L 1 . 3 Mutations of the catalytic subunit of class 1 A PI-3 K are constitutively active in hematopoietic cells in vitro and in vivo

Signaling pathways play critical roles in cancer initiating cell (CIC) growth, differentiation, drug resistance and sensitivity to various therapeutic approaches. This presentation will discuss the key roles that the PI3K/ PTEN/Akt/mTOR/Gsk-3b pathway plays in therapeutic sensitivity of breast cancer. We isolated CIC from three different breast cancer cell lines (MCF-7, T47D and MDA-MB-231) by culture in doxorubicin followed by selection of non-adherent cells and FACS sorting with anti-CD44 and CD24 Abs. Breast CICs displayed increased drug resistance, mammosphere formation capacity and activated Akt expression than parental cells. The effects of the PI3K/PTEN/Akt/mTOR/Gsk-3b pathway on the CIC formation, and drug/hormonal resistance were further examined by the introduction of activated (Act) or dominant negative (DN) PTEN, Akt or Gsk-3b. Ectopic expression of Akt (Act) increased drug and hormonal (tamoxifen) resistance and sensitivity to the mTOR inhibitor rapamycin. Ectopic expression of DN PTEN lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to rapamycin. Synergistic inhibitory interactions were observed when doxorubicin or tamoxifen were combined with rapamy-cin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. Gsk-3b, a kinase which lies downstream of Akt/mTOR, can phosphorylate and inactivate many proteins important in cellular metabolism and cancer and it can be phos-phorylated and inactivated by Akt. Ectopic expression of Gsk-3b DN increased resistance to doxorubicin and tamoxifen, however, expression of Gsk-3b Act increased sensitivity to doxorubicin and tamoxifen. In contrast, the opposite effects were observed with rapamycin as Gsk-3b DN increased sensitivity while Gsk-3b Act increased therapeutic resistance. These studies indicate that disruption of the normal activity of the PI3K/PTEN/ Akt/mTOR/Gsk-3b pathway can have dramatic effects on the frequency of breast cancer CIC formation and therapeutic sensitivity. The serine/threonine kinases Akt and mammalian target of rapamycin (mTOR), which are downstream effec-tors of phosphatidylinositol 3-kinase (PI3K), are known to play an important role in antiapoptotic signaling and have been implicated in the aggressiveness of acute my-elogenous leukemia (AML) and acute T-lymphoblas-tic leukemia (TALL), where they strongly contributes to proliferation, survival, and drug-resistance of leukemic cells. Here, we describe the efficacy of novel small molecule drugs targeting this pathway in leukemic cells. We have investigated the therapeutic potential of two Akt inhibitors, such as perifosine or A443654, as well as the dual PI3K/mTOR inhibitor, PI-103, on human AML and …