molecules

Review Sigma-2 Receptor—A Potential Target for Cancer/Alzheimer’s Disease Treatment via Its Regulation of Cholesterol Homeostasis

1, , 1 1 1 2,3, , 1,4, Kai Yang * †, Cheng Zeng , Changcai Wang , Meng Sun , Dan Yin * † and Taolei Sun * 1 School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, 122 Luoshi Road, Wuhan 430070, China; [email protected] (C.Z.); [email protected] (C.W.); [email protected] (M.S.) 2 State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Science, Hubei University, Wuhan 430062, China 3 Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, National & Local Joint Engineering Research Center of High-Throughput Drug Screening Technology, Hubei University, Wuhan 430062, China 4 State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, 122 Luoshi Road, Wuhan 430070, China * Correspondence: [email protected] (K.Y.); [email protected] (D.Y.); [email protected] (T.S.) These authors contributed equally to this work. †


Received: 23 October 2020; Accepted: 19 November 2020; Published: 20 November 2020


Abstract: The sigma receptors were classified into sigma-1 and sigma-2 receptor based on their different pharmacological profiles. In the past two decades, our understanding of the biological and pharmacological properties of the sigma-1 receptor is increasing; however, little is known about the sigma-2 receptor. Recently, the molecular identity of the sigma-2 receptor has been identified as TMEM97. Although more and more evidence has showed that sigma-2 ligands have the ability to treat cancer and Alzheimer’s disease (AD), the mechanisms connecting these two diseases are unknown. Data obtained over the past few years from human and animal models indicate that cholesterol homeostasis is altered in AD and cancer, underscoring the importance of cholesterol homeostasis in AD and cancer. In this review, based on accumulated evidence, we proposed that the beneficial roles of sigma-2 ligands in cancer and AD might be mediated by their regulation of cholesterol homeostasis.

Keywords: sigma-2 receptor; TMEM97; cholesterol homeostasis; Alzheimer’s disease; cancer

1. Introduction The sigma receptors were identified in 1976 and initially recognized as a member of the opiate receptors; later studies revealed that they were different receptors [1]. The sigma receptors were classified into sigma-1 and sigma-2 receptors based on their different pharmacological profiles [2]. The sigma-1 receptor acts as a molecular chaperon and primarily localizes at the mitochondria-associated endoplasmic reticulum membrane (MAM) [3]. Upon stimulation by its ligands, the sigma-1 receptor translocates from MAM to plasma membrane (PM) [3], where it interacts with ion channels and G protein coupled receptors (GPCRs). The sigma-1 receptor plays important roles in many CNS diseases including drug addiction, depression, schizophrenia, Parkinson’s disease (PD), and Alzheimer’s disease (AD) [4–7]. The structure of the sigma-1 receptor has been recently reported, which exists as a trimer with one single transmembrane topology for each protomer [8]. In contrast, the molecular identity of the sigma-2 receptor was only recently revealed [9]. In 2006, Colabufo et al. proposed

Molecules 2020, 25, 5439; doi:10.3390/molecules25225439 www.mdpi.com/journal/molecules Molecules 2020, 25, 5439 2 of 22 that sigma-2 receptors are histone proteins, since a specific sigma-2 ligand PB28 could pull down histone proteins [10]. However, this hypothesis was discarded. It showed that fluorescent ligands of the sigma-2 receptor are not found in the nucleus, which was not consistent with this histone hypothesis [11].

1.1. PGRMC1/Sigma-2 Receptor In 2011, Xu et al. identified the sigma-2 receptor as a component of the progesterone receptor membrane component 1 (PGRMC1), because PGRMC1 could directly link to WC-21, which is a photoaffinity ligand for the sigma-2 receptor. PGRMC1 knockdown cells abolished this ligand binding to the sigma-2 receptor, while PGRMC1 overexpression increased the interaction between ligand and sigma-2 receptor [12]. However, later-accumulated evidence has suggested that the sigma-2 receptor is distinct from PGRMC1 [13]. The knockdown of PGRMC1 using siRNA or the overexpression of this protein using its cDNA in MCF7 cells did not alter the binding of [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG)to the sigma-2 receptor [14]. Knocking out PGRMC1 using CRISPR/Cas9 techniques in NSC34 cells also drew the same conclusion [15]. Furthermore, the molecular size of PGRMC1 is different from that of the sigma-2 receptor. Using photoaffinity labeling technique, the labeling with [3H] DTG in rat livers reveals that the sigma-2 receptor is about 21.5 kDa, while the molecular size of PGRMC1 is 25 kDa [16]. In addition, the sigma-2 receptor could still be detected by the photolabeling using [125I]-iodoazido-fenpropimorph ([125I]-IAF) in the PGRMC1 knockout cells [14]. Fluorescent sigma-2 ligands still have ability to bind to their receptor without the expression of PGRMC1 [17].

