TGF-β upregulates miR-181a expression to promote breast cancer metastasis
Molly A. Taylor, … , David Danielpour, William P. Schiemann
Molly A. Taylor, … , David Danielpour, William P. Schiemann
Published December 17, 2012
Citation Information: J Clin Invest. 2013;123(1):150-163. https://doi.org/10.1172/JCI64946.
View: Text | PDF
Research Article Oncology

TGF-β upregulates miR-181a expression to promote breast cancer metastasis

Abstract

Late-stage breast cancer metastasis is driven by dysregulated TGF-β signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-β. Here we establish miR-181a as a TGF-β–regulated “metastamir” that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer.

Authors

Molly A. Taylor, Khalid Sossey-Alaoui, Cheryl L. Thompson, David Danielpour, William P. Schiemann

×

Figure 5

miR-181a inhibits anoikis by targeting the proapoptotic protein Bim for downregulation.

Options: View larger image (or click on image) Download as PowerPoint
miR-181a inhibits anoikis by targeting the proapoptotic protein Bim for ...
(A and B) NMuMG and 4T1 cells with manipulated miR-181a activity were suspended over poly-HEMA–coated culture dishes for 0–48 hours to induce anoikis. Caspase-3 cleavage was monitored by immunoblotting detergent-solubilized whole-cell extracts with anti–caspase-3 antibodies, while differences in protein loading were controlled with anti–β-actin antibodies. (C) miRanda alignment of mmu-miR-181a to the wild-type and mutant Bim 3′ UTR sequences demonstrates perfect complementation between the miR-181a seed sequence and the Bim 3′UTR. Asterisks indicate mutated miR-181a seed sequence binding bases. (D and E) Immunoblotting NMuMG and 4T1 cell extracts demonstrated that elevated miR-181a activity decreased Bim protein levels (D), while diminished miR-181a activity elicited increased Bim protein levels. Differences in protein loading were assessed by β-actin immunoblotting. (F and G) NMuMG (F) or 4T1 (G) cells were transiently transfected with a renilla luciferase reporter gene whose expression was driven by either wild-type or mutant (mut) Bim–3′ UTR seed sequences (C). Stable miR-181a expression suppressed that of luciferase containing the wild-type Bim–3′ UTR, an event that was lacking in cells transfected with mut–Bim–3′ UTR vectors. (H) NMuMG cells were transiently transfected with miR-181a Mimics with or without a 3′ UTR–deficient Bim cDNA. Afterward, the transfectants were suspended over poly-HEMA–coated culture dishes for 24 hours to induce anoikis. Bim expression (left panel) and caspase-3 cleavage (right panel) were monitored as described above. All data are representative of 3 independent experiments or are the mean ± SEM. n = 3; *P < 0.05; Student’s t test. Lanes in D and E were run on the same gel, but were noncontiguous (white lines).