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  • R E S E A R C H A R T I C L E

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    MicroRNAs Differentially Regulated byAkt Isoforms Control EMT and Stem CellRenewal in Cancer CellsDimitrios Iliopoulos,1* Christos Polytarchou,2* Maria Hatziapostolou,2 Filippos Kottakis,2

    Ioanna G. Maroulakou,2 Kevin Struhl,1 Philip N. Tsichlis2

    (Published 13 October 2009; Volume 2 Issue 92 ra62)

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    Although Akt is known to play a role in human cancer, the relative contribution of its three isoforms to on-cogenesis remains to be determined. We expressed each isoform individually in anAkt1//Akt2//Akt3/

    cell line. MicroRNA profiling of growth factorstimulated cells revealed unique microRNA signatures forcells with each isoform. Among the differentially regulated microRNAs, the abundance of the miR-200family was decreased in cells bearing Akt2. Knockdown of Akt1 in transforming growth factorb(TGFb)treated MCF10A cells also decreased the abundance of miR-200; however, knockdown of Akt2,or of both Akt1 and Akt2, did not. Furthermore, Akt1 knockdown inMCF10A cells promoted TGFb-inducedepithelial-mesenchymal transition (EMT) and a stem celllike phenotype. Carcinomas developing inMMTV-cErbB2/Akt1/mice showed increased invasiveness because ofmiR-200 down-regulation. Finally,the ratio ofAkt1 toAkt2 and theabundanceofmiR-200 andof themessengerRNAencodingE-cadherin in aset of primary and metastatic human breast cancers were consistent with the hypothesis that in manycases, breast cancer metastasis may be under the control of the AktmiR-200E-cadherin axis. We con-clude that induction of EMT is controlled by microRNAs whose abundance depends on the balance be-tween Akt1 and Akt2 rather than on the overall activity of Akt.

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    INTRODUCTION

    Akt is a serine-threonine protein kinase that is activated by phospho-rylation at two sites (Thr308 and Ser473) through a phosphatidylinositol3-kinase (PI3K)dependent process. Specifically, growth factor signalsand other stimuli that activate PI3K promote the accumulation of D3-phosphorylated phosphoinositides at the plasma membrane. The interac-tion of these phosphoinositides with the pleckstrin homology (PH) domainof Akt promotes the translocation of the kinase to the plasma membrane,where it undergoes phosphorylation by PI3K-dependent kinase1 (PDK1)at Thr308 and by mTOR (mammalian target of rapamycin) complex2(TORC2) at Ser473 (13).

    Ample evidence links Akt with the induction and progression ofhuman cancer (4). However, there are three Akt isoforms and informa-tion regarding their specific oncogenic activities is limited. The Akt1and Akt2 isoforms appear to play different roles in mammary adeno-carcinomas induced in mice by transgenes encoding the oncoproteinspolyoma middle T and ErbB2 (also known as Neu), both of which acti-vate the PI3K-Akt pathway. Indeed, ablation of Akt1 inhibitswhereasablation of Akt2 acceleratestumor induction and growth by both poly-oma middle T and ErbB2 (5, 6). However, the tumors developing in theAkt1 knockout mice are more invasive than the tumors developing inAkt2 knockout and wild-type mice (5), suggesting that ablation of Akt1may also enhance tumor invasiveness, a process separate from and inde-pendent of tumor induction and growth (7). These findings were con-sistent with observations showing that Akt1 knockdown promotes

    1Department of Biological Chemistry and Molecular Pharmacology, HarvardMedical School, Boston, MA 02115, USA. 2Molecular Oncology ResearchInstitute, Tufts Medical Center, Boston, MA 02111, USA.*These authors contributed equally to this work.To whom correspondence should be addressed. E-mail: ptsichlis@tuftsmedicalcenter.org

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    migration and invasiveness of human mammary epithelial cells in culture(810), perhaps by promoting epithelial-mesenchymal transition (EMT),a process that plays important roles in both development and onco-genesis. During EMT, epithelial cells acquire a mesenchymal phenotypecharacterized by the loss of intercellular junctions and increased cell mi-

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    Fig. 1. Strategy for generating lung fibroblasts based on abundance of dif-ferent Akt isoforms and for the identification of IGF-1induced microRNAs.(A) Cells were transduced to express different Akt isoforms. (B) Westernblots of cell lysates were probed with antibody directed against the myctag or with antibodies directed against Akt1, Akt2, or Akt3. Tubulin wasused as a loading control. (C) Cell lysates harvested before and afterIGF-1 treatment were screened with a 365 microRNA array.

