The Biological Function of Sigma-2 Receptor/TMEM97 and Its Utility in PET Imaging Studies in Cancer

The sigma-2 receptor was originally defined pharmacologically and recently identified as TMEM97. TMEM97 has been validated as a biomarker of proliferative status and the radioligand of TMEM97, [18F]ISO-1, has been developed and validated as a PET imaging biomarker of proliferative status of tumors and as a predictor of the cancer therapy response. [18F]ISO-1 PET imaging should be useful to guide treatment for cancer patients. TMEM97 is a membrane-bound protein and localizes in multiple subcellular organelles including endoplasmic reticulum and lysosomes. TMEM97 plays distinct roles in cancer. It is reported that TMEM97 is upregulated in some tumors but downregulated in other tumors and it is required for cell proliferation in certain tumor cells. TMEM97 plays important roles in cholesterol homeostasis. TMEM97 expression is regulated by cholesterol-regulating signals such as sterol depletion and SREBP expression levels. TMEM97 regulates cholesterol trafficking processes such as low density lipoprotein (LDL) uptake by forming complexes with PGRMC1 and low density lipoprotein receptor (LDLR), as well as cholesterol transport out of lysosome by interacting with and regulating NPC1 protein. Understanding molecular functions of TMEM97 in proliferation and cholesterol metabolism will be important to develop strategies to diagnose and treat cancer and cholesterol disorders using a rich collection of TMEM97 radiotracers and ligands.


Introduction
Sigma receptors represent a class of proteins that were originally classified as a subtype of the opiate receptors [1]. Subsequent studies revealed that sigma receptor binding sites represented a distinct class of receptors that are located in the central nervous system as well as in a variety of tissues and organs [2]. Radioligand binding studies and biochemical analyses have shown that there are two types of sigma receptors [3,4]. Sigma-1 receptors (σ 1 Rs) have a molecular weight of 25 kD, whereas sigma-2 receptors (σ 2 Rs) have a molecular weight ranging from 18 to 21.5 kD. For many years, the sigma receptors were described pharmacologically through the binding of the radioligands [ 3 H](+)-pentazocine and [ 3 H]DTG ( Figure 1). The radioligand [ 3 H](+)-pentazocine has a high (3 nM) affinity for the σ 1 R and a low (>1000 nM) affinity for the σ 2 R, whereas [ 3 H]DTG is equipotent at both σ 1 and σ 2 receptors. The σ 1 R was detected and measured by [ 3 H](+)-pentazocine, whereas the σ 2 R by [ 3 H]DTG in the presence of 100 nM unlabeled (+)-pentazocine to mask binding to σ 1 Rs. The σ 1 R was cloned in 1996 [5] and is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein that allosterically modulates the activity of its associated proteins [6]. In contrast, the gene that codes for the σ 2 R remained unknown until recently.

Molecular Cloning of the σ2R
The σ2R was recently identified as transmembrane protein 97 (TMEM97, also known as MAC30) in 2017 [7]. Alon and colleagues synthesized a σ2R ligand and covalently coupled the ligand to agarose beads to prepare an affinity chromatography resin that was used to capture σ2Rs extracted from calf liver. Candidate proteins were isolated and identified by mass spectrometry. The candidate proteins were ectopically expressed and screened by σ2R binding assay using [ 3 H]DTG. Among the candidate proteins tested, only TMEM97 resulted in a significant increase of specific [ 3 H]DTG binding. Expression of TMEM97 in cells lacking σ2Rs increased σ2R binding and siRNA knockdown of TMEM97 proportionally reduced σ2R binding, confirming that TMEM97 is the σ2R. This work also demonstrated that Asp29 and Asp56 are essential for ligand recognition. Cloning the σ2R allows the use of modern biological methods to study its functions as a therapeutic and diagnostic target.
Previously, Xu et al. proposed that the binding site for the σ2R resided in a protein complex containing the progesterone receptor membrane component 1 (PGRMC1) [8]. Subsequent studies challenged the validity of these results and the recent identification of TMEM97 as the gene for the σ2R seemed to substantiate these reports [9,10]. However, Riad et al. recently reported that TMEM97 formed a complex with PGRMC1 and the low density lipoprotein receptor (LDLR) and this trimeric complex was responsible for the rapid internalization of the low density lipoprotein (LDL) in HeLa cells [11]. These data demonstrate that PGRMC1 associates with TMEM97 physically and corroborates our previous report that the σ2R represents a binding site in the PGRMC1 protein complex.

