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.