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:Tamoxifen, an antagonist to estrogen receptor (ER), is a first line drug used in breast cancer treatment. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying the resistance to tamoxifen, we established a tamoxifen-resistant cell line by treating the MCF7 breast cancer cell line with tamoxifen for over 6 months. We showed that this cell line exhibited resistance to tamoxifen both in vitro and in vivo. In order to quantify the phosphorylation alterations associated with tamoxifen resistance, we performed SILAC-based quantitative phosphoproteomic profiling on the resistant and vehicle-treated sensitive cell lines where we identified >5,600 unique phosphopeptides. We found phosphorylation levels of 1,529 peptides were increased (>2 fold) and 409 peptides were decreased (<0.5-fold) in tamoxifen resistant cells compared to tamoxifen sensitive cells. Gene set enrichment analysis revealed that focal adhesion pathway was the top enriched signaling pathway activated in tamoxifen resistant cells. We observed hyperphosphorylation of the focal adhesion kinases FAK1 and FAK2 in the tamoxifen resistant cells. Of note, FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. Suppression of FAK2 by specific siRNA knockdown could sensitize the resistant cells to the treatment of tamoxifen. We further showed that inhibiting FAK activity using the small molecule inhibitor PF562271 repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 significantly associated with short metastasis-free survival of ER-positive breast cancer patients treated with tamoxifen-based hormone therapy. Our studies suggest that FAK2 is a great potential target for the development of therapy for the treatment of hormone refractory breast cancers.

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