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

Abstract

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.functional screen | estrogen receptor T he two thirds of women with estrogen receptor-(ER) or progesterone receptor-positive breast cancers are excellent candidates for antihormone therapy. Selective ER modulators (SERMs), like tamoxifen, block ER activation and have impacted both therapy and survival. However, the success of tamoxifen therapy is limited by intrinsic and acquired drug resistance. Several pathways have been implicated in antiestrogen resistance, including: the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway, which is implicated in cell survival; the EGFR family; and the RAS/RAF/MEK1/2/ERK1/2 family, which regulate cell proliferation (1, 2). Loss of ER expression or function may also be an important mechanism of de novo resistance to tamoxifen, either through relatively rare ER mutations or changes in coactivators and corepressors (3).Several groups have used gene-expression analysis to identify genes regulated through ER (4) that are affected by SERMs in breast cancer cells (5, 6). Others have used tumor samples to develop gene signatures that can predict clinical responses to tamoxifen (7-10). Genetic strategies have also been used to identify genes that drive tamoxifen resistance. Receptor tyrosine kinases and MAPK signaling were detected using expression of pooled cDNA libraries in ZR-75-1, an approach often biased toward the most abundantly expressed genes and which requires recovery of hits by PCR (11). The analysis of antiestrogen-sensitive and -resistant MCF7 cells by SNP and comparative genomic hybridization pointed to changes in protein abundance rather than somatic genomic changes (12). An RNA interference screen of kinases identified CDK10, CRK7, and MAP2K7, whose knockdown cause tamoxif...

Gene Regulation by Cohesin in Cancer: Is the Ring an Unexpected Party to Proliferation?

“…The screen identified 15 genes, including RAD21, that when overexpressed led to antiestrogen resistance. A study that compared the gene expression profiles of MCF7 cells with acquired resistance to tamoxifen showed a significant increase in RAD21 expression (and a 3-fold decrease in PDS5B expression) in tamoxifen-resistant versus tamoxifen-sensitive cells (138). These studies are consistent with the idea that increased cohesin activity leads to resistance to selective ER modulators.…”

Network-based approach to identify prognostic biomarkers for estrogen receptor–positive breast cancer treatment with tamoxifen

“…To validate this hypothesis, 2 datasets, GSE33366 10 and GSE26459, 11 were Figure 2. Elevated expression of the turquoise ME, a group of coexpressed genes indicates poor outcome in ERC breast cancer patients treated with tamoxifen.…”

Autophagy and Cancer Therapy

“…Some papers demonstrate that autophagy is required for tamoxifen resistance (Qadir et al, 2008;Samaddar et al, 2008;Cook et al, 2012), i.e., autophagy protects breast cancer cells; however, another study examining tamoxifen resistance mechanisms (also in MCF7 cells) performed a high throughput screen for kinases that confer resistance to tamoxifen in sensitive MCF7 cells. This identified a kinase called HSPB8 and showed that its ability to protect against tamoxifeninduced death was associated with its ability to induce autophagy (Gonzalez-Malerva et al, 2011). The conclusion from this latter study was that tamoxifen resistance could be overcome by increasing autophagy.…”