Chenbo Zeng

Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumor cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor binding site. WC-21, a sigma-2 ligand containing both a photoactive moiety azide and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was then identified as PGRMC1 (progesterone receptor membrane component-1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalizes with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.

Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes.

We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4′,6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.

Functional assays to define agonists and antagonists of the sigma-2 receptor

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC50 values of the sigma-2 ligands using the cell viability assay ranged from 11.4μM to >200μM, which were comparable with the EC50 values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.

Sigma-2 Receptor as Potential Indicator of Stem Cell Differentiation

PurposeThe sigma-2 (σ2) receptor is a potential biomarker of proliferative status of solid tumors. Specific synthetic probes using N-substituted-9-azabicyclo [3.3.1]nonan-3α-yl carbamate analogs have been designed and implemented for experimental cancer diagnosis and therapy.ProceduresWe employed the fluorescently labeled σ2 receptor probe, SW120, to evaluate σ2 receptor expression in human stem cells (SC), including: bone marrow stromal, neural progenitor, amniotic fluid, hematopoetic, and embryonic stem cells. We concurrently evaluated the intensity of SW120 and 5-ethynyl-2′-deoxyuridine (EdU) relative to passage number and multi-potency.ResultsWe substantiated significantly higher σ2 receptor density among proliferating SC relative to lineage-restricted cell types. Additionally, cellular internalization of the σ2 receptor in SC was consistent with receptor-mediated endocytosis and confocal microscopy indicated SW120 specific co-localization with a fluorescent marker of lysosomes in all SC imaged.ConclusionThese results suggest that σ2 receptors may serve to monitor stem cell differentiation in future experimental studies.

Abstract 2131: Functional assays to define agonists and antagonists of sigma-2 receptors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The sigma receptor was originally defined pharmacologically as the specific binding site for a group of compounds named as sigma ligands. The sigma-2 receptor has been identified as a biomarker in proliferating tumors. It regulates cell growth and is an emerging target for cancer diagnosis and therapeutics. Up to date numerous sigma-2 selective ligands have been developed. However, there is no well-established functional assay for defining agonist/antagonist for sigma-2 receptors. Many sigma-2 ligands with diverse structures have been shown to induce cytotoxicity in a variety of cancer cells by triggering caspase-independent and caspase-dependent apoptosis. Therefore, in the current study, we propose to use cell viability and caspase 3 activity, a hallmark of apoptosis, as functional assays to define agonist/antagonist for sigma-2 receptors. Three classes of sigma-2 ligands developed in our laboratory were tested in these two assays in two tumor cell lines: mouse breast cancer cell line EMT6 and human melanoma cell line MDA-MB435. Based on the ratios of the EC50 values of our sigma-2 ligands to the EC50 of siramesine, we categorized the azabicyclononane analogs as agonists (e.g. SV 119) or partial agonists (e.g. WC 26 and SV 95), and benzamide analogs as non-canonical antagonists (e.g. RHM-1) since they do not trigger cell death, and they do not block sigma-2 agonist-induced cell death. SV 95 partially blocked SV 119-induced cytotoxicity. The establishment of functional assays for defining agonist/antagonist will facilitate functional characterization of sigma-2 ligands and sigma-2 receptors. Citation Format: Robert H. Mach, Chenbo Zeng, Justin M. Rothfuss, Jun Zhang, Suwanna Vangveravong, Wenhua Chu, Zhude Tu, Shihong Li. Functional assays to define agonists and antagonists of sigma-2 receptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2131. doi:10.1158/1538-7445.AM2013-2131

