miR-17/20 sensitization of breast cancer cells to chemotherapy-induced apoptosis requires Akt1

Abstract

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Herein, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse, demonstrates a requirement for Akt1 in miRNA-mediated cellular apoptosis. The miR-17/20 cluster is known to inhibit breast cancer cellular proliferation through G1/S cell cycle arrest via binding to the cyclin D1 3'UTR. Here we show that miR-17/20 overexpression sensitizes cells to apoptosis induced by either Doxorubicin or UV irradiation in MCF-7 cells via Akt1. miR-17/20 mediates apoptosis via increased p53 expression which promotes Akt degradation. Akt1⁻/⁻ mammary epithelial cells which express Akt2 and Akt3 demonstrated increased apoptosis to DNA damaging agents. Akt1 deficiency abolished the miR-17/20-mediated apoptosis. These results demonstrated a novel pathway through which miR17/20 regulate p53 and Akt controlling breast cancer cell apoptosis.

Figure 1. miR-17/20 sensitizes doxorubicin-induced apoptosis, and increase p53 expression in breast cancer cells

A, Tunnel Assay on miR-17/20 or control transduced MCF-7 cells after treatment with doxorubicin at 0.05uM for 24h. B, Tunnel Assays on anti-miR-17-, anti-miR-20a- and negative control-transfected MCF-7 cells after treatment with doxorubicin (0.05μM) for 24h. C, Western blots showing the increased p53 and p27KIP1 in miR-17/20 transduced MCF-7 cells. GDI served as loading control. D, Western blots showing the increased p53 expression in miR-17/20 transduced MCF-7 cells in the presence (lane 1 and 2) or absence (lane 3 and 4) of doxorubicin treatment. E, Western blot showing decreased p53 in anti-miR-17 and anti-miR-20a- transduced MCF-7 cells. F, miR-17/20 sensitizes NAFA cells to doxorubicin-induced apoptosis. G, Western blot showing increased p27KIP1 BAX, p-γ/H2A, PARP and Caspase 9 in miR-17/20 transduced NAFA cells. β-Tubulin served as loading control.

Figure 2. miR-17/20 increases tamoxifen sensitivity of MCF-7 cells

A, Western blot showing the regulation of apoptosis pathway-related genes in miR-17/20 transduced MCF-7 cells. B, Phase contrast images of miR-17/20 or control transduced MCF-7 cells after 36h treatment (15uM tamoxifen). Survived cells are indicated with solid arrow, apoptotic cells are indicated with dashed arrow. C, MTT assays measuring the relative survival rate of MCF-7 cells after treatment with tamoxifen (15uM) for the indicated times. The data are mean +SEM (n=5), **p<0.01.

Figure 3. miR-17/20 increases the sensitivity of MCF-7 cells to doxorubicin

A, Cell survival of miR-17/20 or control transduced MCF-7 cells after treatment with doxorubicin at indicated concentrations for 48h and 72h. B, Cell survival curves of miR-17/20 and control transduced MCF-7 treated with 0.5 μM doxorubicin for 0, 24, 48, and 72 hours. The data represent mean + SEM (n=5).

Figure 4. Akt1 is required for miR-17/20 sensitization of breast tumor cells to doxorubicin-induced apoptosis

A, Annexin V staining showing the increased apoptosis in Akt1−/− murine ErbB2 breast tumor cells treated with doxorubicin at 0.5μM for 24h. B, Annexin V staining as a marker of apoptosis in miR-17/20 transduced Akt1+/+ or Akt1−/− murine ErbB2 breast tumor cells, **p<0.01. C, Tunnel assays for apoptosis in Akt1-/ - vs. Akt1+/+ murine ErbB2 breast tumor cells. D, Western blot of miR-17/20 transduced Akt1−/− and Akt+/+ murine ErbB2 breast tumor cells for p53. β-Tubulin is a loading control. E, Western blot of Akt1, Akt2 and Akt3 expression in Akt1−/− murine ErbB2 mammary tumor cells. GDI served as loading control. F: Western blot of Akt1, total Akt, Cyto C and p53 expression in multiple WT and Akt1−/− murine ErbB2 mammary tumors.

Figure 5. Schematic representation of the molecular mechanisms by which miR-17/20 and Akt1 regulate p53 abundance and thereby apoptosis.