Imaging the Sigma-2 Receptor for Diagnosis and Prediction of Therapeutic Response

Sigma (σ) receptors represent a class of proteins that were initially classified as a subtype of the opiate receptors. Subsequent studies revealed that σ binding sites are a distinct class of receptors that are located in the central nervous system as well as in a variety of tissues and organs [1, 2]. Two σ binding site subtypes were distinguished based on differences in their drug-binding profiles and molecular weight. The two binding sites are known as σ1 and σ2 receptors. σ1 receptors have a molecular weight of ~25 kDa, whereas σ2 receptors have a molecular weight of ~21.5 kDa [3]. The σ1 receptor has been cloned and displays a 30% sequence homology with the enzyme, yeast C8-C7 sterol isomerase [4, 5], but this receptor lacks C8-C7 isomerase activity. Recent studies have shown that neuroactive steroids bind with moderate affinity to σ1 sites, and suggest that σ1 receptors may modulate the activity of GABA and NMDA receptors in the CNS [6-8]. The σ2 receptor has not been cloned, and most of what is known regarding the σ2 receptor has been obtained through the use of in vitro receptor binding studies aimed at the pharmacological characterization of this receptor.


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proliferative status (i.e., high P:Q ratio) typically respond better to hyperfractionated radiation therapy versus conventional radiation therapy. Similarly, tumors having a high P:Q ratio will respond better to cell cycle specific agents such as Ara-C and gemcitabine, whereas tumors having a lower proliferative status will respond better to non-cell cycle specific agents such as cisplatin and BCNU [11]. Also, a change in the proliferative status of a tumor during or after treatment has the potential to serve as a predictor of response and allow further tailoring of therapy. In order to investigate the relationship between the density of σ 2 receptors and the proliferative status of tumors, Wheeler and colleagues used the mouse mammary adenocarcinoma cell line 66 [12,13] to determine if there was a difference in the density of σ 2 receptors in proliferating (66P) and quiescent (66Q) tumor cells in cell culture or in solid tumor xenografts. This group demonstrated that the density of σ 2 receptors in 66P cells was about 10 times greater than the density observed in 66Q cells ( Figure 1A) [14]. The density of σ 2 receptors in the 66P cells was found to be quite high, ~900,000 copies/cell versus ~90,000 receptors/cell in the 66Q cells. This group also reported that the expression kinetics of σ 2 receptors follows the growth kinetics of the 66 cells ( Figure 1B) [15]. A subsequent study in solid tumor xenografts of the same tumor cell lines demonstrated the identical P:Q ratio to that measured in the cell culture condition [16]. The agreement between the solid tumor and tissue culture data indicates that the σ 2 receptor is a receptor-based biomarker of cell proliferation in breast tumors. Thus the  2 receptor possesses properties similar to Ki-67, a marker of proliferation [17]. Ki-67 expression level is determined by immunohistochemistry using Ki-67 antibody. This method requires biopsy or surgical specimens of tumor. In contrast, radiotracers having a high affinity and high selectivity for σ 2 receptors have the potential to assess the proliferative status of human breast tumors using noninvasive imaging techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT). In addition, it is likely that this approach can be extended to assess the proliferative status of other human tumors, such as head and neck, melanoma, and lung tumors, which are known to express a high density of σ 2 receptors [10]. Imaging the proliferative status with σ 2 receptors radiotracers can provide useful information regarding the prognosis and aggressiveness of tumors, and this information can be used to guide treatment of cancer in clinical practice. Rapidly proliferating tumors requires aggressive initial treatment. A reduction in the proliferative status of a tumor can serve as a predictor of the tumor's response to therapy.

