AKT-Induced Tamoxifen Resistance Is Overturned by RRM2 Inhibition

Abstract

Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)-mediated approaches significantly reversed the tamoxifenresistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis.Implications: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies. Mol Cancer Res; 12(3); 394-407. Ó2013 AACR. IntroductionBreast cancer is the most common cancer in American women with an estimated 232,340 new cases of invasive and 64,640 new cases of noninvasive breast cancer this year alone. In 2013, 39,620 women were expected to die from breast cancer, and about 1 in 8 U.S. women would develop invasive breast cancer over the course of her lifetime (1). Breast cancer is frequently classified on the basis of hormone receptor status where 60% of premenopausal and 75% of postmenopausal cancers are estrogen receptor-positive (ER þ ; ref. 2). The ER acts as a master regulator of gene expression in breast cancer and promotes tumor progression via upregulating genes for proliferation and cell survival

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

“…Western blotting was performed as previously described (11,23). Briefly, cells were disrupted in RIPA buffer (Sigma) or tumors were homogenized in lysis buffer (Cell Signaling) supplemented with aprotinin, leupeptin, and okadaic acid (Sigma).…”

“…At study termination, tumors were harvested and either snap-frozen in liquid nitrogen and stored at À80 C (for Western analysis) or fixed with 10% neutral-buffered formalin for immunohistochemical analyses. Tumors were processed for immunoblot or immunohistochemical (by UC Davis VMTH) analyses as previously described (11). Representative data are shown from three independent xenograft experiments.…”

“…Didox can be tolerated by patients with cancer at high dosages without major side effects, making didox attractive for use in clinical applications. We previously reported that the combination of didox and tamoxifen significantly reduced cell proliferation in AKT-induced tamoxifen resistance (11).…”

“…We have previously shown that breast cancer cells overexpressing activated AKT exhibit tamoxifen-stimulated cell proliferation and enhanced cell motility. Moreover, we identified that RRM2 was a key contributor to AKT-induced tamoxifen resistance (11).…”

“…RRM2 is an attractive target for combination therapy, as it is overexpressed in breast cancer and contributes to development of resistance (11). Didox (3,4-dihydroxybenzohydroxamic acid) is a strong inhibitor of ribonucleotide reductase that interferes with DNA synthesis and repair by blocking the production of deoxyribonucleotides and has demonstrated antitumor effects for decades (12,(18)(19)(20).…”