1). Open in a separate window Figure 1 Epithelial-mesenchymal transition is induced by the MEK/ERK signaling pathway in mouse ovarian carcinoma cells. pathway-targeted therapy, PD98059, PI3K/Akt/mTOR, rapamycin, tumor resistance Progress in the development of effective therapies is hindered by a lack of understanding about the complexities of biochemical pathways that are required for tumor maintenance and the absence of defined experimental systems that simulate altered biochemical processes that occur in cancer initiation and progression. We have designed a system in which multiple genetic alterations can be Carbendazim introduced simultaneously or sequentially into mouse ovarian surface epithelial cells.1,2 We have recently used this system to test the effectiveness of mammalian target of rapamycin (mTOR) inhibition when several major pathways, such as c-myc, Ras, and Akt, are activated in the tumor cells.3 mTOR inhibitors specifically target mTOR, a downstream mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which plays a critical role in regulating cell growth and proliferation. Most of the upstream and downstream components of this pathway are directly implicated in tumor initiation and progression. Moreover, mTOR receives input from several biochemical pathways that are altered in cancer cells. Thus, tumors with a number of distinct molecular changes should, in theory, be sensitive to mTOR inhibition. mTOR inhibitors, such as rapamycin, CCI-779, RAD001 and AP23576, have been tested as suppressors of tumor growth in preclinical models4C14 and are currently tested as anti-tumor agents in several clinical trials. These agents inhibit the activity of mTOR by forming a complex with the FK binding protein 12 (FKBP-12), which in turn binds to mTOR. This association results in the inactivation of the ribosomal protein S6 kinase (S6K) and the hypophosphorylation of the eukaryotic initiation factor 4E binding protein (4E-BP1). Hypophosphorylated 4E-BP1 associates with the eukaryotic initiation factor 4E (eIF4E),15,16 thereby inhibiting mRNA translation. Hypersensitivity to mTOR inhibitors may be induced by the loss of PTEN phosphatase7,8 or p53 tumor suppressor function,17 or amplification of the GLI oncogene.18 Although in most preclinical models mTOR inhibitors were potent suppressors of tumor growth, the failure to see robust responses in clinical tests and some preclinical models11C13 taught us that multiple redundant pathways in cancer are capable of overcoming mTOR inhibition. Using ovarian malignancy cell lines with defined combinations of alterations in p53, c-myc, Akt, K-ras, H-ras, and Her-2 genes, we shown that rapamycin efficiently inhibits the growth of cells and tumors that rely on Akt signaling for proliferation and growth. However, cells and tumors in which Akt signaling is not the driving push in proliferation are resistant to rapamycin. We then launched Carbendazim additional genetic alterations to the rapamycin-resistant and rapamycin-sensitive cell lines. First, we explored whether rapamycin-resistant cells become sensitive to rapamycin when they are transduced with constitutively activated Akt. In contrast to the hypothesis that high levels of Akt sensitize tumor cells to rapamycin inhibition,7,8,14,19 we proven that the intro of activated Akt to the rapamycin-resistant cells does not render the cells sensitive to rapamycin if they can utilize alternate pathways for survival and proliferation. These results indicate that mTOR inhibitors may be effective inside a subset of tumors that depend on Akt activity for survival, however, not effective in all tumors that show Akt activation.3 This is a delicate, but important, point that brings into question the selection of individuals for clinical tests that is currently based on the detection of activated Akt and/or the loss of PTEN expression. Second, we explored whether the intro of alternative survival and proliferation pathways Carbendazim induces resistance in rapamycin-sensitive cell lines. Since the Ras and Akt signaling pathways are known to cooperate in facilitating global translation20 and tumor growth,1,21 we explored the ability of oncogenic Ras to modulate the level of sensitivity of tumor cells to rapamycin.3 We demonstrated the rapamycin-sensitive tumors develop resistance to rapamycin when presented with alternative survival pathways, such as the Ras signaling pathway. Transmission transduction downstream of oncogenic Ras is definitely mediated through several effectors: the serine/threonine kinase Raf activates the mitogen-activated extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; PI3K activates several target proteins including the Akt kinase and the GTPase Rac; the nucleotide exchange element Ral-GDS activates the Ras-related protein Ral; and the phospholipase C pathway activates protein kinase C and induces calcium mobilization from intracellular stores.22 The exact functional contributions of these individual Ras-activated pathways to cell cycle deregulation and cellular transformation remain unclear. In order to determine whether the MEK/ERK pathway plays a role in cell resistance to rapamycin inhibition, we treated the rapamycin-resistant cells with the MEK inhibitor PD98059 only or in combination with rapamycin. We showed that cell lines that are resistant to the individual inhibitors respond to the Rabbit Polyclonal to E2F6 combination of rapamycin and PD98059 by.