3.1. The cholesterol epoxide hydrolase (ChEH).
ChEH is responsible for the hydration of 5,6-ECs to give cholestane-3β,5α,6β-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 in vivo and 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 LXRα/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 3β-hydroxyterol-Δ8-Δ7-isomerase (D8D7I) and of the 3β-hydroxyterol-Δ7-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 Δ8- and/or Δ7-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). Δ8-cholesterol precursors such as zymostenol (cholest-8-ene-3β-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 Δ8 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, Δ8- and Δ7-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).