Concomitant suppression of three target genes can explain the impact of a microRNA on metastasis

Abstract

It remains unclear whether a microRNA (miRNA) affects a given phenotype via concomitant down-regulation of its entire repertoire of targets or instead by suppression of only a modest subset of effectors. We demonstrate that inhibition of breast cancer metastasis by miR-31-a miRNA predicted to modulate >200 mRNAs-can be entirely explained by miR-31's pleiotropic regulation of three targets. Thus, concurrent re-expression of integrin-alpha5, radixin, and RhoA abrogates miR-31-imposed metastasis suppression. These effectors influence distinct steps of the metastatic process. Our findings have implications concerning the importance of pleiotropy for the biological actions of miRNAs and provide mechanistic insights into metastasis.

Figure 1.

Individual suppression of ITGA5, RDX, or RhoA impairs metastasis in vivo. (A) Primary tumor growth upon orthotopic injection of the indicated GFP-labeled 231 cells into NOD/SCID mice. The assay was terminated after 11 wk due to primary tumor burden. n = 5 per time point. (B, top panels) Fluorescent images of murine lungs to visualize 231 cells 76 d after orthotopic implantation. (Bottom panel) Quantification of metastatic burden. n = 5. (C, top panels) H&E stain of 231 cell primary mammary tumors 57 d after injection. (Bottom panel) Quantification of local invasion. n = 5. All P-values are >0.67 relative to shLuciferase. (D) Prevalence of GFP-labeled 231 cells in the lungs 1 d after intravenous introduction into NOD/SCID mice. n = 4. (E) Fluorescent images of murine lungs to visualize 231 cells 89 d after intravenous injection. (Arrows) Micrometastases. shRNAs used in these assays were shITGA5 #4, shRDX #3, and shRhoA #5. All error bars represent mean ± SEM.

Figure 2.

Simultaneous re-expression of ITGA5, RDX, and RhoA abrogates miR-31-imposed metastasis suppression in vivo. (A) Primary tumor growth upon orthotopic injection of the indicated GFP-labeled 231 cells into NOD/SCID mice. The assay was terminated after 11 wk due to primary tumor burden. n = 5 per time point. (B, top panels) Fluorescent images of murine lungs to visualize 231 cells 67 d after orthotopic implantation. (Bottom panel) Quantification of metastatic burden. n = 5. All error bars represent mean ± SEM.

Figure 3.

Re-expression of ITGA5, RDX, and/or RhoA affords both unique and partially overlapping reversal of miR-31-evoked inhibition of spontaneous metastasis in vivo. (A) Primary tumor growth upon orthotopic implantation of the indicated GFP-labeled 231 cells into nude mice. The assay was terminated after 13 wk due to primary tumor burden. n = 5. (B,top panels) Fluorescent images of murine lungs to visualize 231 cells 88 d after orthotopic injection. (Bottom panel) Quantification of metastatic burden. n = 5. (C) H&E stain of 231 cell primary mammary tumors 54 d after injection. (Bottom panel) Quantification of local invasion. n = 5. All error bars represent mean ± SEM.

Figure 4.

Re-expression of ITGA5, RDX, and/or RhoA affords both unique and partially overlapping reversal of miR-31-mediated inhibition of experimental metastasis in vivo. (A) Prevalence of the indicated GFP-labeled 231 cells in the lungs 1 d after intravenous introduction into NOD/SCID mice. n = 4. (B) Fluorescent images of murine lungs to visualize 231 cells 84 d after tail vein injection. (C) Lung metastatic burden 84 d subsequent to intravenous injection. n = 5. All error bars represent mean ± SEM.