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Targeting Ribonucleotide Reductase M2 and NF-κB Activation with Didox to Circumvent Tamoxifen Resistance in Breast

Khyati N Shah¹, Elizabeth A Wilson¹, Ritu Malla¹

¹Department of Physiology and Pharmacology, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, California.

Molecular Cancer Therapeutics

|September 4, 2015

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.

Insights

Overexpression of ribonucleotide reductase M2 (RRM2) drives tamoxifen resistance in breast cancer by altering estrogen receptor variants and activating key pathways. Inhibiting RRM2 with didox reverses resistance and enhances tamoxifen efficacy.

Area of Science:

Molecular Biology
Cancer Research
Pharmacology

Background:

Tamoxifen is a standard adjuvant therapy for estrogen receptor (ERα)-positive breast cancer.
Acquired tamoxifen resistance limits its long-term clinical benefit.
Ribonucleotide reductase M2 (RRM2) has been implicated in cancer progression and drug resistance.

Purpose of the Study:

To investigate the role of RRM2 in tamoxifen resistance in breast cancer.
To identify molecular pathways affected by RRM2 overexpression in tamoxifen-resistant cells.
To evaluate the therapeutic potential of RRM2 inhibition in overcoming tamoxifen resistance.

Main Methods:

Overexpression of RRM2 in MCF-7 breast cancer cells.
Analysis of estrogen receptor (ERα) variants (ERα66 and ERα36).
Pathway analysis (NF-κB, HIF1α, MAPK/JNK) and protein profiling.
Time-lapse microscopy and in vitro/in vivo assays.
Inhibition of RRM2 using siRNA (siRRM2) and the RR inhibitor didox.

Main Results:

RRM2 overexpression reduced tamoxifen effectiveness by downregulating ERα66 and upregulating ERα36.
RRM2 overexpression activated NF-κB, HIF1α, and MAPK/JNK pathways, increasing protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50.
RRM2-overexpressing cells showed increased migration and invasion.
Inhibition of RRM2 with siRRM2 or didox prevented and reversed tamoxifen resistance.
Didox combined with tamoxifen synergistically reversed ERα alterations and inhibited NF-κB activation.
Didox treatment reversed tamoxifen-resistant in vivo tumor growth and reduced invasiveness.

Conclusions:

RRM2 plays a critical role in the development of tamoxifen resistance in ERα-positive breast cancer.
Targeting RRM2, particularly with the inhibitor didox, is a promising strategy to overcome tamoxifen resistance.
Combination therapy with didox and tamoxifen offers a potential approach to improve treatment outcomes for patients with tamoxifen-resistant breast cancer.

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