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Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97.

ABSTRACT: Sigma-2 receptor (S2R) is a S2R ligand-binding site historically associated with reportedly 21.5 kDa proteins that have been linked to several diseases, such as cancer, Alzheimer's disease, and schizophrenia. The S2R is highly expressed in various tumors, where it correlates with the proliferative status of the malignant cells. Recently, S2R was reported to be the transmembrane protein TMEM97. Prior to that, we had been investigating the translocator protein (TSPO) as a potential 21.5 kDa S2R candidate protein with reported heme and sterol associations. Here, we investigate the contributions of TMEM97 and TSPO to S2R activity in MCF7 breast adenocarcinoma and MIA PaCa-2 (MP) pancreatic carcinoma cells. Additionally, the role of the reported S2R-interacting partner PGRMC1 was also elucidated. Proximity ligation assays and co-immunoprecipitation show a functional association between S2R and TSPO. Moreover, a close physical colocalization of TMEM97 and TSPO was found in MP cells. In MCF7 cells, co-immunoprecipitation only occurred with TMEM97 but not with PGRMC1, which was further confirmed by confocal microscopy experiments. Treatment with the TMEM97 ligand 20-(S)-hydroxycholesterol reduced co-immunoprecipitation of both TMEM97 and PGRMC1 in immune pellets of immunoprecipitated TSPO in MP cells. To the best of our knowledge, this is the first suggestion of a (functional) interaction between TSPO and TMEM97 that can be affected by S2R ligands.

SUBMITTER: Thejer BM

PROVIDER: S-EPMC10093951 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97.

Thejer Bashar M BM Infantino Vittoria V Santarsiero Anna A Pappalardo Ilaria I Abatematteo Francesca S FS Teakel Sarah S Van Oosterum Ashleigh A Mach Robert H RH Denora Nunzio N Lee Byung Chul BC Resta Nicoletta N Bagnulo Rosanna R Niso Mauro M Contino Marialessandra M Montsch Bianca B Heffeter Petra P Abate Carmen C Cahill Michael A MA

International journal of molecular sciences 20230328 7

Sigma-2 receptor (S2R) is a S2R ligand-binding site historically associated with reportedly 21.5 kDa proteins that have been linked to several diseases, such as cancer, Alzheimer's disease, and schizophrenia. The S2R is highly expressed in various tumors, where it correlates with the proliferative status of the malignant cells. Recently, S2R was reported to be the transmembrane protein TMEM97. Prior to that, we had been investigating the translocator protein (TSPO) as a potential 21.5 kDa S2R ca ...[more]

PMID: 37047353

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Project description:Sigma-2 receptor/transmembrane protein 97 (TMEM97) is upregulated in cancer cells compared to normal cells. Traditional sigma-2 receptor agonists induce apoptosis and autophagy, making them of interest in cancer therapy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor, showing increased 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and stimulation of glycolytic hallmarks. 6-Substituted analogs of the canonical sigma-2 receptor antagonist, 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79), produce both metabolically stimulative and cytotoxic effects. Here, we compare the activities of two related compounds: 6-amino-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (CM571), the 6-amino derivative of SN79, which binds with high affinity to both sigma-1 and sigma-2 receptors, and 1,3-bis(3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-2-oxo-2,3-dihydrobenzo[d]oxazol-6-yl)thiourea (MAM03055A), a homo-bivalent dimer of CM571. MAM03055A resulted from the degradation of 3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one (CM572), the cytotoxic 6-isothiocyanato SN79 derivative. MAM03055A exhibited high affinity and strong preference for sigma-2 receptors (sigma-1 Ki = 3371 nM; sigma-2 receptor Ki = 55.9 nM). Functionally, MAM03055A treatment potently induced cell death in SK-N-SH neuroblastoma, MDA-MB-231 breast, and both SW48 and SW480 colorectal cancer cell lines, causing proapoptotic BH3 interacting-domain death agonist (BID) cleavage in SK-N-SH cells. Conversely, CM571 induced metabolic stimulation. CM571 bound reversibly to both receptors, while MAM03055A bound pseudo-irreversibly to sigma-2 receptors and caused residual cytotoxic activity after acute exposure and removal of the compound from the media. Interestingly, MAM03055A induced a time-dependent loss of sigma-2 receptor/TMEM97 protein from cells, whereas monomer CM571 had no effect on receptor levels. These results suggest that monovalent and bivalent sigma-2 receptor ligands in this series interact differently with the receptor, thus resulting in divergent effects.