1.2. TMEM97/Sigma-2 Receptor Recently transmembrane protein 97 (TMEM97) was identified as a sigma-2 receptor identity using mass spectrometry after affinity purification from the liver [9]. TMEM97 is an ER-resident transmembrane protein, also known as meningioma-associated protein (MAC30) [18]. The reduction in TMEM97 expression using siRNA decreased the binding of the sigma-2 receptor to its ligand [3H] DTG. In addition, the overexpression of TMEM97 in cells lacking the sigma-2 receptor demonstrated a similar sigma-2 receptor binding profile. The affinity of ligands for TMEM97 is identical to the sigma-2 receptor binding affinity [9]. Moreover, TMEM97 ligands bind sigma-2 receptors: Asp29 and Asp56 are identified as ligand binding sites [9]. TMEM97 is expressed in many cell types and play important functions in neurons and cancer cells [19–22]. It has been shown that TMEM97 is involved in alcohol withdrawal behaviors. JVW-1034 (a TMEM97 ligand) prevented withdrawal-induced behavioral impairments in worms and blunted withdrawal-induced excessive alcohol drinking in rats [21]. TMEM97 is also involved in neuropathic pain: the TMEM97 receptor agonist, UKH-1114, reduces mechanical hypersensitivity in an animal model of neuropathic pain [20]. CM398, a selective sigma-2 ligand, also showed anti-inflammatory analgesic effects in formalin model of inflammatory pain in mice [23]. Further, the activation of the sigma-2 receptor is neuroprotective: a novel sigma-2 receptor/TMEM97 modulator, DKR-1677, protects neurons from death and cognitive impairment after blast-mediated traumatic brain injury (TBI) [22]. Additionally several compounds with sigma-1/sigma-2 mixed selectivity were able to counteract the neurotoxicity induced by oxidative stress [24]. These evidences indicate that the sigma-2 receptor/TMEM97 may be a promising target for the treatment of neurological diseases. TMEM97 is also involved in cancer: its agonist, PB221, significantly inhibited the migration and invasion of ALTS1C1 cells (a murine brain tumor cell line). In addition, a high dose of PB221 induced cell death of ALTS1C1 cells; these effects all involved mitochondrial oxidative stress. Consistent with this notion, PB221 effectively retarded tumor growth in tumor models [19]. Using CRISPR/Cas9 technology to remove TMEM97 in HeLa cells, one recent study has found that TMEM97 does not mediate cytotoxicity induced by the sigma-2 ligand [25]. It is possible that this cytotoxicity might be caused Molecules 2020, 25, x FOR PEER REVIEW 3 of 22 Molecules 2020, 25, 5439 3 of 22 Molecules1.3. The 20Complex20, 25, x FORof Sigma PEER- 2REVIEW Receptors 3 of 22 Molecules 2020, 25, x FOR PEER REVIEW 3 of 22 1.3.by sigma-2-ligand-inducedTheIn fact,Complex TMEM97/sigma of Sigma-2 Receptors- lysosomal2 receptor dysfunction forms a trimeric and reactive complex oxygen with PGRMC1 species (ROS)and low production,-density 1.3.lipoproteinbut The whether Complex (LDL) it relies of receptorSigma on sigma-2-2 Receptors(LDLR), receptor which remains is responsible unstudied. for the efficient uptake of LDL into the cells [26]. In This fact, complex TMEM97/sigma is also necessary-2 receptor for forms the internalization a trimeric complex of apolipoprotein with PGRMC1 E and (ApoE) low - anddensity Aβ 1.3. The Complex of Sigma-2 Receptors lipoproteinmonomersIn fact, (LDL)and TMEM97/sigma oligomers receptor (LDLR), [27].-2 receptor These which findings forms is responsible a explaintrimeric for why complex the PGRMC1 efficient with uptake could PGRMC1 be of stillLDLand photoaffinity into low -thedensity cells [26].lipoprotein This complex (LDL) receptor is also (LDLR), necessary which for theis responsible internalization for the of efficient apolipoprotein uptake of E LDL (ApoE) into the and cells Aβ taggedIn with fact, a TMEM97 sigma-2 /ligand,sigma-2 although receptor PGRMC1 forms a is trimeric not the complexsigma-2 receptor with PGRMC1 [12]. and low-density monomers[26]. This complex and oligomers is also [27]. necessary These for findings the internalization explain why PGRMC1 of apolipoprotein could be E still (ApoE) photoaffinity and Aβ lipoproteinConsistently (LDL) the receptor activation (LDLR), of TMEM97 which is by responsible its ligands for could the e affectfficient PGRMC1 uptake- ofdependent LDL into cell the taggedmonomers with and a sigma oligomers-2 ligand, [27]. although These findingsPGRMC1 explain is not the why sigma PGRMC1-2 receptor could [12]. be still photoaffinity processescells [26]. This[21,28]. complex For example, is also necessary JVW-1034 for reduces the internalization withdrawal- ofinduced apolipoprotein impairments E (ApoE) in ethanol and A-β taggedConsistently with a sigma the-2 activationligand, although of TMEM97 PGRMC1 by is its not ligands the sigma could-2 receptor affect PGRMC1 [12]. -dependent cell treatedmonomers worms and via oligomers PGRMC1/VEM [27]. These-1 findings(a PGRMC1 explain ortholog why PGRMC1 in worms), could although be still whether photoaffi JVWnity tagged-1034 processesConsistently [21,28]. the For activation example, ofJVW TMEM97-1034 reduces by its withdrawalligands could-induced affect PGRMC1 impairments-dependent in ethanol cell- bindswith a directly sigma-2 to ligand, PGRMC1 although is not PGRMC1 studied [21]. is not The the Sigma sigma-2-2/TMEM97 receptor [ 12ligand,]. SAS-0132, modulates treatedprocesses worms [21,28]. via ForPGRMC1/VEM example, JVW-1 (a-1034 PGRMC1 reduces ortholog withdrawal in worms),-induced although impairments whether in JVW ethanol-1034- PGRMC1Consistently-dependent the mechanisms activation ofto TMEM97reduce cell by death, its ligands cognitive could deficits, affect and PGRMC1-dependent neuroinflammation cellin bindstreated directly worms to via PGRMC1 PGRMC1/VEM is not studied-1 (a PGRMC1 [21]. The ortholog Sigma- 2/TMEM97in worms), ligand,although SAS whether-0132, modulJVW-1034ates ADprocesses mice [28]. [21,28 ]. For example, JVW-1034 reduces withdrawal-induced impairments in ethanol-treated PGRMC1binds directly-dependent to PGRMC1 mechanisms is not studiedto reduce [21]. cell The death, Sigma cognitive-2/TMEM97 deficits, ligand, and neuroinflammation SAS-0132, modulates in worms via PGRMC1/VEM-1 (a PGRMC1 ortholog in worms), although whether JVW-1034 binds ADPGRMC1 mice [28].-dependent mechanisms to reduce cell death, cognitive deficits, and neuroinflammation in 1.4.directly The Pharmacology to PGRMC1 of isSigma not-2 studied Receptors [21 ]. The Sigma-2/TMEM97 ligand, SAS-0132, modulates AD mice [28]. 1.4.PGRMC1-dependent TheTo Pharmacologydate, numerous of mechanisms Sigmasigma--22 Receptors selective to reduce ligands cell have death, been cognitive developed deficits, and andcharacterized neuroinflammation by receptor in AD mice [28]. 1.4.bindi Theng Pharmacology assays (Table of Sigma 1). The-2 Receptors definition of a sigma-2/TMEM97 agonist and antagonist remains undefined.To date, Zeng numerous et al. proposed sigma-2 selective that sigma ligands-2 selective have been compounds developed with and cytotoxic characterized effects by on receptor cancer 1.4. The Pharmacology of Sigma-2 Receptors bindicells Tongare date, assayscategorized numerous (Table as 1). sigmaagonists, The-2 definitionselective since siramesine, ligands of a sigma have a commonly-been2/TMEM97 developed accepte agonist andd characterizedsigma and antagonist-2 agonist, by receptor remainsinduces undefined. Zeng et al. proposed that sigma-2 selective compounds with cytotoxic effects on cancer bindicytotoxicityTong date, assays in numerous cancer (Table cells 1). sigma-2 [29]. The Using definition selective this approach, ligands of a sigma have sigma-2/TMEM97 been-2 ligands developed agonist have and been and characterized divided antagonist into by agonists, remains receptor cellsundefined. are categorized Zeng et al. as proposed agonists, thatsince sigma siramesine,-2 selective a commonly compounds accepte withd cytotoxic sigma-2 effectsagonist, on induces cancer partialbinding agonists, assays (Tableand antagonists1). The definition [29]. However, of a sigma-2molecular/TMEM97 basis for agonistpharmacological and antagonist mechanism remains of cytotoxicitycells are categorized in cancer cellsas agonists, [29]. Using since this siramesine, approach, asigma commonly-2 ligands accepte haved been sigma divided-2 agonist, into agonists, induces actionundefined. of sigma Zeng-2 ligands et al. proposed is not understood. that sigma-2 selective compounds with cytotoxic effects on cancer partialcytotoxicity agonists, in cancer and antagonistscells [29]. Using [29]. this However, approach, molecular sigma-2 basis ligands for havepharmacological been divided mechanism into agonists, of cells are categorized as agonists, since siramesine, a commonly accepted sigma-2 agonist, induces actionpartial ofagonists, sigma-2 and ligands antagonists is not understood. [29]. However, molecular basis for pharmacological mechanism of cytotoxicity in cancer cells [29Table]. Using 1. The this pharmacology approach, sigma-2 of sigma ligands-2 ligands. have been divided into agonists, action of sigma-2 ligands is not understood. partial agonists, and antagonists [29]. However, molecular basis for pharmacological mechanism of Table 1. The pharmacologyPutative of sigma-2 ligands. action of sigma-2Compound ligands is not understood. Assays Used Reference Table 1. The pharmacologyAction of sigma-2 ligands. Putative CompoundCB-184 Assays Used Reference Table 1. The pharmacologyPutativeAction of sigma-2 ligands. Compound Assays Used Reference CB-184 Action Compound PutativeAgonist Action Apoptosis Assays assay, Used Lactate Reference CB-184 CB-184 (sigma- dehydrogenase (LDH) [30,31] 2/TMEM97)Agonist Apoptosisrelease assay, Lactate (sigma- dehydrogenase (LDH) [30,31] Agonist ApoptosisApoptosis assay, assay, Lactate Lactate Agonist 2/TMEM97)(sigma- dehydrogenasedehydrogenaserelease (LDH) (LDH) [30,31][30,31] (sigma-2/TMEM97) CM398 2/TMEM97) releaserelease

CM398 CM398CM398 ? (sigma- [23] 2/TMEM97) ? (sigma- [23] ?2/TMEM97) (sigma-2? (sigma/TMEM97)- [23] [23] 2/TMEM97)

CT1812

CT1812CT1812 Antagonist (sigmaAntagonist- Trafficking assay [32,33] CT1812 Trafficking assay [32,33] (sigma-22/PGRMC1)Antagonist/PGRMC1) Antagonist(sigma- Trafficking assay [32,33]

2/PGRMC1)(sigma- Trafficking assay [32,33] 2/PGRMC1)

