Unknown,Transcriptomics,Genomics,Proteomics

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In vivo gene expression changes in EW5 Ewing sarcoma xenografts after IGF-1R or mTOR blockade

ABSTRACT: Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent IGF-1R or mTOR inhibition might suggest a number of therapeutic combinations that could improve their clinical activity. Male non-obese diabetic (NOD)-SCID-IL-2Rgnull mice were used to generate EW5 explants (2 mm). Mice bearing subcutaneous tumors were randomized into treatment and control groups when their tumors reached a diameter of 6 mm and received MK-8669 (mTOR inhibitor, 5mg/kg per dose, once weekly), MK-0646 (IGF-1R inhibitor monoclonal antibody, 0.5mg IP twice weekly), or a placebo control (sterile buffer). Animals were treated either until their tumors reached 1500 mm3 in volume. Affymetrix Geneship profiling of EW5 xenografts treated in vivo either with MK-0646, MK-8669, and control and compared each other using extracted RNA and hybridized on Affymetrix microrrays ( Affymetrix Human Genome U133A 2.0 cartridge arrays).

ORGANISM(S): Homo sapiens

SUBMITTER: Joseph Ludwig

PROVIDER: E-GEOD-67529 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent IGF-1R or mTOR inhibition might suggest a number of therapeutic combinations that could improve their clinical activity. Male non-obese diabetic (NOD)-SCID-IL-2Rgnull mice were used to generate EW5 explants (2 mm). Mice bearing subcutaneous tumors were randomized into treatment and control groups when their tumors reached a diameter of 6 mm and received ridaforolimus (MK-8669, mTOR inhibitor, 5mg/kg per dose, once weekly), dalotuzumab (MK-0646, IGF-1R inhibitor monoclonal antibody, 0.5mg IP twice weekly), a placebo control (sterile buffer) or a combination of both treatments. Animals were treated either until their tumors reached 1500 mm3 in volume. RPPA profiling of EW5 xenografts treated in vivo either with MK-0646, MK-8669, control and combination and compared each other were performed simultaneously using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 179 validated primary antibodies, and detected using a DakoCytomation-catalyzed system with secondary antibodies. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Log2 values were media-centered by protein to account for variability in signal intensity by time and were calculated using the formula log2 signal â log2 median. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between treatment and control groups. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs). Biostatistical analyses comparing two groups were performed using an unpaired t-test with Gaussian distribution followed by the Welch correction. To distinguish between treatment groups, we used one-way ANOVA with the Geisser-Greenhouse correction. Differences with p values <0.05 were considered significant. Within clustered image maps (CIM), unsupervised double hierarchical clustering used the Pearson correlation distance and Wardâs linkage method as the clustering algorithm to link entities (proteins or genes) and samples.

Project description:Introduction: Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERÎ±) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option. Methods: Using hormone-sensitive breast cancer cells stably transfected with the aromatase gene (MCF-7/AC-1), works presented herein describe the in vitro and in vivo antitumor efficacy of the following compounds: dalotuzumab (DALO; âMK-0646â; anti-IGF-1R antibody), ridaforolimus (RIDA; âMK-8669â; mTORC1 small molecule inhibitor) and letrozole (âLETâ, aromatase inhibitor). Results: With the exception of MK-0646, all single agent and combination treatment arms effectively inhibited xenograft tumor growth, albeit to varying degrees. Correlative tissue analyses revealed MK-0646 alone and in combination with LET induced insulin receptor alpha A (InsR-A) isoform upregulation (both mRNA and protein expression), thereby further supporting a triple therapy approach. Conclusion: These data provide preclinical rationalization towards the combined triple therapy of LET plus MK-0646 plus MK-8669 as an efficacious anti-tumor strategy for ER-positive breast tumors. 46 samples, 28 days post treatment

Project description:Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent IGF-1R inhibition might suggest a number of therapeutic combinations that could improve its clinical activity. TC32 and TC71 ES clones with acquired resistance to OSI-906 or NVP-ADW-742 were generated by maintaining the corresponding parental cell lines with increasing concentrations of the agents (up to 2.3 Î¼M for OSI-906, 1.5 Î¼M for NVP-ADW-742) for 7 months. All parental and acquired drug resistant cell lines were tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.) according to the manufacturerâs protocol and validated using short-tandem repeat fingerprinting with an AmpFLSTR Identifier kit as previously described. Herein, we determine subtle differences in acquired mechanism of resistance by two promising small molecule inhibitors of IGF-1R/IR-Î±. OSI-906, which inhibits IGF-1R and IR, and NVP-ADW-742, which inhibits only IGF-1R, were evaluated using in vitro assays to decipher the mechanism(s) by which IGF-1R inhibition induces drug resistance in Ewing sarcoma cells. The preparation of extracted proteins from sensitive and acquired resistant Ewing sarcoma cells to OSI-906 and NVP-ADW-742 for reverse-phase protein lysate array (RPPA) analysis were prepared using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 115 validated primary antibodies, and detected using a DakoCytomation-catalyzed system with secondary antibodies. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Log2 values were media-centered by protein to account for variability in signal intensity by time and were calculated using the formula log2 signal â log2 median. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between sensitive and resistant cell lines to drug treatments. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs). Biostatistical analyses comparing two groups were performed using an unpaired t-test with Gaussian distribution followed by the Welch correction. To distinguish between treatment groups, we used one-way ANOVA with the Geisser-Greenhouse correction. Differences with p values <0.05 were considered significant. Within clustered image maps (CIM), unsupervised double hierarchical clustering used the Pearson correlation distance and Wardâs linkage method as the clustering algorithm to link entities (proteins) and samples.

Project description:Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent mTOR inhibition might suggest a number of therapeutic combinations that could improve its clinical activity. TC32 and TC71 ES clones with acquired resistance to ridaforolimus (MK8669, mTOR inhibitor) were generated by maintaining the corresponding parental cell lines with increasing concentrations of the agents (up to 50 Î¼M using ridaforolimus) for 7 months. All parental and acquired drug resistant cell lines were tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.) according to the manufacturerâ??s protocol and validated using short-tandem repeat fingerprinting with an AmpFLSTR Identifier kit as previously described. Herein, we determine subtle differences in acquired mechanism of resistance by promising small molecule inhibitor of mTOR, were evaluated using in vitro assays to decipher the mechanism(s) by which IGF-1R inhibition induces drug resistance in Ewing sarcoma cells. The preparation of extracted proteins from sensitive and acquired resistant Ewing sarcoma cells to ridaforolimus for reverse-phase protein lysate array (RPPA) analysis were prepared using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 115 validated primary antibodies, and detected using a DakoCytomation-catalyzed system with secondary antibodies. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Log2 values were media-centered by protein to account for variability in signal intensity by time and were calculated using the formula log2 signal â?? log2 median. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between sensitive and resistant cell lines to drug treatments. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs). Biostatistical analyses comparing two groups were performed using an unpaired t-test with Gaussian distribution followed by the Welch correction. To distinguish between treatment groups, we used one-way ANOVA with the Geisser-Greenhouse correction. Differences with p values <0.05 were considered significant. Within clustered image maps (CIM), unsupervised double hierarchical clustering used the Pearson correlation distance and Wardâ??s linkage method as the clustering algorithm to link entities (proteins) and samples.