured miRs integrate into the RNA - induced silencing complex (RISC) and direct it to target mRNAs through partial sequence complementarity. miRs down regulate the target gene expression by translation repression (48% through RISC complex), or mRNA degradation (29%), or by both phenomena (23%)². In addition to their involvement in many other physiological processes, they play crucial roles in mammary gland development and lactation, and their deregulation can lead to breast cancer³. This review provides an overview on the recent findings concerning the role of miRs in the developmental stages of the mammary gland and breast cancer progression.

miRs in mammary glands

The study of miRs in mammary gland development is essential, as miRs in milk are derived from the mammary gland and can be a biomarker of a healthy lactating gland, as well as protecting infants and promoting their development⁴.

miRs in milk during lactation are putative markers of activities in mammary gland and act as functional signals for proper growth and development of the young. The highly abundant miRs in milk during lactation cycle are miR-191, 184, 181, 148, 375, and miRs of let-7 family (7f, 7a and 7i) as evidenced from studies in the marsupial tammar wallaby⁵. Comparative analysis of the miRNome of bovine milk, mammary tissue, milk fat, and whey revealed 188 miRs in common, with some novel miRs discovered⁶. The miR signature of milk fat was similar to that of mammary gland tissue miRs. Functional studies of highly expressed miRs in those fractions indicated their role as regulators of mammary gland functions contributing to healthy milk production⁶.

miRs controlling lactation

It was observed that miR-126 plays a role in mammary epithelial cells (MECs) by modulating lipid synthesis⁷. FASN (fatty acid synthase) gene expression is increased when miR-126-3p is inhibited, suggesting its involvement in lipid metabolism in the mammary gland. In addition, estradiol and progesterone enhanced lipid synthesis by downregulating the levels of miR-126-3p⁷. Similarly, miR-150 hampers lipogenesis in MECs and reduces secretory activity⁸, while miR-145 facilitates milk fat synthesis in lactating goats by targeting the gene INSIG1 (insulin induced gene 1)⁹.

The role that miRs play as regulators of lipogenesis was confirmed by overexpression studies, which showed that there was more synthesis of fat droplets, accumulating triacylglycerols, and a higher proportion of unsaturated fatty acids in lactating MECs. For instance, miR-24 was found to be expressed at a much higher level during peak lactation in goats and affects triacylglycerol content, unsaturated fatty acid concentration, and expression of target genes such as FASN, SREBF1 (sterol regulatory element binding transcription factor1), SCD (stearoyl-CoA desaturase), GPAM (glycerol-3-phosphate acyltransferase; mitochondrial), and ACACA (acetyl-CoA carboxylase)¹⁰.

miR expression in MECs is regulated by lactogenic hormones (dexamethasone, insulin, and prolactin) as evidenced by an increased miR-148a level in bovine MECs, which is probably associated with increased milk production during lactation in cows¹¹. The miR-29 family affects lactation by regulating the DNA methylation of target genes DNMT3A and DNMT3B (DNA (cytosine-5)-methyltransferase 3A and 3B) in MECs of dairy cows¹². Moreover, inhibition of the miR-29 family resulted in hypermethylation of promoters of lactation–related genes leading to decreased secretion of triglycerides, proteins, and lactose by the epithelial cells. miR-486 facilitates lactation by downregulating PTEN (phosphatase and tensin homolog) target gene. Downregulation of this gene affects the expression of downstream genes, such as AKT, mTOR and β-casein, which have crucial roles in mammary gland development and lactation¹³. Additionally, diet restriction has been shown to affect the miRNome of lactating mammary glands, indicating the role of miRs in regulating milk composition¹⁴. Lin et al. (2013)¹⁵ observed group of miRs (such as miR-23a, 27b, 103, and 200a) affect milk fat synthesis with synergistic action. miR profiling of lactating and nonlactating bovine mammary gland revealed miR-125b, 181a, and 199b expression level reduced in non-lactation period; whereas miR-141, 484, and 500 expression level was higher in lactation period¹⁶. Highly expressed miRs such as let-7f-5p is found to target many genes involved in protein, carbohydrate, and triglyceride synthesis. The human milk enriched miR such as miR-22-3P regulates development and differentiation of T-lymphocytes. The miR-181a-5p and miR-182-5p have crucial role in immune cell differentiation, miR-375 is required for glucose homeostasis, and miR-148a-3p is a tumour suppressor and involved in liver development¹⁷.

miRs controlling involution and breast cancer

Identification and characterization of miRs involved in breast cancer will facilitate targeting miRs for possible therapy. Improper involution possibly contributes towards tumour development¹⁸. miR-424(322)/503 regulates mammary involution in humans by targeting BCL-2 (B-cell lymphoma 2; anti-apoptotic) and IGF1R (insulin like growth factor-1-receptor) genes. The loss of this miR leads to malignancy and nonresponse to chemotherapy, demonstrating its role as a tumour suppressor¹⁸. By contrast, some miRs, such as miR-660-5p, promote tumour development and metastasis, and the level of miR-660-5p was found to be increased in MCF7 breast cancer cell lines¹⁹. This miR’s tumour promoting activity was confirmed by observation of reduced invasion of MCF7 cells at reduced miR-660-5p levels.