Development of σ 2 receptor selective ligands
A number of structurally-diverse compounds have been shown to possess a high affinity to σ receptors [2]. However, most of these compounds bind selectively to σ 1 receptor or have similar affinities to both σ 1 and σ 2 receptors. One of the first σ 2 selective ligands reported was the benzomorphan-7-one analog, CB-64D [18]. This compound was identified as part of a structure-activity relationship (SAR) study aimed at refining the affinity of (−)-2-methyl-5-(3-hydroxyphenyl)morphan-7-one for μ versus κ opioid receptors [19]. A second series of compounds having a high affinity for σ 2 receptors are the 3-(ω-aminoalkyl)-1H-indole analogs [20,21]. SAR studies with compounds that were originally designed to be serotonin 5-HT 1A agonists, resulted in the synthesis of Lu 28-179 [22], also known as siramesine， which has a subnanomolar affinity for σ 2 receptors and a 140-fold selectivity for σ 2 versus σ 1 www.intechopen.com Molecular Probes for Imaging the Sigma-2 Receptor 305 receptors. Other compounds that were found to have a higher affinity for σ 2 versus σ 1 receptors are: 1) the hallucinogen, ibogaine [23,24]; 2) the mixed serotonin 5-HT 3 antagonist/5-HT 4 agonist BIMU-1 [25]; 3) the tropane analog SM-21, an acetylcholine releaser that has been utilized as an antinociceptic agent [26,27]; 4) the trishomocubane analog ANSTO-19 [28]; and 5) the piperazine analog PB28 [29]. A number of SAR studies using BIMU-1 as the lead compound have identified high affinity, high selectivity σ 2 receptor ligands [30][31][32]. BIMU-1 is an ideal lead compound for SAR studies since it provides a variety of regions where structural modifications can be made to optimize the σ 2 receptor affinity and reduce the affinity for serotonin 5-HT 3 and 5-HT 4 receptors ( Figure 2). The structures of BIMU-1 were altered in three different regions: 1) replacement of the urea linkage with a comformationally-flexible carbamate moiety; 2) replacement of the N-methyl group with an N-benzyl group to diminish serotonin receptor affinity; and 3) preparation of both tropane (i.e., [3.2.1]azabicyclonoctane) and granatane (i.e., [3.3.1]azabicyclononane) ring systems. The most interesting analog from this initial SAR study was the compound ABN-1, which had a σ 2 receptor affinity of ~ 3 nM and a σ 2 : σ 1 selectivity of ~30 ( Figure 2) [31]. Compound ABN-1 was used as a secondary lead compound for a series of subsequent SAR studies aimed at producing second-generation granatane analogs having an improved σ 2 receptor affinity and high σ 2 : σ 1 selectivity ratio. Consequently, this led to the development of a number of fluorescent probes, K05-138, SW120, SW107 and SW116, that have proven useful in two photon and confocal microscopy studies of σ 2 receptors in tumor cells growing under cell culture conditions [33,34]. A second class of compounds having a high affinity for σ 2 receptors and excellent σ 1 : σ 2 selectivity ratios are the conformationally-flexible benzamide analogs. These compounds were identified in an SAR study of the benzamide analog, YUN250, that was developed as a dopamine D 3 -selective ligand ( Figure 3A) [35]. The relatively high lipophilicity of YUN250 (log P = 5.76) suggests that it is not capable of readily crossing the blood-brain barrier and being active in behavioral studies. In order to reduce the lipophilicity of YUN250, the 4-(2,3dichlorophenyl) piperazine moiety of YUN250 was replaced with other aromatic amine groups. Although this strategy resulted in a number of useful dopamine D 3 receptor ligands [36], it was also observed that replacement of the 4-phenylpiperazine moiety with a 6,7dimethoxy-1,2,3,4-tetrahydroisoqinoline ring resulted in compounds having a high affinity and excellent selectivity for σ 2 versus σ 1 receptors, and a dramatic reduction in affinity for dopamine receptors [35]. As discussed in greater detail below, the conformationally-flexible benzamide analogs have proven to be an important class of σ 2 -selective compounds for the preparation of radiolabeled probes to image this receptor in vitro and in vivo.

