DISCUSSION
TAM is widely prescribed in breast cancer patients due to ERα-mediated anti-proliferative and pro-apoptotic effects on tumor cells. TAM cytotoxic activity is also exploited in off-target indications, such as infections, in accordance with repurposing strategies. We here demonstrate that TAM triggers macrophage immune activation, without inducing macrophage cell death, and potentiates cell responses to inflammatory signals by ERs-independent mechanisms that involve NRF2 and inflammasome activation. These results extend our knowledge on the molecular and biological activity of TAM and indicate the immune system as a pharmacological target for this drug, with relevant therapeutic implications for human diseases, such as cancer and infections, that may benefit from TAM-induced immune activation.
The limited number of reports published so far on TAM activity in inflammatory cells mainly focused on lipid trafficking and outlined ERα-independent effects of high TAM concentrations, mediated by the interference with transcription factors, such as GR, PPARγ, and STAT1 (Lee et al., 2000; Bowie et al., 2004; Jiang et al., 2013; Liu et al., 2015; Bekele et al., 2016). We here extend this knowledge and indicate novel mechanism and activity of TAM in macrophages. In fact, we show that TAM regulates the expression of VEGFα, IL1β and Arg1, that are related to cell immune activation, and increases phagocytosis. Moreover, TAM alters the macrophage response to inflammatory signals, by increasing the effects of LPS on IL1b protein secretion and altering the endotoxin-induced mRNA levels encoding inflammatory mediators, such as TNFα, IL1β and IL6. The response to TAM is still detected using ERαKO macrophages and differs from that induced by the GPER1-specific ligand, G1. Altogether, this evidence led us to exclude the involvement of estrogen receptors in the molecular mechanism of TAM action, also considering that ERβ is not expressed in macrophages (Villa et al., 2015; Pepe et al., 2018). Conversely, we ascribed TAM transcriptional response in macrophages to the activation of NRF2 by using Nrf2-reporter and target gene expression assays. Indeed, classic NRF2 activators induce antioxidant, phagocytic and inflammatory responses that are similar to those here described for TAM in macrophages, such as the inhibition of the LPS-induced expression of IL1β and IL6 and increase in LPS-positive effects on TNFα (Harvey et al., 2011; Kobayashi et al., 2016; Wang et al., 2017b; Bewley et al., 2018; Mornata et al., 2020). Activation of NRF2 by TAM has been previously described in epithelial cells (Feng et al., 2017). Thus, we demonstrate that NRF2 is a key molecular mediator of TAM immunomodulatory activity and suggests Nrf2 to be a candidate target for novel therapeutic interventions aimed at regulating macrophage responses and TAM therapeutic efficacy.
Consistent evidence has previously reported that TAM induces cell apoptosis in non-macrophagic cells, such mammary epithelial cells, hepatocytes and retinal cells. This effect has been reconciled with the induction of oxidative stress, formation of active caspase-1 and transcription of NRF2-target genes (Lee et al., 2000; Bowie et al., 2004; Liu et al., 2015; Bekele et al., 2016).
Instead, we here show that TAM does not induce cell death in macrophages despite our data indicate oxidative stress as a primary event in TAM activity, as revealed by caspase-1 activation and induction of ARE-driven and Nrf2-target gene expression. The reasons for this different outcome are unknown. However, macrophages contain regulatory systems that limit oxidative and inflammasome activation from damaging macrophages themselves, although these processes are highly activated in macrophages and are essential for killing pathogens and activating inflammation. These protective systems may also be involved in the observed macrophage-specific effects of TAM, uncoupling the oxidative and inflammatory responses induced by this drug from cell death programs.
Conventional dosages of TAM in breast cancer patients lead to drug concentrations within the mammary gland that are similar to those used in the present study (Kisanga et al., 2004); higher dosages are used for off-target indications, supposedly reaching micromolar drug concentrations in patients serum (Kisanga et al., 2004). Our data show that these pharmacological doses of TAM triggers immunomodulatory effects, which may also be potentiated by high E2 concentrations (>1-100nM) that are reached in the peritoneal fluid following ovulation in the breast adipose tissue (Koninckx et al., 1998; Lønning et al., 2011b, 2011a). This leads us to hypothesize that TAM immune activity may contribute to its clinical outcome. Indeed, TAM antitumor efficacy is also observed in ERα-negative cancers and appears not to be limited to tumor cells. On the other hand, macrophages are key players in the defense against cancer and TAM use in oncology is associated with modifications in immune cell composition (Bekele et al., 2016; Larsson et al., 2019). Thus, existing evidence support a possible contribution of inflammatory cells in TAM efficacy in breast cancer. From the data shown here, we speculate that the TAM-induced potentiation of the inflammatory response and increase in cellular uptake induce a more efficient disposal of apoptotic cancer cells. Moreover, long-term TAM therapy is associated with the acquisition of drug resistance, that eventually leads to disease relapse and the appearance of side effects, such as retinopathy. Interestingly, TAM resistance has been associated with non-cell autonomous processes that may involve NRF2, as this transcription factor is implicated in chemotherapeutics and TAM resistance in epithelial cells (Kim et al., 2008; Bekele et al., 2016; Sanghvi et al., 2019). On the other hand, the oxidative toxicity of TAM, which leads to ERα-independent degeneration of retinal cells, has recently been shown to be counterbalanced by TAM action on retinal microglia, which can rescue retinal cell loss in murine models of photoreceptor degeneration (Wang et al., 2017a). Thus, the role of inflammatory cells in mediating both the therapeutic as well as adverse effects of TAM needs to be more deeply investigated by future studies.
Due to its chemical scaffold, low cost and safety profile, TAM is a highly challenging molecule for repurposing strategies. At higher than standard anti-estrogen doses, it has been used as ERα-independent, off target therapeutic option for a wide range of immune-dependent pathologic conditions, although its mechanism of action on immunity remains unknown (Behjati and Frank, 2009; Vaglio et al., 2011; Dellê et al., 2012). One of its major exploitation pertains a broad range of human infections (Vargas-Villavicencio et al., 2007; Nicolao et al., 2014; Sik Jang et al., 2015; Montoya and Krysan, 2018; Weinstock et al., 2019). We here suggest that TAM may potentiate the macrophage response to infective agents and improve microbicidal activity by activating phagocytosis and modulating macrophage phenotypic activation, particularly against pathogens persisting within macrophages. Indeed, TAM has been shown to increase intracellular killing ofMycobacterium tuberculosis in macrophages (Sik Jang et al., 2015) and it has been used with clinical success in association with classic antifungal drugs which, differently from TAM, do not diffuse through the macrophage cell membrane.
In summary, our study demonstrates that pharmacological concentrations of TAM act in macrophages independently of ERα and are able to skew macrophage polarization induced by inflammatory conditions. These results provide novel hypothesis for TAM pharmacology in breast cancer and other off-target clinical indications and provide molecular targets for future drug development strategies.