Molecules 2020, 25, x FOR PEER REVIEW 4 of 22

Molecules 2020, 25, xDKR FOR -PEER1677 REVIEW 4 of 22

Molecules 2020, 25, x DKRFOR PEER-1677 REVIEW 4 of 22 ? (sigma- Molecules 2020, 25, x FOR PEER REVIEW [22]4 of 22 DKR-1677 2/TMEM97) ? (sigma- Molecules 2020, 25, x DKRFOR PEER-1677 REVIEW [22]4 of 22 2/TMEM97) MoleculesMolecules2020 2020, 25, 2,5 5439, x FOR PEER REVIEW ? (sigma- 4 of 22 DKR-1677 [22] DTG 2/TMEM97) ? (sigma- Molecules 2020, 25, x FORDKR PEER-1677 REVIEW [22]4 of 22 2/TMEM97)TableAgonist 1. Cont. DTG ? (sigma- DKR-1677 (sigma-1 Apoptosis assay, LDH [22] Compound2/TMEM97) PutativeAgonist Action Assays Used Reference[34–36] DTG and? (sigma sigma-- release DKR-1677 (sigma-1 Apoptosis assay, LDH [22] DTG 2/TMEM97)2/TMEM97) [34–36] andAgonist? (sigma sigma - - release [22] ?2/TMEM97) (sigma-22/TMEM97)(sigmaAgonist/TMEM97)-1 Apoptosis assay, LDH [22] DTG [34–36] JVW-1034 and(sigma sigma-1- Apoptosisrelease assay, LDH [34–36] DTG 2/TMEM97)andAgonist sigma - release JVW-1034 (sigma-1 Apoptosis assay, LDH DTG 2/TMEM97)? Agonist(sigma- [34–36] DTG and sigma- release [21] JVW-1034 2/TMEM97)(sigma-1 Apoptosis assay, LDH Agonist2/TMEM97)Agonist (sigma-1 and Apoptosis assay, LDH [34–36] JVW-1034 and? (sigma sigma- - release [34–36] sigma-2(sigma/TMEM97)-1 Apoptosis releaseassay, LDH [21] 2/TMEM97)2/TMEM97) [34–36] and? (sigma sigma- - release JVW-1034 [21] PB221 2/TMEM97)2/TMEM97)? (sigma- JVW-1034JVW-1034 [21] 2/TMEM97) PB221 ? (sigma- JVW-1034 [21] ?2/TMEM97) (sigma-2/TMEM97) [21] Agonist? (sigma - (3-(4,5-dimethylthiazol-2- PB221 [21] 2/TMEM97)(sigma- yl)-2,5-diphenyltetrazolium [19] PB221 ?Agonist (sigma- (3-(4,5-dimethylthiazol-2- 2/TMEM97) bromide (MTT) assay [21] PB221 2/TMEM97)(sigma- yl)-2,5-diphenyltetrazolium [19] PB221 2/TMEM97)Agonist (3-(4,5bromide-dimethylthiazol (MTT) assay-2 - PB221 (sigmaAgonist- yl)(3-2,5-(4,5-diphenyltetrazolium-dimethylthiazol-2- [19] (3-(4,5-dimethylthiazol-2-yl)-2, 2/TMEM97)(sigmaAgonist- yl)bromide-2,5-diphenyltetrazolium (MTT) assay [19] 5-diphenyltetrazolium [19] PB221PB28 (sigma-2Agonist/TMEM97) (3-(4,5-dimethylthiazol-2- 2/TMEM97) bromidebromide (MTT) (MTT) assay assay (sigma- yl)-2,5-diphenyltetrazolium [19] PB28 AgonistAgonist (3-(4,5-dimethylthiazol-2- 2/TMEM97) Apoptosisbromide (MTT) assay, assay in vivo (sigma(sigma-- yl)-2,5-diphenyltetrazolium [35,37][19] PB28 2/TMEM97)AgonistAgonist (3-(4,5bromidetumor-dimethylthiazol xenografts (MTT) assay -2 - 2/TMEM97) Apoptosis assay, in vivo PB28 (sigma(sigma-- yl)-2,5-diphenyltetrazolium [35,37][19] tumor xenografts 2/TMEM97)Agonist bromide (MTT) assay 2/TMEM97) Apoptosis assay, in vivo PB28 (sigmaAgonistAgonist- Apoptosis assay, in vivo [35,37] RHM-4 Apoptosistumor xenografts assay, in vivo [35,37] (sigma-22/TMEM97)(sigma/TMEM97)- 3-(4,5-dimethylthiazoltumor xenografts-2-yl)- [35,37] PB28 tumor xenografts RHM-4 2/TMEM97)Agonist 5-(3- Apoptosis assay, in vivo PB28 (sigma- 3carboxymethoxyphenyl)-(4,5-dimethylthiazol-2-yl)-2-- [35,37] AntagonistAgonist tumor xenografts RHMRHM-4-4 2/TMEM97) Apoptosis5- assay,(3- in vivo (sigma- (4-sulfophenyl)-2H- [35,37][29] (sigma-2) 3-carboxymethoxyphenyl)(4,5-3-(4,5-dimethylthiazol-2-yl)-5dimethylthiazoltumor xenografts-2- yl)-2-- RHM-4 Antagonist2/TMEM97)Agonist tetrazolium (MTS) cell 3-(4,5Apoptosis(4-(3-carboxymethoxyphenyl)-2-dimethylthiazol-sulfophenyl)5 -assay,(3- in-2H vivo-2--yl) - [29] Antagonist(sigma(sigma (sigma-2)--2) viability-(4-sulfophenyl)-2H-tetrazolium assay, Caspase-3 [35,37][29] RHM-4 carboxymethoxyphenyl)tumor xenografts ( -2- Antagonist2/TMEM97) tetrazolium(MTS)activation5 cell-(3 viability-MTS) assay cell assay, 3-(4,5(4-dimethylthiazol-sulfophenyl)-2H-2--yl)- [29] (sigma-2) carboxymethoxyphenyl)viabilityCaspase-3 assay, activation Caspase assay--23- RHM-4 Antagonist 5-(3 (- SAS-0132 3-tetrazolium(4,5(4-activation-sulfophenyl)dimethylthiazolMTS) assay-2H cell -2 -yl)- [29] (sigma-2) carboxymethoxyphenyl)-2- SAS-0132RHM-4 Antagonist viabilitytetrazolium assay,5- (3( MTS)Caspase- cell-3 SAS-0132 3-(4,5(4--activationsulfophenyl)dimethylthiazol assay-2H --2-yl)- [29] (sigmaAntagonist-2) viabilitycarboxymethoxyphenyl) assay, Caspase-3- 2- Antagonist tetrazolium (MTS) cell (sigmaAntagonist- activationCa52+- (3assay- assay [28] (4-sulfophenyl)Ca2+ assay-2H- [28[29]] Molecules 2020, 25, xSAS FOR- 0132PEER REVIEW (sigma-2Antagonist(sigma/PGRMC1)-2) viabilitycarboxymethoxyphenyl) assay, Caspase-3-2 - 5 of 22 2/PGRMC1)Antagonist tetrazolium (MTS) cell SAS-0132 (sigma- (4activation-sulfophenyl)Ca2+ assay assay -2H - [29][28] (sigma-2) viability assay, Caspase-3 Siramesine Antagonist2/PGRMC1) tetrazoliumactivation (MTS) assay cell SAS-0132 (sigma- Ca2+ assay [28] Siramesine Antagonist viability assay, Caspase-3 Agonist LDH release, apoptosis SAS-0132 2/PGRMC1)(sigma- activationCa2+ assay assay [28] Agonist LDH release, apoptosis assay, Antagonist(sigma- assay, in vivo tumor [38[38–4040]] (sigma-22/PGRMC1)/TMEM97) in vivo tumor xenografts SAS-0132 2/TMEM97)(sigma- Caxenografts2+ assay [28] Antagonist 2/PGRMC1) (sigma- Ca2+ assay [28] Antagonist 2/PGRMC1) SV119 (sigma- Ca2+ assay [28] 2/PGRMC1) Agonist LDH release, apoptosis

(sigma- assay, in vivo tumor [41–43] 2/TMEM97) xenografts

UKH-1114

Agonist (sigma- Pain relief [20] 2/TMEM97)

WC-26

Agonist Apoptosis assay, LDH (sigma- [42–44] release 2/TMEM97)

2. Sigma-2 Receptor—A Novel Regulator of Cholesterol Homeostasis

2.1. Cholesterol Synthesis Cholesterol is required for not only membrane integrity and fluidity but also the production of hormones including steroids and vitamins [45,46]. Cholesterol biosynthesis begins with acetyl-CoA in the cytoplasm. Acetoacetyl-CoA is produced from two acetyl-CoA using acetyl-CoA acyltransferase 2 and then reacts with the third acetyl-CoA by the HMG-CoA synthase to produce 3- hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG-CoA reductase (HMGCR) reduces HMG-CoA to mevalonate, which is the primary rate-limiting step in cholesterol synthesis [45,46]. Mevalonate is converted to farnesyl pyrophosphate (FPP) by a series of enzymatic reactions, then two FPPs are condensed to squalene, which commits to sterol production. Most of squalene is converted to lanosterol, and through several additional enzymatic reactions, lanosterol is converted to cholesterol. FPP is also converted to geranylgeranyl pyrophosphate (GGPP); both FPP and GGPP modify oncogenic proteins, such as Ras, via enzymatic prenylation and activate them [45,46] (Figure 1). The synthesis of cholesterol is regulated by feedback control involving sterol regulatory element- binding protein 2 (SREBP2). SREBP is an ER membrane-bound protein, where it interacts with SREBP cleavage-activating protein (SCAP) [47]. When the sterol level at the ER is low, the conformation of SCAP is changed, allowing SREBP2 to translocate from the ER to the Golgi. At the Golgi, SREBP2 undergoes two sequential cleavages by site 1 protease (S1P) and site 2 protease (S2P), liberating the active soluble fragment of SREBP2 from the membrane. The processed SREBP2 enters the nucleus and increases expressions of many genes involving sterol biosynthetic pathway including HMGCR. Conversely the accumulation of cholesterol in the ER inactivates the SREBP2 pathway via insulin- induced genes (INSIGs). INSIGs interact with SCAP and promote the ER retention of SCAP, which blocks the ER-to-Golgi transportation of SREBP2 and reduces cholesterol biosynthesis [47] (Figure 2).

