MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer

Abstract

Dysregulation of microRNAs (miRNAs) is involved in the initiation and progression of several human cancers, including breast cancer (BC), as strong evidence has been found that miRNAs can act as oncogenes or tumor suppressor genes. This review presents the state of the art on the role of miRNAs in the diagnosis, prognosis, and therapy of BC. Based on the results obtained in the last decade, some miRNAs are emerging as biomarkers of BC for diagnosis (i.e., miR-9, miR-10b, and miR-17-5p), prognosis (i.e., miR-148a and miR-335), and prediction of therapeutic outcomes (i.e., miR-30c, miR-187, and miR-339-5p) and have important roles in the control of BC hallmark functions such as invasion, metastasis, proliferation, resting death, apoptosis, and genomic instability. Other miRNAs are of interest as new, easily accessible, affordable, non-invasive tools for the personalized management of patients with BC because they are circulating in body fluids (e.g., miR-155 and miR-210). In particular, circulating multiple miRNA profiles are showing better diagnostic and prognostic performance as well as better sensitivity than individual miRNAs in BC. New miRNA-based drugs are also promising therapy for BC (e.g., miR-9, miR-21, miR34a, miR145, and miR150), and other miRNAs are showing a fundamental role in modulation of the response to other non-miRNA treatments, being able to increase their efficacy (e.g., miR-21, miR34a, miR195, miR200c, and miR203 in combination with chemotherapy).

Keywords: Breast cancer; circulating biomarker; diagnosis; microRNA/miRNA; prediction and therapy.; prognosis; theranostic.

Figure 1

miRNA biogenesis process. A schematic representation of canonical miRNA biogenesis pathway. Each miRNA is transcribed by RNA polymerase II (pri-miRNA) from genomic DNA within the nucleus; pri-miRNA is recognized by Drosha-DGCR8 and processed to pre-miRNA. Pre-miRNA is exported to the cytoplasm by exportin 5 (XPO5), where it is processed and cleaved by DICER complex to a double strand miRNA (miRNA*-miRNA). The duplex is cleaved, and only the mature miRNA is loaded into the RISC complex. The degree of homology of the miRNA “seed” to the 3′ UTR target sequence of the mRNA determines the mRNA translational repression or degradation.

Figure 2

In vitro validation of miRNA-target direct interaction. Cultured cell lines are transfected with a reporter vector containing firefly (FIR) luciferase gene and the 3′ UTR of the gene of interest (GOI). The level of expression of FIR luciferase is measured in a luminometric assay. Cells are then exposed to the mimic miRNA, which is supposed to enter within the cell and to interact with the 3′ UTR of the GOI. If no interaction between miRNA and the 3′ UTR of GOI happens (a), we could observe no alteration in the level of expression of luciferase, thus no alteration in the emitted chemoluminescence, as FIR gene produced an active, luminescent protein. The complete interaction between the miRNA and the 3′ UTR of the GOI (b) leads to reduced FIR luciferase expression, with a decrease of luminescence levels. Other luminescent genes, such as Renilla (REN) luciferase, are usually used as reference genes for luminescence normalization.

Figure 3

Altered steps in miRNA biogenesis lead to cancer. A schematic representation of altered steps of the miRNA biogenesis pathway, commonly deregulated in cancer: 1. miRNA genes contain upstream regulator elements (enhancers/repressors) and promoter regions, indicating that miRNAs are subjected to CpG methylation (CpG promoter met); 2. The alteration in the copy number of miRNA (due to genomic amplification or deletion, activating or repressing mutation, loss of epigenetic silencing and transcriptional activation) could increase the oncogenic miRNAs or decrease the tumor suppressor miRNAs; 3. Alteration in the miRNA processing machinery, i.e. downregulation of Drosha, could decrease the cropping of pri-miR to pre-miR; 4. XPO5 mutation could prevent pre-miR export to the cytoplasm; 5. Mutation of TARBP2 or downregulation of DICER1 decrease mature miRNA levels, causing finally a loss on tumor suppressor miRNAs; 6 and 7. Accumulation of oncogenic miRNAs or loss of tumor suppressor miRNAs could finally lead to cancer development.

Figure 4

Contribution of transcription to miRNA level alteration in cancer. Several transcription factors are able to control the level of expression of miRNAs. In particular, as described in the text, SMAD, Myc, ATM, BRCA1/2 and p53 influence miRNA transcription. P53 can regulate onco-suppressor miRNAs, which are involved in the control of p53 turnover. SMAD, ATM, BRCA1/2 and Myc could influence the transcription levels of miRNAs involved in cell plasticity, cell proliferation and survival, and cell invasion control. Moreover, SMAD is also involved in miRNA processing, by Drosha expression levels control. Ex: example of miRNA regulated by transcription factors.

Figure 5

Examples of miRNA regulatory networks in BC that promote metastasis. A) Two examples of the role of miR-10b/10b* in the regulation of either migration and invasion (left side) or cell cycle and proliferation (right side) processes. B) Example of let-7 regulatory role in the pro-invasive gene network control.

Figure 6

miRNA biomarkers and BC hallmarks. miRNAs have a role as diagnostic miRNA, prognostic miRNAs (italics), miRNAs predictive of the BC response to therapy (*), or miRNAs with multiple functions (diagnosis, prognosis, prediction of therapy outcome; underlined). Circulating (red) and non-circulating (black) miRNAs of Table 4 are included.