Abstract
Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.
Highlights
ERa plays a fundamental role in the etiology and the progression of human breast cancer [1]
We demonstrate that ribonucleotide reductase M2 (RRM2) is associated with increased NF-kB activity, ERa alterations, HER2 and EGF receptor (EGFR) upregulation, and increases in antiapoptotic pathways
We have previously shown that ZR-75-1 cells exhibit tamoxifen-resistant cell proliferation and that inhibition of RRM2 using siRNA oligos targeting RRM2 (siRRM2) restores tamoxifen sensitivity and represses the DNA repair enzymes that protect these cells from tamoxifen-induced apoptosis [11]
Summary
ERa plays a fundamental role in the etiology and the progression of human breast cancer [1]. Many therapies have been designed to inhibit the tumor-promoting effects of ERa; tamoxifen has been the drug of choice for all stages of ERpositive breast cancer. Tamoxifen arrests cells in G0–G1 and decreases expression of several ERa target genes [3]. Apart from blocking classical ER action, tamoxifen induces DNA damage and apoptosis in ER-positive cells [4]. Despite these benefits, some tumors recur due to acquired tamoxifen resistance giving rise to a subpopulation of unresponsive cells [5]. Several mechanisms of acquired tamoxifen resistance have been reported, including downregulation of ERa expression
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