ku-0063794 and Carcinoma--Renal-Cell

Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 versus Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.

Topics: Actin Cytoskeleton; Actins; Carcinoma, Renal Cell; Cell Adhesion; Cell Line, Tumor; Cell Movement; Coculture Techniques; Focal Adhesion Kinase 1; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Indoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Morpholines; Multiprotein Complexes; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; Purines; Pyrimidines; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

The objective of this study was to characterise the mechanism underlying acquired resistance to temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), in renal cell carcinoma (RCC).. A parental human RCC cell line, ACHN (ACHN/P), was continuously exposed to increasing doses of up to 20 μM of temsirolimus, and a cell line resistant to temsirolimus (ACHN/R), showing a sixfold higher IC50 than that of ACHN/P, was developed.. Following treatment with temsirolimus, phosphorylation of S6 kinase in ACHN/P was markedly inhibited, whereas there was no detectable expression of phosphorylated S6 in ACHN/R before and after temsirolimus treatment. However, AKT and p44/42 mitogen-activated protein kinase (MAPK) were constitutively phosphorylated even after temsirolimus treatment in ACHN/R, but not in ACHN/P. There was no significant difference between the sensitivities of ACHN/P and ACHN/R to KU0063794, a dual inhibitor of mTOR complex 1 (mTORC1) and mTORC2. Similar sensitivities to temsirolimus in ACHN/P and ACHN/R could be achieved by additional treatment with specific inhibitors of AKT- and MAPK-signaling pathways.. The activation of signal transduction pathways via mTORC2, but not via mTORC1, may have an important role in the acquisition of a resistant phenotype to temsirolimus in RCC.

Topics: Animals; Apoptosis; Butadienes; Carcinoma, Renal Cell; Cell Line, Tumor; Chromones; Drug Resistance, Neoplasm; Humans; Kidney Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Morpholines; Multiprotein Complexes; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin analogs, temsirolimus and everolimus, are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). However, the mTOR kinase exists in two distinct multiprotein complexes, mTORC1 and mTORC2, and both complexes may be critical regulators of cell metabolism, growth and proliferation. Furthermore, it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus. We evaluated Ku0063794, which is a small molecule that inhibits both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling proteins downstream of both mTORC1 and mTORC2, including p70 S6K, 4E-BP1 and Akt. Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC cell lines, Caki-1 and 786-O, in vitro by inducing cell cycle arrest and autophagy, but not apoptosis. However, in a xenograft model there was no difference in the inhibition of tumor growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has additional effects on the tumor microenvironment. Consistent with this possibility, temsirolimus, but not Ku0063794, decreased tumor angiogenesis in vivo, and decreased the viability of HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant concentrations. Furthermore, expression levels of VEGF and PDGF were lower in Caki-1 and 786-O cells treated with temsirolimus than cells treated with Ku0063794.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Carcinoma, Renal Cell; Cell Cycle Proteins; Cell Proliferation; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Multiprotein Complexes; Neovascularization, Pathologic; Oncogene Protein v-akt; Phosphoproteins; Pyrimidines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterologous