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Genome-wide association study for biomarker identification of Rapamycin and Everolimus using a lymphoblastoid cell line system.

Jiang J, Fridley BL, Feng Q, Abo RP, Brisbin A, Batzler A, Jenkins G, Long PA, Wang L - Front Genet (2013)

Bottom Line: We found that 16 expression probe sets (genes) that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors.Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line.Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Rochester, MN, USA.

ABSTRACT
The mammalian target of rapamycin (mTOR) inhibitors, a set of promising potential anti-cancer agents, has shown response variability among individuals. This study aimed to identify novel biomarkers and mechanisms that might influence the response to Rapamycin and Everolimus. Genome-wide association (GWA) analyses involving single nucleotide polymorphisms (SNPs), mRNA, and microRNAs microarray data were assessed for association with area under the cytotoxicity dose response curve (AUC) of two mTOR inhibitors in 272 human lymphoblastoid cell lines (LCLs). Integrated analysis among SNPs, expression data, microRNA data and AUC values were also performed to help select candidate genes for further functional characterization. Functional validation of candidate genes using siRNA screening in multiple cell lines followed by MTS assays for the two mTOR inhibitors were performed. We found that 16 expression probe sets (genes) that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors. One hundred and twenty seven and one hundred SNPs had P < 10(-4), while 8 and 10 SNPs had P < 10(-5) with Rapamycin and Everolimus AUC, respectively. Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line. Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs. In summary, this study identified genes and a microRNA that might contribute to response to mTOR inhibitors.

No MeSH data available.


Functional validation of candidate genes with siRNA knockdown in IMR90, U87 and Caki2 cell lines, followed by cytotoxicity assay (A) and colony formation assays (B). Data shown are representative experiments for selected genes in each cell line. siRNA knockdown for each individual gene (dashed line) were compared with negative control siRNA (solid line). (C). Knockdown efficiency was determined by qRT-PCR. Experiments were repeated in triplicate with at least two independent experiments. Significant P-values are listed for every gene. Error bars indicate standard error of the mean (SEM) values. Significance of AUC values between the control and specific siRNA was determined by student t-test.
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Figure 4: Functional validation of candidate genes with siRNA knockdown in IMR90, U87 and Caki2 cell lines, followed by cytotoxicity assay (A) and colony formation assays (B). Data shown are representative experiments for selected genes in each cell line. siRNA knockdown for each individual gene (dashed line) were compared with negative control siRNA (solid line). (C). Knockdown efficiency was determined by qRT-PCR. Experiments were repeated in triplicate with at least two independent experiments. Significant P-values are listed for every gene. Error bars indicate standard error of the mean (SEM) values. Significance of AUC values between the control and specific siRNA was determined by student t-test.

Mentions: In summary, we selected 23 genes based on the strategy shown in Figure 3 to perform siRNA screening, followed by MTS and colony formation assays to determine the effect of these candidate genes on the cytotoxicity of mTOR inhibitors. We performed these studies using Caki2 renal carcinoma cells, U87 glioblastoma cells, and IMR90 primary fibroblast cells. The two cancer cell lines were chosen because mTOR inhibitors are used to treat glioblastoma and renal carcinoma. The IMR90 cell line was chosen to be a normal cell line used in the study. Eleven out of twenty-three genes were verified to have a significant impact on the cytotoxicity of Rapamycin and/or Everolimus using the MTS assays in at least one cell line. Figure 4A shows the data for representative genes with a significant influence on the cytotoxicity of each drug treatment in each cell line. Specifically, knockdown of 5 genes, ECOP, MGLL, SLC39A9, ZNF765, and MAN1B1 sensitized the cells to Rapamycin and/or Everolimus in at least 2 cell lines (P < 0.05). Down-regulation of NDUFAF2 and SLC39A9 desensitized the cells to Rapamycin treatment in IMR90 and U87 cell lines, respectively (P < 0.05). Additional genes that significantly altered cell sensitivities are shown in Supplementary Figures S2, S3, S4 for each cell line. All of the genes that were functionally verified are listed in Table 2.