In vitro binding and in vivo imaging studies of σ 2 receptors
The conformationally-flexible benzamide analogs have been used to develop the σ 2 radioligands for in vitro binding studies. The tritium labeled σ 2 selective ligand, [ 3 H]RHM-1, has been used to conduct Scatchard studies of σ 2 receptors in tumors and normal tissues ( Figure 3A) [37,38]. The in vitro receptor binding studies indicate that [ 3 H]RHM-1 binds with a high affinity to both EMT-6 mouse breast tumors (K d =3.45 nM) and chemicallyinduced rat breast tumors (K d =4.66 nM). The σ2 receptor densities of both tumors are found to be high (B max =2290 fmol/mg of protein for EMT-6 mouse breast tumors and B max =2410 fmol/mg of protein for N-methyl-N-nitrosourea (NMNU)-induced rat breast tumors) ( Figure 3B). The pharmacologic profile of , a most commonly used radioligand for the σ 2 receptor binding assay. These results indicate that [ 3 H]RHM-1 is a useful radioligand for studying  2 receptors in vitro. The high correlation between the density of σ 2 receptors and the proliferative status of solid tumors indicates that σ 2 selective radiotracers can be used for imaging the proliferative status of solid tumors with PET and SPECT. The conformationally-flexible benzamide analogs have been used for the development of PET radiotracers for imaging the σ 2 receptor status of solid tumors. The first PET radiotracers prepared were [ 11 C]1-4 ( Figure 4A), which involves labeling the corresponding ortho-hydroxy group with [ 11 C]methyl iodide [38].
MicroPET and tumor uptake studies were conducted with [ 11 C]1-4; the most promising analog proved to be [ 11 C]4. Although all four analogs had a high affinity for σ 2 receptors, the optimal lipophilicity of [ 11 C]4 was responsible for the high tumor uptake and suitable signal: normal tissue ratios for imaging. These data indicate that both receptor affinity and lipophilicity are important properties for successful receptor-based tumor imaging agents.
MicroPET and MicroCT imaging studies in a murine solid breast tumor EMT-6 clearly show the potential of [ 11 C]4 as a radiotracer for imaging the σ 2 receptor status of breast tumors with PET. Although [ 11 C]4 provides a clear image of breast tumors in microPET imaging studies, the short half life of carbon 11 (t 1/2 = 20.4 min) is not ideal for the development of radiotracers which can be used in clinical PET imaging studies. The longer half-life of 18 F (t 1/2 =109.8 min) compared to 11 C places fewer time constraints on tracer synthesis, allows imaging studies to be conducted up to 2 h after injection of the radiotracers, and usually results in higher tumor: normal tissue ratios. A number of 18 F-labeled radiotracers [ 18 F]5-8 ( Figure 4B) based on the conformationally-flexible benzamide analogs have been generated and evaluated in murine breast tumor models [39]. The strategy involved replacement of the 2methoxy group in the benzamide ring with a 2-fluoroethoxy group. The 2-fluoroethoxy-for methoxy-substitution is a common strategy used in the development of 18 F-labeled radiotracers. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated that all four 18 F -labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 1 and 2 h post-i.v. injection ( Figure 4C). The moderate to high tumor/normal tissue ratios and the rapid clearance from the blood for

Confocal and two photon microscopy studies of σ 2 receptors in tumor cells
Since the σ 2 receptor protein has not been cloned, current knowledge of this receptor is based predominantly on receptor binding studies with radiolabeled probes such as [ 3 H]DTG. The function of the σ 2 receptors has also been investigated by studying their effects on the biochemical and physiological properties of tumor cells. Several studies have shown that σ 2 ligands induced cell death. The proposed mechanisms of cell death include caspase-independent apoptosis [40], lysosomal leakage [41], Ca 2+ release [42,43], oxidative stress [41], ceramide production [44] and autophagy [45]. However, the subcellular localizations where the σ 2 ligands bind and function were not known. Using the fluorescent probes shown in Figure 2, Zeng and colleagues [33,34] recently conducted a series of confocal and two photon microscopy studies that have provided the information regarding the localization of σ 2 receptors in breast tumor cells. MDA-MB-435 cells were incubated with 30 nM SW120 and each of the five subcellular organelle markers: the mitochondria tracker, MitoTracker Red CMXRos (20 nM ), the endoplasmic reticulum tracker, ER-Tracker TM Red (500 nM), the lysosome tracker, LysoTracker Red DND-99 (50 nM), the nuclear marker, DAPI (300 nM), or the plasma membrane tracker, FM 4-64FX (5 g/mL). The results showed that SW120 partially colocalized with MitoTracker, ER-Tracker, LysoTracker and the plasma membrane tracker, suggesting that σ 2 receptors may localize