Evaluation of the σ2R as a Proliferation Biomarker
In a pioneer study, our lab demonstrated that the σ2R is a promising biomarker of the proliferative status in cell culture and mouse models with subcutaneous injection of mouse mammary adenocarcinoma line 66 cells, labeling with BrdU over time and assessing the proliferation status using flow cytometry compared to expression of σ2R using radioligand binding assays [12,13]. Previous studies had shown that line 66 cells displayed exponential growth kinetics

Molecular Cloning of the σ 2 R
The σ 2 R was recently identified as transmembrane protein 97 (TMEM97, also known as MAC30) in 2017 [7]. Alon and colleagues synthesized a σ 2 R ligand and covalently coupled the ligand to agarose beads to prepare an affinity chromatography resin that was used to capture σ 2 Rs extracted from calf liver. Candidate proteins were isolated and identified by mass spectrometry. The candidate proteins were ectopically expressed and screened by σ 2 R binding assay using [ 3 H]DTG. Among the candidate proteins tested, only TMEM97 resulted in a significant increase of specific [ 3 H]DTG binding. Expression of TMEM97 in cells lacking σ 2 Rs increased σ 2 R binding and siRNA knockdown of TMEM97 proportionally reduced σ 2 R binding, confirming that TMEM97 is the σ 2 R. This work also demonstrated that Asp29 and Asp56 are essential for ligand recognition. Cloning the σ 2 R allows the use of modern biological methods to study its functions as a therapeutic and diagnostic target.
Previously, Xu et al. proposed that the binding site for the σ 2 R resided in a protein complex containing the progesterone receptor membrane component 1 (PGRMC1) [8]. Subsequent studies challenged the validity of these results and the recent identification of TMEM97 as the gene for the σ 2 R seemed to substantiate these reports [9,10]. However, Riad et al. recently reported that TMEM97 formed a complex with PGRMC1 and the low density lipoprotein receptor (LDLR) and this trimeric complex was responsible for the rapid internalization of the low density lipoprotein (LDL) in HeLa cells [11]. These data demonstrate that PGRMC1 associates with TMEM97 physically and corroborates our previous report that the σ 2 R represents a binding site in the PGRMC1 protein complex. In a pioneer study, our lab demonstrated that the σ 2 R is a promising biomarker of the proliferative status in cell culture and mouse models with subcutaneous injection of mouse mammary adenocarcinoma line 66 cells, labeling with BrdU over time and assessing the proliferation status using flow cytometry compared to expression of σ 2 R using radioligand binding assays [12,13]. Previous studies had shown that line 66 cells displayed exponential growth kinetics for a period of 4 days, followed by a plateau phase that remained stable up to day 14 [14] (Figure 2A). In vitro studies also revealed that the exponentially growing 66 proliferating (66P) cells had 50% of the population in the G 1 phase of the cell cycle and >90% of the population labeled with [ 3 H]thymidine after 24-h labeling, indicating the cells were actively proliferating, whereas plateau phase 66 quiescent (66Q) cells have >97% of the population in G 1 and 2% of the population labeled with [ 3 H]thymidine after 24-h labeling indicating the cells were not proliferating as indicated by the lack of [ 3 H]thymidine incorporation into newly synthesized DNA, which occurs in the S phase. Furthermore, subsequent studies revealed that 100% of the 66Q cells can be recruited into the P-cell compartment [15]. These data indicate that this cell line is an appropriate in vitro model system for studying σ 2 R expression in proliferating and quiescent cells.

