Abstract

   Tamoxifen (TAM) resistance remains a major obstacle in the treatment of
   advanced breast cancer (BCa). In addition to the competitive inhibition
   of the estrogen receptor (ER) signaling pathway, damping of
   mitochondrial function by increasing reactive oxygen species (ROS) is
   critical for enhancing TAM pharmacodynamics. Here, we showed that RelB
   contributes to TAM resistance by inhibiting TAM-provoked ferroptosis.
   TAM-induced ROS level promoted ferroptosis in TAM-sensitive cells, but
   the effect was alleviated in TAM-resistant cells with high constitutive
   levels of RelB. Mechanistically, RelB inhibited ferroptosis by
   transcriptional upregulating glutathione peroxidase 4 (GPX4).
   Consequently, elevating RelB and GPX4 in sensitive cells increased TAM
   resistance, and conversely, depriving RelB and GPX4 in resistant cells
   decreased TAM resistance. Furthermore, suppression of RelB
   transcriptional activation resensitized TAM-resistant cells by
   enhancing ferroptosis in vitro and in vivo. The inactivation of GPX4 in
   TAM-resistant cells consistently resensitized TAM by increasing
   ferroptosis-mediated cell death. Together, this study uncovered that
   inhibition of ferroptosis contributes to TAM resistance of BCa via
   RelB-upregulated GPX4.

   Keywords: Tamoxifen resistance, Breast cancer, ROS, Ferroptosis, GPX4,
   RelB

Graphical abstract

   [41]Image 1
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1. Introduction

   Tamoxifen (TAM) is the first line of a therapeutic drug to treat
   premenopausal women diagnosed with estrogen receptor-postive (ER^+)
   breast cancer (BCa) [[43]1]. TAM was initially designed to block the
   interaction between estrogen and ER for treating ER^+ BCa [[44]2]. In
   addition, mounting evidence has demonstrated that TAM also perturbs
   mitochondrial function due to increased reactive oxygen species (ROS)
   [[45]3,[46]4]. Thus, TAM not merely induces cell death in ER^+ BCa
   cells but also inhibits many other types of tumor cell independence of
   ER [[47]5,[48]6]. As a result, BCa patients have received therapeutic
   benefits from TAM. Unfortunately, most patients eventually develop more
   aggressive phenotypes that were resistant to TAM [[49]7]. The
   dysfunction of ER signaling is thought to be the main consequence of
   TAM resistance. Nevertheless, several studies highlighted that TAM
   resistance in some patients was not due to the inactivation of or loss
   of ER [[50]8,[51]9]. In this regard, the deficiency of the
   mitochondrial function by excessive ROS is involved in BCa TAM
   resistance [[52]3,[53]10,[54]11].

   Increasing ROS to the threshold level of cell death is the principle
   for the most convenient radio- and chemo-therapeutic strategies
   [[55]12,[56]13]. To date, programmed cell death is widely recognized to
   be involved in anticancer treatment, such as apoptosis and necroptosis
   [[57]14,[58]15]. In addition, ferroptosis is a newly discovered
   iron-dependent regulated cell death involved in cancer progression and
   therapeutic response [[59][16], [60][17], [61][18]]. Ferroptosis
   resulted from extensive phospholipids peroxidation and led to membrane
   structure instability. It is caused by hydroxyl radicals generated
   through the Fenton reaction [[62]19]. Therefore, increasing ROS
   generation and/or reprogramming iron metabolism are essential for
   promoting ferroptosis [[63]20]. Consequently, the cellular antioxidant
   defense systems prevent ROS-induced ferroptosis by inhibiting
   iron-dependent lipid peroxidation [[64]21,[65]22]. Among antioxidant
   enzymes, glutathione peroxidase 4 (GPX4) is critical to block
   ferroptosis via glutathione-dependent inhibition of lipid peroxidation
   [[66]23]. Furthermore, recent evidence revealed that FSP1, a
   glutathione-independent ferroptosis inhibitor, can prevent lipid
   peroxidation via activation of CoQ oxidoreductase [[67]21,[68]24].
   Mechanistically, multiple signaling pathways are relevant to the
   inhibition of ferroptosis in cancer progression and therapeutic
   response, including NF-κB, PI3K/AKT/mTOR, and Stat3 [[69][25],
   [70][26], [71][27]].

   NF-κB, a redox-sensitive transcription factor, participates in the
   adaptive activation of antioxidant response for activating the
   pro-survival pathways [[72]28]. In particular, the activation of the
   NF-κB pathway plays a crucial role in cancer progression and
   therapeutic resistance [[73]29]. Numerous studies have demonstrated
   that the NF-κB activation contributes to BCa endocrine therapy
   resistance, such as TAM [[74]30,[75]31]. The NF-κB pathway contributes
   to inhibition of apoptosis, pyroptosis, and necrosis. Moreover, recent
   studies have shown that the NF-κB pathway is also implicated in
   regulating ferroptosis [[76]32]. In addition to the well-documented
   canonical NF-κB pathway, the RelB-activated noncanonical NF-κB pathway
   also contributes to cancer progression [[77]33]. Previously, we
   reported that RelB enhances prostate cancer radioresistance and immune
   evasion by upregulating MnSOD and PD-L1 [[78]34,[79]35]. Recently, we
   showed that the constitutive level of RelB increased in advanced BCa,
   and repression of RelB transcriptional activation suppressed BCa
   tumorigenesis [[80]36].

