TMEM 97 and PGRMC 1 do not mediate sigma-2 ligand-induced cell death

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 (Ki) 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. Introduction The sigma receptor was originally defined pharmacologically. The sigma receptor was once thought to be a subset of the opioid receptor, but was subsequently revealed to be a distinct class of receptors. There are two subtypes of sigma receptors, sigma-1 and sigma-2 receptors. The molecular weight of sigma-1 and sigma-2 receptors was previously reported to be 25 and 18–21.5 kD, respectively. The sigma-2 receptor has been shown to be expressed in higher density in proliferating versus quiescent tumor cells. Sigma-2 ligands have been developed as molecular probes for imaging solid tumors and also as potential therapeutic agents for treating cancer. A sigma-2 receptor ligand has also shown promise as a novel approach for treating Alzheimer’s disease (AD). Recently, transmembrane protein 97 (TMEM97), a protein implicated in cancer and cholesterol homeostasis, was identified as the sigma-2 receptor. TMEM97 has a © The Author(s) 2019. OpenAccessThis article is licensedunder aCreativeCommonsAttribution 4.0 International License,whichpermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changesweremade. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Correspondence: Robert H. Mach (rmach@pennmedicine.upenn.edu) Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Room 283 231S. 34th St, Philadelphia, PA 19104, USA Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA Edited by E. Sayan Official journal of the Cell Death Differentiation Association 12 34 56 78 90 () :,; 12 34 56 78 90 () :,; 1 2 3 4 5 6 7 8 9 0 () :,; 12 34 56 78 90 () :,; role in cholesterol and lipid metabolism, and it was reported that TMEM97 and cholesterol biosynthesis genes in normal ovarian epithelial cells were coordinately upregulated by progesterone treatment. In another report, TMEM97 was identified as a regulator of cellular cholesterol homeostasis in targeted RNAi screening. TMEM97 knockdown reduced cellular cholesterol levels as well as the rate of internalization of low density lipoprotein (LDL) uptake by the LDL receptor (LDLR). Furthermore, cell cultures growing under sterol-depleted conditions results in an upregulation of TMEM97 mRNA levels. These data indicated that TMEM97 has a key role in the regulation of cholesterol homeostasis. Previously, our group proposed that the binding site for the sigma-2 receptor resided in a protein complex containing the progesterone receptor membrane component 1 (PGRMC1). Subsequent studies challenged the validity of our results, and the recent identification of the TMEM97 as the gene for the sigma-2 receptor seemed to substantiate these reports. However, our group recently reported that TMEM97 formed a complex with PGRMC1 and LDLR, and this trimeric complex was responsible for the rapid internalization of LDL in HeLa cells. These data demonstrated that PGRMC1 associates with TMEM97 physically and functionally, and corroborates our previous report that the sigma-2 receptor represents a binding site in the PGRMC1 protein complex. TMEM97 (initially called MAC30) was first reported as a differentially expressed gene in meningioma. TMEM97 was decreased in meningioma compared to normal leptomeningeal tissues. Subsequent studies showed that TMEM97 was overexpressed in gastric cancer, colorectal cancer, breast cancer, glioma, and ovarian cancer cells. Overexpression of TMEM97 was positively correlated with tumor stage, metastasis, and shorter survival time of patients with various cancers. The differential expression of TMEM97 in cancer cells suggest that this protein may have a role in tumor development, growth, and proliferation. The sigma-2 receptor is a potential target for cancer therapeutics. Sigma-2 ligands have been shown to induce cytotoxicity in cancer cells as a single agent, a drug delivery agent and a sensitizer to other anticancer drugs in cell culture and in animal models. Sigma-2 ligands trigger cell death by inducing lysosome dysfunction, ROS production, caspase-independent, and caspasedependent events. However, it is not known how sigma-2 ligands interact with sigma-2 receptors and induce subsequent cell death. Sigma-2 ligands have nanomolar binding affinities to sigma-2 receptors but require micromolar range of concentrations to exhibit cell-killing effects. The factors responsible for the large difference between sigma-2 receptor affinity and potency in the cell-kill assays are currently unknown. In the current study, we studied whether TMEM97 or PGRMC1 mediated sigma-2 ligand-induced cell death. We show that CRISPR/Cas9 gene editing of TMEM97 and PGRMC1 did not affect the cytotoxicity of a panel of structurally diverse sigma-2 ligands. We also examined whether the alternative residual binding site of DTG, a gold standard sigma-2 ligand, in TMEM97 knockout (KO) and TMEM97/PGRMC1 double KO cells could be responsible for sigma-2 ligand cytotoxicity. Our data showed that this residual binding site of DTG does not appear to be responsible for the cytotoxicity of sigma-2 ligands, and raises a question regarding the mechanism of action of these ligands as anticancer drugs. Results Knockout of TMEM97, PGRMC1, or both proteins did not affect EC50 values of sigma-2 ligands Sigma-2 ligands were previously reported as potential therapeutic drugs in various cancer cells in cell culture and in animal models. In order to study whether the cytotoxicity of sigma-2 ligands was mediated by TMEM97 or PGRMC1, we compared cell-killing potency of sigma-2 ligands in control, TMEM97 KO, PGRMC1 KO, and double KO HeLa cells. These cell lines were previously created in our laboratory using CRISPR/Cas9 technology and western blot data demonstrated that TMEM97 or/ and PGRMC1 proteins were completely removed in the knockout cell lines. Three sigma-2 agonists SW43, siramesine and PB28, and three sigma-2 antagonists RHM4, RHM-1, and ISO-1 were studied in this study. The diverse chemical structures of these ligands and their binding affinities obtained by the competition assay for sigma-1 and sigma-2 receptors were shown in Fig. 1 and Table 1. The control and knockout cells were incubated with sigma-2 ligands at increasing concentrations for 24 h and cell viability assay was performed. Dose–response curves were generated as shown in Fig. 2a. EC50 values of sigma-2 ligands in each cell line determined from the dose–response curves are shown in Fig. 2b and Table 2. The data showed that knockout of TMEM97, PGRMC1, or both proteins did not affect EC50 values of sigma-2 agonists. Similarly, sigma-2 antagonists exhibited little cytotoxicity (EC50 > 200 μM) in all the four cell lines as expected (Fig. 2a and Fig. S2A). These data suggest that sigma-2 ligand-induced cytotoxicity is not mediated by TMEM97, PGRMC1 or both of the proteins. The aforementioned TMEM97 KO cell line was generated by CRISPR/Cas9 technology using guide RNA1. We tested whether TMEM97 KO cell lines generated using different guide RNAs resulted in the same results. We performed cell viability assays on TMEM97 KO cell lines generated using guide RNA2 (TMEM97 KO-g2) and guide RNA3 (TMEM