Evaluation of the σ 2 R PET Radiotracer for Assessing Proliferative Status and Predicting
Cancers 2020, 12, x 3 of 14 for a period of 4 days, followed by a plateau phase that remained stable up to day 14 [14] ( Figure  2A). In vitro studies also revealed that the exponentially growing 66 proliferating (66P) cells had 50% of the population in the G1 phase of the cell cycle and >90% of the population labeled with [ 3 H]thymidine after 24-h labeling, indicating the cells were actively proliferating, whereas plateau phase 66 quiescent (66Q) cells have >97% of the population in G1 and 2% of the population labeled with [ 3 H]thymidine after 24-h labeling indicating the cells were not proliferating as indicated by the lack of [ 3 H]thymidine incorporation into newly synthesized DNA, which occurs in the S phase. Furthermore, subsequent studies revealed that 100% of the 66Q cells can be recruited into the P-cell compartment [15]. These data indicate that this cell line is an appropriate in vitro model system for studying σ2R expression in proliferating and quiescent cells.  [16,17].
In cell culture models, σ2R Scatchard studies revealed that 66P cells had a 10 fold increase the σ2Rs/cell than 66Q cells [13] ( Figure 2B). The kinetics study of the expression of σ2Rs in 66 cells revealed a rapid increase in the σ2R density during the exponential growth phase that leveled off during the early plateau phase, followed by a decrease during the late plateau phase. Despite the kinetics of σ2Rs expression and cell growth kinetics being similar, the decreased density in the late plateau phase was delayed until day 10-12 after subculturing the Q-cells. Taken together, these results indicated a prolonged quiescent period is necessary to maximize σ2R loss from these 66 cells. In the mouse model of the 66 tumors, the ratio of the number of σ2R per P-cell to the number of σ2R per Q-cell was 10.6, consistent with the result obtained in the cell culture model [12]. These data indicate that the σ2R is a biomarker of the proliferative status of solid tumors and radioligands In cell culture models, σ 2 R Scatchard studies revealed that 66P cells had a 10 fold increase the σ 2 Rs/cell than 66Q cells [13] ( Figure 2B). The kinetics study of the expression of σ 2 Rs in 66 cells revealed a rapid increase in the σ 2 R density during the exponential growth phase that leveled off during the early plateau phase, followed by a decrease during the late plateau phase. Despite the kinetics of σ 2 Rs expression and cell growth kinetics being similar, the decreased density in the late plateau phase was delayed until day 10-12 after subculturing the Q-cells. Taken together, these results indicated a prolonged quiescent period is necessary to maximize σ 2 R loss from these 66 cells. In the mouse model of the 66 tumors, the ratio of the number of σ 2 R per P-cell to the number of σ 2 R per Q-cell was 10.6, consistent with the result obtained in the cell culture model [12]. These data indicate that the σ 2 R is a biomarker of the proliferative status of solid tumors and radioligands targeting the σ 2 R have the potential to noninvasively assess the proliferative status of tumors and the transition from proliferating to quiescent status using positron emission tomography (PET).

Development of σ 2 R Radioligands
The Mach group has developed numerous σ 2 R radioligands for in vivo imaging of proliferation status and in vitro σ 2 R binding studies [18]. A σ 2 R selective PET radioligand, N-(4-(6,7-dimethoxy-3,-4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-18 F-fluoroethoxy)-5-methylbenzamide ([ 18 F]ISO-1; Figure 1) has been validated as a PET imaging biomarker of the proliferation status in rodent models and human clinical studies (as described in the sections below). Another iodine-125 labeled σ 2 R radioligand [ 125 I]RHM-4 ( Figure 1) has been developed for in vitro binding studies. HeLa cells were used for these studies as they express TMEM97 and PGRMC1 and are able to be genetically edited efficiently via CRISPR/Cas9. In vitro σ 2 R binding studies using two different radioligands,  [19]. Thirty patients with primary breast cancer (n = 13), head and neck cancer (n = 10) and lymphoma (n = 7) underwent [ 18 [20]. Twenty-eight women with 29 primary invasive breast cancers were evaluated. The [ 18 F]ISO-1 uptake quantitated by SUVmax at 55-60 min post-i.v. injection was correlated with the Ki-67 score determined by immunohistochemistry. Tumors stratified into the high Ki-67 (≥20%) group had SUVmax greater than the low Ki-67 (<20%) group (p = 0.02). SUVmax exhibited a positive correlation with Ki-67 across all breast cancer subtypes (ρ = 0.46, p = 0.01, n = 29; Figure 3). The data demonstrated that [ 18 F]ISO-1 uptake in breast cancer modestly correlates with Ki-67 and can be used to provide an in vivo measure of tumor proliferative status. tumor volume between consecutive MR imaging sessions. These data suggest that a given value of [ 18 F]ISO-1 uptake provides a predictive measure of an expected change in tumor volume (i.e., growth rate), which attests to the tumor's aggressiveness.  [21]. The [ 18 F]ISO-1 uptake was correlated with the in vivo proliferative status determined by flow cytometric measures of BrdU-labeled tumor cells ( Figure 2B,C). Data showed a strong linear correlation between the [ 18 F]ISO-1 uptake and the proliferation status (i.e., ratio of proliferating cells to quiescent cells), indicating that [ 18 F]ISO-1 can be used to image the proliferative status. In addition, [ 18 F]ISO-1 was evaluated for assessing the tumor growth rate in a chemically-induced (N-methyl-N-nitrosourea (MNU)) model of rat mammary carcinoma. Each rat was imaged with [ 18 F]ISO-1 over a 10-week period at 2-week intervals to assess the σ2R status. MRI was used to monitor the tumor volume. The data showed that there was a significant correlation (R = 0.68, p < 0.003) between [ 18 F]ISO-1 uptake and changes in tumor volume between consecutive MR imaging sessions. These data suggest that a given value of [ 18 F]ISO-1 uptake provides a predictive measure of an expected change in tumor volume (i.e., growth rate), which attests to the tumor's aggressiveness.

