14q32-encoded microRNAs mediate an oligometastatic phenotype

Abstract

Oligometastasis is a clinically distinct subset of metastasis characterized by a limited number of metastases potentially curable with localized therapies. We analyzed pathways targeted by microRNAs over-expressed in clinical oligometastasis samples and identified suppression of cellular adhesion, invasion, and motility pathways in association with the oligometastatic phenotype. We identified miR-127-5p, miR-544a, and miR-655-3p encoded in the 14q32 microRNA cluster as co-regulators of multiple metastatic pathways through repression of shared target genes. These microRNAs suppressed cellular adhesion and invasion and inhibited metastasis development in an animal model of breast cancer lung colonization. Target genes, including TGFBR2 and ROCK2, were key mediators of these effects. Understanding the role of microRNAs expressed in oligometastases may lead to improved identification of and interventions for patients with curable metastatic disease, as well as an improved understanding of the molecular basis of this unique clinical entity.

Figure 1. Predicted pathways targeted by oligometastasis-associated microRNAs

(A) Heatmap diagram demonstrating one-way unsupervised hierarchical clustering of 35 microRNAs up-regulated in surgically resected lung oligometastases as compared to lung polymetastases (surgical dataset) and predicted KEGG target pathways grouped by functions (AIM: adhesion/invasion/motility; ICS: intracellular signaling; CSS: cancer-specific signaling). Values represents −log₁₀ of p-value where higher values correspond to lower p. (B) Most significant predicted microRNA-regulated pathways comprising each functional group of the surgical dataset. Red, blue, and orange colors denote AIM, ICS, and CSS functional groups. (C) Heatmap diagram depicting 13 microRNAs over-expressed in oligometastases treated by SBRT and predicted KEGG target pathways. (D) Most significant predicted miRNA-regulated pathways from the SBRT dataset. (E) A disproportionate number of differentially expressed microRNAs in both datasets are located in the 14q32 genomic locus comprised of two microRNA clusters and one snoRNA cluster (red rectangles are protein-coding genes within locus). (F) Over-expression of 14q32-encoded microRNAs (miR-127-5p, miR-369-3p, miR-544a and miR-655-3p) is associated with improved recurrence-free survival in patients undergoing surgical resection of lung metastases (blue: top 50% by expression; red: bottom 50% by expression; n = 24 per group).

Figure 2. 14q32-encoded microRNAs co-regulate target pathways

(A) Venn diagram illustrating the number of overlapping genes suppressed 1.4-fold or greater (FDR ≤ 5%) after ectopic expression of each microRNA in the metastatic breast cancer cell line MDA-MB-231. (B) Functional clusters altered by individual microRNAs. (C) Network analysis of 28 pathways related to adhesion/invasion/motility and intracellular signaling altered by two or more miRNAs.

Figure 3. Co-repression of target genes by 14q32-encoded miRNAs

Left column (A, D, G, J): luciferase reporter activity assays of HEK 293T cells co-transfected with a luciferase vector containing the 3′ untranslated region (UTR) of the specified gene and each of the tested miRNAs. Right column (B, E, H, K): microarray gene expression values of MDA-MB-231 cells transfected with miR-127-5p, miR-369-3p, miR-544a, miR-655-3p or a non-targeting control. Schematics (C, F, I, L) depict the distribution of binding sites of tested miRNAs in the 3′ UTR of tested genes. (A–C) TGFBR2. (D–F) ROCK2. (G–I) ICK. (J–L) CDH11. Data represent mean ± SD (n = 5 per group). *p ≤ 0.05 compared to control.

Figure 4. Oligometastatic microRNAs suppress in vitro metastatic phenotype

Adhesion of MDA-MB-231 (A) or MDA-MB-435 (B) cells to Matrigel after transient transfection of microRNAs (n = 5 per group). (C) MDA-MB-231 invasion through Matrigel after ectopic expression of microRNAs (n = 3 per group). Data represent mean ± SD. *p ≤ 0.05 compared to control.

Figure 5. Ectopic expression of 14q32-encoded miRNAs limits lung colonization of metastatic tumor cells

(A) MDA-MB-231 polymetastatic breast cancer cells co-labeled with luciferase and GFP and transfected with miR-127-5p, miR-369-3p, miR-544a, miR-655-3p or a non-targeting control were injected into the tail veins of NOD/SCID mice. (B) In vivo whole-animal luciferase imaging at 3 weeks (n = 4–5 per group). (C) Number of metastases per 10 high-powered fields (HPFs) based on fluorescence imaging of GFP at 2 weeks post-injection demonstrating significantly fewer lung metastases after transfection with miR-127-5p, miR-544a and miR-655-3p (n = 3 per group). (D) Ex vivo photomicrographs of lung colonies at 2 weeks. (E) Micro-colony diameter at 2 weeks showing a significant growth delay in miR-544a and miR-655-3p transfected cells. (F) Number of metastases per 10 HPFs at 3 weeks post-injection demonstrating significantly reduced lung metastases after transfection with miR-127-5p, miR-544a or miR-655-3p (n = 6–8 per group). (G) Ex vivo photomicrographs of lungs at 3 weeks. Arrows denote individual metastatic colonies. (H) Micro-colony diameter at 3 weeks showed no significant differences in size among groups. Data represent mean ± SD. *p ≤ 0.05 compared to control.

Figure 6. Stable suppression of microRNA target genes TGFBR2 and ROCK2 phenocopies oligometastatic state

(A) Adhesion and invasion of MDA-MB-231 cells to Matrigel after stable suppression of TGFBR2 or ROCK2 using small hairpin RNA (shRNA) targeting each gene (n = 3 per group). (B) MDA-MB-231 polymetastatic breast cancer cells transfected with shTGFBR2, shROCK2, or a scrambled control and co-labeled with luciferase and GFP were injected into the tail veins of NOD/SCID mice. At 3 weeks, stable suppression of both genes led to a significant reduction in luciferase lung activity when compared to control. (C) Number and size of lung metastases per 10 HPFs at 3 weeks post-injection demonstrating significantly reduced numbers of lung metastases as a result of TGFBR2 and ROCK2 suppression (n = 5–7 per group). Micro-colony diameter at 3 weeks showed a growth delay due to TGFBR2, but not ROCK2, suppression when compared to control. Data represent mean ± SD. *p ≤ 0.05 compared to control. (D) Schematic of a proposed 14q32 microRNA-dependent regulation of an oligometastatic phenotype through co-repression of target genes.