Research ArticleCancer

MicroRNAs Differentially Regulated by Akt Isoforms Control EMT and Stem Cell Renewal in Cancer Cells

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Science Signaling  13 Oct 2009:
Vol. 2, Issue 92, pp. ra62
DOI: 10.1126/scisignal.2000356
  • Fig. 1

    Strategy for generating lung fibroblasts based on abundance of different Akt isoforms and for the identification of IGF-1–induced microRNAs. (A) Cells were transduced to express different Akt isoforms. (B) Western blots of cell lysates were probed with antibody directed against the myc tag or with antibodies directed against Akt1, Akt2, or Akt3. Tubulin was used as a loading control. (C) Cell lysates harvested before and after IGF-1 treatment were screened with a 365 microRNA array.

  • Fig. 2

    Akt isoforms have different microRNA gene signatures. (A) IGF-1 treatment induces the phosphorylation of all Akt isoforms at Ser473 and Thr308 (in Akt1 or the equivalent sites in Akt2 and Akt3) in murine lung fibroblasts engineered to express Akt1, Akt2, or Akt3. After overnight serum starvation, cells were treated with IGF-1 (50 ng/ml) for 10 min and cell lysates were analyzed by Western blot. Tubulin was used as a loading control. (B) Heat map of differentially expressed microRNAs in untreated and IGF-1–treated (1, 4, and 16 hours) fibroblasts carrying individual Akt isoforms. Red indicates up-regulation, and blue indicates down-regulation. (C) Overlap between the microRNA signatures of IGF-1–treated (16 hours) Akt1, Akt2, Akt3, and TKO fibroblasts. (D) The abundance of miR-200 family members was measured by real-time RT-PCR 1, 4, and 16 hours after IGF-1 treatment. MicroRNA abundance before the IGF-1 treatment was set to 0. MiR-200 family members were down-regulated after IGF-1 treatment only in Akt2-expressing fibroblasts. (E) Down-regulation of miR-200a and miR-200c in Akt2-expressing MEFs. The expression of miR-200 family members was measured at 16 hours after IGF-1 treatment by real-time RT-PCR. The experiments were performed in triplicate and data are presented as mean ± SD.

  • Fig. 3

    Knockdown of Akt1 promotes EMT by decreasing abundance of the miR-200 microRNA family. (A) Knockdown of Akt1 and Akt2 in MCF10A cells. Western blots of cell lysates after transfection of Akt1, Akt2, or Akt1 and Akt2 siRNAs probed with the indicated antibodies. (B) TGFβ synergizes with Akt1 knockdown to increase Zeb1 abundance. Zeb1 abundance measured by real-time PCR in cells transfected with the indicated siRNAs and treated with TGFβ. (C) Akt1 knockdown promotes EMT. (Upper panel) The knockdown synergizes with TGFβ to decrease E-cadherin abundance. Lysates of cells transfected with the indicated siRNAs and treated with TGFβ, probed with antibodies against E-cadherin and β-actin. (Lower panel) The knockdown synergizes with TGFβ to stimulate cell migration. Cell migration of TGFβ-stimulated cells transfected with the indicated siRNAs. (D) (Upper panel) Chromosomal map of the miR-200 microRNA family. (Lower panel) The knockdown synergizes with TGFβ to decrease abundance of miR-200a and miR-200c. MicroRNA abundance was measured by real-time PCR. (E) The abundance of miR-200c and the mRNA encoding E-cadherin in cells transfected with Akt1 siRNA returned to pretransfection values as the effect of the siRNA on Akt1 abundance wane. Time course analysis by real-time RT-PCR in cells transfected with Akt1 siRNA and treated with TGFβ.

  • Fig. 4

    Overexpression of miR-200a, miR-200c, and miR-200a plus miR-200c blocks the up-regulation of Zeb1 in MCF10A cells transfected with Akt1 siRNA and treated with TGFβ. (A and B) Overexpression of miR-200a or miR-200c inhibits the up-regulation of Zeb1 and Zeb2 in MCF10A cells transfected with Akt1 siRNA and treated with TGFβ. (C) Overexpression of miR-200a or miR-200c inhibits the down-regulation of E-cadherin in MCF10A cells transfected with Akt1 siRNA and treated with TGFβ. Abundance of the mRNAs encoding Zeb1, Zeb2, and E-cadherin was measured by real-time PCR in lysates of TGFβ-treated MCF10A cells transfected with the indicated siRNAs. (D) Overexpression of miR-200a or miR-200c inhibits cell migration in MCF10A cells transfected with Akt1 siRNA and treated with TGFβ. Cell migration was measured in TGFβ-stimulated MCF10A cells 24 hours after transfection with the indicated siRNAs. Experiments were performed in triplicate and data are presented as mean ± SD.