Evaluation of [ 18 F]ISO-1 for the Monitoring and Predicting Response to Therapy
[ 18 F]ISO-1 was used to assess the response of MNU-induced tumors to bexarotene, a retinoid X receptor agonist and vorozole, an aromatase inhibitor therapeutic [21].  [ 18 F]ISO-1 was used to assess the response of MNU-induced tumors to bexarotene, a retinoid X receptor agonist and vorozole, an aromatase inhibitor therapeutic [21]. Cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) in combination with endocrine-therapy has emerged as an important regimen of care for estrogen receptor (ER)-positive metastatic breast cancer [22]. Despite the efficacy of this combined approach, not all patients benefit from the combination therapy. Biomarkers that can monitor changes in tumor proliferation early in the course of therapy can be used to identify patients most likely to benefit from this combinational therapy versus endocrine can be used to identify patients most likely to benefit from this combinational therapy versus endocrine therapy alone. Recently, Elmi et al. assessed the ability of two PET-proliferation tracers, [ 18 F]ISO-1 and [ 18 F]FLT, for evaluating the response to palbociclib, a CDK4/6i and fulvestrant, an ER-antagonist [23]. PET imaging of MCF7 xenografts showed a significant decrease in [ 18 F]FLT on day 3 and maintained low uptake on day 14 after treatment in all treatment groups (palbociclib, fulvestrant and the combination of both). The decrease in [ 18 F]FLT uptake correlated with S phase depletion (Figure 4). On the other hand, the PET imaging showed no changes in [ 18 F]ISO-1 uptake in all treated mice on day 3 and a significant decrease in [ 18 F]ISO-1 uptake upon the combination therapy (but not single agents therapy) on day 14 ( Figure 4)

Subcellular Localization of σ 2 R/TMEM97
TMEM97 exists in multiple subcellular locations. (1) TMEM97 localizes in cellular membranes. Before the σ 2 R was cloned, it was detected and measured by the σ 2 R binding assay in membrane preparations of various tissues and cell lines [4,24]. In fact, the σ 2 R was purified and identified as TMEM97 from calf liver membrane fractions [7]. Based on the amino acid sequence of TMEM97, this protein is predicted to have a four-pass transmembrane topology. Together, these data indicate that TMEM97 is a membrane-bound protein.
(2) TMEM97 localizes in lipid rafts. Gebreselassie and Bowen demonstrated that the σ 2 R, detected using [ 3 H]DTG and a lipid raft biomarker flotillin-2, detected by immunoblotting, colocalized in lipid raft fractions after equilibrium centrifugation of extracts of rat liver membranes in discontinuous sucrose density gradients [25]. The data indicate that the σ 2 R localizes in lipid rafts. (3) TMEM97 localizes in the endoplasmic reticulum (ER), lysosomes and plasma membranes. TMEM97 sequence analysis shows that the C-terminus contains the predicted ER-retention sequence "KRKKK" [7]. Transient expression of TMEM97-YFP in HeLa cells revealed a predominant localization of the protein to reticular ER-like membranes and the nuclear envelope, consistent with ER localization. On the other hand, in sterol-depleted cells, a prominent fraction of TMEM97-YFP localized to lysosome and the plasma membrane. Sterol depletion-induced TMEM97 translocation is believed to regulate lysosomal cholesterol export. A confocal microscopy study showed that σ 2 R fluorescent ligands were colocalized with ER, lysosome and plasma membrane trackers, consistent with the aforementioned findings [26]. Subcellular localization of TMEM97 should provide clues to study TMEM97 biological functions.

Differential Expression of TMEM97 in Normal and Cancer Tissues
TMEM97 expression in normal and cancer tissues has been studied using an σ 2 R binding assay and immunohistochemistry to measure protein levels and using northern-blot, quantitative RT-PCR, DNA microarray or RNAseq methods to determine mRNA levels. Using σ 2 R binding assays, Vilner et al. first demonstrated that the σ 2 R exists in a number of rodent and human tumor cell lines [24]. Numerous studies demonstrated that TMEM97 was ubiquitously expressed in a wide variety of normal human tissues and cancer. TMEM97 mRNA and/or proteins was significantly increased in breast, colon, gastric, esophageal, lung, ovarian cancer and prostate cancer [27][28][29][30][31][32][33][34][35][36], but decreased in meningiomas, pancreatic and renal cancer [27,37]. TMEM97 upregulation in certain tumors and downregulation in others suggests that this protein plays a distinct role in human malignancies.

Potential Requirement of TMEM97 in Cancer Proliferation
It is reported that stable knockout of TMEM97 by CRISPR/Cas9 gene editing decreased cell proliferation in prostate cancer LNCaP cells [36]. It is also reported that transient knockdown of TMEM97 by siRNA decreased cell viability and migration/invasion in breast cancer, gastric cancer and glioma cell [38][39][40]. These data suggest that TMEM97 may be required for cell proliferation in certain tumor cell lines.

Regulation of TMEM97 Expression in Cancer
The TMEM97 gene is located on chromosome band 17q11.2. The coding nucleotides of TMEM97 in 39 ovarian cancer samples were sequenced [41]. No coding sequence variations were found, suggesting that the TMEM97 coding sequence is not responsible for ovarian cancer development.
Limited research examined the regulation of TMEM97 expression in cancer. (1) TMEM97 was found to be a transcriptional target of the BRCA1 gene [42]. Inherited mutations of the BRCA1 gene predispose to breast, ovarian and other cancers. The role of the BRCA1 gene in the maintenance of chromosomal integrity is linked to transcriptional regulation function of the BRCA1 protein. In one study, differentially expressed genes were identified by comparing control MCF7 breast cancer cells with MCF7 cells ectopically expressing BRCA1. TMEM97 was among a set of BRCA1-induced genes. The data suggest that TMEM97 may be related to BRCA1 function in breast cancer cells.
(2) The epidermal growth factor (EGF) transmitted signal transduction pathways play a major role in carcinogenesis and proliferation of human bladder cancer cells. One study demonstrated that treatment with 10 ng/mL EGF for 5 days downregulated TMEM97 mRNA expression in the human bladder cancer cell line J82, but had no effect on TMEM97 mRNA expression in another human bladder cancer cell line RT4 [43]. The data suggest that TMEM97 expression is differentially regulated by EGF in different bladder cancer cell lines. (3) TGF-β is known to be overexpressed in pancreatic cancer. The effects of TGF-β on TMEM97 mRNA expression were examined in pancreatic cancer cell lines [27]. TMEM97 was transiently downregulated by TGF-β in two pancreatic cancer cell lines Colo-357 and Panc-1. In contrast, TMEM97 expression was not affected by TGF-β in five other pancreatic cancer cell lines. The data suggest that TMEM97 expression is differentially regulated by TGF-β expression in different pancreatic cancer cell lines. (4) MicroRNAs are globally downregulated in prostate cancer, especially in poorly differentiated tumors. miR-152-3p was identified as a common epigenetically regulated onco-suppressor in prostate cancer [36]. miR-152-3p was under-expressed in prostate cancer. Overexpression of miR-152-3p suppressed cell viability and invasion potential and downregulated TMEM97 mRNA expression in prostate cancer LNCaP cells. Knockout of TMEM97 inhibited proliferation of LNCaP cells. The data indicate that TMEM97 is a target gene of miR-152-3p and may be related to prostate cancer development and proliferation. Taken together, TMEM97 is regulated by oncogenic factors in certain cancer cells and may be related to cancer development and proliferation in certain tumors.  [44]. The data showed that knockout of TMEM97, PGRMC1 or both by CRISPR/Cas9 gene editing did not affect EC 50 values, the concentrations of σ 2 R ligands by which 50% of cell death was induced ( Table 1), suggesting that cytotoxic effects of these compounds are not mediated by TMEM97 and/or PGRMC1. It is of interest to note that not all σ 2 R ligands display cytotoxicity. For example, the σ 2 R ligands siramesine, SW43 and PB28 display similar cytotoxicity in the above described cell lines, whereas the benzamide analogs RHM-4 and ISO-1 are not cytotoxic under any conditions (Table 1) [42]. This work will facilitate elucidating mechanisms of σ 2 R ligand cytotoxicity.

Biological Roles of TMEM97 in Cholesterol Homeostasis
Several lines of evidence indicate that TMEM97 plays important roles in cholesterol homeostasis. Ovarian cancer most often derives from ovarian surface epithelial cells. Increased exposure to progesterone (P4) protects women against developing ovarian cancer. Wilcox et al. determined P4-induced gene expression in ovarian surface epithelial cells with oligonucleotide microarrays [41]. The data showed that TMEM97 and cholesterol biosynthesis genes were coordinately upregulated upon P4 treatment, suggesting that TMEM97 associates with cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in ovarian surface epithelial cells might play a role in conferring protection against ovarian cancer.
Elevated plasma cholesterol levels result in excess cholesterol deposition in arterial vessel walls and are a major risk factor for atherosclerosis. In order to understand cellular cholesterol regulation, Bartz identified cholesterol-regulating genes by cultivating HeLa cells in sterol-depleted serum to activate cellular sterol homeostatic regulatory machinery and then performing genome-wide gene expression analysis with cDNA microarrays and targeted RNAi screening [45]. One of 20 genes identified with this strategy is TMEM97. The data showed that TMEM97 mRNA was regulated by upstream cholesterol regulatory factors: sterol depletion upregulated TMEM97 mRNA; low density lipoprotein receptor (LDLR) knockdown by siRNAs upregulated TMEM97 mRNA under control condition (in the presence of sterol) and sterol regulatory-element binding protein (SREBP-2) knockdown by siRNAs downregulated TMEM97 mRNA in sterol-depleted cells. The data also demonstrated that TMEM97 regulated downstream cholesterol homeostasis: knockdown of TMEM97 by siRNAs considerably inhibited cellular low density lipoprotein (LDL) uptake under sterol depletion condition and total free cholesterol under control condition. TMEM97 interacted with LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). NPC1 knockdown induced a robust cholesterol storage, whereas TMEM97 knockdown increased NPC1 protein level, reduced lysosomal lipid storage and restored cholesterol trafficking to the endoplasmic reticulum in NPC1-knockdown cells [46]. Thus, TMEM97 was identified as the cholesterol-regulating gene.
Riad et al. reported that TMEM97 and/or PGRMC1 knockout in HeLa cells reduced LDL internalization [11]. Immunocytochemistry and proximity ligation assay studies indicated that TMEM97 interacted with PGRMC1 and LDLR physically, suggesting that this ternary complex was responsible for this rapid rate of internalization of LDL.
By a computational protein sequence analysis, Sanchez-Pulido and Ponting identified a new domain, the expanded EBP superfamily (EXPERA) domain, which is conserved among TMEM97, transmembrane 6 superfamily member (TM6SF1 and TM6SF2), emopamil binding protein (EBP) and emopamil binding protein-like protein (EBPL) families [47]. This EXPERA domain family of proteins is implicated in cholesterol homeostasis. Carriers of the Glu167Lys coding variant in the TM6SF2 gene are more susceptible to non-alcoholic fatty liver disease (NAFLD). EBP is an enzyme with ∆8-∆7 sterol isomerase activity and regulates key steps in the final cholesterol biosynthesis pathway. EBP mutations are the cause of chondrodysplasia punctata 2 X-linked dominant (CDPX2), a defective cholesterol biosynthesis disorder. These evidences further indicate that TMEM97, like other EXPERA domain-containing proteins, is involved in cholesterol homeostasis.

TMEM97 in the Interplay of Cancer and Cholesterol Homeostasis
The involvement of TMEM97 in cholesterol homeostasis is consistent with the findings that TMEM97 is upregulated in proliferating versus quiescent breast cancer cells and TMEM97 is differentially expressed in normal and cancer tissues. Cholesterol is a key component of the cell membrane. Accelerated cholesterol and lipid metabolism are the hallmarks of cancer [48]. The metabolic dependency of cancer cells on cholesterol and other lipids is tightly regulated by the cholesterol homeostasis network, including (1) SREBP, master transcriptional regulators of cholesterol and fatty acid pathway genes, (2) nuclear sterol receptors (liver X receptors, LXR), which regulate intracellular cholesterol level through the expression of cholesterol efflux proteins such as ABC transporters and (3) lipid particle receptors, such as LDLR, providing exogenous sterol and lipids to cancer cells. Besides the well-established structural role of cholesterol in membranes, the deregulation of metabolite production at different points in the cholesterol bio-synthesis pathway has been reported to promote cancer or cause resistance to therapies in different in vitro and in vivo models described in [49]. TMEM97 is a target gene of SREBP and mediates LDL uptake, cholesterol trafficking and cellular cholesterol level. Given the metabolic dependency of cancer cells on cholesterol and various effects of cholesterol metabolites on cancer, TMEM97 may play roles in cancer via involvement in cholesterol homeostasis ( Figure 5). ABC transporters and (3) lipid particle receptors, such as LDLR, providing exogenous sterol and lipids to cancer cells. Besides the well-established structural role of cholesterol in membranes, the deregulation of metabolite production at different points in the cholesterol bio-synthesis pathway has been reported to promote cancer or cause resistance to therapies in different in vitro and in vivo models described in [49]. TMEM97 is a target gene of SREBP and mediates LDL uptake, cholesterol trafficking and cellular cholesterol level. Given the metabolic dependency of cancer cells on cholesterol and various effects of cholesterol metabolites on cancer, TMEM97 may play roles in cancer via involvement in cholesterol homeostasis ( Figure 5).

Conclusions
TMEM97 has been validated as a biomarker of proliferative status in in vitro and in vivo studies. [ 18 F]ISO-1, which targets TMEM97, has been validated as a PET imaging biomarker of proliferative status in human breast cancer patients. [ 18 F]ISO-1 is also a promising predictor of the drug response in preclinical studies. The proliferative status of a tumor has a profound effect on the outcome of both chemotherapy and radiotherapy treatments. Thus, [ 18 F]ISO-1 should be useful to guide treatment plan for cancer patients.
TMEM97 plays distinct roles in cancer. TMEM97 is upregulated in some tumors but downregulated in other tumors. TMEM97 may be required for cell proliferation in certain tumor cells. TMEM97 is regulated by upstream oncogenic signals such as EGF, TGF-β and microRNA in some but not other tumor cells. The data suggest that TMEM97 is associated with cancer proliferation. Emerging evidence also demonstrates that TMEM97 plays important roles in cholesterol homeostasis. TMEM97 expression is regulated by upstream cholesterol-regulating factors such as sterol depletion and SREBP expression levels. TMEM97 regulates the downstream

Conclusions
TMEM97 has been validated as a biomarker of proliferative status in in vitro and in vivo studies. [ 18 F]ISO-1, which targets TMEM97, has been validated as a PET imaging biomarker of proliferative status in human breast cancer patients. [ 18 F]ISO-1 is also a promising predictor of the drug response in preclinical studies. The proliferative status of a tumor has a profound effect on the outcome of both chemotherapy and radiotherapy treatments. Thus, [ 18 F]ISO-1 should be useful to guide treatment plan for cancer patients.
TMEM97 plays distinct roles in cancer. TMEM97 is upregulated in some tumors but downregulated in other tumors. TMEM97 may be required for cell proliferation in certain tumor cells. TMEM97 is regulated by upstream oncogenic signals such as EGF, TGF-β and microRNA in some but not other tumor cells. The data suggest that TMEM97 is associated with cancer proliferation. Emerging evidence also demonstrates that TMEM97 plays important roles in cholesterol homeostasis. TMEM97 expression is regulated by upstream cholesterol-regulating factors such as sterol depletion and SREBP expression levels. TMEM97 regulates the downstream processes such as LDL uptake by forming complexes with PGRMC1 and LDLR, as well as cholesterol transport out of lysosome by interacting and regulating the NPC1 protein. Moreover, TMEM97 belongs to the EXPERA domain-containing protein family, which is implicated in cholesterol biology. Understanding molecular functions of TMEM97 in proliferation and cholesterol metabolism will be important to elucidate the interplay between cancer proliferation and cholesterol metabolism and to develop strategies to diagnose and treat cancer and cholesterol disorders using a rich collection of existing σ 2 R/TMEM97 radiotracers and ligands [18,50,51].