Compositions and methods for diagnosis and prognosis of cancer and progression, and for screening anti-cancer agents

#Compositions and methods for diagnosing and prognosing cancer appearance and development related to miRNA species, particularly Akt-1, -2 and -3 imbalances, and methods for screening potential agents that alter these imbalances are provided.

#Akt proteins are serine-threonine protein kinases characterized by activation of phosphorylation at two sites (Thr308 and Ser473) through a phosphatidylinositol 3-kinase (PI3K)-dependent process. Specifically, Akt proteins refer to growth factor signals and other stimuli that activate PI3K and promote accumulation of D3-phosphorylated phosphoinositides at the plasma membrane. The interaction of these phosphoinositides with the pleckstrin homology (PH) domain of Akt promotes the translocation of the kinase to the plasma membrane, for phosphorylation by PI3K-dependent kinase-1 (PDK1) at Thr308 and by mTOR (mammalian target of rapamycin) complex-2 (TORC2) at Ser473 (Bellacosa et al., 1991 Science 254: 274-277; Franke et al., 1995 Cell 81: 727-736; Manning et al., 2007 Cell 129: 1261-1274).

#Akt is linked with the induction and progression of human cancer (Altomare et al., 2005 Oncogene 24: 7455-7464). However, there are three Ala isoforms and information regarding their specific oncogenic activities is limited. The Akt1 and Akt2 isoforms appear to play different roles in mammary adenocarcinomas induced in mice by transgenes encoding the oncoproteins polyoma middle T and ErbB2 (also known as Neu), both of which activate the PI3K-Akt pathway. Ablation of Akt1 inhibits—whereas ablation of Akt2 accelerates—tumor induction and growth by both polyoma middle T and ErbB2 (Maroulakou et al., 2007 Cancer Res. 67: 167-177; Ju et al., 2007 Proc. Natl. Acad. Sci. U.S.A. 104: 7438-7443). However, the tumors developing in the Akt1 knockout mice are more invasive than the tumors developing in Akt2 knockout and wild-type mice (Maroulakou et al., 2007 Cancer Res. 67: 167-177). Ablation of Akt1 may also enhance tumor invasiveness, a process separate from and independent of tumor induction and growth (Chiang et al., 2008 N. Engl. J. Med. 359: 2814-2823). These findings were consistent with observations showing that Akt1 knockdown promotes migration and invasiveness of human mammary epithelial cells in culture (Irie et al., 2005 J. Cell Biol. 171:1023-1034; Yoeli-Lerner et al., 2005 Mol. Cell 20: 539-550; Liu et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103: 4134-4139), perhaps by promoting epithelial-mesenchymal transition (EMT), a process that plays important roles in both development and oncogenesis. During EMT, epithelial cells acquire a mesenchymal phenotype characterized by the loss of intercellular junctions and increased cell migration. On the molecular level, cells undergoing EMT decrease the abundance of epithelial cell-specific proteins, such as E-cadherin, and increase the abundance of mesenchymal cell-specific proteins, such as vimentin. The developmental switch characteristic of EMT renders tumor cells undergoing this process more invasive and metastatic. Thus, inhibition of individual Akt isoforms may have both desirable and undesirable effects during oncogenesis.

#FIGS. 1A-1C are a drawing, a set of photographs of immunoblots and a photograph of a microarray showing strategy for generating lung fibroblasts according to abundance of different Akt isoforms and for the identification of IGF1-induced microRNAs.

#FIG. 1A shows a method to obtain cells that express different Akt isoforms.

#FIG. 1B shows photographs of Western blots of cell lysates probed with antibody specific for a myc tag or with antibodies specific for Akt1, Akt2, or Akt3. Tubulin was used as a loading control.

#Akt is known to play a role in human cancer, however, the relative contribution of its three isoforms to oncogenesis remains to be determined. Examples herein identify and characterize the downstream targets of Akt isoforms, which are shown herein to discriminate the beneficial from the detrimental effects of isoform-specific Akt inhibition. Each isoform was expressed individually in an Akt1−/−/Akt2−/−/Akt3−/− cell line. MicroRNA profiling of growth factor-stimulated cells showed unique microRNA signatures for cells with each isoform. Among the differentially regulated microRNAs, the abundance of the miR-200 family was decreased in cells bearing Akt2. Knockdown of Akt1 in transforming growth factor-β (TGFβ)-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 in MCF10A cells promoted TGFβ-induced epithelial-mesenchymal transition (EMT) and a stem cell-like phenotype. Carcinomas developing in MMTV-cErbB2/Akt1−/− mice showed increased invasiveness because of miR-200 downregulation. Without being limited by any particular theory or mechanism, the ratio of Akt1 to Akt2 and the abundance of miR-200 and of the messenger RNA encoding E-cadherin in a set of primary and metastatic human breast cancers showed that in many cases, breast cancer metastasis may be under the control of the Akt-miR-200-E-cadherin axis. It is shown herein that induction of EMT is controlled by microRNAs the abundance of which depends on the balance between Akt1 and Akt2 rather than on the overall activity of Akt.

#A set of microRNAs was shown to be differentially regulated by the three Akt isoforms in cells stimulated with insulin-like growth factor 1 (IGF1), a treatment that activates Akt. In addition, it was shown herein that a decrease in the abundance of the miR-200 microRNA family in cells in which the ratio of Akt1 to Akt2 was decreased promotes EMT and the acquisition of a stem cell-like phenotype in cultured cells, as well as in mouse and human tumors. Downregulation of the miR-200 microRNA family in these cells depends on the balance between Akt1 and Akt2 and does not depend on Akt activity per se.

#The examples herein focus on the role of different Akt isoforms in regulation of microRNA gene expression in IGF1 and TGFβ-treated cells. A number of different microRNAs were shown to be differentially regulated by the different Akt isoforms, including members of the miR-200 microRNA family. Examples show that Akt1 and Akt2 had opposing activities on the expression of the miR-200 family, Akt1 as an activator and Akt2 as a repressor, and that the expression of miR-200 microRNAs depended upon the balance between Akt1 and Akt2, not overall Akt levels. Data show that selective inhibition of Akt1 leads to an invasive cancer stem cell-like phenotype, which was promoted by the downregulation of miR-200 microRNAs. Without being limited by any particular theory or mechanism of action an imbalance in the expression or activity of Akt1 and Akt2, not necessarily overall Akt levels influences the invasiveness and oncogenic potential of human carcinomas.