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  • R E S E A R C H A R T I C L E

    gration. On the molecular level, cells undergoing EMT decrease theabundance of epithelial cellspecific proteins, such as E-cadherin, andincrease the abundance of mesenchymal cellspecific proteins, such asvimentin. The developmental switch characteristic of EMT renders tu-mor cells undergoing this process more invasive and metastatic. Thus,inhibition of individual Akt isoforms may have both desirable and un-desirable effects during oncogenesis. We therefore wished to identify andcharacterize the downstream targets of Akt isoforms, which may dis-

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    criminate the beneficial from the detrimental effects of isoform-specificAkt inhibition.

    MicroRNAs are a class of molecules that regulate gene expression byvarious mechanisms and play important roles in oncogenesis (11, 12).Like conventional oncogenes and tumor suppressor genes, microRNAsmay either promote or inhibit oncogenesis. Also, like conventional cancergenes, their expression is selectively increased or decreased in various hu-man and animal tumors. The selective deregulation of microRNA gene

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    miR-200a miR-200c

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    miR-200a miR-200b miR-200c miR-141 miR-429

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    Akt1 Akt2 Akt3

    miR-27amiR-149miR-145miR-20amiR-17-5pmiR-375miR-15miR-16miR-455miR-34amiR-34bmiR-34cmiR-200amiR-200bmiR-200cmiR-141miR-429miR-449miR-433miR-532miR-331miR-365miR-10bmiR-127

    miR-27amiR-149miR-145miR-20amiR-17-5pmiR-375miR-15miR-16miR-455miR-34amiR-34bmiR-34cmiR-200amiR-200bmiR-200cmiR-141miR-429miR-449miR-433miR-532miR-331miR-365miR-10bmiR-127

    miR-27amiR-149miR-145miR-20amiR-17-5pmiR-375miR-15miR-16miR-455miR-34amiR-34bmiR-34cmiR-200amiR-200bmiR-200cmiR-141miR-429miR-449miR-433miR-532miR-331miR-365miR-10bmiR-127

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    -3.65 2.230 -5.43 4.560 -4.35 5.110

    16 hours IGF

    Fig. 2. Akt isoforms have different microRNA gene signatures. (A) IGF-1treatment induces the phosphorylation of all Akt isoforms at Ser473 andThr308 (in Akt1 or the equivalent sites in Akt2 and Akt3) in murine lungfibroblasts engineered to express Akt1, Akt2, or Akt3. After overnightserum starvation, cells were treated with IGF-1 (50 ng/ml) for 10 minand cell lysates were analyzed by Western blot. Tubulin was used asa loading control. (B) Heat map of differentially expressed microRNAsin untreated and IGF-1treated (1, 4, and 16 hours) fibroblasts carryingindividual Akt isoforms. Red indicates up-regulation, and blue indicatesdown-regulation. (C) Overlap between the microRNA signatures of IGF-

    1treated (16 hours) Akt1, Akt2, Akt3, and TKO fibroblasts. (D) Theabundance of miR-200 family members was measured by real-timeRT-PCR 1, 4, and 16 hours after IGF-1 treatment. MicroRNA abundancebefore the IGF-1 treatment was set to 0. MiR-200 family members weredown-regulated after IGF-1 treatment only in Akt2-expressing fibro-blasts. (E) Down-regulation of miR-200a and miR-200c in Akt2-expressing MEFs. The expression of miR-200 family members wasmeasured at 16 hours after IGF-1 treatment by real-time RT-PCR. Theexperiments were performed in triplicate and data are presented asmean SD.

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    expression may be due to deletions, amplifications, or mutations target-ing the microRNAs themselves or their regulatory sequences, as well asto dysregulation of transcription factors and epigenetic regulators target-ing the genes encoding them (13). Understanding the regulation andfunctional activities of individual microRNA families in cancer and oth-er human diseases could thus lead to new opportunities for therapeuticintervention.

    Here, we describe a set of microRNAs that are differentially regulatedby the three Akt isoforms in cells stimulated with insulin-like growth fac-tor 1 (IGF-1), a treatment that activates Akt. In addition, we show that adecrease in the abundance of the miR-200 microRNA family in cells inwhich the ratio of Akt1 to Akt2 was decreased promotes EMT and theacquisition of a stem celllike phenotype in cultured cells, as well as inmouse and human tumors. Down-regulation of the miR-200 microRNAfamily in these cells appears to depend not on Akt activity per se, but onthe balance between Akt1 and Akt2.

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    RESULTS

    Akt isoforms have different microRNA gene signaturesLung fibroblast and kidney epithelial cells from Akt1fl/fl/Akt2//Akt3/

    mice were immortalized as described in Materials and Methods. The im-mortalized lung fibroblasts were transduced with myc-tagged Akt1, Akt2,or Akt3 retroviral constructs or with the empty retroviral vector. Knockingout the floxed Akt1 allele in these cells with Cre recombinase gave riseto cell lines that expressed only one of the three Akt isoforms. Knock-ing out the floxed Akt1

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