in mitochondria, endoplasmic reticulum, lysosomes and the plasma membrane ( Figure 5). The data also showed that SW120 did not co-localize with the nuclear marker, DAPI, suggesting that either the σ 2 receptor does not exist in the nucleus or SW120 does not enter the nucleus. The similar results were obtained for the other σ 2 fluorescent probes (SW116, K05-138 and SW107) [33,34]. The kinetic studies of internalization of σ 2 fluorescent probes in MDA-MB-435 cells were conducted using confocal microscopy [33,34]. σ 2 fluorescent probes enter tumor cells rapidly. For example, K05-138 and SW120 reach 50% of the maximal fluorescent intensity (T 1/2 ) in 16 seconds and 11 minutes, respectively ( Figure 6A). The internalization of SW120 can be blocked by the σ 2 selective ligands, SW43 and siramesine, by 52% and 44%, respectively ( Figure 6B). This internalization can not be blocked by the σ 1 selective ligand, (+)-pentazocine. The data suggest that the internalization of the σ 2 selective ligand is partially mediated by σ 2 receptor. In order to study whether the internalization of the σ 2 ligand is mediated by endocytosis, Zeng et al examined the effect of phenylarsine oxide (PAO), a well-characterized endocytosis inhibitor [46], on the internalization of SW120. MDA-MB-435 cells were pretreated with 5 or 10 M PAO for 30 min, and then treated with 10 nM SW120 in the absence or presence of PAO for an additional 30 min. Flow cytometric analysis ( Figure 6C) showed that 10 M PAO significantly blocked internalization of SW120 by 30%. These data demonstrate that 30% of the  2 receptor ligand was internalized by an endocytosis-mediated mechanism, while the remaining 70 % was internalized by other mechanisms such as passive diffusion. The rapid internalization of σ 2 receptors via endocytosis suggests that σ 2 selective ligands may potentially serve as receptor-mediated probes for delivering cytotoxic agents to solid tumors. The two photon and confocal microscopy studies conducted by Zeng et al. [33,34] have provided useful information for the interpretation of studies demonstrating that σ 2 receptor ligands may have a role as cancer chemotherapeutic agents. Mitochondria are a key organelle to regulate the intrinsic apoptotic pathway. Apoptotic signals such as UV irradiation or treatment with chemotherapeutic agents cause the release of cytochrome C from the mitochondria and the subsequent activation of caspase-3 leading to an apoptotic cell death [47]. The subcellular localization of σ 2 ligands in mitochondria is consistent with the previous studies that  2 ligands trigger apoptosis in tumor cells by acting on mitochondria [48]. The data are also consistent with our observation by transmission electron microscopy that the  2 ligand siramesine induces distortion of mitochondria (unpublished data). The endoplasmic reticulum (ER) serves as a dynamic Ca 2+ storage pool [49]. σ 2 selective ligands have been reported to induce transient Ca 2+ release from the ER, which may be responsible for σ 2 ligand-induced cell death [42]. The presence of the σ 2 fluorescent probes in the ER is consistent with these results. Additional research will be required to determine how σ 2 receptors regulate the Ca 2+ release channels in the ER. Lysosomal proteases, such as cathepsins, calpains and granzymes, have been reported to contribute to apoptosis [50]. Under physiological conditions, these proteases are found within the lysosomes but are released into the cytoplasm upon exposure to cell damaging agents, thereby triggering a cascade of intracellular events leading to cell death. The σ 2 selective ligand siramesine has been reported to cause lysosomal leakage and induce cell death by caspase-independent mechanisms [41,45]. The localization of fluorescent σ 2 receptor probes in the lysosomes is consistent with the hypothesis that siramesine induces cell death partially by targeting lysosomes to cause lysosomal damage, the release of proteases, and eventually cell death. Evidence has also been reported that σ 2 receptors exist in lipid rafts which are mainly found in the plasma membrane [51]. Lipid rafts play an important role in the signaling associated with a variety of cellular events including adhesion, motility, and membrane trafficking [52,53]. The observation that σ 2 fluorescent ligands are co-localized with cytoplasmic membrane markers, and undergo receptor mediated endocytosis, is consistent with their localization in lipid rafts. How the σ 2 receptor is involved in lipid raft function deserves further investigation. The inhibition of SW120 internalization by phenylarsine oxide (PAO). MDA-MB-435 cells were preincubated with 5 or 10 M PAO for 30 min at 37C, and then incubated with 10 nM SW120 for an additional 30 min. The cells were analyzed by flow cytometry. The internalization of SW120 was significantly reduced by 10 μM PAO (* P < 0.05).

