Unknown,Transcriptomics,Genomics,Proteomics

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Systematic drug perturbations of cancer cells reveal diverse exit paths from the proliferative state

ABSTRACT: Analysis of the time courses of gene expression profiles of breast cancer cell line MCF7 treated by 16 differentiation-inducing drugs at day 1, day 3 and day 5. The drugs are the screening results from the the JHCCL library (1,500 drugs). The hypothesis tested in the present study was that cancer cells exit the proliferative state via multiple paths. Results showed that cell state transition trajectories firstly diverged and later converged to a quiescient differentiated state MCF7 cells were cultured in 150mm dishes and treated 1/5/10 Î¼M of each of the 16 drugs (see details in Table S1-2). 14 plates of cells were left untreated as control samples. Cells were collected after 1, 3 and 5 days of drug treatment in RNeasy (Qiagen) lysis buffer and RNA was isolated according to the manufactureâs protocol and sent to Vancouver Prostate Center for transcript profiling.

ORGANISM(S): Homo sapiens

SUBMITTER: Joseph ZHOU

PROVIDER: E-GEOD-74281 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Resistance to tamoxifen in breast cancer patients is a serious therapeutic problem and major efforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene-expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones, which also predicts disease-free survival in tamoxifen-treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicted poor clinical outcome in one cohort of patients. Further studies revealed that HSPB8 protected MCF7 cells from tamoxifen and blocked autophagy. Moreover, silencing HSBP8 induced autophagy and caused cell death. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones, and tamoxifen-resistant cells were sensitive to the induction of autophagy by other drugs. These results may point to an important role for autophagy in the sensitivity to tamoxifen. For defined estrogen culture experiments, sensitive (MCF7-B7TamS) and resistant (MCF7-G11TamR) subclones, were plated in 10-cm dish (Nunc) in DMEM supplemented with 5% FBS. The day after plating, the media was changed to steroid depleted media (phenol red-free DMEM) supplemented with 5% charcoal-dextran treated FBS and grown for 2 d. On day 3, cells were rinsed three times with PBS and treated in triplicate using one of four different conditions: (i) estrogen-depleted medium (control); (ii) estrogen (E) at 10 to 9 M; (iii) 4-OHT at 1 uM; and (iv) E plus 4-OHT, both drugs at the same concentrations as in (ii) and (iii). After 4 h, RNA was isolated and processed for gene-expression profiling according to the Affymetrix protocol (Human Genome U133 plus 2.0 Array) at the Microarray Core Facility at the Dana Farber Cancer Institute. The gene-expressionCEL files were normalized using dChip, using the invariant method and PM-MM difference methods for background subtraction. We identified genes that are differentially regulated by tamoxifen using the following criteria: (i) Genes that responded to estrogen in both MCF7-B7TamS andMCF7-G11TamR were identified if cells under tamoxifen treatment were more than 1.1-fold either up- or down-regulated [90% confidence interval (CI)] relative to its corresponding control cells; (ii) Genes unresponsive to tamoxifen in resistant cells: genes that showed more than 1.1-fold up- or down-regulation (90% CI) in MCF7-G11TamR but not in MCF7-B7TamS when comparing cells treated with both estradiol and tamoxifen with those treated with drug only.

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Systematic drug perturbations of cancer cells reveal diverse exit paths from the proliferative state

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