\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{The 5,6-epoxycholesterol metabolic pathway in cancer: emergence of new
pharmacological targets}



\author[1]{Philippe de Medina}%
\author[1]{Khadijetou Diallo}%
\author[1]{Emilie Huc-Claustre}%
\author[1]{Mehdi Attia}%
\author[2]{Régis Soulès}%
\author[1]{Sandrine Silvente-Poirot}%
\author[2]{Marc Poirot}%
\affil[1]{UMR 1037}%
\affil[2]{Cancer Research Center of Toulouse}%


\vspace{-1em}



  
  \date{June 10, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Metabolic pathways have emerged as cornerstones in carcinogenic
deregulation providing new therapeutic strategies for cancer management.
This is illustrated by the recent discovery of a cholesterol metabolic
branch involving the biochemical transformation of 5,6-epoxycholesterol
(5,6-ECs). 5,6-ECs have been shown to be differentially metabolized in
breast cancers (BC) compared to normal breast tissue. 5,6-ECs are
metabolized into the tumour promoter oncosterone in BC, while they are
transformed into the tumour suppressor metabolite dendrogenin A (DDA) in
normal breast tissue. Blocking oncosterone's mitogenic and invasive
potential will represent new opportunities for BC treatment. The
reactivation of DDA biosynthesis, or its use as a drug, represents
promising therapeutic approaches such as DDA-deficiency complementation,
activation of BC cell re-differentiation and BC chemoprevention. This
review presents current knowledge as to the 5,6-EC metabolic pathway in
BC focusing on the 5,6-EC metabolic enzymes ChEH and HSD11B2, and on
5,6-EC metabolite targets LXR\selectlanguage{greek}β \selectlanguage{english}and GR.%
\end{abstract}%



\sloppy




\textbf{1. Introduction.}

Metabolic reprogramming has emerged as a hallmark in cancer, impacting
gene expression, cell de-differentiation and tumour microenvironment
(Pavlova \& Thompson, 2016). It is well established that cancer cells
must rewire the cellular metabolism to satisfy the demands of growth and
proliferation, most notably by providing energy, reducing equivalents
and building blocks while several metabolites exert a signalling
functions promoting tumour growth and progression (Danhier et al.,
2017). The exploration of cancer metabolism for clinical benefits is
required to identify metabolic pathways that are limiting for tumour
progression (Vander Heiden \& DeBerardinis, 2017). Cholesterol is a
major lipid that is crucial as a building block for membrane formation
and protein structuration (Grouleff, Irudayam, Skeby \& Schiott, 2015).
It is also the precursor of steroid hormones, bile acids and oxysterols
(Schroepfer, 2000). Cholesterol biosynthesis is a multi-step process
(Nes, 2011) that is subject to homeostasis and finely regulated in cells
(Luo, Yang \& Song, 2020). In cancer cells several deregulations have
recently emerged, opening up new therapeutic strategies (Huang, Song \&
Xu, 2020; Kuzu, Noory \& Robertson, 2016).

Recent epidemiological studies have shown that breast cancer (BC) still
represent the world leading female cancer in terms of incidence and
mortality (Bray, Ferlay, Soerjomataram, Siegel, Torre \& Jemal, 2018;
Global Burden of Disease Cancer et al., 2019). Thus, there is an urgent
need to find and validate new therapeutic targets in order to improve
patient survival and tumour recurrence. BC is a heterogeneous pathology
and several molecular BC subtypes have been described driving
therapeutic treatments. We can distinguish the following three major
subtypes: 1) Estrogen receptor positive breast cancers (ER(+)BC) are the
most frequent BC and are treated with selective ER modulators (SERM) and
aromatase inhibitors (AI). SERM block the mitogenic effects of
17\selectlanguage{greek}β-\selectlanguage{english}oestradiol (E2) at the ER level, and AI inhibit E2 neosynthesis in
BC (Jordan \& Brodie, 2007). 2) ER(-)negative and HER2(+) BC are treated
using anti-HER2 therapeutic antibodies that block the activation of
HER2-dependent mitogenic pathways with or without conventional
chemotherapy (Goldhirsch et al., 2011); 3) triple negative BC (TNBC)
that do not express steroid hormone receptors and HER2 are treated by
conventional chemotherapy with non-selective cytotoxic drugs (Goldhirsch
et al., 2011).

Epidemiological population studies have identified links between
cholesterol and cancer. Meta-analysis of clinical trials have shown an
inverse relationship between circulating cholesterol levels and BC
(Touvier et al., 2015), while hypercholesterolemia has been proposed as
a risk factor for BC recurrence (Nelson, 2018), implying that
cholesterol metabolism deregulations occured in BC and that targeting
cholesterol metabolism deregulations may be of interest for BC treatment
and chemoprevention (Garcia-Estevez \& Moreno-Bueno, 2019).

At the molecular level, recent studies have shpwn that certain
oxysterols display either tumour promoter but also tumour suppressor
properties (Fig 1A). 27-hydroxycholesterol (27-HC) has been shown to
stimulate ER(+)BC proliferation and invasiveness through the modulation
of ER and LXR\selectlanguage{greek}β \selectlanguage{english}respectively (Nelson et al., 2013) . It has also been
shown that the pro-metastatic action of 27-HC in mice required myeloid
immune cell functions such as polymorphonuclear-neutrophils and \selectlanguage{greek}γδ-\selectlanguage{english}T
cells at distal metastatic sites (Baek et al., 2017). In mice, it has
been shown that the CXCR2 receptor was involved in this effect (Baek et
al., 2017; Raccosta et al., 2013; Raccosta, Fontana, Traversari \&
Russo, 2013) . It has been shown that other side-chain oxysterols
displayed similar properties, possibly after sulfation by the
sulfotransferase SULT2B1b (Moresco et al., 2018; Raccosta et al., 2013).
These observations led to the proposal that combination therapies
associating the inhibition of 27-HC biosynthesis at the 27-hydroxylase
level (CYP27A1) and the use of ER\selectlanguage{greek}α \selectlanguage{english}and LXR antagonist could increase the
efficacy of treatments against ER(+)-BC (Nelson, 2018). In human,
clinical studies from the EPIC-Heidelberg cohort showed that high level
of circulating 27-HC were associated with a decreased BC risk in
postmenauposal women suggesting that 27-HC could prevent BC in these
cases (Le Cornet et al., 2020; Lu, Le Cornet, Sookthai, Johnson, Kaaks
\& Fortner, 2019). In contrast, ~strategies aiming to target 27HC
biosynthesis as well as its effectors, as proposed by Nelson et al,
should be limited to ER(+)-BC patients after an endocrine therapy to
protect them against BC recurrence .

Certain B-ring oxysterols such as 7-hydroperoxycholesterol and
5,6-epoxycholesterol (5,6-EC) (Fig.1A) have retained the attention of
researchers during the last century as they are major autoxidation and
photo-oxidation products of cholesterol (Smith, 1981; Smith \& Johnson,
1989), and are suspected to be alkylating substances and thus possibly
mutagenic and carcinogenic. These oxysterols were shown to induce
mutagenicity in some yeast strains (Ansari, Walker, Smart \& Smith,
1982; Smith, Smart \& Ansari, 1979) and chinese hamster V79 cells
(Chang, Jone, Trosko, Peterson \& Sevanian, 1988; Peterson, Peterson,
Spears, Trosko \& Sevanian, 1988; Sevanian \& Peterson, 1984; Sevanian
\& Peterson, 1986) \emph{in vitro.} However \emph{in vivo} tests failed
to show any carcinogenic potencies for 5,6-EC (el-Bayoumy et al., 1996).
Meanwhile, recent studies have revealed that 5,6-EC are involved in a
metabolic branch clearly involved in carcinogenesis and identified new
5,6-EC metabolites with opposite properties regarding BC oncogenesis. 1)
5,6\selectlanguage{greek}α-\selectlanguage{english}EC can give metabolites with antiproliferative and cancer cell
redifferentiation properties: 5,6\selectlanguage{greek}α-\selectlanguage{english}EC can be sulphated by the
sulfotransferase SULT2B1b in BC cells to produce
5,6\selectlanguage{greek}α-\selectlanguage{english}epoxy-cholesterol-3\selectlanguage{greek}β-\selectlanguage{english}sulfate (5,6-ECS) (Fig 1A) and 5,6-ECS was
shown to induce BC cell death and BC cell redifferentiation
activities\emph{in vitro} . In normal breast tissue 5,6\selectlanguage{greek}α-\selectlanguage{english}EC was shown to
be conjugated to histamine to give Dendrogenin A (Fig 1A,B), a steroidal
alkaloid that displays tumour suppressive properties (de Medina et al.,
2013; Poirot \& Silvente-Poirot, 2018; Segala et al., 2017;
Silvente-Poirot \& Poirot, 2014) (Fig 2A). 2) 5,6\selectlanguage{greek}α-\selectlanguage{english}EC can be transformed
into a tumour promoter: 5,6-ECs were shown to give a secondary
metabolite named oncosterone (6-oxo-cholestan-3,6- diol,
cholestan-3,6-diol-6-one, OCDO) (Fig 1A,C) with tumour promoter
properties in ER(+)BC and TN BC (Poirot, Soules, Mallinger, Dalenc \&
Silvente-Poirot, 2018; Silvente-Poirot, Dalenc \& Poirot, 2018; Voisin
et al., 2017) (Fig 2B).