Project description:Sigma-2 receptors have been implicated in both tumor proliferation and neurodegenerative diseases. Recently the sigma-2 receptor was identified as transmembrane protein 97 (TMEM97). Progesterone receptor membrane component 1 (PGRMC1) was also recently reported to form a complex with TMEM97 and the low density lipoprotein (LDL) receptor, and this trimeric complex is responsible for the rapid internalization of LDL. Sigma-2 receptor ligands with various structures have been shown to induce cell death in cancer cells. In the current study, we examined the role of TMEM97 and PGRMC1 in mediating sigma-2 ligand-induced cell death. Cell viability and caspase-3 assays were performed in control, TMEM97 knockout (KO), PGRMC1 KO, and TMEM97/PGRMC1 double KO cell lines treated with several sigma-2 ligands. The data showed that knockout of TMEM97, PGRMC1, or both did not affect the concentrations of sigma-2 ligands that induced 50% of cell death (EC50), suggesting that cytotoxic effects of these compounds are not mediated by TMEM97 or PGRMC1. Sigma-1 receptor ligands, (+)-pentazocine and NE-100, did not block sigma-2 ligand cytotoxicity, suggesting that sigma-1 receptor was not responsible for sigma-2 ligand cytotoxicity. We also examined whether the alternative, residual binding site (RBS) of 1,3-Di-o-tolylguanidine (DTG) could be responsible for sigma-2 ligand cytotoxicity. Our data showed that the binding affinities (K i) of sigma-2 ligands on the DTG RBS did not correlate with the cytotoxicity potency (EC50) of these ligands, suggesting that the DTG RBS was not fully responsible for sigma-2 ligand cytotoxicity. In addition, we showed that knocking out TMEM97, PGRMC1, or both reduced the initial internalization rate of a sigma-2 fluorescent ligand, SW120. However, concentrations of internalized SW120 became identical later in the control and knockout cells. These data suggest that the initial internalization process of sigma-2 ligands does not appear to mediate the cell-killing effect of sigma-2 ligands. In summary, we have provided evidence that sigma-2 receptor/TMEM97 and PGRMC1 do not mediate sigma-2 ligand cytotoxicity. Our work will facilitate elucidating mechanisms of sigma-2 ligand cytotoxicity.

Project description:BackgroundThere are limited effective drugs that can reach the brain to target brain tumors, in particular glioblastoma, which is one of the most difficult cancers to be cured from. Because the overexpression of the sigma-2 receptor is frequently reported in glioma clinical samples and associated with poor prognosis and malignancy, we herein studied the anti-tumor effect of the sigma-2 receptor agonist PB221 (4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperidine) on an anaplastic astrocytoma tumor model based on previous encouraging results in pancreatic cancer and neuroblastoma SK-N-SH cells.MethodsThe expression of the sigma-2 receptor, transmembrane protein 97 (TMEM97), in ALTS1C1 and UN-KC6141 cell lines was measured by RT-PCR and quantitative RT-PCR. The binding of sigma-2 receptor fluorescent ligands PB385 (6-[5-[3-(4-cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronaphthalen-5-yloxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine) and NO1 (2-{6-[2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one-5-yloxy]hexyl}-5-(dimethylamino)isoindoline-1,3-dione) was examined by flow cytometry and the fluorescent plate reader. The antitumor activity of PB221 was initially examined in the murine brain tumor cell line ALTS1C1 and then in the murine pancreatic cell line UN-KC6141. The potential therapeutic efficacy of PB221 for murine brain tumors was examined by in vitro migration and invasion assays and in vivo ectopic and orthotopic ALTS1C1 tumor models.ResultsThe IC50 of PB221 for ALTS1C1 and UN-KC6141 cell lines was 10.61 ± 0.96 and 13.13 ± 1.15 μM, respectively. A low dose of PB221 (1 μM) significantly repressed the migration and invasion of ALTS1C1 cells, and a high dose of PB221 (20 μM) resulted in the apoptotic cell death of ALTS1C1 cells. These effects were reduced by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine, suggesting mitochondrial oxidative stress is involved. The in vivo study revealed that PB221 effectively retarded tumor growth to 36% of the control tumor volume in the ectopic intramuscular tumor model and increased the overall survival time by 20% (from 26 to 31 days) in the orthotopic intracerebral tumor model.ConclusionsThis study demonstrates that the sigma-2 receptor agonist PB221 has the potential to be an alternative chemotherapeutic drug for brain tumors with comparable side effects as the current standard-of-care drug, temozolomide.

Dataset Information

Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97.

Publications

Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97.

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