Molecules 2020, 25, x FOR PEER REVIEW 5 of 22

Molecules 2020, 25, Siramesinex FOR PEER REVIEW 5 of 22

Siramesine Molecules 2020, 25, x FOR PEER REVIEW Agonist LDH release, apoptosis 5 of 22 (sigma- assay, in vivo tumor [38–40] Agonist LDH release, apoptosis Siramesine 2/TMEM97) xenografts (sigma- assay, in vivo tumor [38–40] 2/TMEM97) xenografts Agonist LDH release, apoptosis SV119 (sigma- assay, in vivo tumor [38–40] Molecules 2020, 25, 5439 5 of 22 2/TMEM97) xenografts SV119 Agonist LDH release, apoptosis

Table(sigma 1.-Cont. assay, in vivo tumor [41–43] Agonist LDH release, apoptosis SV119 2/TMEM97) xenografts Compound Putative(sigma Action- assay, in Assays vivo Usedtumor Reference[41–43] SV119 2/TMEM97) xenografts Agonist LDH release, apoptosis Agonist LDH release, apoptosis assay, UKH-1114 (sigma- assay, in vivo tumor [41[41–43]–43] (sigma-2/TMEM97) in vivo tumor xenografts 2/TMEM97) xenografts UKH-1114 Agonist (sigma- Pain relief [20] UKH-1114 Agonist 2/TMEM97) UKH-1114 (sigmaAgonist- Pain relief [20] Pain relief [20] (sigma-22/TMEM97)/TMEM97) Agonist WC-26 (sigma- Pain relief [20] WC-26 WC-26 2/TMEM97)Agonist Apoptosis assay, LDH (sigmaAgonist- Apoptosis assay, LDH [42–44] [42–44] (sigma-2Agonist/TMEM97) releaserelease WC-26 2/TMEM97) Apoptosis assay, LDH (sigma- [42–44] release 2/TMEM97) Agonist Apoptosis assay, LDH (sigma- [42–44] 2. Sigma-2 Receptor—A Novel Regulator of Cholesterol Homeostasisrelease 2. Sigma-2 Receptor—A Novel Regulator 2/TMEM97)of Cholesterol Homeostasis 2.1. Cholesterol Synthesis 2.1.2. Sigma Cholesterol-2 Receptor Synthesis—A Novel Regulator of Cholesterol Homeostasis Cholesterol is required for not only membrane integrity and fluidity but also the production Cholesterol is required for not only membrane integrity and fluidity but also the production of 2.1.of hormonesCholesterol Synthesis including steroids and vitamins [45,46]. Cholesterol biosynthesis begins with 2.hormones Sigma-2 Receptorincluding— steroidsA Novel and Regulator vitamins of [45,46] Cholesterol. Cholesterol Homeostasis biosynthesis begins with acetyl-CoA acetyl-CoACholesterol in the is cytoplasm.required for Acetoacetyl-CoA not only membrane is produced integrity fromand fluidity two acetyl-CoA but also the using production acetyl-CoA of in the cytoplasm. Acetoacetyl-CoA is produced from two acetyl-CoA using acetyl-CoA 2.1.hormonesacyltransferase Cholesterol including Synthesis 2 and steroids then reacts and vitamins with the [45,46] third acetyl-CoA. Cholesterol by biosynthesis the HMG-CoA begins synthase with acetyl to produce-CoA acyltransferase 2 and then reacts with the third acetyl-CoA by the HMG-CoA synthase to produce 3- in3-hydroxy-3-methylglutaryl-CoA the cytoplasm. Acetoacetyl-CoA (HMG-CoA). is produced HMG-CoA from reductase two acetyl (HMGCR)-CoA usingreduces acetyl HMG-CoA-CoA hydroxyCholesterol-3-methylglutaryl is required-C foroA not (HMG only-CoA). membrane HMG -integrityCoA reductase and fluidity (HMGCR) but also reduces the production HMG-CoA of to acyltransferaseto mevalonate, 2 which and then is the reacts primary with rate-limitingthe third acetyl step-CoA in cholesterolby the HMG synthesis-CoA synthase [45,46 ].to Mevalonateproduce 3- hormonesmevalonate, including which issteroids the primary and vitamins rate-limiting [45,46] step. Cholesterol in cholesterol biosynthesis synthesis begins [45,46]. with Mevalonate acetyl-CoA is hydroxyis converted-3-methylglutaryl to farnesyl pyrophosphate-CoA (HMG-CoA). (FPP) HMG by a- seriesCoA reductase of enzymatic (HMGCR) reactions, reduces then HMG two- FPPsCoA areto inconverted the cytoplasm. to farnesyl Acetoacetyl pyrophosphate-CoA (FPP) is produced by a series from of enzymatic two acetyl reactions,-CoA usingthen two acetyl FPPs-CoA are mevalonate,condensed to which squalene, is the which primary commits rate to-limiting sterol production. step in cholesterol Most of squalenesynthesis is [45,46]. converted Mevalonate to lanosterol, is acyltransferasecondensed to 2 squalene, and then whichreacts with commits the third to sterol acetyl production.-CoA by the MostHMG - ofCoA squalene synthase is to converted produce 3 to- convertedand through to severalfarnesyl additional pyrophosphate enzymatic (FPP) reactions, by a series lanosterol of enzymatic is converted reactions, to cholesterol. then two FPPFPPs is are also hydroxylanosterol,-3-methylglutaryl and through several-CoA (HMGadditional-CoA). enzymatic HMG-CoA reactions, reductase lanosterol (HMGCR) is converted reduces HMGto cholesterol.-CoA to cconvertedondensed to to geranylgeranyl squalene, which pyrophosphate commits to (GGPP); sterol production. both FPP and Most GGPP of modifysqualene oncogenic is converted proteins, to mevalonate,FPP is also which converted is the to primary geranylgeranyl rate-limiting pyrophosphate step in cholesterol (GGPP); synthesis both FPP [45,46]. and Mevalonate GGPP modify is lanosterol,such as Ras, and via through enzymatic several prenylation additional and enzymatic activatethem reactions, [45,46 lanosterol] (Figure1 is). converted to cholesterol. convertedoncogenic to proteins, farnesyl such pyrophosphate as Ras, via enzymatic (FPP) by prenylationa series of enzymatic and activate reactions, them [45,46] then (Figuretwo FPPs 1). are FPP is also converted to geranylgeranyl pyrophosphate (GGPP); both FPP and GGPP modify condensedTheThe synthesis synthesis to squalene, of cholesterol of cholesterol which commitsis regulated is regulated to by sterol feedback by production. feedback control Mostinvolving control of involving squalene sterol regulatory is sterol converted regulatoryelement to - oncogenic proteins, such as Ras, via enzymatic prenylation and activate them [45,46] (Figure 1). lanosterol,bindingelement-binding protein and through 2 protein(SREBP2). several 2 (SREBP2). SREBP additional is SREBPan ER enzymatic membrane is an ER reactions, membrane-bound-bound protein,lanosterol where protein, is converted it interacts where to it cholesterol.with interacts SREBP with The synthesis of cholesterol is regulated by feedback control involving sterol regulatory element- FPPcleavageSREBP is also cleavage-activating-activating converted protein to geranylgeranyl protein(SCAP) (SCAP) [47]. When [pyrophosphate47]. the When sterol the level sterol (GGPP); at level the both atER the is FPP ERlow, is andthe low, conformation GGPPthe conformation modify of binding protein 2 (SREBP2). SREBP is an ER membrane-bound protein, where it interacts with SREBP oncogenicSCAPof SCAP is changed, isproteins, changed, allowingsuch allowing as Ras, SREBP2 SREBP2via enzymatic to translocate to translocate prenylation from from the and the ER activate ER to tothe the Golgi.them Golgi. [45,46] At At the the (Figure Golgi, Golgi, SREBP1). SREBP2 2 cleavage-activating protein (SCAP) [47]. When the sterol level at the ER is low, the conformation of undergoesundergoesThe synthesis two two sequential sequential of cholesterol cleavages cleavages is regulated by by site site by1 protease feedback 1 protease (S1P) control (S1P) and involving andsite site2 protease 2sterol protease regulatory(S2P), (S2P), liberating element liberating the- SCAP is changed, allowing SREBP2 to translocate from the ER to the Golgi. At the Golgi, SREBP2 bindingactivethe active soluble protein soluble fragment 2 (SREBP2). fragment of SREBP2SREBP of SREBP2 i sfrom an ER the frommembrane membrane. the membrane.-bound The protein,processed The where processed SREBP2 it interacts enters SREBP2 with the enters nucleusSREBP the undergoes two sequential cleavages by site 1 protease (S1P) and site 2 protease (S2P), liberating the cleavageandnucleus increases-activating and increasesexpressions protein expressions of (SCAP) many [47].genes of many When involving genes the sterol sterol involving level biosynthetic at sterol the ER biosynthetic pathway is low, the including pathway conformation HMGCR. including of active soluble fragment of SREBP2 from the membrane. The processed SREBP2 enters the nucleus SCAPConverselyHMGCR. is changed, Conversely the accumulation allowing the accumulationSREBP2 of cholesterol to translocate of cholesterol in the fromER inactivates in the the ER ER to inactivates the SREBP2Golgi. theAt pathway SREBP2the Golgi, via pathway SREBP insulin2 via - and increases expressions of many genes involving sterol biosynthetic pathway including HMGCR. undergoesinducedinsulin-induced genes two sequential(INSIGs). genes (INSIGs). INSIGscleavages interact INSIGs by site with interact 1 protease SCAP with and (S1P) SCAP promote and and site promotethe 2 proteaseER retention the (S2P), ER retentionof liberating SCAP, of which SCAP,the Conversely the accumulation of cholesterol in the ER inactivates the SREBP2 pathway via insulin- activeblockswhich soluble the blocks ER -fragmentto the-Golgi ER-to-Golgi transpo of SREBP2rtation transportation from of SREBP2the membrane. of and SREBP2 reduces The and cholesterolprocessed reduces SREBP2 cholesterolbiosynthesis enters biosynthesis [47] the (Figure nucleus 2). [47 ] induced genes (INSIGs). INSIGs interact with SCAP and promote the ER retention of SCAP, which and(Figure increases2). In expressions addition to SREBP2, of many liver genes X receptorsinvolving (LXRs) sterol bi [ 48osynthetic,49] and nuclearpathway factor including erythroid HMGCR. 2 related blocks the ER-to-Golgi transportation of SREBP2 and reduces cholesterol biosynthesis [47] (Figure 2). Converselyfactor-1 (NRF1) the accumulation [50] are also involvedof cholesterol in the in regulation the ER inactivates of cholesterol the synthesis.SREBP2 pathway High cholesterol via insulin levels- activate LXRs, resulting in cholesterol synthesis inhibition [48,49]. induced genes (INSIGs). INSIGs interact with SCAP and promote the ER retention of SCAP, which blocks the ER-to-Golgi transportation of SREBP2 and reduces cholesterol biosynthesis [47] (Figure 2).

Molecules 2020, 25, x FOR PEER REVIEW 6 of 22

In addition to SREBP2, liver X receptors (LXRs) [48,49] and nuclear factor erythroid 2 related factor-

1Molecules (NRF1)2020 [50], 25 ,are 5439 also involved in the regulation of cholesterol synthesis. High cholesterol levels6 of 22 activate LXRs, resulting in cholesterol synthesis inhibition [48,49].

Figure 1. Cholesterol synthesis in the cytoplasm. Cholesterol biosynthesis begins with Figure 1. Cholesterol synthesis in the cytoplasm. Cholesterol biosynthesis begins with acetyl-CoA. acetyl-CoA. See the text for the detailed information. HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA; See the text for the detailed information. HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA; HMGCR: HMGCR: HMG-CoA reductase; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate. HMG-CoA reductase; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate.

Molecules 2020, 25, 5439 7 of 22 Molecules 2020, 25, x FOR PEER REVIEW 7 of 22

FigureFigure 2. The 2. The synthesis synthesis of of cholesterol cholesterol isis regulatedregulated by feedback feedback control control involving involving sterol sterol regulatory regulatory element-bindingelement-binding protein protein 2 (SREBP2). 2 (SREBP2). See See the the texttext for the detailed detailed information. information.

2.2. Cholesterol2.2. Cholesterol Transport Transport In additionIn addition to de to novo de novo cholesterol cholesterol synthesis, synthesis, cells cells can can acquire acquire cholesterol cholesterol from from an an external external source. Cholesterolsource. Cholesterol can be obtained can be obtained from LDL, from which LDL, which is the is major the major cholesterol-carrier cholesterol-carrier in in the the bloodblood via via the the clathrin-mediated endocytosis of LDLR. In the brain, neurons acquire their cholesterol mainly clathrin-mediated endocytosis of LDLR. In the brain, neurons acquire their cholesterol mainly from from apolipoprotein E (ApoE), the major lipoprotein in the CNS, via multiple ApoE receptors apolipoproteinincluding LDLR, E (ApoE), LDLR the-related major protein lipoprotein 1 (LRP1), in the and CNS, the very via- multiplelow-density ApoE lipoprotein receptors receptor including LDLR,(VLDLR) LDLR-related [51]. Once protein in the late 1 (LRP1), endosomes/lysosomes and the very-low-density in the cytosol, lipoproteinLDL is released receptor from LDLR, (VLDLR) then [51]. OnceLDLR in the is laterecycled endosomes back to the/lysosomes cell surface, in while the cytosol, LDL is degraded LDL is releasedin the lysosome, from LDLR,and cholesterol then LDLR is is recycledreleased. back When to the the cell cell’s surface, cholesterol while demand LDL is is degraded no longer high, in the proprotein lysosome, convertase and cholesterol subtilisin/kexin is released. Whentype the 9 cell’s (PCSK9) cholesterol is secreted, demand which is directs no longer LDLR high, to the proprotein lysosome convertase for degradation subtilisin [52,53]./kexin After type 9 (PCSK9)cholesterol is secreted, is released, which it is directs then transported LDLR to theto the lysosome destined membranes for degradation to meet [52 the,53 ne].ed After of cells cholesterol [54]. is released,When intracellular it is then transported cholesterol is toin theexcess, destined it is either membranes exported out to meetof cells the by needATP-binding of cells cassette [54]. When intracellular(ABC) transporters cholesterol including is in excess, ABCA1 it is and either ABCG1 exported or becomes out of esterified cells by ATP-bindingto form cholesteryl cassette esters (ABC) (CE) by acyl-coenzyme A: cholesterol acyltransferases (ACATs) [45]. Extracellularly high-density transporters including ABCA1 and ABCG1 or becomes esterified to form cholesteryl esters (CE) by lipoproteins (HDL) remove excess cholesterol from tissues to the liver through reverse cholesterol acyl-coenzymetransport (RCT), A: cholesterol where it is acyltransferases subsequently eliminated (ACATs) with [45 bile]. Extracellularly [55]. high-density lipoproteins (HDL) removeCholesterol excess can cholesterol also be oxidize fromd tissuesvia enzymatic to the or liver non through-enzymatic reverse reactions cholesterol to produce transport oxysterols (RCT), where[56]. it is Cholesterol subsequently yields eliminated 7-ketocholesterol with bile (7KC) [55 and]. 7β-hydroxycholesterol (7βHC) by autoxidation, Cholesterolwhich does not can need also the help be oxidizedof enzymes. via Other enzymatic oxysterols orare non-enzymaticproduced enzymatically reactions by memb to produceers oxysterolsof the [56 cytochrome]. Cholesterol P450 yields family; 7-ketocholesterol for example, 25-hydroxycholesterol (7KC) and 7β-hydroxycholesterol (25-HC) is produced (7β HC) by by autoxidation,cholesterol which 25-hydroxylase does not need (CH25H), the help 24 of-hydroxycholesterol enzymes. Other oxysterols (24-HC) areby produced CYP46A1, enzymatically and 27- by membershydroxycholesterol of the cytochrome (27-HC) by CYP27A1, P450 family; respectively for example,[57]. These 25-hydroxycholesteroloxysterols could act as ligands (25-HC) to is producedactivate by LXRs, cholesterol which regulate 25-hydroxylase lipid metabolism (CH25H), and efflux 24-hydroxycholesterol [58]. In addition, oxysterols (24-HC) also have by CYP46A1, roles in immunity and inflammation. In particular, oxysterols, such as 25-HC, have been shown to have and 27-hydroxycholesterol (27-HC) by CYP27A1, respectively [57]. These oxysterols could act as both pro- and anti-inflammatory effects on immune response. On the one hand, it can act as a ligandssignaling to activate molecule LXRs, to which amplify regulate inflammatory lipid metabolism activation in and macrophages efflux [58 ]. [59]: In addition, 25-HC increases oxysterols the also haveproduction roles in immunity of many pro and-inflammatory inflammation. cytokines In particular, and chemokine oxysterols, including such IL as-6 25-HC, and IL- have8 [60]. been On the shown to haveother both hand, pro- the and production anti-inflammatory of 25-HC can eff preventects on immuneabsent in response.melanoma 2On (AIM2) the one inflammasome hand, it can act as a signaling molecule to amplify inflammatory activation in macrophages [59]: 25-HC increases the production of many pro-inflammatory cytokines and chemokine including IL-6 and IL-8 [60]. On the other hand, the production of 25-HC can prevent absent in melanoma 2 (AIM2) inflammasome Molecules 2020, 25, 5439 8 of 22

activationMolecules in 20 macrophages20, 25, x FOR PEER [ REVIEW61]. In conclusion, the roles of 25-HC in inflammation is quite8 complex,of 22 and further studies are strongly required. Lipoproteinactivation in macrophages LDL can also [61]. be In modified conclusion, by the oxidation. roles of 25 This-HC in modified inflammation LDL is (oxLDL) quite complex, is a ligand and further studies are strongly required. for Toll-like receptors (TLRs) in macrophages, which directly activates pro-inflammatory signaling Lipoprotein LDL can also be modified by oxidation. This modified LDL (oxLDL) is a ligand for pathways [62]. Further, macrophages also engulf oxLDL and cause the accumulation of cellular Toll-like receptors (TLRs) in macrophages, which directly activates pro-inflammatory signaling cholesterolpathways in macrophages, [62]. Further, macrophages resulting in the also amplification engulf oxLDL of and TLR cause signaling the accumulation [63,64]. of cellular cholesterol in macrophages, resulting in the amplification of TLR signaling [63,64]. 2.3. The Involvement of the Sigma-2 Receptor in Cholesterol Homeostasis Substantial2.3. The Involvement evidence of the has Sigma shown-2 Receptor that the in Cholesterol sigma-2 receptor Homeostasis/TMEM97 is involved in the synthesis of cholesterol.Substantial It was evidence reported has shown that, afterthat the treatment sigma-2 receptor/TMEM97 with progesterone, is involved TMEM97 in the and synthesis cholesterol biosynthesisof cholesterol. genes are It was coordinately reported that, upregulated after treatment in normal with ovarian progesterone, surface TMEM97epithelial and cells cholesterol [65]. TMEM97 sharesbiosynthesis EXPERA functional genes are coordinately domain with upregulated other cholesterol-related in normal ovarian genes surface including epithelial transmembrane cells [65]. 6 superfamilyTMEM97 member shares 2 EXPERA (TM6SF) functional and emopamil domain binding with protein other cholesterol (EBP) [18],-related implicating genes that including TMEM97 is transmembrane 6 superfamily member 2 (TM6SF) and emopamil binding protein (EBP) [18], a cholesterol synthesis gene. Consistently, using an RNAi screening technique, TMEM97 was identified implicating that TMEM97 is a cholesterol synthesis gene. Consistently, using an RNAi screening as a regulator of cholesterol homeostasis. The knockdown of TMEM97 reduced cholesterol contents technique, TMEM97 was identified as a regulator of cholesterol homeostasis. The knockdown of as wellTMEM97 as the reduced internalization cholesterol of contents LDLR. as Furthermore, well as the internalization under sterol-depleted of LDLR. Furthermore, conditions, under TMEM97 mRNAsterol is upregulated,-depleted conditions, indicating TMEM97 TMEM97 mRNA plays is a upregulated,very important indicating role in the TMEM97 regulation plays of a cholesterol very homeostasisimportant [66 role]. in the regulation of cholesterol homeostasis [66]. TMEM97TMEM97 also also controls controls the the tra ffitraffickingcking of cholesterol.of cholesterol. TMEM97 TMEM97 could could form form a a trimeric trimeric complexcomplex with PGRMC1with andPGRMC1 LDLR and and LDLR mediate and cholesterol mediate cholest uptakeerol via uptake its interaction via its interaction with PGRMC1 with PGRMC1 and LDLR: and the loss orLDLR: inhibition the loss of or eitherinhibition one of of either these one proteins of these resultsproteins in results a decreased in a decreased uptake uptake of cholesterol of cholesterol [26 ]. In addition,[26]. TMEM97 In addition, is TMEM97a Niemann-Pick is a Niemann C1 (NPC1)-Pick C1 binding (NPC1) protein, binding which protein, is requiredwhich is required for transporting for transporting cholesterol out of lysosomes [66]. The loss of this protein results in NPC, a fatal cholesterol out of lysosomes [66]. The loss of this protein results in NPC, a fatal lysosomal storage lysosomal storage disorder. In NPC cellular model, TMEM97 knockdown upregulates NPC1 disorder. In NPC cellular model, TMEM97 knockdown upregulates NPC1 expression, reduces expression, reduces cholesterol accumulation, and restores the trafficking of cholesterol out of the cholesterollysosome accumulation, [67] (Figure 3). and restores the trafficking of cholesterol out of the lysosome [67] (Figure3).

FigureFigure 3. Sigma-2 3. Sigma receptor-2 receptor controls controls the the tra traffickingfficking of cholesterol. TMEM97 TMEM97 forms forms a trimeric a trimeric complex complex withwith progesterone progesterone receptor receptor membrane membrane component component 1 (PGRMC1) and and low low-density-density lipoprotein lipoprotein receptor receptor (LDLR),(LDLR), which which is responsible is responsible for for the the internalization internalization of LDL. LDL. In In addition, addition, TMEM97 TMEM97 is a isNiemann a Niemann-Pick-Pick C1 (NPC1)C1 (NPC1) binding binding protein protein and and controls controls the the tra traffickingfficking of cholesterol cholesterol out out of oflysosomes. lysosomes.

Molecules 2020, 25, 5439 9 of 22

3. Anti-Cancer Effects of Sigma-2 Receptor Ligands

3.1. Increased Cholesterol Synthesis and Uptake Cellular cholesterol is usually acquired from both synthetic pathways and diet; most cancer cells exhibit increased cholesterol synthesis and uptake [46]. In cancer, multiple enzymes involved in mevalonate pathway are upregulated, including HMGCR and SREBP, which produce more cholesterol for cell proliferation [68]. Cancers are characterized by a gain of oncogenes and loss of tumor suppressors. P53, a tumor suppressor, could block SREBP activation and reduce cholesterol synthesis [69]. In addition to de novo cholesterol biosynthesis, some cancer cells increase cholesterol uptake, which is more efficient and requires less ATP consumption. Usually for these cells, they have a high expression of LDLR [70].

3.2. Enriched Cholesterol-Derived Metabolites: Oxysterols In the presence of excessive cholesterol, a high amount of oxysterol is produced in cancer cells. However, the exact effect of oxysterols on carcinogenesis and cancer progression is quite complicated. Many studies have showed that oxysterols can play precancerous and pro-proliferative roles in cancer cells. In patients with estrogen-receptor-positive breast cancer, oxysterol 27-HC is elevated [71]. In addition, the application of 27-HC reduces the expression of E-cadherin and β-catenin in breast carcinoma MCF7 cells, implicating that 27-HC is involved in the epithelial–mesenchymal transition (EMT) [72]. In addition, 27-HC promotes cell proliferation in MCF7 cells [73]. These effects can be explained by the modulation of signaling pathways by oxysterols; 27-HC could reduce p53 activation by enhancing the function of p53 E3 ligase, murine double minute 2 (MDM2), and promoting cell proliferation [73]. It could activate signal transducer and activator of transcription 3 (STAT3)-vascular endothelial growth factor (VEGF) signaling and facilitate angiogenesis [74]. Oxysterols also have anti-cancer effects and induce the death of tumor cells. The pro-apoptotic effect of oxysterols is caused by the overproduction of ROS and/or the increase in Ca2+ level in the cells [75]. Indeed, 27-HC could inhibit gastric cancer cell proliferation and migration via the modulation of LXR signaling [76]. Similarly, 27-HC treatment impedes cell proliferation in colorectal cancer cells, but this effect is mediated by the dephosphorylation of the kinase Akt [77].

3.3. The Involvement of Sigma-2 Receptor in Cancer

3.3.1. Sigma-2 Receptor as an Imaging Target for Cancer Diagnosis The sigma-2 receptor is expressed in higher density in proliferating tumor cells compared to quiescent tumor cells. Using the mouse mammary tumor 66 cell line, the density of sigma-2 receptors was found to be 10-fold higher in proliferating 66 versus quiescent 66 cells in vitro [78], later this conclusion is confirmed in solid tumor xenografts [79], suggesting that the sigma-2 receptor might be a promising marker for the proliferative solid tumors. Up to now, both 11C- and 18F-radiolabeled sigma-2 receptor ligands have been developed and validated in a variety of tumor models [80,81]. [18F]ISO-1 has been tested in humans for tumor positron emission tomography (PET) imaging [80,82] and used as a predictor of the cancer therapy response [83]. In 28 breast cancer patients, the uptake of [18F] ISO-1 was significantly correlated with expression of tumor proliferation marker Ki-67 [82].

3.3.2. Sigma-2 Ligands as Anticancer Agents Sigma-2 ligands can inhibit cancer cell proliferation, suppress tumor growth, and induce tumor cell death [19,84]. Many studies have shown that they are cytotoxicity in cancer cells. For example, using propidium iodide (PI) staining technique, it has been shown that NO1, a fluorescent sigma-2 receptor ligand, enhances apoptosis in breast cancer cell lines. Further in cells with overexpression of TMEM97, more death of cancer cells can be induced by NO1. In addition, NO1 reduces cell proliferation and migration in triple-negative breast cancer cells [84]. It is proposed that NO1 can downregulate Molecules 2020, 25, 5439 10 of 22 stromal interaction molecule (STIM1)-Orai1 interaction and reduce store-operated calcium entry (SOCE) in cancer cells [84]. Additionally, PB221, a sigma-2/TMEM97 receptor ligand, significantly enhances cell death and has antiproliferation activity against brain tumor cells [19]. This effect is mediated by mitochondrial oxidative stress. It also retards the migration and invasion of invasive murine astrocytoma cells in vitro. Furthermore, in vivo study reveals that PB221 effectively inhibits tumor growth [19]. However, in these studies, if the sigma-2 receptor is involved, it is not studied. Recently, Zeng et al. reported that neither sigma2/TMEM97 nor PGRMC1 mediates sigma2-ligand-induced cytotoxicity. Instead, sigma-2-ligand-induced lysosomal dysfunction and ROS production may be responsible for sigma-2-ligand-induced cytotoxicity [25]. However, whether TMEM97 or PGRMC1 mediates sigma-2-ligand-induced lysosome dysfunction and ROS production needs to be studied. Considering a large difference in the affinity of ligands for the sigma-2 receptor (nM) and their efficacy in cytotoxicity assays (µM), the anti-cancer mechanism of action of putative sigma2 selective compounds remains unclear.

3.3.3. Sigma-2 Receptor Ligands as Anticancer Drug Delivery Vehicles In order to reduce side effects caused by non-targeted chemotherapeutic agents, the sigma-2 ligand has also been used as a vesicle to deliver anticancer drug to cancer cells precisely [85–87], it could deliver small molecules by its internalization into cancer cells. Two different approaches using sigma-2 ligand-based drug delivery have been developed. First, sigma-2 ligands are conjugated with various nanoparticles, which are filled with cytotoxic agents. Telmisartan (TEL) is a cytotoxic agent that could inhibit the prostate cancer by the augmentation of apoptosis [88], but it has several dose-dependent side-effects including renal dysfunction and myocardial infarction [89], which hamper its wide acceptance. In order to improve the targeting of TEL, sigma-2 receptor ligand, 3-(4-cyclohexylpiperazine-1-yl) propyl amine (CPPA), was linked to nanostructured lipid particles containing TEL (CPPA-TEL-NLPs) (Figure4A), CPPA-TEL-NLPs enter the PC-3 cells via sigma-2-receptor-mediated endocytosis and subsequently activate multiple apoptosis pathways to kill cancer cells. This construct demonstrated superior cytotoxicity and great cellular uptake in PC-3 cells [87]. Second, sigma-2 ligands were covalently linked to antisense oligonucleotides or antitumor peptides. Erastin is a small molecule capable of inducing ferroptosis, it inhibits the cystine-glutamate antiporter system Xc- and prevents cells from synthesizing the antioxidant glutathione, which results in excessive lipid peroxidation and cell death [90]. Although Erastin and its analogues have cancer-selective cytotoxic activity, they lack effectiveness for pancreatic cancer patients; this might be caused by a deficiency in cellular drug uptake. In order to solve this issue, the Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to the sigma-2 ligand SV119 to create SW V-49 (Figure4B). This conjugation increases the killing capacity of dm-Erastin in vitro. Further, SW V-49 overcomes the cellular internalization block of dm-Erastin and reduces tumor sizes [86]. In addition, SW IV-134 is constructed by the conjugation of a sigma-2 receptor ligand (SW43) and small molecule second mitochondria-derived activator of caspases (SMAC) mimetic compound (SMC) (Figure4C). SMC is also called inhibitor of the apoptosis protein (IAP) antagonist; it has the ability to suppress IAPs and reestablish the apoptotic pathways [91]. SW IV-134 is tested in triple-negative breast cancer. It showed SW IV-134 can induce cytotoxicity that exceeds the most commonly used drug in breast cancer therapy [85]. Molecules 2020, 25, 5439 11 of 22 Molecules 2020, 25, x FOR PEER REVIEW 11 of 22

FigureFigure 4. The 4. The structures structures of of CPPA-TEL-NLP, CPPA-TEL-NLP, SW SW V-49,V-49, and SW SW IV IV 134. 134. (A ()A The) The structure structure of CPPA of CPPA and and Telmisartan:Telmisartan: CPPA-TEL-NLP CPPA-TEL-NLP was was prepared prepared byby anchoringanchoring CPPA CPPA to to nanostructured nanostructured lipid lipid particles particles of of Telmisartan.Telmisartan. (B) The(B) The sigma-2 sigma ligand-2 ligand SV119 SV119 was was chemicallychemically conjugated conjugated to todm dm-Erastin,-Erastin, resulting resulting in the in the sigma-2sigma/dm-Erastin-2/dm-Erastin conjugate conjugate SW SW V-49. V-49. ( C(C)) SW SW IVIV 134 was was constructed constructed by by sigma sigma-2-2 ligand ligand SW43 SW43 and and smallsmall molecule molecule SMAC SMAC mimetic mimetic compound compound(SMC). (SMC). CPPA-TEL-NLP:CPPA-TEL-NLP: 3- 3-(4-cyclohexylpiperazine-1-yl)(4-cyclohexylpiperazine-1-yl) propylpropyl amine amine (CPPA) (CPPA) anchored anchored nanostructured nanostructured lipid lipid particlesparticles (NLP) (NLP) of of telmisartan telmisartan (TEL). (TEL).

3.4. Mechanisms3.4. Mechanisms of Anti-Cancer of Anti-Cancer by by Sigma-2 Sigma-2 Ligands Ligands Targeting Cholesterol Cholesterol Homeostasis Homeostasis It hasIt beenhas been proposed proposed that that the the anti-cancer anti-cancer eeffectsffects of sigma sigma-2-2 ligands ligands might might depend depend both both on the on the sigma-2sigma ligand-2 ligand used usedand on and the on cell the type, cell they type, involve they involve caspase-dependent caspase-dependent and -independent and -independent apoptosis, apoptosis, Ca2+ overload, ROS generation, lysosomal membrane permeabilization (LMP), and Ca2+ overload, ROS generation, lysosomal membrane permeabilization (LMP), and autophagy [92–95]. autophagy [92–95]. In fact, more and more recent evidence provides another possibility that sigma-2 In fact,ligands more might and more target recent cholesterol evidence homeostasis provides to treat another cancer possibility [96]. that sigma-2 ligands might target cholesterolIn homeostasiscancer cells, accumulated to treat cancer cholesterol [96]. could form more lipid rafts and activate various cellular Insignaling cancer pathways, cells, accumulated which promote cholesterol cancer development could form [97,98]. more Lipid lipid rafts rafts are and highly activate ordered various cellularmembrane signaling domains pathways, consisting which of cholesterol promote and cancer sphingolipids. development They [have97,98 the]. Lipidability raftsto modulate are highly orderedmembrane membrane fluidity, domains lateral consisting movement of of cholesterol proteins as and well sphingolipids. as signal transduction They have [99]. the The ability to modulatecompartmentalization membrane fluidity, of signaling lateral pathway movement enhances of proteins the efficiency as well of assignal signal transduction. transduction For [99]. The compartmentalizationexample, cholesterol activates of signaling the sonic pathway hedgehog enhances (SHH) the pathway efficiency and promotes of signal cell transduction. cycle For example,progression, cholesterol which contributes activates to cancer the sonic development hedgehog [98]. (SHH) Cholesterol pathway also increases and promotes transforming cell cycle growth factor β (TGF-β) signaling. TGF-β is required for the induction of EMT in cancer cells. Further, progression, which contributes to cancer development [98]. Cholesterol also increases transforming TGF-β receptor in lipid raft activates the mitogen-activated protein kinase (MAPK) pathway, which β β β growthfavors factor cancer (TGF-cell proliferation) signaling. and migration TGF- is [97]. required Sigma-2 for ligands the inductioncould disorganize of EMT the in lipid cancer rafts cells. Further, TGF-β receptor in lipid raft activates the mitogen-activated protein kinase (MAPK) pathway, which favors cancer cell proliferation and migration [97]. Sigma-2 ligands could disorganize the lipid rafts in the membrane by displacing cholesterol molecules and dampen the lipid raft Molecules 2020, 25, 5439 12 of 22 microdomain-mediated signaling. Consistent with this notion, the depletion of cholesterol from these lipid rafts enhances apoptotic death of cancer cells [100]. Furthermore, the tumor microenvironment is very important for cancer development. Cholesterol metabolites, oxysterols, affect immune cells in the tumor microenvironment. Oxysterols inhibit T cell anti-tumor ability via LXR activation [101]. Furthermore, oxysterols promote tumor metastasis. In addition, 25-HC interacts with EBI2 (a GPCR that directs the migration of immune cells in response to oxysterols) and triggers migration of both macrophages and monocytes [102]. In a breast cancer model, 27-HC has been found to attract polymorphonuclear neurotrophils and γδ T cells [103]. Sigma-2 ligands might play an anti-cancer role by reducing the production of oxysterols and inhibit cancer development.

4. Sigma-2 Receptor Ligands May Target Cholesterol Homeostasis to Treat AD AD is the most common neurodegenerative disease in the elderly [104]. It is characterized by extracellular accumulation of Aβ and intracellular deposits of hyper-phosphorylated tau protein [104]. Aβ is produced via a two-step cleavage of the amyloid precursor peptide (APP) by β secretase and γ secretase (also called presenilin (PS)) [105]. Abnormal lipid metabolism has been observed in AD [106]. Many genes associated with the regulation of lipid metabolism are also linked to the risk of developing sporadic AD, including clusterin (CLU), ATP-binding cassette subfamily A member 7 (ABCA7), sortilin-related receptor 1 (SORL1), and triggering receptor expressed on myeloid cells 2 (TREM2) [107]. In addition, the ε4 allele of ApoE is identified as the most important risk gene for late-onset sporadic AD [108].

4.1. Cellular Cholesterol Accumulation in AD The blood–brain barrier (BBB) consists of tight junctions between the endothelial cells of brain and blood vessels, which prevents cholesterol uptake from the periphery, so brain cholesterol is synthesized in situ in the brain. Although all types of brain cells can synthesize cholesterol during development, but neurons in the adult do not efficiently synthesize cholesterol, they rely on the input from astrocytes as an external source [109]. In AD, cholesterol synthesis is reduced. Aβ fibrils reduce cholesterol synthesis in cultured neurons [110]. In addition, Aβ42 prevented the cleavage of SREBP-2 by protease and inhibited the transcription of many proteins including HMGCA, which are required for cholesterol synthesis [111]. Surprisingly, despite the reduced cholesterol synthesis, cellular cholesterol content is increased in AD [112]. It might be caused by the reduction in cholesterol conversion to 24-HC or increased cholesterol uptake. High levels of cholesterol have been suggested as a risk factor for AD [113]. For patients taking statins, which are cholesterol lowering drugs, the prevalence of AD is reduced [114,115], but other studies have obtained the opposite conclusion [116].

4.2. Altered Level of Oxysterols in AD To date, the oxysterols including 24-HC and 27-HC are implicated in the pathogenesis of AD. In neurons, a brain-specific enzyme CYP46A1 converts excessive cholesterol into 24-HC, then it is exported out of the brain and carried by LDL to the liver for degradation, while another oxysterol, 27-HC, is produced by CYP27A1 in the periphery and moves into the brain by circulation. Then, 27-HC is converted into 7α-hydroxy-3-oxo-4-cholestenoic acid (7 OH-4-C) by the enzyme CYP7B, which then − diffuses out of the brain through the BBB and moves into the periphery for degradation [109] (Figure5). Molecules 2020, 25, 5439 13 of 22 Molecules 2020, 25, x FOR PEER REVIEW 13 of 22

Figure 5. Cholesterol transport and metabolism in brain. Neurons in the adult do not efficiently Figure 5. Cholesterol transport and metabolism in brain. Neurons in the adult do not efficiently synthesize cholesterol; they rely on the input from astrocytes as an external source. In neurons, CYP46A1 synthesize cholesterol; they rely on the input from astrocytes as an external source. In neurons, converts excessive cholesterol into 24-HC, then it is exported out of the brain and carried by LDL to the CYP46A1 converts excessive cholesterol into 24-HC, then it is exported out of the brain and carried liver for degradation, while another oxysterol, 27-HC, is produced by CYP27A1 in the periphery and by LDL to the liver for degradation, while another oxysterol, 27-HC, is produced by CYP27A1 in the moves into the brain by circulation. Then, 27-HC is converted into 7 OH-4-C by the enzyme CYP7B, periphery and moves into the brain by circulation. Then, 27-HC is −converted into 7−OH-4-C by the which then diffuses out of the brain through the BBB and moves into the periphery for degradation. enzyme CYP7B, which then diffuses out of the brain through the BBB and moves into the periphery Similarfor degradation. to cholesterol, the levels of oxysterols are also altered in AD. It has been shown that 24-HC can both increase [117] and decrease [118] in the brain. It is proposed that 24-HC is elevated in early Similar to cholesterol, the levels of oxysterols are also altered in AD. It has been shown that 24- AD but decreases later when neurons that express CYP46A1 die. In addition, 27-HC is elevated in AD HC can both increase [117] and decrease [118] in the brain. It is proposed that 24-HC is elevated in brains [119]: accumulation of 27-HC in the brain is due to the increased flux of this oxysterol from early AD but decreases later when neurons that express CYP46A1 die. In addition, 27-HC is elevated the periphery across the BBB. Further, 27-HC could be synthesized in situ, because CYP27A1 is also in AD brains [119]: accumulation of 27-HC in the brain is due to the increased flux of this oxysterol expressed in astrocytes and oligodendrocytes [120]. Additionally, because neurons expressing CYP7B from the periphery across the BBB. Further, 27-HC could be synthesized in situ, because CYP27A1 is die, reduced degradation of 27-HC also contributes to its accumulation [121]. also expressed in astrocytes and oligodendrocytes [120]. Additionally, because neurons expressing 4.3.CYP7B The die, Involvement reduced of degradation Sigma-2 Receptor of 27- inHC AD also contributes to its accumulation [121].

4.3.1.4.3. The Sigma-2 Involvement Receptor of Sigma Ligands-2 Receptor as Therapeutic in AD Target of AD Using an in vitro quantitative receptor autoradiography technique, both sigma-1 and sigma-2 4.3.1. Sigma-2 Receptor Ligands as Therapeutic Target of AD receptors are found to be widely distributed in the rat brain, but the expression levels of sigma-2 receptorUsing are an generally in vitro quantitative lower than thosereceptor of sigma-1autoradiography receptor [technique,122]. In addition, both sigma high-1 and amounts sigma of-2 sigma-2receptors receptors are found are to located be widely in the distributed substantia nigrain the parsrat brain, reticulata but (SNr),the expression cerebellum, levels and of the sigma motor-2 cortexreceptor [122 are]. generally lower than those of sigma-1 receptor [122]. In addition, high amounts of sigma- 2 receptorsIn AD, are the located expression in the of substantia the sigma-2 nigra receptor pars reticulata is altered. (SNr), In cerebellum, female mice and with the amotor double cortex Aβ deposition[122]. (APP/PS1), sigma-2 expression was significantly reduced compared to that in control [123]. However,In AD, another the expression study found of that the theresigma is-2 no receptor difference is altered. in sigma-2 In expressionfemale mice in with postmortem a double brain Aβ tissuedeposition from late-stage (APP/PS1), AD sigma patients-2 expression [33]: how sigma-2 was significantly expression is reduced changed compared in AD remains to that further in control study. [123].Sigma-2 However, receptor another antagonists study found can that be used there to is treat no difference AD. In 2014, in sigma the role-2 expression of sigma-2 receptorin postmortem in AD wasbrain first tissue studied from by late Izzo.-stage He ADidentified patients several [33]: compounds how sigma-2 that expression could reverse is changed Aβ oligomer-induced in AD remains synapsefurther study. loss in neuronal culture, as well as the memory deficits in AD mouse models [124]. Later, these Sigma-2 receptor antagonists can be used to treat AD. In 2014, the role of sigma-2 receptor in AD was first studied by Izzo. He identified several compounds that could reverse Aβ oligomer-induced

Molecules 2020, 25, 5439 14 of 22 compounds were identified as sigma-2 receptor ligands. In his other paper, the sigma-2 receptor was shown to act as an Aβ oligomer receptor in neurons; its specific antagonists and antibodies prevented the binding of Aβ oligomer to synaptic puncta in vitro [33]. In addition, the expression of the sigma-2 receptor is upregulated upon the treatment of Aβ oligomers [33]. Consistent with this notion, sigma-2 receptor antagonist, SAS-0132, protects the C. elegan AD model from neurodegeneration. Further, it rescues memory deficits in the AD mouse model [28]. Another sigma-2 receptor antagonist, CT1812, also prevents and displaces the binding of Aβ oligomers to neurons and has entered clinical trial II to treat AD [32].

4.3.2. The Potential Mechanism of Sigma-2 Receptor Ligands in AD Although multiple mechanisms have been proposed to explain the anti-AD effects of sigma-2 ligands including its neuroprotection and anti-inflammation. They may work via its effects on cholesterol homeostasis to modulate lipoprotein trafficking, Aβ production, tau hyperphosphorylation, and neuroinflammation. The disruption of lipoprotein trafficking may contribute to the actions of sigma-2 ligands in AD. In the brain, ApoE binds cholesterol to form lipoprotein particles, then enters into neurons by LDLR and LRP1 [51]. ApoE is also associated with Aβ42. Recently, it has been shown that the inhibition of TMEM97/sigma-2 reduces Aβ42 and ApoE uptake in primary rat cortical neurons [27]. Increased uptake of Aβ42 results in accumulation and aggregation within neurons eventually leading the formation of plaques and neuronal death [125], which contributes to AD pathogenesis. This study indicates that TMEM97/sigma-2–PGRMC1-LDLR trimeric complex might be a potential target to reduce Aβ42 accumulation in neurons [27]. Sigma-2 ligands might also modulate Aβ production via cholesterol. Cholesterol has been shown to directly modulate APP cleavage in neuronal cultures by promoting β- and γ-secretase activity [126]. Decreasing membrane cholesterol levels using β-methyl-cyclodextrin (βMCD) reduces Aβ generation via the inhibition of β secretase and γ-secretase [127]. In addition, using cell-free assay, it has been demonstrated that cholesterol could directly regulate the activities of recombinant β secretase and γ-secretase [128]. Cholesterol could affect the activities of secretases via its modulation on lipid rafts. The increase in membrane cholesterol promotes the association of APP, β-, and γ-secretases within the lipid rafts and increases Aβ production [129,130]. Similarly, sigma-2 ligands also affect tau phosphorylation via cholesterol. Cholesterol controls Aβ-induced tau proteolytic cleavage by calpain [131]. In addition, hyperphosphorylated tau is also present in lipid rafts, indicating cholesterol might have ability to modulate tau hyperphosphorylation [132]. Furthermore, sigma-2 ligands could modulate neuroinflammation via oxysterols in AD. LXRs activated by oxysterols inhibit inflammatory gene expression, since LXRs could bind and inactivate pro-inflammatory genes [133]. Moreover, LXR activation may prevent neuroinflammation by indirectly down-regulating TLR target genes [134]. However, LXR-activating oxysterols might promote inflammation independently of LXRs; 27-HC, 24-HC, and 7β-HC enhanced inflammatory gene expression in human neuroblastoma SH-SY5Y cells via TLR4/cyclooxygenase-2/prostaglandin E synthase [135].

5. Conclusions More and more evidence has shown that the sigma-2 receptor might present a potential avenue for treating cancer and AD; the mechanisms connecting these two diseases are unknown. There is increasing proofs that they converge on a common pathological hub that involves cholesterol homeostasis. Data obtained over the past few years from human and animal models indicate that cholesterol metabolism is altered in AD and cancer, underscoring the importance of cholesterol homeostasis in AD and cancer. Future studies should aim to resolve the question of whether sigma-2 ligands could modulate cholesterol homeostasis to treat AD and cancer. In addition, the molecular and cellular mechanism of their potential benefits in AD and cancer also needs to be studied. Although the molecular identity Molecules 2020, 25, 5439 15 of 22 of the sigma-2 receptor has been identified as TMEM97, the lack of its structural information has still severely hindered the understanding of its physiological roles, its signaling pathways, and the development of more selective sigma-2 ligands. In addition, sigma-2 ligands (usually its antagonists) are claimed to be neuroprotective in AD, while its agonists are reported to be cytotoxic in cancer. The basis for these opposing outcomes remains unknown.

Author Contributions: D.Y., K.Y., and C.Z. reviewed the literature and drafted and wrote the manuscript; C.W. and M.S. revised the manuscript; T.S. proposed the topic of this manuscript and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Fundamental Research Funds for the Central Universities to D.Y. (193343009) and the National Natural Science Foundation of China to T.S. (51873168). Conflicts of Interest: The authors declare no conflict of interest.

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