\textbf{2. The 5,6-epoxycholesterols (5,6-ECs)}

5,6-ECs exist as two different diastereoisomers: the 5,6\selectlanguage{greek}α-\selectlanguage{english}EC and the
5,6\selectlanguage{greek}β-\selectlanguage{english}EC (Fig 1A). They stem from the mono-oxygenation of cholesterol on
its \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{5,6}-double bond. In biological systems they can
be produced as a mixture via a free radical lipid peroxidation process
(Iuliano, 2011; Poirot \& Silvente-Poirot, 2013; Porter, Xu \& Pratt,
2020; Yin, Xu \& Porter, 2011). The stereo-selective synthesis of
5,6\selectlanguage{greek}β-\selectlanguage{english}EC can be achieve using porphyrins, that mimic cytochrome p450
monooxygenases(Poirot \& Silvente-Poirot, 2013), and a stereoselective
biosynthesis of 5,6\selectlanguage{greek}α-\selectlanguage{english}EC by a yet unidentified cytochrome p450 have been
described in bovine adrenals (Watabe \& Sawahata, 1979). 5,6-EC are
known as major autoxidation and photo-oxidation products of cholesterol
(Smith, 1981). They are present in biological fluids and solid tissues
from mammals (Poirot \& Silvente-Poirot, 2013; Schroepfer, 2000).

The epoxide ring, is well known by chemists for its reactivity towards
nucleophilic groups and is widely used for this reason in organic
chemistry (Gorzynski Smith, 1984; Parker \& Isaacs, 1959). 5,6\selectlanguage{greek}α -\selectlanguage{english}EC was
suspected to be involved in skin photocarcinogenesis as it accumulated
in skin after UV exposure (Black \& Douglas, 1973; Black \& Lo, 1971; Lo
\& Black, 1973; Lo \& Black, 1972) and activated a ChEH activity to
accelerate the hydrolysis and elimination of 5,6-EC (Chan \& Black,
1974). Meanwhile, 5,6-EC were found to be chemically stable towards
nucleophiles ruling out a direct carcinogenic activity (Paillasse,
Saffon, Gornitzka, Silvente-Poirot, Poirot \& de Medina, 2012).

In the presence of a catalyst 5,6\selectlanguage{greek}α-\selectlanguage{english}EC was the only of the two 5,6-ECs
that reacts with nucleophiles to give a single product of addition with
a 5\selectlanguage{greek}α-\selectlanguage{english}6\selectlanguage{greek}β \selectlanguage{english}stereochemistry through a trans-diaxial ring opening (Paillasse,
Saffon, Gornitzka, Silvente-Poirot, Poirot \& de Medina, 2012; Poirot \&
Silvente-Poirot, 2013) (Fig 1B). 5,6-EC were reported to be modulators
of nuclear receptors such as Liver-X-Receptors \selectlanguage{greek}α \selectlanguage{english}(LXR\selectlanguage{greek}α\selectlanguage{english}) and \selectlanguage{greek}β \selectlanguage{english}(LXR\selectlanguage{greek}β\selectlanguage{english})
(Berrodin, Shen, Quinet, Yudt, Freedman \& Nagpal, 2010; Song, Hiipakka
\& Liao, 2001) showing that these oxysterols are biologically active
oxysterols. The metabolism of 5,6-EC has been previously reviewed and
showed that 5,6\selectlanguage{greek}α-\selectlanguage{english}EC and 5,6\selectlanguage{greek}β-\selectlanguage{english}EC were differentially metabolized in
mammals (Poirot \& Silvente-Poirot, 2013). Interestingly, 5,6\selectlanguage{greek}β-\selectlanguage{english}EC was
reported to accumulate in breast nipple fluids from patients with BC and
preneoplastic breast lesions supporting that the 5,6-EC metabolism was
linked to breast carcinogenesis (Gruenke, Wrensch, Petrakis, Miike,
Ernster \& Craig, 1987; Petrakis, Gruenke \& Craig, 1981; Wrensch et
al., 1989).

\textbf{3. Metabolism of 5,6-epoxycholesterols}

\textbf{3.1. The cholesterol epoxide hydrolase (ChEH).}

ChEH is responsible for the hydration of 5,6-ECs to give
cholestane-3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α,\selectlanguage{english}6\selectlanguage{greek}β-\selectlanguage{english}triol (CT)(Aringer \& Eneroth, 1974; Chan \& Black,
1974; Nashed, Michaud, Levin \& Jerina, 1985; Sevanian \& McLeod, 1986)
(Fig 1C). Like other epoxide hydrolases, ChEH was initially considered
as a type II detoxification enzyme to eliminate toxicants (Morisseau \&
Hammock, 2005). However, the biological functions epoxide hydrolases
have been extended to the control of the production of bioactive lipids
(Kodani \& Hammock, 2015; Morisseau, 2013; Newman, Morisseau \& Hammock,
2005) as observed for other families of type two detoxification enzymes
such as glutathione transferases (Board \& Menon, 2013). CT was shown to
display certain biological properties suggesting it may have a
physiological role in mammals. CT is an oxysterol that can induce
cytotoxicity \emph{in vivo} and \emph{in vitro} in normal and cancerous
cells (Carvalho, Silva, Moreira, Simoes \& Sa e Melo, 2010; Carvalho,
Silva, Moreira, Simoes \& Sa, 2011; Imai, Werthessen, Subramanyam,
LeQuesne, Soloway \& Kanisawa, 1980; Kandutsch, Chen \& Heiniger, 1978)
and hypocholesterolemia in animals (Aramaki, Kobayashi, Imai, Kikuchi,
Matsukawa \& Kanazawa, 1967). As opposed to other oxysterols such as
25-hydroxycholesterol, CT is not an inhibitor of the HMG-coA reductase,
the rate-controlling enzyme of the cholesterol pathway, (Cavenee,
Gibbons, Chen \& Kandutsch, 1979). This hypocholesterolemic property of
CT might be due to its inhibition of post-lanosterol cholesterogenic
enzymes such as DHCR7 (Witiak, Parker, Dempsey \& Ritter, 1971) and C4
lanosterol demethylase (Scallen, Dhar \& Loughran, 1971), and to a
modulation of the LXR\selectlanguage{greek}α/\selectlanguage{english}SREBP2 axis (Lin et al., 2013). CT stimulates
phospholipid biosynthesis and CTP-phosphocholine Cytidyltransferase in
mammalian cells (Mahfouz, Smith, Zhou \& Kummerow, 1996). CT was shown
to inhibit osteoblastic differentiation and the induction of bone marrow
stromal cell apoptosis (Liu, Yuan, Xu, Wang \& Zhang, 2005). CT was also
reported to suppress prostate cancer cell proliferation, migration and
invasion (Lin et al., 2013), and to inhibit voltage-gated sodium
channels (Tang et al., 2015; Tang et al., 2018). CT displays chaperone
properties for the Niemann-Pick C1 protein, which is an intracellular
sterol transporter (Ohgane, Karaki, Noguchi-Yachide, Dodo \& Hashimoto,
2014)and is also a blood marker for Niemann-Pick C1 Disease (Porter et
al., 2010). This illustrates the fact that CT displays certain
biological properties.

ChEH was characterized in 2010 as being carried out by a
multiproteinaceous hetero-oligomeric complex. This complex includes
enzymes involved in the late stages of cholesterol biosynthesis (de
Medina, Paillasse, Segala, Poirot \& Silvente-Poirot, 2010). ChEH is
composed of the 3\selectlanguage{greek}β-\selectlanguage{english}hydroxyterol-\selectlanguage{greek}Δ\selectlanguage{english}8-\selectlanguage{greek}Δ\selectlanguage{english}7-isomerase (D8D7I) and of the
3\selectlanguage{greek}β-\selectlanguage{english}hydroxyterol-\selectlanguage{greek}Δ\selectlanguage{english}7-reductase (DHCR7). D8D7I also known as the emopamyl
binding protein (EBP) is the catalytic subunit, and DHCR7 is a
regulatory subunit of ChEH (Fig 3A). ChEH was characterized as a
pharmacological target of the antitumour drugs tamoxifen and tesmilifene
(de Medina, Paillasse, Segala, Poirot \& Silvente-Poirot, 2010; de
Medina et al., 2013; Segala et al., 2013; Sola et al., 2013). The
different structural classes of drugs that inhibit the ChEH activity
were also found to be inhibitors of EBP and/or DHCR7 leading to the
accumulation of \selectlanguage{greek}Δ\selectlanguage{english}8- and/or \selectlanguage{greek}Δ\selectlanguage{english}7-cholesterol intermediates in the
biosynthesis of cholesterol (Kedjouar et al., 2004; Korade et al., 2016;
Segala et al., 2017; Silvente-Poirot \& Poirot, 2012) (Fig 3B).
\selectlanguage{greek}Δ\selectlanguage{english}8-cholesterol precursors such as zymostenol (cholest-8-ene-3\selectlanguage{greek}β-\selectlanguage{english}ol) were
shown to induce BC cell arrest in the G1 phase of the cell cycle (Payre
et al., 2008), lysosome biogenesis and autophagy (de Medina et al.,
2009; de Medina, Silvente-Poirot \& Poirot, 2009; Segala et al., 2017;
Sola et al., 2013). Inhibition of EBP has been shown to enhance
oligodendrocyte formation and myelination (Allimuthu et al., 2019;
Hubler et al., 2018) suggesting that \selectlanguage{greek}Δ\selectlanguage{english}8 intermediates in cholesterol
biosynthesis play a role in cell differentiation. Truncated
APC-selective inhibitors (TASIN) including EBP and DHCR7 inhibitors that
are under development for colorectal cancer treatment applications
(Cully, 2016; Theodoropoulos et al., 2020; Wang, Zhang, Morlock,
Williams, Shay \& De Brabander, 2019; Zhang, Kim, Luitel \& Shay, 2018;
Zhang et al., 2016). Interestingly, \selectlanguage{greek}Δ\selectlanguage{english}8- and \selectlanguage{greek}Δ\selectlanguage{english}7-sterols including
zymostenol are unstable sterols that are prone to autoxidation (Kedjouar
et al., 2004; Lamberson, Muchalski, McDuffee, Tallman, Xu \& Porter,
2017; Payre et al., 2008; Porter, Xu \& Pratt, 2020) to produce B-ring
oxysterols (Lamberson, Muchalski, McDuffee, Tallman, Xu \& Porter,
2017). On the other hand, B-ring oxysterols are known as endogenous ChEH
inhibitors (de Medina, Paillasse, Segala, Poirot \& Silvente-Poirot,
2010). This and the fact that EBP carries the ChEH activity, suggests
that it will be important to consider these parameters in the cell
differentiation effects of TASIN compounds especially if they are
developed for clinical applications. The recent elucidation of the EBP
structure complexed with tamoxifen (Long, Hassan, Thompson, McDonald,
Wang \& Li, 2019) by X-ray crystallography will encourage
structure-function studies to identify amino acid residues responsible
for ChEH activity.