Genome-wide association study for biomarker identification of Rapamycin and Everolimus using a lymphoblastoid cell line system.

Jiang J, Fridley BL, Feng Q, Abo RP, Brisbin A, Batzler A, Jenkins G, Long PA, Wang L - Front Genet (2013)

Functional validation of candidate genes with siRNA knockdown in IMR90, U87 and Caki2 cell lines, followed by cytotoxicity assay (A) and colony formation assays (B). Data shown are representative experiments for selected genes in each cell line. siRNA knockdown for each individual gene (dashed line) were compared with negative control siRNA (solid line). (C). Knockdown efficiency was determined by qRT-PCR. Experiments were repeated in triplicate with at least two independent experiments. Significant P-values are listed for every gene. Error bars indicate standard error of the mean (SEM) values. Significance of AUC values between the control and specific siRNA was determined by student t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757297&req=5

Figure 4: Functional validation of candidate genes with siRNA knockdown in IMR90, U87 and Caki2 cell lines, followed by cytotoxicity assay (A) and colony formation assays (B). Data shown are representative experiments for selected genes in each cell line. siRNA knockdown for each individual gene (dashed line) were compared with negative control siRNA (solid line). (C). Knockdown efficiency was determined by qRT-PCR. Experiments were repeated in triplicate with at least two independent experiments. Significant P-values are listed for every gene. Error bars indicate standard error of the mean (SEM) values. Significance of AUC values between the control and specific siRNA was determined by student t-test.
Mentions: In summary, we selected 23 genes based on the strategy shown in Figure 3 to perform siRNA screening, followed by MTS and colony formation assays to determine the effect of these candidate genes on the cytotoxicity of mTOR inhibitors. We performed these studies using Caki2 renal carcinoma cells, U87 glioblastoma cells, and IMR90 primary fibroblast cells. The two cancer cell lines were chosen because mTOR inhibitors are used to treat glioblastoma and renal carcinoma. The IMR90 cell line was chosen to be a normal cell line used in the study. Eleven out of twenty-three genes were verified to have a significant impact on the cytotoxicity of Rapamycin and/or Everolimus using the MTS assays in at least one cell line. Figure 4A shows the data for representative genes with a significant influence on the cytotoxicity of each drug treatment in each cell line. Specifically, knockdown of 5 genes, ECOP, MGLL, SLC39A9, ZNF765, and MAN1B1 sensitized the cells to Rapamycin and/or Everolimus in at least 2 cell lines (P < 0.05). Down-regulation of NDUFAF2 and SLC39A9 desensitized the cells to Rapamycin treatment in IMR90 and U87 cell lines, respectively (P < 0.05). Additional genes that significantly altered cell sensitivities are shown in Supplementary Figures S2, S3, S4 for each cell line. All of the genes that were functionally verified are listed in Table 2.

Bottom Line: We found that 16 expression probe sets (genes) that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors.Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line.Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Rochester, MN, USA.

ABSTRACT
The mammalian target of rapamycin (mTOR) inhibitors, a set of promising potential anti-cancer agents, has shown response variability among individuals. This study aimed to identify novel biomarkers and mechanisms that might influence the response to Rapamycin and Everolimus. Genome-wide association (GWA) analyses involving single nucleotide polymorphisms (SNPs), mRNA, and microRNAs microarray data were assessed for association with area under the cytotoxicity dose response curve (AUC) of two mTOR inhibitors in 272 human lymphoblastoid cell lines (LCLs). Integrated analysis among SNPs, expression data, microRNA data and AUC values were also performed to help select candidate genes for further functional characterization. Functional validation of candidate genes using siRNA screening in multiple cell lines followed by MTS assays for the two mTOR inhibitors were performed. We found that 16 expression probe sets (genes) that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors. One hundred and twenty seven and one hundred SNPs had P < 10(-4), while 8 and 10 SNPs had P < 10(-5) with Rapamycin and Everolimus AUC, respectively. Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line. Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs. In summary, this study identified genes and a microRNA that might contribute to response to mTOR inhibitors.

No MeSH data available.