Background:
microRNA genes are known to be involved in cellular transformation as well as cancer progression and metastasis. We sought to identify novel microRNAs causally involved in breast cancer metastasis. Using different mouse models of breast cancer metastasis we identified microRNA-342 (miR-342) as down-regulated during breast cancer metastasis. High miR-342 expression in whole primary tumours was associated with longer patient survival in multiple combined clinical datasets.
Aims:
1. To determine the role of miR-342 in breast cancer metastasis using clinically-relevant mouse models.
2. To identify RNA targets of miR-342 that might serve as nodes for the development of future anti-cancer therapies.
Methods:
microRNA microarrays (Affymetrix) were employed to identify differentially expressed microRNAs in primary mammary tumours from an isogenic mouse model of spontaneous breast cancer metastasis. Ectopic miR-342 expression was produced in the MDA-MB-231_HM (highly-metastatic) human breast cancer cell line using a retroviral expression vectors. Spontaneous metastasis assays were conducted by intra-mammary fat pad (IMFP) inoculation of tumour cells into female Nod.Scid.Gamma-null (NSG) mice. Differential gene expression between 231_HM-miR-342 cells and control cells cultured in Matrigel was determined using a novel modified next-generation RNA sequencing technique developed in our laboratories named poly-A tail RNA-Seq (PAT-Seq).
Results:
miR-342 was down-regulated in the more highly metastatic cell lines and tumours from the '4T1' Balb/c syngeneic (mouse-in-mouse) model of breast cancer metastasis, and miR-342 was also down-regulated in the highly metastatic MDA-MB-231_HM (231_HM) cell line compared to less metastatic MDA-MB-231 isogenic variants. In agreement with these findings, elevated miR-342 expression was associated with more favourable outcome in breast cancer patients using three different web-based cancer databases; namely BreastMark (http://glados.ucd.ie/BreastMark/miRNA_analysis.html),
PROGMIR (http://watson.compbio.iupui.edu/chirayu/progmir/database/?url=progmir), and MIRUMIR (http://www.bioprofiling.de/GEO/MIRUMIR/mirumir.html). miR-342 was ectopically expressed in the 231_HM line and stable transfectants evaluated using in vitro proliferation and cellular motility assays. Primary 231_HM-miR-342 mammary tumours grew at the same rate as control tumours but produced significantly lower spontaneous metastasis to distant organs including lung, liver, and spleen. 231HM_HM-miR-342 and control cells were cultured in 3D (Matrigel) in triplicate and PAT-Seq conducted. Down-regulation of cadherin-11, and the trefoil factor genes TFF1 and TFF3, was found in miR-342 over-expressing cells.
Conclusion:
The results demonstrate that miR-342, a microRNA gene strongly associated with favourable outcome in breast cancer patients, may negatively influence breast cancer metastasis through effects on cadherin-11, TFF1 and TFF3 expression.