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. 2014 Aug 8;289(32):21888-95.
doi: 10.1074/jbc.M114.582866. Epub 2014 Jun 25.

The microRNA-23b/27b/24 cluster promotes breast cancer lung metastasis by targeting metastasis-suppressive gene prosaposin

Brian Ell  1 , Qiong Qiu  1 , Yong Wei  1 , Laura Mercatali  2 , Toni Ibrahim  2 , Dino Amadori  2 , Yibin Kang  3
Affiliations

The microRNA-23b/27b/24 cluster promotes breast cancer lung metastasis by targeting metastasis-suppressive gene prosaposin

Brian Ell et al. J Biol Chem. .

Abstract

MicroRNAs (miRNAs) have been shown to function as key regulators of tumor progression and metastasis. Recent studies have indicated that the miRNAs comprising the miR-23b/27b/24 cluster might influence tumor metastasis, although the precise nature of this regulation remains unclear. Here, expression of the miR-23b/27b/24 cluster is found to correlate with metastatic potential in mouse and human breast cancer cell lines and is elevated in metastatic lung lesions in human breast cancer patients. Ectopic expression of the miRNAs in the weakly metastatic mouse 4TO7 mammary tumor cell line had no effect on proliferation or morphology of tumor cells in vitro but was found to increase lung metastasis in a mouse model of breast cancer metastasis. Furthermore, gene expression profiling analysis of miRNA overexpressing 4TO7 cells revealed the direct targeting of prosaposin (PSAP), which encodes a secreted protein found to be inversely correlated with metastatic progression in human breast cancer patients. Importantly, ectopic expression of PSAP was able to suppress the metastatic phenotype in highly metastatic 4T1 and MDA-MB-231 SCP28 cells, as well as in cells ectopically expressing miR-23b/27b/24. These findings support a metastasis-promoting function of the miR-23b/27b/24 cluster of miRNAs, which functions in part through the direct inhibition of PSAP.

Keywords: Breast Cancer; Invasion; Metastasis; MicroRNA (miRNA); Tumor Metastases.

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Figures

FIGURE 1.
FIGURE 1.
The miR-23a/27a/24 and miR-23b/27b/24 clusters correlate with breast cancer metastasis. A, upper panel, schematic representing the chromosomal locations of human miR-23ab/27ab/24. Lower panel, sequence alignment of the miR-23ab/27ab/24 clusters. Seed sequences are highlighted in red, and miRNA members with identical seed sequences are color-coded. B, box plots showing normalized expression levels of indicated miRNAs in human primary (Pri) tumor and metastasis (Met) samples (n = 10). p values were based on Student's t test. C–E, normalized qRT-PCR expression of miR-23ab, miR-27ab, and miR-24 in 4T1 (C), MCF10A (D), or MDA-MB-231 (E) cell line series, as shown by heat maps. Data represent average, U6-normalized expression.
FIGURE 2.
FIGURE 2.
Analysis of ectopic miR-23b/27b/24 expression in 4TO7 cells. A, qRT-PCR miRNA expression levels of indicated miRNAs in 4TO7-miR-23b/27b/24 cells. Data represent average ± S.D. normalized to vector-transduced cells. ***, p < 0.001. B, phase contrast images of 4TO7 pMSCV-puro vector control or miR-23b/27b/24-overexpressing cells. C, in vitro growth curves showing similar growth rates for either vector or miRNA-overexpressing cells during the exponential growth phase. Data represent average ± S.D. with triplicate for each line at each time point. D, migration and invasion of 4TO7 vector or miRNA-overexpressing cells toward serum-containing media. Data represent average number of cells counted from bottom well from triplicate experiments ± S.D. **, p < 0.01; ***, p < 0.001. E, quantification of overt metastatic lesions on lungs from BALB/c mice injected with the indicated cell lines (105 cells per injection) and allowed to grow for 14 days. p value from Student's t test (n = 10). F, representative images of Bouin's fixed lungs from mice in E. G, normalized qRT-PCR miRNA expression levels of indicated miRNAs in overt lung lesions isolated from mice injected with indicated cells or non-injected miRNA-overexpressing cells in culture. Data represent average ± S.D. H, in vivo primary mammary tumor volume from mice injected with 106 4TO7 vector control or miR-23b/27b/24 overexpressing cells (n = 10). Data represent average ± S.D. I, representative images of resected primary tumors from experiment in H at the indicated time point. J, representative crystal violet-stained cell culture plates showing tumor colonies derived from dissociated lungs from mice in H. K, quantification of dissociated lung colonies from J. Data represent average ± S.D. No significance (n.s.) by Student's t test.
FIGURE 3.
FIGURE 3.
Identification of putative miR-23b/27b/24 cluster targets. A, average gene expression (expressed as fold change) for candidate target genes from microarray analysis of 4TO7-miR-23b/27b/24 cells relative to vector control. B, qRT-PCR mRNA expression levels for putative target genes in 4TO7 vector cells or miR-23b/27b/24 overexpressing cells. **, p < 0.05; **, p < 0.01 by Student's t test. Data represent average ± S.D. C, normalized firefly luciferase expression from reporter plasmids containing indicated 3′-UTR 24 h after co-transfection with control or miR-23b/27b/24 miRNA oligonucleotides. **, p < 0.01. Data represent average ± S.D. D, schematic representing the predicted miR-27ab and miR-24 binding sites within the human PSAP 3′-UTR. MiR-24 contains two predicted sites, referred to here as 1 and 2, whereas miR-27ab contains one predicted binding site. E, normalized firefly luciferase (Luc) expression from PSAP reporter constructs similar to following site-directed mutagenesis of predicted miRNA binding sites and co-transfection with miR-27b or miR-24 as indicated. *, p < 0.05; **, p < 0.01 by Student's t test. Data represent average ± S.D.
FIGURE 4.
FIGURE 4.
PSAP decreases in cells with elevated metastatic potential. A and B, qRT-PCR expression levels of Psap in indicated cell lines from 4T1 (A) and MCF10A (B) series. Data represent average ± S.D. C and D, Western blot analysis of PSAP or β-actin from indicated cells lines as in A and B.
FIGURE 5.
FIGURE 5.
PSAP is a metastasis suppressor and a poor prognosis factor in breast cancer. A, qRT-PCR (upper panel) and Western blot analysis of the expression levels of PSAP in 4T1 or SCP28 cells after transfection of the expression plasmid or vector (Vec). Data represent average ± S.D. B, lung lesion quantification and representative images of lungs from mice injected with vector or PSAP-overexpressing cells from A (p values by Student's t test). Data represent average ± S.D. C, lung lesion quantification and representative images of lungs from mice injected with indicated SCP28 cells. D, Kaplan-Meier curves showing the distant metastasis free survival of patients with high or low expression of PSAP in breast tumors. p values were computed by a log rank test. E, box plots showing PSAP expression levels in 10 primary and metastasis (Met) samples as quantified by qRT-PCR analysis. p values computed by Student's t test. Data represent mean ± S.E. normalized to GAPDH expression. ER, estrogen receptor; HR, hazard ratio.
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