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 Akt1^fl/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