Abstract 5690: Validating sigma-2 receptor ligand as a novel tumor-targeted drug delivery agent for treating ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The sigma-2 receptor is overexpressed in various human tumors. Sigma-2 receptor selective radiotracers have been shown to target various solid tumors in rodents and in human patients using positron emission tomography. Sigma-2 receptor ligands can be internalized into tumor cells by the endocytotic pathway. Therefore, sigma-2 receptor ligands are excellent candidates as tumor-targeted delivery agents when covalently attached to drugs. Smac is a protein released from mitochondria into the cytosol in response to apoptotic stimuli. Small-molecule Smac mimetic compounds (SMC) have been developed by several laboratories as anticancer drugs. In this study we validate sigma-2 ligand as a tumor-targeting drug delivery agent for treating ovarian cancer. We have synthesized sigma-2 ligand-conjugated SMC (SSMC), SW III-123. Our data show that SW III-123 has adequate sigma-2 receptor binding affinities (Kiα2 =190 nM and Kiα1 =2046 nM). SW III-123 exhibits potent antitumor activities with EC50 values at 4.0 μM, 2.3 μM and 2.4 μM in human ovarian cancer cell lines SKOV-3, CaOV-3, and BG-1, respectively. In contrast, unconjugated SMC, SW IV-52s, shows little cytotoxicity in these cell lines (EC50 > 200 μM). These results suggest that the sigma-2 ligand has successfully delivered SMC into ovarian cancer cells. Western blot results show that SW III-123 degrades inhibitor of apoptosis proteins (IAPs), XIAP and cIAP-1, in SKOV-3 cells, possibly by interacting with the baculovirus IAP repeat (BIR) domains of IAPs. SW III-123 cleaves caspase-3, 8, and 9 dose-dependently in SKOV-3 cells, indicating that SW III-123 kills ovarian cancer cells by activating both intrinsic and extrinsic apoptotic pathways. In conclusion, sigma-2 ligand is a promising tumor-targeting drug delivery agent. SSMC will likely offer a novel class of therapeutic drugs for treating ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5690. doi:1538-7445.AM2012-5690

Abstract 1054: Identification of progesterone receptor membrane component-1 as the putative sigma-2 receptor

Sigma binding sites represent a unique class of receptors and are highly expressed in tumor cells. Two sigma binding site subtypes, known as sigma-1 and sigma-2 receptors, were distinguished based on differences in their pharmacological profiles and molecular weights. The sigma-1 receptor gene has been cloned and well characterized in the CNS and cancer biology. However, the sigma-2 receptor has not yet been cloned. The purpose of the current study is to identify and clone sigma-2 receptor. We have synthesized an irreversible sigma-2 receptor selective photoaffinity probe, WC-II-21, containing a fluorescein isothiocyanate (FITC) group. This ligand has high binding affinity for sigma-2 receptor (Ki = 13 nM) and relatively low binding affinity for sigma-1 receptor (Ki > 1,000 nM). This ligand irreversibly labeled sigma-2 receptors in the rat liver membrane fractions after UV irradiation. The ligand-labeled proteins were solubilized, enriched and then separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The position of a predominant band of the ligand-labeled protein in the gel was determined by western blot analysis using FITC antibody. MALDI-mass spectrometry analysis for the ligand-labeled proteins indentified the protein progesterone receptor membrane component-1 (PGRMC-1) with a convincingly high score of 81. Based on the similarities of the reported biological characteristics and functions between PGRMC1 and sigma-2 receptor, PGRMC1 was investigated in the current study. Immunocytochemistry revealed that PGRMC1 colocalized with molecular markers of endoplasmic reticulum and mitochondria in human melanoma cell line MDA-MB435. The subcellular localization of sigma-2 receptor also colocalized with fluorescent markers of endoplasmic reticulum and mitochondria as reported previously from our group (Cancer Res 2007; 67, 6708-6716). Thus, PGRMC1 and sigma-2 receptor appear to localize in the same subcellular organelles in the cell. PGRMC1 was knocked down with PGRMC1 siRNA in MDA-MB435 cells. Sigma-2 radioligand binding activity was significantly reduced in the PGRMC1 knocked down cells relative to the nontargeting siRNA transfected cells. These data suggest that PGRMC1 is the putative sigma-2 receptor. Further validation of PGRMC1 as the sigma-2 receptor is ongoing in our group. (Supported by CA 102869). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1054.