Project description:The mTOR (mammalian Target of Rapamycin) pathway is constitutively activated in Diffuse Large B-Cell Lymphoma (DLBCL). mTOR inhibition has been shown to have clinical activity in patients with DLBCL, although overall response rates remain low. We therefore evaluated differences in the transcriptome between DLBCL cell lines with differential sensitivity to the mTOR inhibitor Rapamycin, to (A) identify gene-expression patterns(GEP) capable of identifying sensitivity to Rapamycin, (B) understand the underlying mechanisms of resistance to Rapamycin in DLBCL and (C) identify bioactive molecules likely to synergize with mTOR inhibitors. Using Affymetrix HuGene ST 1.0 microarrays, we were able to identify a gene expression signature capable of accurately predicting sensitivity and resistance to Rapamycin in DLBCL cell lines. Pathway analysis identified the serine/threonine kinase Akt as central to the differentially-expressed gene network. Connectivity mapping of our datasets identified compounds targeting the AKT pathway with a high likelihood of reversing the GEP associated with resistance to Rapamycin. Specifically, we evaluated the HIV protease inhibitor (PI) Nelfinavir, which is known to have anti-cancer and Akt-inhibitory properties, as well as the small molecule Akt inhibitor MK-2206, for their potential to synergize with to Rapamycin in DLBCL. Nelfinavir and MK-2206 caused profound inhibition of cell viability in combination with Rapamycin in DLBCL cell lines. Low nanomolar concentrations of Rapamycin inhibited phosphorylation of Akt and also downstream targets of activated mTOR when used in combination with these Akt inhibitors. These findings have the potential to significantly improve patient selection for mTOR inhibitor therapy, and to improve rates and depths of response. More broadly, they support the use of global RNA expression and connectivity mapping to improve patient selection and identify synergistic drug combinations for cancer therapy. DLBCL cell lines were tested for Rapamycin sensitivity and classified as "sensitive" or "resistant." Genome-wide analysis of all cell lines were performed using the Affymetrix HuGene ST 1.0 Array Platform. Genes with differential expression between sensitive and resistant cell lines were analyzed using Statistical Analysis of Microarrays (SAM) software, and a signature of genes determnined. This signature was found to accurately predict sensitivity or resistance of other DLBCL cell lines, and to identify the protein kinase Akt as central to resistance.

Project description:The mTOR (mammalian Target of Rapamycin) pathway is constitutively activated in Diffuse Large B-Cell Lymphoma (DLBCL). mTOR inhibition has been shown to have clinical activity in patients with DLBCL, although overall response rates remain low. We therefore evaluated differences in the transcriptome between DLBCL cell lines with differential sensitivity to the mTOR inhibitor Rapamycin, to (A) identify gene-expression patterns(GEP) capable of identifying sensitivity to Rapamycin, (B) understand the underlying mechanisms of resistance to Rapamycin in DLBCL and (C) identify bioactive molecules likely to synergize with mTOR inhibitors. Using Affymetrix HuGene ST 1.0 microarrays, we were able to identify a gene expression signature capable of accurately predicting sensitivity and resistance to Rapamycin in DLBCL cell lines. Pathway analysis identified the serine/threonine kinase Akt as central to the differentially-expressed gene network. Connectivity mapping of our datasets identified compounds targeting the AKT pathway with a high likelihood of reversing the GEP associated with resistance to Rapamycin. Specifically, we evaluated the HIV protease inhibitor (PI) Nelfinavir, which is known to have anti-cancer and Akt-inhibitory properties, as well as the small molecule Akt inhibitor MK-2206, for their potential to synergize with to Rapamycin in DLBCL. Nelfinavir and MK-2206 caused profound inhibition of cell viability in combination with Rapamycin in DLBCL cell lines. Low nanomolar concentrations of Rapamycin inhibited phosphorylation of Akt and also downstream targets of activated mTOR when used in combination with these Akt inhibitors. These findings have the potential to significantly improve patient selection for mTOR inhibitor therapy, and to improve rates and depths of response. More broadly, they support the use of global RNA expression and connectivity mapping to improve patient selection and identify synergistic drug combinations for cancer therapy.

Project description:Spontaneous metastasis of gastric carcinoma to secondary organs is seldom reproduced in the mice. In this study, we established highly reproducible, experimental mice model in which gastric carcinoma progressively grows in the site of gastric walls and metastasize to secondary sites. A highly tumorigenic GC cells were engineered to express type 2 luciferase and injected via orthotopic route into BALB/c nude mice. The mice developed highly progressive GC tumors in the gastric wall where implanted and slow metastases to liver, spleen, lung, kidney and ovary, with rate of metastases to ovary being 63.6% of implanted mice. The tumors colonized in the ovary expressed a GI marker MUC5A, suggestive of typical human Krukenberg tumor. Immuno-histochemical stainings revealed that many mesenchymal markers were strongly positive in motile tumor cells in the vein of ovary and became gradually weak during extravasations to colonize in the ovary. When routes of metastasis were further investigated, the mesenchymal marker SMA stained low in the tumor cells of the primary site of stomach wall, became high in intravasating vein in the stomach wall, remained high in extravasating veins in the liver and ovary and finally returned low in the colonized tumor in the liver and ovary. However, expression of an epithelial marker Claudin did not show in the opposite profile to SMA, indicating that acquisition of mesenchymal phenotypes rather than loss of epithelial characteristics may more give rise to metastatic potential. Dissociated cells derived from tumors in the implanted mouse stomach or metastasized to ovaries retain their tumorigenicity and metastatic potentials. The mRNA microarray and Western blot analyses showed that metastatic tumor-derived cells showed significantly higher expression of GAGE12 gene family than orthotopic tumors-derived cells. The shRNA-mediated knock-down of the GAGE12 family in the metastatic tumors-derived cells abolished the tumor formation in gastric wall and metastasis as well. In conclusion, we established an in vivo orthotopic gastric cancer mouse model spontaneously metastasizing to secondary organs including ovary, which exhibits typical characteristics of a Krukenberg tumor. Expression of mesenchymal markers should be functionally associated with the gain of metastatic ability in this study. A hightly tumorigenic gastric carcinoma SNU-16 cells were orthotpically injected into stomach wall. Tumors formed in the stomach wall (16S) and mestasized to ovary (16O) were dissociated and cultured for 7 day. These cells were re-injected orthotopically and tumors formed in the stomach from 16S (16SS) and ovary from 16O injection (16OO) were dissociated. Total RNAs were isolated from 16S, 16SS, 16O and 16OO2 and subjected to mRNA microarrays

			Action	DRS
	SRR11119135_1.fastq.gz	Fastqsanger.gz
	SRR11119135_2.fastq.gz	Fastqsanger.gz
	SRR11119136_1.fastq.gz	Fastqsanger.gz
	SRR11119136_2.fastq.gz	Fastqsanger.gz
	SRR11119137_1.fastq.gz	Fastqsanger.gz

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Stomach-specific c-Myc overexpression drives intestinal-type gastric cancer in mice via AKT/mTOR signaling

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