Nuclear hormone receptors that are not eliminated by small molecule Sigma1 inhibitor (IPAG).

Sigma1 is required for DHT-induced AR-mediated LD accumulation in prostate cancer cells. A, Confocal micrograph showing LD accumulation in LNCaP cells cultured in CSS containing medium for 3 days and then treated for 1, 2, 3, and 6 days of DHT (1 nmol/L). HCS LipidTOX stained LDs (red). DAPI stained nuclei (blue). Quantification of LD number per cell and average area of LD particles/cell. Data represent mean values from at least three independent determinations, and error bars represent SEM. LD particle numbers and lipid area were quantified using ImageJ. Statistical analysis was performed using ANOVA and Bonferroni after test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. B, Confocal micrograph showing LD accumulation in LNCaP transduced with nonspecific control and Sigma1 shRNA. Two distinct Sigma1 shRNA clones were tested and produced comparable results. Cells were cultured in CSS containing medium for 3 days and then treated for 3 days with DHT (1 nmol/L). LD number per cell was determined as in A, above. C, Immunoblots of whole-cell protein extracts from LNCaP cells infected with Sigma1 shRNA #4 and #5 and treated, serum starved for 3 days, and treated with 3 days of DHT. D, SRS confirming lipid content of LDs in LNCaP cells following 3 days of 1 nmol/L DHT treatment in CSS medium, conditions described above. E, LD numbers per cell in panel of AR-driven (C4-2, C4-2B), ARV-driven (22Rv1), and AR-negative, independent (PC3, DU145) prostate cancer cell lines. Data represent mean values from at least three independent determinations, and error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ns = no significance. F, Confocal micrograph of LDs in PC3 cells (endogenous AR-negative prostate cancer cell line), transfected with empty vector (pcDNA) or recombinant AR plasmid, then treated with DHT (1 nmol/L, 3 days). Quantification of the average number of LDs per cell. Right, Quantification of the mean number of particles per cell ± SE. *, P < 0.05; **, P < 0.01. G, Confocal micrograph showing that LDs accumulate only in AR-transduced PC3 cells. AR (green), LDs (red), DAPI stained nucleus (blue). H, Immunoblot further confirming transduction and expression of recombinant AR in PC3 cells. I, ARV7-induced LDs require Sigma1. LDs (red) in 22Rv1 cells transduced with nonspecific control shRNA or Sigma1 shRNA #5. Magnified inset (white boxes) shown below. LD stain (red), DAPI stain (blue). J, Immunoblot confirmation of Sigma1 shRNA KD in 22Rv1 cells. K, Control confirming that only ARV7-positive cells are also LD-positive. ARV7 immunostain (green), LD stain (red), DAPI stain (blue). L, LDs (red) in PC3 cells transduced with nonspecific control shRNA or Sigma1 shRNA #5 and subsequently transfected with ARV7. Magnified inset (white boxes) shown below. DAPI stain of nuclei (blue). Average number of LDs per cell calculated and analyzed as above. M, Immunoblot confirmation of Sigma1 shRNA KD and transfected ARV7 expression in PC3 cells.

RNAi mediated knockdown of ATG5 does not prevent IPAG induced decrease in AR levels.

CT-189 Chemical, ADME, and Pharmacokinetics Properties.

Quantification of immunoblot band densities in Figure 3B.

AbstractLipid droplets (LD) are dynamic organelles that serve as hubs of cellular metabolic processes. Emerging evidence shows that LDs also play a critical role in maintaining redox homeostasis and can mitigate lipid oxidative stress. In multiple cancers, including prostate cancer, LD accumulation is associated with cancer aggressiveness, therapy resistance, and poor clinical outcome. Prostate cancer arises as an androgen receptor (AR)-driven disease. Among its myriad roles, AR mediates the biosynthesis of LDs, induces autophagy, and modulates cellular oxidative stress in a tightly regulated cycle that promotes cell proliferation. The factors regulating the interplay of these metabolic processes downstream of AR remain unclear. Here, we show that Sigma1/SIGMAR1, a unique ligand-operated scaffolding protein, regulates LD metabolism in prostate cancer cells. Sigma1 inhibition triggers lipophagy, an LD selective form of autophagy, to prevent accumulation of LDs which normally act to sequester toxic levels of reactive oxygen species (ROS). This disrupts the interplay between LDs, autophagy, buffering of oxidative stress and redox homeostasis, and results in the suppression of cell proliferation in vitro and tumor growth in vivo. Consistent with these experimental results, SIGMAR1 transcripts are strongly associated with lipid metabolism and ROS pathways in prostate tumors. Altogether, these data reveal a novel, pharmacologically responsive role for Sigma1 in regulating the redox homeostasis required by oncogenic metabolic programs that drive prostate cancer proliferation.Significance:To proliferate, cancer cells must maintain productive metabolic and oxidative stress (eustress) while mitigating destructive, uncontrolled oxidative stress (distress). LDs are metabolic hubs that enable adaptive responses to promote eustress. Targeting the unique Sigma1 protein can trigger distress by disrupting the LD-mediated homeostasis required for proliferation.