In the clinic, Kaplan-Meier analyses of several BC transcriptome patient
datasets showed that EBP and DHCR7 expression were positively correlated
and were overexpressed in all BC subtypes compared to normal breast
tissue (Voisin et al., 2017). In addition, it was reported that high
levels of expression of EBP and DHCR7 were associated with a lower
survival rate of patients (Voisin et al., 2017). ChEH can therefore
represent an interesting target for the development of anticancer
compounds which deserves further exploration with regard to
redifferentiation therapies (Bizzarri, Giuliani, Cucina \& Minini,
2020).

\textbf{3.2. Oncosterone}

\textbf{3.2.1. Oncosterone an oncometabolite and a tumour promoter in
BC}

Studies on the 5,6-ECs metabolism in BC cell lines have shown that they
are metabolized into CT by ChEH and that CT was subsequently transformed
into a substance structurally characterized as the
6-oxo-cholestan-3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α-\selectlanguage{english}diol (OCDO, Oncosterone) (Fig 1C). Since ChEH is
known to be inhibited by antiproliferative substances (de Medina,
Paillasse, Segala, Poirot \& Silvente-Poirot, 2010; Silvente-Poirot \&
Poirot, 2012), and that CT has been reported to display certain
carcinogenic properties after oxydation (Cheng, Kang, Shih, Lo \& Wang,
2005). This suggested that oncosterone could promote BC cell
proliferation. Indeed, oncosterone was shown to dose-dependently
stimulate the proliferation of human and mouse ER(+) and TNBC cell lines
suggesting a receptor-mediated effect of oncosterone. Importantly,
oncosterone was shown to stimulate the growth of BC tumours \emph{in
vivo} . The inhibition of ChEH inhibited BC tumour growth, and
oncosterone addition rescued tumour growth. Together these data
establish that oncosterone is a tumour promoter. Thus blocking
oncosterone biosynthesis or neutralising oncosterone receptor(s)
provides a new rational for the pharmacological control of BC growth
which is extremely attractive especially for TNBC for which no targeted
therapy has been developed to date.

Only a few recent studies have included the dosage of oncosterone (OCDO)
in addition to other oxysterols (Dalenc et al., 2017; Iuliano et al.,
2015; Soules et al., 2017). Since 5,6-EC and CT have been linked to
several pathologies such as cancer and Niemann-Pick C1 disease, it will
be interesting to include the dosage of oncosterone in future studies to
determine its potential role in these pathologies

\selectlanguage{greek}\textbf{3.2.2. Oncosterone is produced by the 11β-hydroxysteroid
dehydrogenases type 2 (HSD11B2)}\selectlanguage{english}

Oncosterone (OCDO) is an enzymatic oxidation product of CT and is not an
autoxidation product of cholesterol (Poirot, Soules, Mallinger, Dalenc
\& Silvente-Poirot, 2018). Oncosterone was shown to be generated from CT
by the 3\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase type 2 (HSD11B2) (Voisin et al.,
2017). HSD11B2 is expressed in BC from patients and BC cell lines, while
weakly or not expressed in normal breast tissue, and the expression of
HSD11B2 paralleled OCDO levels, establishing that oncosterone is an
oncometabolite (Voisin et al., 2017). HSD11B2's primary function is the
inactivation of cortisol by oxidation to produce cortisone (Chapman,
Holmes \& Seckl, 2013). This reaction is known to be reverted by
HSD11B1, and HSD11B1 was earlier reported to reduce 7-ketocholesterol
into 7\selectlanguage{greek}β-\selectlanguage{english}hydroxycholesterol (Mitic et al., 2013). HSD11B1 was also found
to catalyse the conversion of OCDO into CT (Voisin et al., 2017).
HSD11B1 requires hexose-6-phosphate dehydrogenase (H6PD) for the
regeneration of its cofactor NADPH in order to achieve a reductase
activity. In the absence of H6PD, it was reported that HSD11B1 functions
as an oxidase like HSD11B2 (Odermatt \& Klusonova, 2015). HSD11B1 and
H6PD were found weakly or not expressed in the breast and in BC as well
as in BC cell lines showing that the eventual equilibrium of CT
oncosterone conversion was completely displaced in favour of oncosterone
production in BC (Voisin et al., 2017) .

The knock down of HSD11B2 in the ER(+)-BC cell line MCF-7 blocks the
endogenous production of oncosterone, cell proliferation and
clonogenicity \emph{in vitro} and tumour growth \emph{in vivo} .
Addition of exogenous oncosterone reverts the effect of HSD11B2 knock
down (Voisin et al., 2017). These data suggest that the oncosterone
biosynthesis enzyme HSD11B2 may constitute a target for the development
of inhibitors in BC treatment. Natural and synthetic inhibitors of
HSD11B2 have been described and it would be of interest to define their
impact on OCDO biosynthesis (Latif, Pardo, Hardy \& Morris, 2005;
Morris, Latif, Hardy \& Brem, 2007; Vitku et al., 2016; Zhou, Ye, Wu, Ye
\& Chen, 2017). An open question and an exciting challenge will be to
develop HSD11B2 inhibitors that block oncosterone biosynthesis with
little or no impact on the cortisol metabolism to limit side effects
such as hypertension. Such inhibitors could display anticancer
properties in all BC-subtypes, offering new targeted therapies for TN
BC. Such a hypothesis should deserve further exploration.

Analysis of HSD11B2 expression in patient BC samples showed that it was
significantly higher than in normal matched tissue. Kaplan-Meier
analyses of several BC transcriptome patient datasets showed that high
levels of HSD11B2 mRNA were associated with a poor prognosis. High
expression levels of both ChEH subunits and HSD11B2 were associated with
the risk highest risk of patient mortality (Voisin et al., 2017). These
data strongly support the development of ChEH and HSD11B2 inhibitors for
BC treatment.

\textbf{3.2.3. Oncosterone is a ligand and a modulator of nuclear
receptors.}

Glucocorticoid receptor (GR) and oxysterol receptors LXR have been
identified as oncosterone receptors. Oncosterone was shown to activate
the nuclearization of GR in MDA-MB231 TN BC cells as observed with
cortisol. Oncosterone was found to antagonize cortisol in its
stimulation of the expression of two GR-dependent SGK1 and MKP1 genes.
Gene reporter assays further confirmed that oncosterone did not modulate
other oxysterol receptors such as RORs or FXR (Voisin et al., 2017), and
binding assays showed that oncosterone interacted with LXRs and GR. GR
knock down in BC cell lines induced a loss of the proliferative effects
of oncosterone. A similar effect was also observed with GR ligands such
as mifepristone (RU38486) and dexamethasone showing that GR mediates
oncosterone mitogenicity. Structural differences between cortisol and
oncosterone are key elements that drive GR activation differently in
terms of gene regulation and cell proliferation. This suggests that
conformational modifications of GR complexes will affect differently the
recruitment of co-regulators, GR dimerization states (Kadmiel \&
Cidlowski, 2013; Vandewalle, Luypaert, De Bosscher \& Libert, 2018) and
even GR heterodimerization with other nuclear receptors (De Bosscher,
Desmet, Clarisse, Estebanez-Perpina \& Brunsveld, 2020).

In contrast, the LXR knock down did not affect OCDO-mediated BC cell
proliferation (Voisin et al., 2017). Since LXR was found to control 27HC
invasiveness, it was postulated that LXR could mediate the pro-invasive
effects of oncosterone. Further investigations will be required to show
that genetic and pharmacological inhibition of LXR can block the effects
of oncosterone. These observations, in addition to the links that have
already established between GR, glucocorticoids and BC (Kanai et al.,
2020; Obradovic et al., 2019; Perez Kerkvliet et al., 2020;
Tonsing-Carter et al., 2019), highlight the pharmacological interest of
GR targeting for the control of BC development. The discovery of
oncosterone as an endogenous GR ligand in BC will add a new rational to
consider the implication of GR in BC development. Together these data
underline the pharmacological importance of GR and LXR as effector of
oncosterone and give a new rational for the targeted therapy of BC (Fig
3).