  • Fig. 5

    Cells undergoing EMT through miR-200 down-regulation in response to TGFβ treatment and Akt1 knockdown exhibit stem cell properties. (A) MCF10A cells transfected with control siRNA or siRNAs for Akt1, Akt2, or Akt1 and Akt2 were cultured for 6 days in suspension in the presence or absence of recombinant TGFβ (20 ng/ml). The bar graph shows the number of mammospheres per 1000 plated cells in each culture (mean ± SD) at the end of the experiment. (B) Phase-contrast images (low and high magnification) of mammospheres described in (A). Scale bar, 100 μm. (C) Replating efficiency of mammospheres derived from MCF10A cultures transfected with siRNA control, and siRNAs for Akt1, Akt2, and Akt1 plus Akt2. (D) Primary mammospheres of MCF10A cells transfected with Akt1 siRNA have less miR-200a, miR-200c, and E-cadherin than do MCF10A cells transfected with control, Akt2, or Akt1 plus Akt2 siRNAs. MicroRNA and E-cadherin expression were measured in 6-day mammospheres by real-time RT-PCR. Experiments were performed in triplicate, and data are presented as mean ± SD.

  • Fig. 6

    Mammary adenocarcinomas developing in MMTV-cErbB2/Akt1−/− mice have low amounts of microRNAs of the miR-200 family, high amounts of Zeb1, and low amounts of E-cadherin. (A) Mammary adenocarcinomas developing in MMTV-cErbB2/Akt1−/− mice are more invasive than mammary adenocarcinomas arising in MMTV-cErbB2/WT and MMTV-cErbB2/Akt2−/− mice. (B) MMTV-cErbB2/Akt1−/− mammary adenocarcinomas have lower abundance of miR-200 microRNAs than do mammary adenocarcinomas developing in MMTV-cErbB2/Akt1+/+ and MMTV-cErbB2/Akt2−/− mice. MicroRNA abundance was measured by real-time RT-PCR. (C) MMTV-cErbB2/Akt1−/− mammary adenocarcinomas have more abundant Zeb1 and vimentin and less abundant E-cadherin than do mammary adenocarcinomas developing in MMTV-cErbB2/Akt1+/+ and MMTV-cErbB2/Akt2−/− mice. Western blots of primary tumor cell lysates were probed with the indicated antibodies. (D) In situ hybridization (for microRNA) and immunofluorescence (for E-cadherin) confirmed that MMTV-cErbB2/Akt1−/− mammary adenocarcinomas have low abundance of miR-200 family members and E-cadherin (microphotographs at 40× magnification). Scale bar, 10 μm.

  • Fig. 7

    The Akt–miR-200–E-cadherin axis contributes to the metastatic phenotype in most human mammary adenocarcinomas. (A) The abundance of Akt1, Akt2, and E-cadherin mRNAs and microRNAs were measured by real-time RT-PCR in primary and metastatic tumors. The data represent triplicate measurements from each RNA sample normalized to GAPDH. Gray lines connect each primary tumor with the corresponding metastatic tumor. Asterisks are used to mark two cases in which the ratio of Akt1 to Akt2 remained high in the metastatic tumor sample despite the fact that miR-200 and E-cadherin abundances were lower than in the primary tumor samples from the same patients. The indicated P value (P = 0.013) was calculated with all tumors included. P values were calculated with a paired two-population Student’s t test. The mean value for each set is shown as a horizontal black line. (B) Plotting the Akt1/Akt2 ratio and the expression of miR-200a, miR-200c, and E-cadherin in the six metastatic tumor samples characterized by low Akt1/Akt2 ratios [see (A)] revealed an excellent correlation between these parameters. Spearman rank correlation coefficients and the P values in parentheses are shown (lower panel).