B C
The σ 2 receptor has been validated as a proliferation marker in cell culture and in solid tumors. Therefore it is possible that σ 2 fluorescent probes could preferentially label proliferating cells versus non-proliferating cells and serve as agents to image cell proliferation in vivo. This hypothesis was tested in nude mice implanted with murine mammary adenocarcinoma 66 cells and BALB/C mice implanted with mouse mammary carcinoma cell line EMT6 [34]. The mice were treated with SW120 (50 g in 100 L PBS) for one hour. The peripheral blood mononuclear cells (PBMC), which are commonly used as controls for non-proliferative cells, and tumor cells were prepared. These cells were analyzed by flow cytometry for SW120 uptake and for Ki67 expression, a commonly-used proliferation marker. Our data showed that PBMC were Ki67 negative, whereas a large portion of the tumor cells were Ki-67 positive (Figure 7). The data also showed that PBMC were not labeled by SW120, whereas a portion of the tumor cells were labeled with SW120.
The trend of the positive correlation between Ki67 expression and SW120 labeling implies that the fluorescent may possess in vivo selectivity toward proliferating cells versus nonproliferative cells. These data suggest that σ 2 fluorescent probes could be used as imaging agents for monitoring cell proliferation in mice. The data also suggest that σ 2 selective ligands hold a potential to serve as therapeutic agents to selectively target tumor cells in vivo. Fig. 7. Ki67 expression and SW120 fluorescent intensity in solid mouse breast tumors and peripheral blood mononuclear cells (PBMC) of mice by flow cytometric analysis. The female nude mice were implanted with murine mammary adenocarcinoma 66 cells. BALB/C mice were implanted with mouse mammary carcinoma cell line EMT6. The mice were i.v. injected with SW120 (50 µg/ mouse) and tissues harvested after 1 hour. The 66 cells and EMT6 cells were dissociated from the solid tumors. PBMC were prepared from the blood of mice. The cells were analyzed for the fluorescent intensity of SW120 and the Ki67 expression determined by Ki67 immunostaining using a flow cytometer. The data indicate the trend of the positive correlation between Ki67 expression and SW120 labeling.

Conclusions
The  2 receptor continues to be an important molecular target in the field of tumor biology. The high expression of this receptor in proliferating versus quiescent breast tumors indicates that the  2 receptor is an important clinical biomarker for determining the proliferative status of solid tumors using the functional imaging techniques PET and SPECT. The  2 receptor fluorescent probes are useful to study the subcellular localization and the function of  2 receptors using confocal and two photon microscopy techniques, and may be used to image tumor proliferation in mice using optical imaging techniques. The full utility of the  2 receptor in the diagnosis and prediction of therapeutic response will rely on the cloning of the  2 receptor and elucidation of its functional role in normal and tumor cell biology.
Note: While this article is accepted, a paper from our group is published in Nature Communication [54].
In this paper we have identified progesterone receptor membrane component 1(PGRMC1) as the putative 2 receptor. This finding will greatly facilitate investigations of the functions of the 2 receptor in normal and tumor cells.