\textbf{3.3. Dendrogenin A (DDA)}

\textbf{3.3.1. DDA is enzymatically produced in normal tissues}

Researchers have hypothesized that 5,6-EC could be metabolized into
inducers of cell-differentiation such as dendrogenin A for several
reasons: 1) ChEH contains the microsomal anti-oestrogen binding site
(AEBS) which controls BC cell death and differentiation, showing that
5,6-EC and 5,6-EC metabolites mediated BC cell differentiation(de Medina
et al., 2009; Payre et al., 2008; Segala et al., 2013; Silvente-Poirot
\& Poirot, 2012); 2) ChEH subunits EBP and DHCR7 are required for human
development programs (Herman, 2003; Porter \& Herman, 2011) opening up
the possibilities that ChEH activity and 5,6-EC metabolites could
contribute to development programs as inducers of cell differentiation;
3) at the chemical level, 5,6\selectlanguage{greek}α-\selectlanguage{english}EC was found to be reactive towards
nucleophilic groups such as mercaptant and amines only in the presence
of a catalyst and give a single conjugated stereoisomer amongst multiple
possibilities(Silvente-Poirot, de Medina, Record \& Poirot, 2016). This
supported the possible existence of a new metabolic branch based on
5,6\selectlanguage{greek}α-\selectlanguage{english}EC stereospecific conjugation since homochirality is a common
occurence in life (Blackmond, 2019; Fujii \& Saito, 2004); 4) the AEBS
binds 5,6-EC and nucleophilic substances such as histamine or polyamines
opening up a possibility of conjugation reaction at the ChEH level
(Leignadier, Dalenc, Poirot \& Silvente-Poirot, 2017). The chemical
synthesis of 5,6\selectlanguage{greek}α-\selectlanguage{english}EC conjugation products with certain biogenic amines
including histamine and polyamine has been performed(de Medina,
Paillasse, Payre, Silvente-Poirot \& Poirot, 2009). As expected, 5,6\selectlanguage{greek}α-\selectlanguage{english}EC
conjugates displayed strong cell differentiation properties \emph{in
vitro} (de Medina, Paillasse, Payre, Silvente-Poirot \& Poirot, 2009).
Amongst these conjugates, the histaminic adduct called dendrogenin A
(DDA), was found to induce cell differentiation properties in
pluripotent undifferentiated cells (de Medina, Paillasse, Payre,
Silvente-Poirot \& Poirot, 2009) and normal progenitor cells (Khalifa,
de Medina, Erlandsson, El-Seedi, Silvente-Poirot \& Poirot, 2014). DDA
was reported to induce cell differentiation and death in mouse and human
cells of various tissue origins (Bauriaud-Mallet et al., 2019; de
Medina, Paillasse, Payre, Silvente-Poirot \& Poirot, 2009; de Medina et
al., 2013). More specifically DDA was found to induce BC cell
re-differentiation to produce breast epithelial-like cells
(Bauriaud-Mallet et al., 2019; de Medina, Paillasse, Payre,
Silvente-Poirot \& Poirot, 2009; de Medina et al., 2013). The production
of chemical tools such as radio- and deuterium-labelled DDA, and the
development of analytical methods has made possible the detection and
quantification of DDA in mammalian tissues which has led to the
demonstration that DDA exists in mammals (de Medina et al., 2013; Soules
et al., 2019). DDA can be formed in mammalian tissue from 5,6\selectlanguage{greek}α-\selectlanguage{english}EC and
histamine and this reaction was shown to require a proteinaceous enzyme
that remains to be identified (de Medina et al., 2013). Cultured normal
breast epithelial cells produced DDA, while DDA was not detectable in BC
cell lines (de Medina et al., 2013). This suggests that a deregulation
in DDA metabolism could occur during oncogenesis and it may result from
an alteration of the DDA metabolism in epithelial cells. This was
confirmed in patients by comparing DDA levels in BC tumours and normal
adjacent tissues (de Medina et al., 2013). Interestingly, thanks to the
improvement of analytical methods for DDA quantification, dendrogenin B
which is a 5,6\selectlanguage{greek}α-\selectlanguage{english}EC conjugate of spermidine with neurodifferentiation and
neurone regeneration properties (Dalenc, Poirot \& Silvente-Poirot,
2015; de Medina, Paillasse, Payre, Silvente-Poirot \& Poirot, 2009;
Fransson, de Medina, Paillasse, Silvente-Poirot, Poirot \& Ulfendahl,
2015; Khalifa, de Medina, Erlandsson, El-Seedi, Silvente-Poirot \&
Poirot, 2014) was found to exist as a mammalian metabolite (Soules et
al., 2019). This showed that a new metabolic branch existed in the
cholesterol pathway and this branch is centered around 5,6\selectlanguage{greek}α-\selectlanguage{english}EC
conjugation and produces inducers of cell differentiation.

\textbf{3.3.2. DDA is an oxysterol with chemopreventive and
oncosuppressive properties}

DDA is a mammalian metabolite present in mammalian tissue including the
breast, the levels of which decreased drastically during oncogenesis.
Tested on preclinical models of BC in immunocompetent mice, DDA was
found to inhibit the growth of aggressive syngeneic tumours at low doses
(0.035 \selectlanguage{greek}µ\selectlanguage{english}g/kg). This effect was observed on a chemopreventive setting and
after 10 days of post implantation of BC cells in mice. Analysis of
tumours from treated animals showed that cancer cells harboured features
of normal epithelial mammary cells, and that tumours were infiltrated
with T lymphocytes and dendritic cells (de Medina et al., 2013). This
data suggested that the immune system could contribute to the
anti-cancer action of DDA. This was further supported by the fact that
the same treatment with the same dose of DDA was inefficient to cure or
prevent BC development in immunodepressed nude mice (Sandrine
Silvente-Poirot et al, unpublished observations). It has been reported
that DDA inhibits ChEH (de Medina et al., 2013) and OCDO production in
BC cells (Voisin et al., 2017). This explains the control of OCDO
mitogenicity \emph{in vitro} and \emph{in vivo} in BC tumors implanted
in immunocompromised mice at DDA doses that induced ChEH inhibition
(Voisin et al., 2017). This confirmed the existence of a metabolic
balance involving on 5,6-EC and controlled by ChEH (Silvente-Poirot \&
Poirot, 2014; Voisin et al., 2017). Whether the DDA/oncosterone ratio
reflects a pre-cancerous or a cancerous situation in the breast deserves
further investigations. Together these data suggest that the use of DDA
could be an interesting alternative strategy for BC treatment through a
metabolic deficiency complementation- and redifferentiation-therapy.

\textbf{3.3.3. DDA is an LXR modulator that induces lethal autophagy in
cancer cells.}

DDA was found to induce cell death and differentiation in mouse and
human cancer cells. Further work was done to study the molecular
mechanisms involved in DDA cytotoxicity. It was found that DDA induced
cytotoxicity in cultured cancer cells in a dose- and time-dependent
manner suggesting the implication of a receptor, death was not
apoptotic, and required gene expression and protein neosynthesis (Segala
et al., 2017), as observed with other ChEH inhibitors (de Medina et al.,
2009; Leignadier, Dalenc, Poirot \& Silvente-Poirot, 2017; Payre et al.,
2008). However, as opposed to other ChEH inhibitors, DDA cytoxicity was
not inhibited by anti-oxidants because 5,6-EC do not accumulate and are
not second messengers of DDA (de Medina et al., 2009; Leignadier,
Dalenc, Poirot \& Silvente-Poirot, 2017; Payre et al., 2008; Segala et
al., 2017). Studies on the DDA molecular mechanism of cytotoxicity
showed that it induced lethal autophagy (Segala et al., 2017) as opposed
to other ChEH inhibitors that induced a protective autophagy
(Leignadier, Dalenc, Poirot \& Silvente-Poirot, 2017). ChEH contributed
to autophagy through the accumulation of pro-autophagic \selectlanguage{greek}Δ\selectlanguage{english}8-sterols due
the inhibition of its EBP subunit (de Medina, Silvente-Poirot \& Poirot,
2009; Silvente-Poirot, Segala, Poirot \& Poirot, 2018). DDA was found to
be a ligand of LXR\selectlanguage{greek}α \selectlanguage{english}and LXR\selectlanguage{greek}β \selectlanguage{english}receptors as opposed to other ChEH
inhibitors (Segala et al., 2013). Genetic and pharmacological evidences
has been given to confirm that LXR\selectlanguage{greek}β \selectlanguage{english}was required for lethal autophagy.
Although DDA was a poor modulator of canonical LXR-dependent genes, it
activated via LXR\selectlanguage{greek}β \selectlanguage{english}the transcription of genes encoding master regulators
of lysosome biogenesis and autophagy such as the transcription factor EB
(TFEB) (Segala et al., 2017). DDA is, to our knowledge, the first
example of an LXR ligand that induces TFEB expression. Such an effect
has not been reported to date by other cytotoxic natural LXR ligands
belonging to the oxysterols family. It was further established that DDA
did not modulate other common nuclear receptors establishing its
selectivity to LXRs (Segala et al., 2017).

It was next established that DDA, at cytotoxic doses, actively inhibit
the growth of human and mouse tumours implanted into immunocompromized
mice with different administration modes, including primary tumours from
patients (Segala et al., 2017). Knock down of LXR receptors in cancer
cells using small interfering RNA (siRNA) or single hairpin RNA (shRNA)
approaches strongly impaired DDA induction of autophagy and its
anticancer activities \emph{in vitro} and \emph{in vivo} (Segala et al.,
2017). This showed that LXR\selectlanguage{greek}β \selectlanguage{english}was required to control the anticancer
activity of DDA. The particular cell death induced by DDA compared to
conventional ChEH inhibitors and LXR modulators is probably due to its
specific LXR\selectlanguage{greek}β-\selectlanguage{english}dependent regulation of gene expression and induction of
\selectlanguage{greek}Δ\selectlanguage{english}8-sterols accumulation as discussed earlier (Poirot \& Silvente-Poirot,
2018). Moreover, this suggests that the control of autophagy might be a
specific physiological function of the LXR\selectlanguage{greek}β \selectlanguage{english}isoform when activated by
specific ligands such as DDA. This data shows the importance of LXR in
cancer cells as targets for anticancer strategies. Due to the
established importance of LXR in the tumour micro-enrironment (Ma \&
Nelson, 2019) and in the control of the immune response (Fessler, 2016),
it will be interesting to determine how autophagy and LXR from immune
cells can contribute to the anti-tumour action of DDA observed in
immunocompetent mice (de Medina et al., 2013). It would be of interest
to determine the impact of combination treatments of DDA with other
chemotherapeutic agents acting through different molecular mechanisms,
and in particular with drugs that have a limited therapeutic outcome or
resistance through the induction of protective autophagy.