Pharmacologic Sigma1 inhibitor eliminates DHT-induced AR-mediated LDs by lipophagy. Treatment with a small-molecule Sigma1 inhibitor eliminates AR and ARV7 mediated LDs. Confocal image of HCS LipidTox stained LDs (red) in LNCaP cells (A), in C4-2 cells (B), and in VCaP cells (C) treated with drug vehicle (DMSO), DHT (1 nmol/L, 3 days), and treatment with DHT (1 nmol/L, 3 days) combined with Sigma1 inhibitor (IPAG, 10 µmol/L, added for the final 16 hours of the 3-day DHT treatment), DAPI stained nuclei (blue). Quantification of LDs expressed as the mean number of LDs per cell ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Confocal image of LDs (red) in AR-transfected PC3 cells [PC3 (+AR)] treated with DHT (1 nmol/L, 3 days) and with Sigma1 inhibitor (IPAG, 10 µmol/L, added for the final 16 hours). E, ARV7-transfected PC3 cells [PC3 (+ARV7)] treated with drug vehicle (DMSO) and Sigma1 inhibitor (IPAG, 10 µmol/L, 16 hours). Quantification of LDs immediately to the right of micrographs. Data expressed as the mean number of particles per cell ± SEM. ****, P < 0.0001. F, Confocal micrographs showing colocalization of GFP-LC3 (LC3, green) and HCS LipidTox labeled LDs (red) in LNCaP (GFP-LC3) cells that were cultured in CSS medium for 3 days and treated with 1 nmol/L DHT for 3 days alone or in combination with 10 µmol/L IPAG and 10 nmol/L bafilomycin A1 (Baf A1) for the final 8 hours prior to fixing the cells. G, Inset from column 4, white boxed area, Merge from E showing magnified view of autophagosome (LC3, green) colocalization with LD (red). Overlapping, colocalization events indicated by white arrows. H, Box and whisker plot of Mander overlap coefficients. Data are presented as mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance. I, Immunoblots of whole-cell protein extracts from parallel cell culture and experimental conditions used in J. J,In vitro cell proliferation assay of LNCaP cells precultured in CSS medium for 3 days and then treated for 3 days with 1 nmol/L DHT and IPAG (10 µmol/L) was added for the final 16 hours. Live cells were counted by Trypan blue exclusion assay. Error bars represent SEM. **, P < 0.01. K, LNCaP and C4-2 colony formation is suppressed by IPAG in a dose-responsive manner. Data presented as relative number of colonies compared with no drug treatment (as % control).

LNCaP DHT + NAC + Sigma1 shR proliferation

Multiple Sigma1 shRNA and absence of general protein level changes - evidence of selective actions.

Abstract Lipid droplets (LDs) are dynamic organelles that serve as hubs of cellular metabolic processes. Emerging evidence shows that LDs also play a critical role in maintaining redox homeostasis and can mitigate lipid oxidative stress. In multiple cancers, including prostate cancer (PCa), LD accumulation is associated with cancer aggressiveness, therapy resistance, and poor clinical outcome. PCa arises as an androgen receptor (AR) driven disease. Among its myriad roles, AR mediates the biosynthesis of LDs, induces autophagy, and modulates cellular oxidative stress in a tightly regulated cycle that promotes cell proliferation. The factors regulating the interplay of these metabolic processes downstream of AR remain unclear. Here, we show that Sigma1/SIGMAR1, a unique ligand-operated scaffolding protein, regulates LD metabolism in PCa cells. Sigma1 inhibition triggers lipophagy, an LD selective form of autophagy, to prevent accumulation of LDs which normally act to sequester toxic levels of reactive oxygen species (ROS). This disrupts the interplay between LDs, autophagy, buffering of oxidative stress and redox homeostasis, and results in the suppression of cell proliferation in vitro and tumor growth in vivo. Consistent with these experimental results, SIGMAR1 transcripts are strongly associated with lipid metabolism and reactive oxygen species pathways in prostate tumors. Altogether, these data reveal a novel, pharmacologically responsive role for Sigma1 in regulating the redox homeostasis required by oncogenic metabolic programs that drive PCa proliferation. Citation Format: Halley M. Oyer, Alexandra R. Steck, Charles Longen, Sanjana Venkat, Konuralp Bayrak, Eleanor Munger, Dan Fu, Christina Sanders, Justin Myers, Matthew Schiewer, Nan Chen, Elahe Mostaghel, Felix J. Kim. Sigma1 regulates lipid droplet mediated redox homeostasis required for prostate cancer proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 284.