   This present study aims to examine whether ferroptosis inhibition is
   involved in BCa TAM resistance. We found that TAM-induced ROS enhanced
   ferroptosis in BCa cells, and vice versa; activation of GPX4
   resensitized the cells to TAM by inhibiting ferroptosis.
   Mechanistically, RelB activation inhibited ferroptosis by upregulating
   GPX4, and consistently suppression of RelB-activated GPX4 resensitized
   the resistant BCa cells to TAM.

2. Results

2.1. ROS-induced ferroptosis relevant to TAM-induced cell death

   To investigate the TAM-resistant (TAMR) mechanisms, we used ER^+ BCa
   cell lines (MCF7 and T47D) to establish their TAMR cell lines
   (MCF7/TAMR and T47D/TAMR). Additionally, triple-negative breast cancer
   (TNBC) MDA-MB-231 cell line characterized TAM insensitive [[81]37], was
   included as an innate TAMR control. After TAM treatment, MTT and colony
   survival assays confirmed the TAMR phenotype. Although the growth rates
   of TAMR lines were equivalent to their parental cell lines in the
   untreated condition ([82]Fig. S1A), the half-inhibitory concentration
   (IC[50]) of TAM significantly increased in TAMR cell lines compared to
   TAM-sensitive (TAMS) cell lines ([83]Figs. S1B and C). Colony survival
   assay further confirmed the TAMR phenotype ([84]Figs. S1D and E). In
   parallel, ERα decreased but drug-resistant marker BCRP increased in
   TAMR cells ([85]Fig. S1F).

   The programmed cell death mechanisms involve apoptosis, necroptosis,
   autophagy, ferroptosis, and cuproptosis [[86]38]. Numerous studies have
   reported that TAM efficiently induced apoptotic cell death in BCa
   [[87]39,[88]40]. To examine whether other mechanisms are implicated in
   TAM-induced cell death, we pretreated both TAMS and TAMR BCa cell lines
   with multiple cell death inhibitors to block TAM-induced cytotoxicity
   ([89]Fig. 1A and B). Intriguingly, the pan-caspase inhibitor
   (Z-VAD-FMK) was essential but not sufficient to rescue the cells
   against TAM, suggesting that the apoptotic pathway is not only
   responsible for TAM-induced cell death. Notably, inhibitors of
   ferroptosis including deferoxamine (DFO), ferrostatin-1 (Fer-1), and
   N-acetylcysteine (NAC) also contributed to protect the cell survival,
   indicating that ferroptosis is involved in TAM resistance. However,
   there were no significant protective effects observed with other types
   of cell death inhibitors, such as necrostatin-1 (Nec-1), chloroquine
   (CQ), and tetrathiomolybdate (TTM), which target necroptosis,
   autophagy, and cuproptosis, respectively.

Fig. 1.

   [90]Fig. 1
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   TAM-induced cytotoxicity by increasing ROS level. (A) Multiple cell
   death inhibitors were used to examine their protective effects on
   survival of BCa cells against Tamoxifen (TAM), including 20 mM
   necrostatin-1 (Nec-1), 10 mM ferrostatin-1 (Fer-1), 5 mM
   N-acetylcysteine (NAC), 50 μM deferoxamine (DFO), 50 μM Z-VAD-FMK,
   50 μM chloroquine (CQ) or 20 μM tetrathiomolybdate (TTM). The cells
   were pretreated with the inhibitors for 12 h and then treated with
   20 μM TAM for 48 h. MTT assay was performed to quantify cell survival
   as indicated in a heatmap (n = 3). (B) Schematic diagrams of apoptosis,
   autophagy, necroptosis, cuproptosis, and ferroptosis. Inhibitors
   targeting the relative cell death pathways are marked in red colors.
   (C, D) Superoxide anions in the treated cells were quantified using a
   DHE fluorescent probe (DHE fluorescence indicated as blue color and its
   oxidative products indicated as red color). PEG-SOD was used as a
   negative control to remove superoxide anions (n = 3). (E, F) The levels
   of ROS were quantified using an H[2]DCFDA probe with CDCFDA
   normalization. NAC and PEG-catalase were used as negative controls to
   remove specific types of ROS (n = 3). (G) The cytotoxicity of TAM was
   estimated using the MTT assay. (H, I) TAM-mediated cytotoxicity was
   estimated by colony survival assay (n = 3). Data are shown as
   mean ± SD, * (p < 0.05) and **(p < 0.01); t-test (D-right), two-way
   ANOVA (D-left, F–H). (For interpretation of the references to color in