\textbf{4. Conclusion:}

We report herein that 5,6-ECs are at the center of a newly discovered
metabolic branch that controls carcinogenesis (Fig 4). The
identification of oncosterone, its biosynthetic pathway and its
effectors, highlights the existence of interesting targets for the
development of anticancer drugs applicable for TNBC treatment (Fig 5).
This is reminiscent of the development of targeted therapeutic
strategies against the tumour promoter 17\selectlanguage{greek}β-\selectlanguage{english}oestradiol for ER(+)-BC
treatment (Simpson \& Santen, 2015). On the other hand, studies of the
5,6\selectlanguage{greek}α-\selectlanguage{english}EC metabolism in normal tissues led to the identification of DDA.
Surprisingly DDA was found to display tumour suppressor properties on BC
through the activation of cell differentiation and death programs, and
the inhibition of oncosterone biosynthesis, highlighting the existence
of a metabolic balance between the tumour promoteur oncosterone and the
tumour suppressor DDA. This opens up new options for the development of
new anticancer agents targeting the oncosterone pathway in BC and new
strategies for the chemoprevention of BC. This also provides a new
rational at the molecular level to study potential relationships that
may exist between cholesterol, diet and BC oncogenesis programs
(Silvente-Poirot, Dalenc \& Poirot, 2018).

\textbf{References:}

Allimuthu D, Hubler Z, Najm FJ, Tang H, Bederman I, Seibel W\emph{, et
al.} (2019). Diverse Chemical Scaffolds Enhance Oligodendrocyte
Formation by Inhibiting CYP51, TM7SF2, or EBP. Cell Chem Biol
26\textbf{:} 593-599 e594.

Ansari GA, Walker RD, Smart VB, \& Smith LL (1982). Further
investigations of mutagenic cholesterol preparations. Food Chem Toxicol
20\textbf{:} 35-41.

Aramaki Y, Kobayashi T, Imai Y, Kikuchi S, Matsukawa T, \& Kanazawa K
(1967). Biological studies of cholestane-3beta,5alpha,6beta-triol and
its derivatives. 1. Hypocholesterolemic effects in rabbits, chickens and
rats on atherogenic diets. J Atheroscler Res 7\textbf{:} 653-669.

Aringer L, \& Eneroth P (1974). Formation and metabolism in vitro of
5,6-epoxides of cholesterol and beta-sitosterol. J Lipid Res
15\textbf{:} 389-398.

Baek AE, Yu YA, He S, Wardell SE, Chang CY, Kwon S\emph{, et al.}(2017).
The cholesterol metabolite 27 hydroxycholesterol facilitates breast
cancer metastasis through its actions on immune cells. Nat Commun
8\textbf{:} 864.

Bauriaud-Mallet M, Vija-Racaru L, Brillouet S, Mallinger A, de Medina P,
Rives A\emph{, et al.} (2019). The cholesterol-derived metabolite
dendrogenin A functionally reprograms breast adenocarcinoma and
undifferentiated thyroid cancer cells. J Steroid Biochem Mol Biol
192\textbf{:} 105390.

Berrodin TJ, Shen Q, Quinet EM, Yudt MR, Freedman LP, \& Nagpal S
(2010). Identification of 5alpha, 6alpha-epoxycholesterol as a novel
modulator of liver X receptor activity. Mol Pharmacol
78\textbf{:}1046-1058.

Bizzarri M, Giuliani A, Cucina A, \& Minini M (2020). Redifferentiation
therapeutic strategies in cancer. Drug Discov Today 25\textbf{:}731-738.

Black HS, \& Douglas DR (1973). Formation of a carcinogen of natural
origin in the etiology of ultraviolet light-induced carcinogenesis.
Cancer Res 33\textbf{:} 2094-2096.

Black HS, \& Lo WB (1971). Formation of a carcinogen in human skin
irradiated with ultraviolet light. Nature 234\textbf{:} 306-308.

Blackmond DG (2019). The Origin of Biological Homochirality. Cold Spring
Harb Perspect Biol 11\textbf{:} a002147.

Board PG, \& Menon D (2013). Glutathione transferases, regulators of
cellular metabolism and physiology. Biochim Biophys Acta
1830\textbf{:}3267-3288.

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, \& Jemal A
(2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence
and mortality worldwide for 36 cancers in 185 countries. CA Cancer J
Clin 68\textbf{:} 394-424.

Carvalho JF, Silva MM, Moreira JN, Simoes S, \& Sa e Melo ML (2010).
Sterols as anticancer agents: synthesis of ring-B oxygenated steroids,
cytotoxic profile, and comprehensive SAR analysis. J Med Chem
53\textbf{:} 7632-7638.

Carvalho JF, Silva MM, Moreira JN, Simoes S, \& Sa EMML (2011).
Selective cytotoxicity of oxysterols through structural modulation on
rings A and B. Synthesis, in vitro evaluation, and SAR. J Med Chem
54\textbf{:} 6375-6393.

Cavenee WK, Gibbons GF, Chen HW, \& Kandutsch AA (1979). Effects of
various oxygenated sterols on cellular sterol biosynthesis in Chinese
hamster lung cells resistant to 25-hydroxycholesterol. Biochim Biophys
Acta 575\textbf{:} 255-265.

Chan JT, \& Black HS (1974). Skin carcinogenesis:
cholesterol-5alpha,6alpha-epoxide hydrase activity in mouse skin
irradiated with ultraviolet light. Science 186\textbf{:} 1216-1217.

Chang CC, Jone C, Trosko JE, Peterson AR, \& Sevanian A (1988). Effect
of cholesterol epoxides on the inhibition of intercellular communication
and on mutation induction in Chinese hamster V79 cells. Mutat Res
206\textbf{:} 471-478.

Chapman K, Holmes M, \& Seckl J (2013). 11beta-hydroxysteroid
dehydrogenases: intracellular gate-keepers of tissue glucocorticoid
action. Physiol Rev 93\textbf{:} 1139-1206.

Cheng YW, Kang JJ, Shih YL, Lo YL, \& Wang CF (2005).
Cholesterol-3-beta, 5-alpha, 6-beta-triol induced genotoxicity through
reactive oxygen species formation. Food Chem Toxicol
43\textbf{:}617-622.

Cully M (2016). Anticancer drugs: Exploiting a weakness in colorectal
cancers. Nat Rev Drug Discov 15\textbf{:} 820-821.

Dalenc F, Iuliano L, Filleron T, Zerbinati C, Voisin M, Arellano
C\emph{, et al.} (2017). Circulating oxysterol metabolites as potential
new surrogate markers in patients with hormone receptor-positive breast
cancer: Results of the OXYTAM study. J Steroid Biochem Mol Biol
169\textbf{:} 210-218.

Dalenc F, Poirot M, \& Silvente-Poirot S (2015). Dendrogenin A: A
Mammalian Metabolite of Cholesterol with Tumor Suppressor and
Neurostimulating Properties. Curr Med Chem 22\textbf{:} 3533-3549.

Danhier P, Banski P, Payen VL, Grasso D, Ippolito L, Sonveaux P\emph{,
et al.} (2017). Cancer metabolism in space and time: Beyond the Warburg
effect. Biochim Biophys Acta Bioenerg 1858\textbf{:} 556-572.

De Bosscher K, Desmet SJ, Clarisse D, Estebanez-Perpina E, \& Brunsveld
L (2020). Nuclear receptor crosstalk - defining the mechanisms for
therapeutic innovation. Nat Rev Endocrinol in press.

de Medina P, Paillasse MR, Payre B, Silvente-Poirot S, \& Poirot M
(2009). Synthesis of new alkylaminooxysterols with potent cell
differentiating activities: identification of leads for the treatment of
cancer and neurodegenerative diseases. J Med Chem 52\textbf{:}7765-7777.

de Medina P, Paillasse MR, Segala G, Poirot M, \& Silvente-Poirot S
(2010). Identification and pharmacological characterization of
cholesterol-5,6-epoxide hydrolase as a target for tamoxifen and AEBS
ligands. Proc Natl Acad Sci U S A 107\textbf{:} 13520-13525.

de Medina P, Paillasse MR, Segala G, Voisin M, Mhamdi L, Dalenc F\emph{,
et al.} (2013). Dendrogenin A arises from cholesterol and histamine
metabolism and shows cell differentiation and anti-tumour properties.
Nat Commun 4\textbf{:} 1840.

de Medina P, Payre B, Boubekeur N, Bertrand-Michel J, Terce F,
Silvente-Poirot S\emph{, et al.} (2009). Ligands of the
antiestrogen-binding site induce active cell death and autophagy in
human breast cancer cells through the modulation of cholesterol
metabolism. Cell Death Differ 16\textbf{:} 1372-1384.

de Medina P, Silvente-Poirot S, \& Poirot M (2009). Tamoxifen and AEBS
ligands induced apoptosis and autophagy in breast cancer cells through
the stimulation of sterol accumulation. Autophagy 5\textbf{:} 1066-1067.

el-Bayoumy K, Ji BY, Upadhyaya P, Chae YH, Kurtzke C, Rivenson A\emph{,
et al.} (1996). Lack of tumorigenicity of cholesterol epoxides and
estrone-3,4-quinone in the rat mammary gland. Cancer Res
56\textbf{:}1970-1973.

Fessler MB (2016). The Intracellular Cholesterol Landscape: Dynamic
Integrator of the Immune Response. Trends Immunol 37\textbf{:} 819-830.

Fransson A, de Medina P, Paillasse MR, Silvente-Poirot S, Poirot M, \&
Ulfendahl M (2015). Dendrogenin A and B two new steroidal alkaloids
increasing neural responsiveness in the deafened guinea pig. Front Aging
Neurosci 7\textbf{:} 145.

Fujii N, \& Saito T (2004). Homochirality and life. Chem Rec
4\textbf{:}267-278.

Garcia-Estevez L, \& Moreno-Bueno G (2019). Updating the role of obesity
and cholesterol in breast cancer. Breast Cancer Res 21\textbf{:} 35.

Global Burden of Disease Cancer C, Fitzmaurice C, Abate D, Abbasi N,
Abbastabar H, Abd-Allah F\emph{, et al.} (2019). Global, Regional, and
National Cancer Incidence, Mortality, Years of Life Lost, Years Lived
With Disability, and Disability-Adjusted Life-Years for 29 Cancer
Groups, 1990 to 2017: A Systematic Analysis for the Global Burden of
Disease Study. JAMA Oncol 5\textbf{:} 1749-1768.

Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn
HJ\emph{, et al.} (2011). Strategies for subtypes--dealing with the
diversity of breast cancer: highlights of the St. Gallen International
Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann
Oncol 22\textbf{:} 1736-1747.

Gorzynski Smith J (1984). Synthetically Useful Reactions of Epoxides.
Synthesis 1984\textbf{:} 629-656.

Grouleff J, Irudayam SJ, Skeby KK, \& Schiott B (2015). The influence of
cholesterol on membrane protein structure, function, and dynamics
studied by molecular dynamics simulations. Biochim Biophys Acta
1848\textbf{:} 1783-1795.

Gruenke LD, Wrensch MR, Petrakis NL, Miike R, Ernster VL, \& Craig JC
(1987). Breast fluid cholesterol and cholesterol epoxides: relationship
to breast cancer risk factors and other characteristics. Cancer Res
47\textbf{:} 5483-5487.

Herman GE (2003). Disorders of cholesterol biosynthesis: prototypic
metabolic malformation syndromes. Hum Mol Genet 12 Spec No
1\textbf{:}R75-88.

Huang B, Song B-l, \& Xu C (2020). Cholesterol metabolism in cancer:
mechanisms and therapeutic opportunities. Nat Metab 2\textbf{:} 132-141.

Hubler Z, Allimuthu D, Bederman I, Elitt MS, Madhavan M, Allan KC\emph{,
et al.} (2018). Accumulation of 8,9-unsaturated sterols drives
oligodendrocyte formation and remyelination. Nature
560\textbf{:}372-376.

Imai H, Werthessen NT, Subramanyam V, LeQuesne PW, Soloway AH, \&
Kanisawa M (1980). Angiotoxicity of oxygenated sterols and possible
precursors. Science 207\textbf{:} 651-653.

Iuliano L (2011). Pathways of cholesterol oxidation via non-enzymatic
mechanisms. Chem Phys Lipids 164\textbf{:} 457-468.

Iuliano L, Crick PJ, Zerbinati C, Tritapepe L, Abdel-Khalik J, Poirot
M\emph{, et al.} (2015). Cholesterol metabolites exported from human
brain. Steroids 99\textbf{:} 189-193.

Jordan VC, \& Brodie AM (2007). Development and evolution of therapies
targeted to the estrogen receptor for the treatment and prevention of
breast cancer. Steroids 72\textbf{:} 7-25.

Kadmiel M, \& Cidlowski JA (2013). Glucocorticoid receptor signaling in
health and disease. Trends Pharmacol Sci 34\textbf{:} 518-530.

Kanai A, McNamara KM, Iwabuchi E, Miki Y, Onodera Y, Guestini F\emph{,
et al.} (2020). Significance of glucocorticoid signaling in
triple-negative breast cancer patients: a newly revealed interaction
with androgen signaling. Breast Cancer Res Treat 180\textbf{:} 97-110.

Kandutsch AA, Chen HW, \& Heiniger HJ (1978). Biological activity of
some oxygenated sterols. Science 201\textbf{:} 498-501.

Kedjouar B, de Medina P, Oulad-Abdelghani M, Payre B, Silvente-Poirot S,
Favre G\emph{, et al.} (2004). Molecular characterization of the
microsomal tamoxifen binding site. J Biol Chem 279\textbf{:}34048-34061.

Khalifa SA, de Medina P, Erlandsson A, El-Seedi HR, Silvente-Poirot S,
\& Poirot M (2014). The novel steroidal alkaloids dendrogenin A and B
promote proliferation of adult neural stem cells. Biochem Biophys Res
Commun 446\textbf{:} 681-686.

Kodani SD, \& Hammock BD (2015). The 2014 Bernard B. Brodie award
lecture-epoxide hydrolases: drug metabolism to therapeutics for chronic
pain. Drug Metab Dispos 43\textbf{:} 788-802.

Korade Z, Kim HY, Tallman KA, Liu W, Koczok K, Balogh I\emph{, et
al.}(2016). The Effect of Small Molecules on Sterol Homeostasis:
Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and
Human Fibroblasts. J Med Chem 59\textbf{:} 1102-1115.

Kuzu OF, Noory MA, \& Robertson GP (2016). The Role of Cholesterol in
Cancer. Cancer Res 76\textbf{:} 2063-2070.

Lamberson CR, Muchalski H, McDuffee KB, Tallman KA, Xu L, \& Porter NA
(2017). Propagation rate constants for the peroxidation of sterols on
the biosynthetic pathway to cholesterol. Chem Phys Lipids
207\textbf{:}51-58.

Latif SA, Pardo HA, Hardy MP, \& Morris DJ (2005). Endogenous selective
inhibitors of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2 of
adrenal origin. Mol Cell Endocrinol 243\textbf{:} 43-50.

Le Cornet C, Walter B, Sookthai D, Johnson TS, Kuhn T, Herpel E\emph{,
et al.} (2020). Circulating 27-hydroxycholesterol and breast cancer
tissue expression of CYP27A1, CYP7B1, LXR-beta, and ERbeta: results from
the EPIC-Heidelberg cohort. Breast Cancer Res 22\textbf{:} 23.

Leignadier J, Dalenc F, Poirot M, \& Silvente-Poirot S (2017). Improving
the efficacy of hormone therapy in breast cancer: The role of
cholesterol metabolism in SERM-mediated autophagy, cell differentiation
and death. Biochem Pharmacol 144\textbf{:} 18-28.

Lin CY, Huo C, Kuo LK, Hiipakka RA, Jones RB, Lin HP\emph{, et
al.}(2013). Cholestane-3beta, 5alpha, 6beta-triol suppresses
proliferation, migration, and invasion of human prostate cancer cells.
PLoS One 8\textbf{:} e65734.

Liu H, Yuan L, Xu S, Wang K, \& Zhang T (2005).
Cholestane-3beta,5alpha,6beta-triol inhibits osteoblastic
differentiation and promotes apoptosis of rat bone marrow stromal cells.
J Cell Biochem 96\textbf{:} 198-208.

Lo W, \& Black HS (1973). Inhibition of carcinogen formation in skin
irradiated with ultraviolet light. Nature 246\textbf{:} 489-491.

Lo WB, \& Black HS (1972). Formation of cholesterol-derived
photoproducts in human skin. J Invest Dermatol 58\textbf{:} 278-283.

Long T, Hassan A, Thompson BM, McDonald JG, Wang J, \& Li X (2019).
Structural basis for human sterol isomerase in cholesterol biosynthesis
and multidrug recognition. Nat Commun 10\textbf{:} 2452.

Lu DL, Le Cornet C, Sookthai D, Johnson TS, Kaaks R, \& Fortner RT
(2019). Circulating 27-Hydroxycholesterol and Breast Cancer Risk:
Results From the EPIC-Heidelberg Cohort. J Natl Cancer Inst
111\textbf{:} 365-371.

Luo J, Yang H, \& Song BL (2020). Mechanisms and regulation of
cholesterol homeostasis. Nat Rev Mol Cell Biol 21\textbf{:} 225-245.

Ma L, \& Nelson ER (2019). Oxysterols and nuclear receptors. Mol Cell
Endocrinol 484\textbf{:} 42-51.

Mahfouz MM, Smith TL, Zhou Q, \& Kummerow FA (1996). Cholestane-3 beta,
5 alpha, 6 beta-triol stimulates phospholipid synthesis and
CTP-phosphocholine cytidyltransferase in cultured LLC-PK cells. Int J
Biochem Cell Biol 28\textbf{:} 739-750.

Mitic T, Shave S, Semjonous N, McNae I, Cobice DF, Lavery GG\emph{, et
al.} (2013). 11beta-Hydroxysteroid dehydrogenase type 1 contributes to
the balance between 7-keto- and 7-hydroxy-oxysterols in vivo. Biochem
Pharmacol 86\textbf{:} 146-153.

Moresco MA, Raccosta L, Corna G, Maggioni D, Soncini M, Bicciato
S\emph{, et al.} (2018). Enzymatic Inactivation of Oxysterols in Breast
Tumor Cells Constraints Metastasis Formation by Reprogramming the
Metastatic Lung Microenvironment. Front Immunol 9\textbf{:} 2251.

Morisseau C (2013). Role of epoxide hydrolases in lipid metabolism.
Biochimie 95\textbf{:} 91-95.

Morisseau C, \& Hammock BD (2005). Epoxide hydrolases: mechanisms,
inhibitor designs, and biological roles. Annu Rev Pharmacol Toxicol
45\textbf{:} 311-333.

Morris DJ, Latif SA, Hardy MP, \& Brem AS (2007). Endogenous inhibitors
(GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2:
derivatives of adrenally produced corticosterone and cortisol. J Steroid
Biochem Mol Biol 104\textbf{:} 161-168.

Nashed NT, Michaud DP, Levin W, \& Jerina DM (1985). Properties of liver
microsomal cholesterol 5,6-oxide hydrolase. Arch Biochem Biophys
241\textbf{:} 149-162.

Nelson ER (2018). The significance of cholesterol and its metabolite,
27-hydroxycholesterol in breast cancer. Mol Cell Endocrinol
466\textbf{:} 73-80.

Nelson ER, Wardell SE, Jasper JS, Park S, Suchindran S, Howe MK\emph{,
et al.} (2013). 27-Hydroxycholesterol links hypercholesterolemia and
breast cancer pathophysiology. Science 342\textbf{:} 1094-1098.

Nes WD (2011). Biosynthesis of cholesterol and other sterols. Chem Rev
111\textbf{:} 6423-6451.

Newman JW, Morisseau C, \& Hammock BD (2005). Epoxide hydrolases: their
roles and interactions with lipid metabolism. Prog Lipid Res
44\textbf{:} 1-51.

