RNA interference (RNAi) screening approach identifies agents that enhance paclitaxel activity in breast cancer cells

Abstract

Introduction: Paclitaxel is a widely used drug in the treatment of patients with locally advanced and metastatic breast cancer. However, only a small portion of patients have a complete response to paclitaxel-based chemotherapy, and many patients are resistant. Strategies that increase sensitivity and limit resistance to paclitaxel would be of clinical use, especially for patients with triple-negative breast cancer (TNBC).

Methods: We generated a gene set from overlay of the druggable genome and a collection of genomically deregulated gene transcripts in breast cancer. We used loss-of-function RNA interference (RNAi) to identify gene products in this set that, when targeted, increase paclitaxel sensitivity. Pharmacological agents that targeted the top scoring hits/genes from our RNAi screens were used in combination with paclitaxel, and the effects on the growth of various breast cancer cell lines were determined.

Results: RNAi screens performed herein were validated by identification of genes in pathways that, when previously targeted, enhanced paclitaxel sensitivity in the pre-clinical and clinical settings. When chemical inhibitors, CCT007093 and mithramycin, against two top hits in our screen, PPMID and SP1, respectively, were used in combination with paclitaxel, we observed synergistic growth inhibition in both 2D and 3D breast cancer cell cultures. The transforming growth factor beta (TGFbeta) receptor inhibitor, LY2109761, that targets the signaling pathway of another top scoring hit, TGFbeta1, was synergistic with paclitaxel when used in combination on select breast cancer cell lines grown in 3D culture. We also determined the relative paclitaxel sensitivity of 22 TNBC cell lines and identified 18 drug-sensitive and four drug-resistant cell lines. Of significance, we found that both CCT007093 and mithramycin, when used in combination with paclitaxel, resulted in synergistic inhibition of the four paclitaxel-resistant TNBC cell lines.

Conclusions: RNAi screening can identify druggable targets and novel drug combinations that can sensitize breast cancer cells to paclitaxel. This genomic-based approach can be applied to a multitude of tumor-derived cell lines and drug treatments to generate requisite pre-clinical data for new drug combination therapies to pursue in clinical investigations.

Figure 1

shRNA screen to identify paclitaxel sensitizers. A. The overlay of druggable genome libraries (Qiagen and Open Biosystems) and genes deregulated in breast cancer resulted in 428 candidate druggable genes. B. Reproducibility of shRNA screen by correlation of the effect of shRNAs on cell growth compared to non-silencing shRNA in vehicle-treated plates of two replicate experiments. Spearman correlation coefficient, r = 0.89. C. Each shRNA was scored for the level of paclitaxel sensitivity using the sensitivity index (SI) as described in Materials and Methods. The SI score ranges from -1 to 1. Positive significant SI scores indicate sensitization and negative significant SI scores indicate antagonism. The scatter plot of all shRNAs is shown in rank order. The dashed lines indicate the relative threshold of significant drug sensitivity.

Figure 2

Novel drug combinations sensitize breast cancer cells to paclitaxel. A. MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cell lines were seeded in six-well plates and treated with vehicle (control), < IC₅₀ concentrations of the putative PPM1D inhibitor, CCT007093 (CCT); paclitaxel (tax); or a combination of both (CCT + tax). Cells were treated for 72 h, washed, trypsinized and counted. The percent of viable cells relative to control was plotted for each drug or combination. B. Same as A with < IC₅₀ concentration the putative SP1-binding inhibitor, mithramycin (mith). Error bars represent standard deviation of triplicates from three independent experiments. * indicates P < 0.05, ** indicates P < 0.01.

Figure 3

Analysis of drug combinations on growth of breast cancer cells grown in 3D cultures. A. Cells were seeded on Matrigel in eight-well chamber slides as described in Materials and Methods. 3D cultures formed after two days and were treated every two to three days with single agents, vehicle (control), 1 nM paclitaxel (tax), 500 nM LY2109761 (LY), 10 μM CCT007093 (CCT), 25 nM mithramycin (mith) (upper panels) or a combination of drugs (lower panels). After 10 to 14 days, mammospheres were visualized using phase-contrast microscopy. Bar scale, 50 μm. B. To count cell numbers, the Matrigel was dissolved, mammospheres were collected, trypsinized and single cells were counted by trypan blue exclusion assay using a hemocytometer. The percent cell number relative to control was plotted for each drug or combination for the two cell lines. Error bars represent standard deviation from replicates from three independent experiments.

Figure 4

Drug combinations to enhance cell death of TNBC cell lines. A. Twenty-two triple-negative cell lines were each seeded in 96-well plates. The next day cells were treated with vehicle control or paclitaxel (0.3 to 30 nM). IC₅₀ values for each cell line were generated based on the median-effect plot from three independent experiments. IC₅₀ values represent the inhibitor concentration required for a 50% reduction in cell viability relative to vehicle-treated controls. Error bars represent standard deviation of four replicates from three independent experiments. B. Cell lines were seeded in 96-well plates and treated with single agents (IC₅₀ values) or a combination of drugs (CCT007093 + paclitaxel or mithramycin + paclitaxel) of the IC₅₀ concentrations of each drug (1:1 ratio) serial-diluted (IC₅₀-IC₂₅-IC_12.5). Combination index (CI) values were calculated using the Chou-Talalay method with CalcuSyn software (Biosoft). CI values significantly > 1 are antagonistic, not significantly different than 1 are additive, and values < 1 are synergistic. Error bars represent standard deviation of quadruplicates from three independent experiments.