Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer

The role of the tumour-suppressor miR-34 family in breast physiology and in mammary stem cells (MaSCs) is largely unknown. Here, we revealed that miR-34 family, and miR-34a in particular, is implicated in mammary epithelium homoeostasis. Expression of miR-34a occurs upon luminal commitment and differentiation and serves to inhibit the expansion of the pool of MaSCs and early progenitor cells, likely in a p53-independent fashion. Mutant mice (miR34-KO) and loss-of-function approaches revealed two separate functions of miR-34a, controlling both proliferation and fate commitment in mammary progenitors by modulating several pathways involved in epithelial cell plasticity and luminal-to-basal conversion. In particular, miR-34a acts as endogenous inhibitor of the Wnt/beta-catenin signalling pathway, targeting up to nine upstream regulators at the same time, thus modulating the expansion of the MaSCs/early progenitor pool. These multiple roles of miR-34a are maintained in a model of human breast cancer, in which chronic expression of miR-34a in triple-negative mesenchymal-like cells (enriched in cancer stem cells—CSCs) could promote a luminal-like differentiation programme, restrict the CSC pool, and inhibit tumour propagation. Hence, activation of miR-34a-dependent programmes could provide a therapeutic opportunity for the subset of breast cancers, which are rich in CSCs and respond poorly to conventional therapies.

For 3D Matrigel culture, 24-well plates were pre-coated with Matrigel (BD Biosciences, 120 uL/well) 15 minutes at 37°C. Cells were then seeded at 5,000 cells/well density and incubated for 14 days, medium was replenished every 2-3 days. L-Wnt3a ligand was produced using mouse fibroblast transfected with Wnt3a expressing vector as described (2).
Transplantation experiments. SUM159PT, previously infected with a lentiviral vector encoding luciferase (Addgene, #17477) and with pSlik34a, was injected in 6-8-week-old NSG female mice. For miR34a overexpression, mice were fed with doxycycline-containing food (625 mg kg -1 ). Tumour growth was monitored weekly using caliper measurements. For measurements using IVIS Illumina System (Caliper Life Sciences), mice were injected by i.p. with D-luciferin (150 mg kg -1 ) and imaged 15 min after injection. Animals were euthanized when the tumours were approximately 0.5-1 cm in the largest diameter. For all transplantation procedures, mice were anaesthetized with 2.5% avertin in PBS (100% avertin: 10 g of tribromoethanol in 10 ml of tertamyl alcohol [Sigma]). Cells were resuspended at the appropriate cell density in 14 µL PBS, 6 µL Matrigel® (BD Biosciences). For second passage transplantation assay, tumour cells were re-transplanted into NSG mice at limiting dilution, with no further treatment with doxy. TIC frequency was calculated with the Extreme Limiting Dilution Analysis (ELDA) web tool. ALDH assay. ALDH activity was measured using Aldefluor Kit (Stem Cell Technologies #01700). Briefly, SUM159pPT were incubated for 45 min at 37°C with enzyme substrate (ALDEFLUOR reagent) or with an enzyme inhibitor (DEAB reagent), and cells were immediately FACS analyzed using MACSQuant Flow cytometer (Milteny). Wnt-Reporter activity. Sca low or Sca high cells transduced with Wnt reporter vector (7TF) were treated with L-Wnt3a and collected after 24 hours. For luciferase activity cells were lysed and assayed in multiple wells using the Dual-Luciferase Reporter Assay System (Promega) and a PHERAstar FS plate reader.
3ʹ UTR luciferase binding assays. HEK293T cells were seeded at 10 000 cells per well in 96-well plate. After 24 h, cells were co-transfected with 100 ng of the indicated 3ʹ UTR reporter construct (pmiR-GLO; Promega) and 40 nM of SCR or miRNA-34a mimics (QIAGEN) using Lipofectamine 3000 (1 µl/well; Invitrogen), according to the manufacturer's instructions. After 24 h, cells were lysed and assayed in quadruplicate wells for Firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) and a Pherastar Plus plate reader.
Virus production-For lentiviral production, HEK293T cells were transfected in 15cm plate with: 10 µg of pRSV-Rev, 10 µg of pMDLg/pRRE (gag&pol), 10 µg of pMD2.G (VSV-G), and 20 µg of the lentiviral vector, 250 µl of 2M CaCl 2 , in a final volume of 2 ml TE 0.1X. The mix was added dropwise to 2 ml of 2X HBS, by bubbling, and then the calciumphosphate precipitates were added to the cells at 70% of confluence. For virus concentration, supernatant was collected 36 h post-transfection, filtered through a 0.45 µm syringe-filter and ultracentrifuged for 2 h at 22000 rpm at 4°C. Viral pellet was resuspended in MEBM medium at 100X concentration. Viral stock was frozen (-80°C) or directly used to infect target cells at ratio: 10-30 µl virus/100,000 target cells in presence of 1µg/mL polybrene.
IHC-Mammary glands were fixed overnight in 4% formaldehyde at room temperature, embedded in paraffin, and sectioned (5µm). Sections were deparaffinized, subjected to antigen retrieval, and then incubated with antibody against Ki67 (rabbit, SP6, ThermoScientific, RM-9106-R7) and detected with MACH1 Universal HRP-Polymer Detection (Biocare Medical). Slides were counterstained with haematoxylin and finally mounted with Eukitt (Kindler GmbH). To assess the extent of the proliferative activity, the number of Ki67-positive cells were counted using 100x microscopic fields (microscope type) centered on mammary epithelial structures.

Transplantation Experiments-WT and miR-34TKO mammary cells were transplanted into the cleared fat pad of 3
week-old syngenic female mice. Transplanted mammary glands were collected 12 weeks after transplantation, fixed in a 4% formaldehyde and colored with carmine alum for whole mount staining (DeOme, 1959).
Images were analyzed at stereoscope.

RNA isolation and RT-qPCR Analysis-Total RNA was purified onto RNeasy columns (Qiagen) and treated on-column
with DNase (Qiagen). For gene expression, cDNA was retro-transcribed using iSCRIPT reverse transcriptase (Biorad).
Real-time RT-PCR was performed with FAST SYBR Green Master Mix (Applied Biosystems). Primer pairs were designed through computer assisted primer design software (Primer3 and 50 million read depth (3x). RNA-seq NGS reads were aligned to the mm9 mouse or hg38 human reference genome using the TopHat aligner (version 2.0.6) with default parameters. Differentially expressed genes (DEGs) were identified using the Bioconductor package DESeq2 based on read counts, considering genes whose q value relative to the control is lower than 0.05 and whose maximum expression is higher than RPKM of 1. Data are deposited on GEO, GSE99401.