Obradovic MMS, Hamelin B, Manevski N, Couto JP, Sethi A, Coissieux
MM\emph{, et al.} (2019). Glucocorticoids promote breast cancer
metastasis. Nature 567\textbf{:} 540-544.

Odermatt A, \& Klusonova P (2015). 11\selectlanguage{greek}β-\selectlanguage{english}Hydroxysteroid dehydrogenase 1:
Regeneration of active glucocorticoids is only part of the story. J
Steroid Biochem Mol Biol 151\textbf{:} 85-92.

Ohgane K, Karaki F, Noguchi-Yachide T, Dodo K, \& Hashimoto Y (2014).
Structure-activity relationships of oxysterol-derived pharmacological
chaperones for Niemann-Pick type C1 protein. Bioorg Med Chem Lett
24\textbf{:} 3480-3485.

Paillasse MR, Saffon N, Gornitzka H, Silvente-Poirot S, Poirot M, \& de
Medina P (2012). Surprising unreactivity of cholesterol-5,6-epoxides
towards nucleophiles. J Lipid Res 53\textbf{:} 718-725.

Parker RE, \& Isaacs NS (1959). Mechanisms of epoxide reactions. Chem
Rev 59\textbf{:} 737-799.

Pavlova NN, \& Thompson CB (2016). The Emerging Hallmarks of Cancer
Metabolism. Cell Metab 23\textbf{:} 27-47.

Payre B, de Medina P, Boubekeur N, Mhamdi L, Bertrand-Michel J, Terce
F\emph{, et al.} (2008). Microsomal antiestrogen-binding site ligands
induce growth control and differentiation of human breast cancer cells
through the modulation of cholesterol metabolism. Mol Cancer Ther
7\textbf{:} 3707-3718.

Perez Kerkvliet C, Dwyer AR, Diep CH, Oakley RH, Liddle C, Cidlowski
JA\emph{, et al.} (2020). Glucocorticoid receptors are required
effectors of TGFbeta1-induced p38 MAPK signaling to advanced cancer
phenotypes in triple-negative breast cancer. Breast Cancer Res
22\textbf{:} 39.

Peterson AR, Peterson H, Spears CP, Trosko JE, \& Sevanian A (1988).
Mutagenic characterization of cholesterol epoxides in Chinese hamster
V79 cells. Mutat Res 203\textbf{:} 355-366.

Petrakis NL, Gruenke LD, \& Craig JC (1981). Cholesterol and cholesterol
epoxides in nipple aspirates of human breast fluid. Cancer Res
41\textbf{:} 2563-2565.

Poirot M, \& Silvente-Poirot S (2013). Cholesterol-5,6-epoxides:
chemistry, biochemistry, metabolic fate and cancer. Biochimie
95\textbf{:} 622-631.

Poirot M, \& Silvente-Poirot S (2018). The tumor-suppressor cholesterol
metabolite, dendrogenin A, is a new class of LXR modulator activating
lethal autophagy in cancers. Biochem Pharmacol 153\textbf{:} 75-81.

Poirot M, Soules R, Mallinger A, Dalenc F, \& Silvente-Poirot S (2018).
Chemistry, biochemistry, metabolic fate and mechanism of action of
6-oxo-cholestan-3beta,5alpha-diol (OCDO), a tumor promoter and
cholesterol metabolite. Biochimie 153\textbf{:} 139-149.

Porter FD, \& Herman GE (2011). Malformation syndromes caused by
disorders of cholesterol synthesis. J Lipid Res 52\textbf{:} 6-34.

Porter FD, Scherrer DE, Lanier MH, Langmade SJ, Molugu V, Gale SE\emph{,
et al.} (2010). Cholesterol Oxidation Products Are Sensitive and
Specific Blood-Based Biomarkers for Niemann-Pick C1 Disease. Sci Transl
Med 2\textbf{:} 56ra81.

Porter NA, Xu L, \& Pratt DA (2020). Reactive Sterol Electrophiles:
Mechanisms of Formation and Reactions with Proteins and Amino Acid
Nucleophiles. Chemistry 2\textbf{:} 390-417.

Raccosta L, Fontana R, Maggioni D, Lanterna C, Villablanca EJ, Paniccia
A\emph{, et al.} (2013). The oxysterol-CXCR2 axis plays a key role in
the recruitment of tumor-promoting neutrophils. J Exp Med
210\textbf{:}1711-1728.

Raccosta L, Fontana R, Traversari C, \& Russo V (2013). Oxysterols
recruit tumor-supporting neutrophils within the tumor microenvironment:
The many facets of tumor-derived oxysterols. Oncoimmunology
2\textbf{:}e26469.

Scallen TJ, Dhar AK, \& Loughran ED (1971). Isolation and
characterization of C-4 methyl intermediates in cholesterol biosynthesis
after treatment of rat liver in vitro with cholestan-3 beta, 5 alpha,6
beta-triol. J Biol Chem 246\textbf{:} 3168-3174.

Schroepfer GJ, Jr. (2000). Oxysterols: modulators of cholesterol
metabolism and other processes. Physiol Rev 80\textbf{:} 361-554.

Segala G, David M, de Medina P, Poirot MC, Serhan N, Vergez F\emph{, et
al.} (2017). Dendrogenin A drives LXR to trigger lethal autophagy in
cancers. Nat Commun 8\textbf{:} 1903.

Segala G, de Medina P, Iuliano L, Zerbinati C, Paillasse MR, Noguer
E\emph{, et al.} (2013). 5,6-Epoxy-cholesterols contribute to the
anticancer pharmacology of tamoxifen in breast cancer cells. Biochem
Pharmacol 86\textbf{:} 175-189.

Sevanian A, \& McLeod LL (1986). Catalytic properties and inhibition of
hepatic cholesterol-epoxide hydrolase. J Biol Chem 261\textbf{:} 54-59.

Sevanian A, \& Peterson AR (1984). Cholesterol epoxide is a
direct-acting mutagen. Proc Natl Acad Sci U S A 81\textbf{:} 4198-4202.

Sevanian A, \& Peterson AR (1986). The cytotoxic and mutagenic
properties of cholesterol oxidation products. Food Chem Toxicol
24\textbf{:} 1103-1110.

Silvente-Poirot S, Dalenc F, \& Poirot M (2018). The Effects of
Cholesterol-Derived Oncometabolites on Nuclear Receptor Function in
Cancer. Cancer Res 78\textbf{:} 4803-4808.

Silvente-Poirot S, de Medina P, Record M, \& Poirot M (2016). From
tamoxifen to dendrogenin A: The discovery of a mammalian tumor
suppressor and cholesterol metabolite. Biochimie 130\textbf{:} 109-114.

Silvente-Poirot S, \& Poirot M (2012). Cholesterol epoxide hydrolase and
cancer. Curr Opin Pharmacol 12\textbf{:} 696-703.

Silvente-Poirot S, \& Poirot M (2014). Cancer. Cholesterol and cancer,
in the balance. Science 343\textbf{:} 1445-1446.

Silvente-Poirot S, Segala G, Poirot MC, \& Poirot M (2018).
Ligand-dependent transcriptional induction of lethal autophagy: a new
perspective for cancer treatment. Autophagy 14\textbf{:} 555-557.

Simpson E, \& Santen RJ (2015). Celebrating 75 years of oestradiol. J
Mol Endocrinol 55\textbf{:} T1-20.

Smith LL (1981) \emph{Cholesterol Autoxidation} . Plenum press: New
York.

Smith LL, \& Johnson BH (1989). Biological activities of oxysterols.
Free Radic Biol Med 7\textbf{:} 285-332.

Smith LL, Smart VB, \& Ansari GA (1979). Mutagenic cholesterol
preparations. Mutat Res 68\textbf{:} 23-30.

Sola B, Poirot M, de Medina P, Bustany S, Marsaud V, Silvente-Poirot
S\emph{, et al.} (2013). Antiestrogen-binding site ligands induce
autophagy in myeloma cells that proceeds through alteration of
cholesterol metabolism. Oncotarget 4\textbf{:} 911-922.

Song C, Hiipakka RA, \& Liao S (2001). Auto-oxidized cholesterol
sulfates are antagonistic ligands of liver X receptors: implications for
the development and treatment of atherosclerosis. Steroids
66\textbf{:}473-479.

Soules R, Audouard-Combe F, Huc-Claustre E, de Medina P, Rives A,
Chatelut E\emph{, et al.} (2019). A fast UPLC-HILIC method for an
accurate quanti fi cation of dendrogenin A in human tissues. J Steroid
Biochem Mol Biol 194\textbf{:} 105447.

Soules R, Noguer E, Iuliano L, Zerbinati C, Leignadier J, Rives A\emph{,
et al.} (2017). Improvement of 5,6alpha-epoxycholesterol,
5,6beta-epoxycholesterol, cholestane-3beta,5alpha,6beta-triol and
6-oxo-cholestan-3beta,5alpha-diol recovery for quantification by GC/MS.
Chem Phys Lipids 207\textbf{:} 92-98.

Tang L, Wang Y, Leng T, Sun H, Zhou Y, Zhu W\emph{, et al.} (2015).
Cholesterol metabolite cholestane-3beta,5alpha,6beta-triol suppresses
epileptic seizures by negative modulation of voltage-gated sodium
channels. Steroids 98\textbf{:} 166-172.

Tang L, Yan M, Leng T, Yin W, Cai S, Duan S\emph{, et al.} (2018).
Cholestane-3beta, 5alpha, 6beta-triol suppresses neuronal
hyperexcitability via binding to voltage-gated sodium channels. Biochem
Biophys Res Commun 496\textbf{:} 95-100.

Theodoropoulos PC, Wang W, Budhipramono A, Thompson BM, Madhusudhan N,
Mitsche MA\emph{, et al.} (2020). A Medicinal Chemistry-Driven Approach
Identified the Sterol Isomerase EBP as the Molecular Target of TASIN
Colorectal Cancer Toxins. J Am Chem Soc 142\textbf{:} 6128-6138.

