(5-(2-4-bis((3s)-3-methylmorpholin-4-yl)pyrido(2-3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol and Carcinoma--Renal-Cell

Renal cell carcinomas (RCC) harboring a TFE3 gene fusion (TfRCC) represent an aggressive subset of kidney tumors. Key signaling pathways of TfRCC are unknown and preclinical in vivo data are lacking. We investigated Akt/mTOR pathway activation and the preclinical efficacy of dual mTORC1/2 versus selective mTORC1 inhibition in TfRCC.. Levels of phosphorylated Akt/mTOR pathway proteins were compared by immunoblot in TfRCC and clear cell RCC (ccRCC) cell lines. Effects of the mTORC1 inhibitor, sirolimus, and the dual mTORC1/2 inhibitor, AZD8055, on Akt/mTOR activation, cell cycle progression, cell viability and cytotoxicity were compared in TfRCC cells. TfRCC xenograft tumor growth in mice was evaluated after 3-week treatment with oral AZD8055, intraperitoneal sirolimus and respective vehicle controls.. The Akt/mTOR pathway was activated to a similar or greater degree in TfRCC than ccRCC cell lines and persisted partly during growth factor starvation, suggesting constitutive activation. Dual mTORC1/2 inhibition with AZD8055 potently inhibited TfRCC viability (IC50 = 20-50 nM) due at least in part to cell cycle arrest, while benign renal epithelial cells were relatively resistant (IC50 = 400 nM). Maximal viability reduction was greater with AZD8055 than sirolimus (80-90% versus 30-50%), as was the extent of Akt/mTOR pathway inhibition, based on significantly greater suppression of P-Akt (Ser473), P-4EBP1, P-mTOR and HIF1α. In mouse xenograft models, AZD8055 achieved significantly better tumor growth inhibition and prolonged mouse survival compared to sirolimus or vehicle controls.. Akt/mTOR activation is common in TfRCC and a promising therapeutic target. Dual mTORC1/2 inhibition suppresses Akt/mTOR signaling more effectively than selective mTORC1 inhibition and demonstrates in vivo preclinical efficacy against TFE3-fusion renal cell carcinoma.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Renal Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

This study aimed to investigate the correlations of stearoyl-coenzyme A desaturase 1 (SCD-1) with clear cell renal cell carcinoma (ccRCC) severity and PI3K-AKT-mTOR signaling pathway. From 2004 to 2006, tumor tissue and normal pericarcinomatous tissue from ccRCC samples were collected from ccRCC patients at Renji Hospital of Shanghai Jiaotong University. The expression of SCD-1 in the collected ccRCC samples and four cell lines (A498, 769-P, 786-O, and CAKI) was detected by Western blot. The correlation between SCD-1 expression and ccRCC severity was also analyzed by immunohistochemistry. Stable 786-O and 769-P ccRCC cells expressing SCD-1 short hairpin RNA (shRNA) were constructed, and the expression of proteins in the PI3K-AKT-mTOR signaling pathway was also detected. Finally, the inhibitory effect of PI3K-AKT-mTOR inhibitors (PI103, MK2206, rapamycin, AZD8055, and RAD001) on ccRCC cells stably expressing SCD-1 shRNA was also measured. Higher SCD-1 expression level was observed in ccRCC tissues compared with normal tissues. SCD-1 expression level was the highest in 786-O. SCD-1 expression was positively correlated with the tumor-node-metastasis (TNM) stage, grade of tumor cells, and lymphatic metastasis. There were no changes in the expression of AKT, ERK, PI3K, and PDK1. Significant differences were observed in the expression of p-AKT (at the Ser473 and Thr308 site), p-ERK, and two mTOR downstream molecules (4E-BP1 and p-P70S6K1) in cells stably expressing SCD-1 shRNA. PI103 and AZD8055 could enhance the inhibitory effect of SCD-1 interference on proliferation and migration of 786-O and 769-P cells. AZD8055 is recommended for the combined ccRCC treatment with shRNA interference.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Everolimus; Female; Furans; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Kidney Neoplasms; Lymphatic Metastasis; Male; Morpholines; Pyridines; Pyrimidines; RNA Interference; Signal Transduction; Stearoyl-CoA Desaturase

To evaluate first-generation rapamycin analogs (everolimus, temsirolimus, and rapamycin) and second-generation drugs inhibiting mTOR kinase (AZD-8055), PI3K (BKM-120) or both (BEZ-235 and GDC-0980) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cells characterized for acquired resistance to sorafenib or sunitinib.. Anti-proliferative (MTT assay) and cell signaling (Western blot) effects of rapamycin analogs (1-20 μM) and second-generation drugs (0.03-20.0 μM) were assessed in human HCC SK-HEP1, RCC 786-0, and sorafenib- (SK-Sora) or sunitinib-resistant (786-Suni) cells.. In SK-HEP1 cells displaying high PTEN and Bcl2 expression, rapamycin analogs had poor anti-proliferative effects. However, SK-Sora cells were more sensitive to rapamycin analogs (≥1 μM) than SK-HEP1 cells. In 786-0 cells, lacking PTEN and Bcl2 expression, ≥1 μM rapamycin analogs blocked mTORC1 signaling, transiently activated Akt, and inhibited cell proliferation. Protracted sunitinib exposure in 786-Suni cells yielded an increase in p27 expression and a decreased sensitivity to rapamycin analogs, although mTORC1 function could be inhibited with rapamycin analogs. Second-generation drugs induced more potent growth inhibition than rapamycin analogs at concentrations >0.03 μM in parental cells, SK-Sora, and 786-Suni cells. Growth inhibitory concentrations of these new drugs also blocked mTORC1 downstream targets.. Rapamycin analogs inhibited mTORC1 downstream targets and yielded anti-proliferative effects in HCC and RCC cells. Second-generation drugs also appeared to be potent inhibitors of mTORC1 signaling; however, they appeared to be far more potent in inhibiting cellular proliferation in parental HCC and RCC cells and in cells developing resistance to sorafenib or sunitinib.

Topics: Aminopyridines; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Everolimus; Humans; Imidazoles; Indoles; Kidney Neoplasms; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Niacinamide; Phenylurea Compounds; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Pyrroles; Quinolines; Signal Transduction; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases