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.

Project description:Multidrug resistance (MDR) to chemotherapeutic drugs remains one of the major impediments to the treatment of cancer. Discovery and development of drugs that can prevent and reverse the acquisition of multidrug resistance constitute a foremost challenge in cancer therapeutics. In this work, we screened a library of 1,127 compounds with known targets for their ability to overcome Pgp-mediated multidrug resistance in cancer cell lines. We identified four compounds (CHIR-124, Elesclomol, Tyrphostin-9 and Brefeldin A) that inhibited the growth of two pairs of parental and Pgp-overexpressing multidrug-resistant cell lines with similar potency irrespective of their Pgp status. Mechanistically, CHIR-124 (a potent inhibitor of Chk1 kinase) inhibited Pgp activity in both multidrug-resistant cell lines (KB-V1 and A2780-Pac-Res) as determined through cell-based Pgp-efflux assays. Other three inhibitors on the contrary, were effective in Pgp-overexpressing resistant cells without increasing the cellular accumulation of a Pgp substrate, indicating that they overcome resistance by avoiding efflux through Pgp. None of these compounds modulated the expression of Pgp in resistant cell lines. PIK-75, a PI3 Kinase inhibitor, was also determined to inhibit Pgp activity, despite being equally potent in only one of the two pairs of resistant and parental cell lines. Strong binding of both CHIR-124 and PIK-75 to Pgp was predicted through docking studies and both compounds inhibited Pgp in a biochemical assay. The inhibition of Pgp causes accumulation of these compounds in the cells where they can modulate the function of their target proteins and thereby inhibit cell proliferation. In conclusion, we have identified compounds with various cellular targets that overcome multidrug resistance in Pgp-overexpressing cell lines through mechanisms that include Pgp inhibition and efflux evasion. These compounds, therefore, can avoid challenges associated with the co-administration of Pgp inhibitors with chemotherapeutic or targeted drugs such as additive toxicities and differing pharmacokinetic properties.

Dataset Information

High-throughput ectopic expression screen for tamoxifen resistance identifies an atypical kinase that blocks autophagy.

Publications

High-throughput ectopic expression screen for tamoxifen resistance identifies an atypical kinase that blocks autophagy.

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