One of the major causes of breast cancer is disturbed estrogen signaling, where the altered expression of estrogen receptor α (ERα) and its cross-talk with the related miR culminates in neoplasia²⁰. Studies on the effect of E2 on the expression pattern of miRs in MCF7 and ZR75 cell lines revealed 172 miRs that were up or downregulated. Notable miRs are miR-206, miR-34a, miR-17-5p, and miR-125 a/b, which act as tumour suppressors, and miR-21, miR-10B, and miR-155, which act as oncogenes²¹. Another study using an ACI rat model for the effect of E2 on the miR signature showed 33 dysregulated miRs²². Additionally, the use of ellagic acid (natural phenol antioxidant) reversed the dysregulation of miR-206, miR-182, miR-375, miR-127, miR-183, and miR-122, subsequently modulating the target proteins ERα, RASD1, cyclin D1, FoxO1, FoxO3a, Bcl-w, Bcl-2 and cyclin G118²². Furthermore, overexpression of the tumour suppressing miR-133a in MCF-7 and MDA-MB-231 cells suppressed phosphorylated Akt (p-Akt) protein and inhibited p-Akt nuclear translocation, and this miR also regulates the cell cycle of cancerous cells by targeting the EGFR (epidermal growth factor receptor) gene²³.

miR-206 is found to play a crucial role in BRCA1 (Breast CAncer susceptibility gene; tumour suppressor) depleted mouse mammary gland²⁴. Overexpression of miR-206 showed no effect on lactation, but did have a role in tissue remodeling through increasing fat tissue and reducing branching morphogenesis. There may be a possibility of increased miR-206 levels due to BRCA1 loss, culminating in mammary gland remodeling and tumour development²⁴. miR-184 is found to be a tumour suppressor by regulating the number of genes in the PI3K/AKT/mTOR pathway, as observed by miR profiling of the pubertal mouse mammary gland²⁵. This pathway is important in mammary gland development and lactation²⁶. miR-184 is only expressed in epithelial cells, and the level increases during differentiation of cells from terminal end bud into ductal epithelial cells²⁵. The miR 126/126* pair is observed to repress the recruitment of mesenchymal stem cells and inflammatory monocytes into the tumour stroma, thus inhibiting breast cancer metastasis²⁷. Urinary miR detection of overexpressed miR-155, and lowered expression of miR – 21, 125b, and 451 can act as non-invasive biomarker for breast cancer detection²⁸. Chan et al. (2013)²⁹ identified the miR signature (miR-1, 92a, 133a, and 133b) in serum which can be used as biomarkers for breast cancer detection as a noninvasive diagnostic strategy. Similar study by Mar-Aguilar et al., (2013)³⁰ identified miR-145, 155, and 382 as potential biomarker for breast cancer detection. Deregulated expression of miR-17, 34a, 155 ,and 373 and their concentration in serum as cell-free miR probably connects to the progression and metastasis of breast cancer³¹.

Exosomes are cell - derived small vesicles (40 – 100 nm) containing mRNA, protein, miR etc. They are crucial mediator of inter cellular signaling during cancer development. Exosomes derived from cancer associated fibroblasts in breast cancer have 3 miRs such as miR-21, -143, and -378e which promote stemness, epithelial-mesenchymal transition, and anchorage-independent cell growth, thus accelerating oncogenic signaling in breast cancer cells³². The exosomes released from cancer cells of breast cancer patients induce the non-tumorigenic epithelial cells to form tumors with the help of dicer endonuclease³³.

Triple negative breast cancer (TNBC) is a subtype of breast cancer where the cancer cells lack the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2)³⁴. Out of total breast cancer cases 10 – 20% are TNBC cases and various studies showed the expression of specific miR pattern in these cases which can act as biomarker for cancer diagnosis^30,35,36. A recent study on paired sera and tissue samples of TNBC patients in India revealed overexpression of 3 oncogenic miRs (miR – 21, 221, and 210) and down regulation of miR – 195 and miR – 145 as compared to that of triple positive breast cancer cases. The TNBC cases were found to be highly prevalent (73.9%) in pre-maunopausal (<35 yr) women and the miR let7a was overexpressed although it is known as a tumour suppressor. Increased serum level of exosomal miR-373 can also be a marker for TNBC cases³⁷. The study of TNBCs with lymphnode metastases showed association of 27 miRs with metastatic capability. This study observed 71 differentially expressed miRs in TNBCs including miR-200 family and miR-17-92 oncogenic cluster³⁸.

Conclusions

miRs have been shown to be one of the major regulators of mammary gland development and neoplasia. miRNome studies of mammary gland in different developmental stages and cancerous tissues will elucidate biomarkers for early cancer diagnosis, and may be used as therapeutic targets. Future studies focusing on the cross-talk between miRs and target genes with the signaling pathways involved in development and neoplasia of mammary gland will open the door to understand mammary gland biology and oncogenesis in molecular detail.