  • Fig. 8

    Schematic diagram of the proposed model: The imbalance between Akt1 and Akt2 dysregulates microRNAs that control EMT and stem cell renewal programs. Red indicates a positive and blue indicates a negative role in the development of EMT. GFs, growth factors.

Additional Files

  • Supplementary Materials for:

    MicroRNAs Differentially Regulated by Akt Isoforms Control EMT and Stem Cell Renewal in Cancer Cells

    Dimitrios Iliopoulos, Christos Polytarchou, Maria Hatziapostolou, Filippos Kottakis, Ioanna G. Maroulakou, Kevin Struhl, Philip N. Tsichlis*

    *To whom correspondence should be addressed. E-mail: ptsichlis{at}tuftsmedicalcenter.org

    This PDF file includes:

    • Fig. S1. Abundance of the three Akt isoforms in spontaneously immortalized lung fibroblasts transduced with retroviral constructs of Akt1, Akt2, or Akt3 and in primary lung fibroblasts from wild type mice.
    • Fig. S2. Heat map of differentially expressed microRNAs in untreated and IGF-1-treated fibroblasts, expressing no Akt (TKO), or individual Akt isoforms.
    • Fig. S3. Validation of microRNA microarray data by SYBR Green real-time RT-PCR analysis in Akt1-, Akt2-, and Akt3-expressing fibroblasts.
    • Fig. S4. Abundance of miR-200a and miR-200c in spontaneously immortalized lung fibroblasts from Akt1fl/fl/Akt2–/–/Akt3–/– mice transduced with MigR1-GFP constructs of Akt1, Akt2, or Akt3 and MigR1-RFP-Cre.
    • Fig. S5. Down-regulation of miR-200 microRNA family members in myrAkt2-expressing cells.
    • Fig. S6. Akt1, Akt2 and Akt3 phosphorylation by IGF-1 is not affected by overexpression of miR-200a, miR-200c, or miR-200a plus miR-200c.
    • Fig. S7. TGFβ treatment (20 ng/ml) induces Akt phosphorylation (at Ser473) in MCF10A mammary epithelial cells and in murine lung fibroblasts expressing each of the three Akt isoforms.
    • Fig. S8. MCF10A cells have similar amounts of Akt1 and Akt2.
    • Fig. S9. Akt1 and Akt2 have opposing effects on the induction of EMT.
    • Fig. S10. The knockdown of Akt1 enhances cell motility, whereas the knockdown of Akt2 does not.
    • Fig. S11. Genomic localization of miR-200 family members. miR-200b, miR-200a and miR-429 map in a cluster on chromosome 1, and miR-200c and miR-141, map in a second cluster on chromosome 12.
    • Fig. S12. The knockdown of Akt1 decreases the abundance of all the members of the miR-200 microRNA family.
    • Fig. S13. The abundance of miR-200a, and the mRNAs encoding Zeb1, and Zeb2 in MCF10A cells transfected with Akt1 siRNA, returned to the pretransfection values as the effects of the Akt1 siRNA on Akt1 abundance wane.
    • Fig. S14. Expression of Akt1 and Akt2 in adherent and non-adherent MCF10A cells treated with siRNAs for Akt1 and/or Akt2 (harvested on the 6th day of culture at passage 5).
    • Fig. S15. E-cadherin and vimentin mRNA abundance in MCF10A cells, harvested at consecutive days of culture in suspension.
    • Fig. S16. MMTV-cErbB2-induced mammary adenocarcinomas express miR-200c and E-cadherin.
    • Fig. S17. MMTV-cErbB2/Akt1–/– mammary adenocarcinomas have more Zeb1 and vimentin and less E-cadherin than mammary adenocarcinomas developing in MMTV-cErbB2/Akt1+/+ and MMTV-cErbB2/Akt2–/– mice.
    • Table S1. The microRNA signatures of immortalized lung fibroblasts expressing a single Akt isoform at a time and responding to IGF-1 differ.
    • Table S2. Comparison of the microRNA signatures of triple Akt knockout (TKO) lung fibroblasts, and their derivatives expressing Akt1, Akt2 or Akt3.
    • References

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    Citation: D. Iliopoulos, C. Polytarchou, M. Hatziapostolou, F. Kottakis, I. G. Maroulakou, K. Struhl, P. N. Tsichlis, MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell renewal in cancer cells. Sci. Signal. 2, ra62 (2009).

    © 2009 American Association for the Advancement of Science

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