Tonsing-Carter E, Hernandez KM, Kim CR, Harkless RV, Oh A, Bowie
KR\emph{, et al.} (2019). Glucocorticoid receptor modulation decreases
ER-positive breast cancer cell proliferation and suppresses wild-type
and mutant ER chromatin association. Breast Cancer Res 21\textbf{:} 82.

Touvier M, Fassier P, His M, Norat T, Chan DS, Blacher J\emph{, et
al.}(2015). Cholesterol and breast cancer risk: a systematic review and
meta-analysis of prospective studies. Br J Nutr 114\textbf{:} 347-357.

Vander Heiden MG, \& DeBerardinis RJ (2017). Understanding the
Intersections between Metabolism and Cancer Biology. Cell
168\textbf{:}657-669.

Vandewalle J, Luypaert A, De Bosscher K, \& Libert C (2018). Therapeutic
Mechanisms of Glucocorticoids. Trends Endocrinol Metab
29\textbf{:}42-54.

Vitku J, Starka L, Bicikova M, Hill M, Heracek J, Sosvorova L\emph{, et
al.} (2016). Endocrine disruptors and other inhibitors of
11beta-hydroxysteroid dehydrogenase 1 and 2: Tissue-specific
consequences of enzyme inhibition. J Steroid Biochem Mol Biol
155\textbf{:} 207-216.

Voisin M, de Medina P, Mallinger A, Dalenc F, Huc-Claustre E, Leignadier
J\emph{, et al.} (2017). Identification of a tumor-promoter cholesterol
metabolite in human breast cancers acting through the glucocorticoid
receptor. Proc Natl Acad Sci U S A 114\textbf{:} E9346-E9355.

Wang W, Zhang L, Morlock L, Williams NS, Shay JW, \& De Brabander JK
(2019). Design and Synthesis of TASIN Analogues Specifically Targeting
Colorectal Cancer Cell Lines with Mutant Adenomatous Polyposis Coli
(APC). J Med Chem 62\textbf{:} 5217-5241.

Watabe T, \& Sawahata T (1979). Biotransformation of cholesterol to
cholestane-3beta,5alpha,6beta-triol via cholesterol alpha-epoxide
(5alpha,6alpha-epoxycholestan-3beta-ol) in bovine adrenal cortex. J Biol
Chem 254\textbf{:} 3854-3860.

Witiak DT, Parker RA, Dempsey ME, \& Ritter MC (1971). Inhibitors and
stimulators of cholesterolgenesis enzymes. A structure-activity study in
vitro of amino and selected N-containing analogs of 5\selectlanguage{greek}α-\selectlanguage{english}cholestane-3\selectlanguage{greek}β ,\selectlanguage{english}5\selectlanguage{greek}α
,\selectlanguage{english}6\selectlanguage{greek}β-\selectlanguage{english}triol. J Med Chem 14\textbf{:} 684-693.

Wrensch MR, Petrakis NL, Gruenke LD, Miike R, Ernster VL, King EB\emph{,
et al.} (1989). Breast fluid cholesterol and cholesterol beta-epoxide
concentrations in women with benign breast disease. Cancer Res
49\textbf{:} 2168-2174.

Yin H, Xu L, \& Porter NA (2011). Free radical lipid peroxidation:
mechanisms and analysis. Chem Rev 111\textbf{:} 5944-5972.

Zhang L, Kim SB, Luitel K, \& Shay JW (2018). Cholesterol Depletion by
TASIN-1 Induces Apoptotic Cell Death through the ER Stress/ROS/JNK
Signaling in Colon Cancer Cells. Mol Cancer Ther 17\textbf{:} 943-951.

Zhang L, Theodoropoulos PC, Eskiocak U, Wang W, Moon YA, Posner B\emph{,
et al.} (2016). Selective targeting of mutant adenomatous polyposis coli
(APC) in colorectal cancer. Sci Transl Med 8\textbf{:} 361ra140.

Zhou C, Ye F, Wu H, Ye H, \& Chen Q (2017). Recent advances in the study
of 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2)Inhibitors.
Environ Toxicol Pharmacol 52\textbf{:} 47-53.

\textbf{List of abbreviations:} DDA: dendrogenin A; 5,6-EC:
5,6-epoxycholesterol; 5,6\selectlanguage{greek}α-\selectlanguage{english}EC: 5,6\selectlanguage{greek}α-\selectlanguage{english}epoxycholesterol; 5,6\selectlanguage{greek}β-\selectlanguage{english}EC:
5,6\selectlanguage{greek}β-\selectlanguage{english}epoxycholesterol; CT: Cholestane-3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α,\selectlanguage{english}6\selectlanguage{greek}β-\selectlanguage{english}triol; OCDO:
6-oxo-cholestan-3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α-\selectlanguage{english}diol ; AEBS: microsomal antioestrogen binding
site; D8D7I: 3\selectlanguage{greek}β-\selectlanguage{english}hydroxyterol-\selectlanguage{greek}Δ\selectlanguage{english}8-\selectlanguage{greek}Δ\selectlanguage{english}7-isomerase; EPB: emopamyl binding
protein; DHCR7: 3\selectlanguage{greek}β-\selectlanguage{english}hydroxyterol-\selectlanguage{greek}Δ\selectlanguage{english}7-reductase; ChEH:
cholesterol-5,6-epoxide hydrolase; HSD11B1: 11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid
dehydrogenase type 1; H6PD, HSD11B2: 11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase
type 2; ER: estrogen receptor; LXR: liver-X-receptor; GR: glucocorticoid
receptor; BC: breact cancer; ER(+)BC: oestrogen receptor positive BC;
TNBC, triple negative BC; HER2: Epithelial Growth Factor Receptor of
type 2; SERM: selective ER modulators; AI: aromatase inhibitors; 27-HC:
27-hydroxycholesterol; CXCR2: C-X-C Motif Chemokine Receptor 2;
SULT2B1b: hydroxysteroid sulfotransferase 2B1b; CYP27A1: Cytochrome P450
Family 27 Subfamily A Member 1; 5,6-ECS:
5,6\selectlanguage{greek}α-\selectlanguage{english}epoxy-cholesterol-3\selectlanguage{greek}β-\selectlanguage{english}sulfate; zymostenol: cholest-8-ene-3\selectlanguage{greek}β-\selectlanguage{english}ol.

\subsubsection*{Aknowledgement: We thank members of the ENOR consortium
for fruitful
discussions.}

{\label{aknowledgement-we-thank-members-of-the-enor-consortium-for-fruitful-discussions.}}

\subsubsection*{Competing interests: The authors declare no competing
financial
interests.}

{\label{competing-interests-the-authors-declare-no-competing-financial-interests.}}

\textbf{Funding:} This work was funded by an internal grant from the
`Institut National de la Sant\selectlanguage{ngerman}é et de la Recherche Médicale' and the
`Université de Toulouse III', the Fondation de France (R11166BB), the
Association pour la Recherche sur le Cancer (PJA 2013 12 00 342), the
INCA translational (PRTK-K15-118), the INCA (PLBIO-2018-145), the
Fondation Toulouse Cancer Santé (2017CS065); and the associations
``Céline'', ``Flo'' and ''Elles'' for their generous support.

\textbf{Figure legends:}

\textbf{Figure 1:} A) Detailed chemical structure of: cholesterol,
27-hysdroxycholesterol (27-HC), 5,6\selectlanguage{greek}α-\selectlanguage{english}epoxycholesterol (5,6\selectlanguage{greek}α-\selectlanguage{english}EC),
5,6\selectlanguage{greek}β-\selectlanguage{english}epoxycholesterol (5,6\selectlanguage{greek}β-\selectlanguage{english}EC), 5,6\selectlanguage{greek}α-\selectlanguage{english}epoxy-cholesterol-3\selectlanguage{greek}β-\selectlanguage{english}sulfate
(5,6-ECS), DDA: dendrogenin A, Cholestane-3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α,\selectlanguage{english}6\selectlanguage{greek}β-\selectlanguage{english}triol (CT),
6-oxo-cholestan-3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α-\selectlanguage{english}diol (oncosterone). B) 5,6\selectlanguage{greek}α-\selectlanguage{english}EC can react with
histamine to give dendrogenin A in the presence of a chemical catalyst
or an enzyme.C) 5,6-EC is hydrated by the cholesterol-5,6-epoxide
hydrolase (ChEH) to give CT. CT is transformed into OCDO by the
11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase type 2 (HSD11B2) to give oncosterone.
The reverse reaction is catalysed by the 11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid
dehydrogenase type 1 (HSD11B1) and the hexose-6-phosphate dehydrogenase
(H6PD), the enzyme that produces the cofactor NADPH necessary for the
reductase activity of HSD11B1.

\textbf{Figure 2:} A) DDA is a mammalian metabolite produced in normal
tissues. DDA is a tumour suppressor metabolite that induces cancer cell
re-differentiation into normal-like cells and kills cancer cells via a
mechanism of lethal autophagy. DDA inhibits oncosterone biosynthesis at
the ChEH step. B) Oncosterone is a tumour promoter that stimulates
cancer cell proliferation.

\textbf{Figure 3:} A) ChEH is made of two subunits involved in the late
steps of cholesterol biosynthesis. ChEH catalyses the trans-hydration of
the epoxide ring 5,6-EC to produce CT which can be subsequently
transformed into oncosterone. B) DDA inhibit ChEH which blocks the
hydration of 5,6-EC and induces the accumulation of zymostenol.

\textbf{Figure 4:} Snapshot of the 5,6-EC metabolism in normal and
pathological breast.

\textbf{Figure 5:} Diagram showing different pharmacological strategies
to block the enzymes responsible of oncosterone biosynthesis from 5,6-EC
and the receptors that are effectors of oncosterone tumour promoter
activity. DDA can control oncosterone biosynthesis and action at the
ChEH and LXR levels.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4-1/Figure-4-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