Sigma1 small-molecule inhibition disrupts GSH ratios and increases ROS levels in LNCaP and C4-2 cells. A, Quantification of CM-H2DCFDA signal per cell in LNCaP cells cultured in CSS medium for 3 days and treated for 3 days with DHT (1 nmol/L) alone or combined with 10 µmol/L IPAG for the last 16 hours. Data are presented as mean ± SEM from three independent experiments, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance. B, Representative fluorescent micrographs showing CM-H2DCFDA levels in LNCaP cells in A. C, Total GSH and GSH:GSSG measurements in LNCaP cells treated as in A. Data are presented as mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

GSEA and correlation analysis of Sigma1/SIGMAR1 in prostate tumors. GSEA using Adipogenesis and ROS Pathway Hallmark gene sets on localized prostate tumors from TCGA (A; 60) and metastatic prostate tumors from SU2C/PCF Dream Team (B; 61). C, Heat map of normalized enrichment scores from primary, metastatic, or benign prostate tissue utilizing TCGA, MSKCC, SU2C/PCF Dream Team, and FHCRC datasets (60, 61, 63). D, Single gene correlation analysis between SIGMAR1 and PNPLA2, BCSL2, and PLIN5 using Z-scores from published primary prostate tumor datasets (60, 63, 64).

Sigma1 KD triggers lipophagy. A, Confocal micrographs showing colocalization of GFP-LC3 (LC3, green) and HCS LipidTox labeled LDs (red) in Sigma1 shRNA-transduced LNCaP (GFP-LC3) cells that were serum-starved for 3 days and treated with 1 nmol/L DHT for 3 days alone or combined with 10 nmol/L bafilomycin A1 (BafA1) for the final 8 hours prior to fixing the cells. B, Inset from column 8, Merge from A showing magnified view of autophagosome (LC3, green) colocalization with LD (red). Overlapping, colocalization events indicated by white arrows. C, Immunoblots of whole-cell protein extracts from parallel cell culture performed in parallel and using same experimental treatment conditions. GFP, GFP-LC3 I, and GFP-LC3 II were detected using an anti-GFP antibody. GFP-LC3 II band represents autophagosomes, similarly to canonical LC3B II immunoblot banding patterns. Independent GFP band indicates active autolysosomal degradation, autophagic flux.

Sigma1 KD suppresses DHT induced prostate cancer cell proliferation and tumor growth despite (or due to) increased autophagy. A,In vitro cell proliferation assay of Sigma1 shRNA-transduced LNCaP and C4-2 cells precultured in CSS medium for 3 days and then treated for 3 and 6 days with 1 nmol/L DHT. Live cells were counted by Trypan blue exclusion assay at the start of the time course (day 0), and 3 and 6 days of DHT treatment. Data are represented as fold induction over cells treated with control shRNA at day 0. Datapoints represent mean fold increase in cell number from at least three independent determinations, and error bars represent SEM. Two distinct Sigma1 shRNA clones, #4 and #5, were tested and produced similar results. B, Immunoblots of whole-cell protein extracts from parallel C4-2 cell culture performed in parallel and using same experimental treatment conditions as in A. Data shown for Sigma1 shRNA clone #5 KD C4-2 cell cultures. C, Immunoblot of Sigma1 shRNA clone #4 and #5 transduced C4-2 cells immediately prior to subcutaneous flank implantation into SCID mice. D, C4-2 cells infected with Sigma1 shRNA (#4, #5) and control shRNA (#1) were injected into the right and left flanks of SCID mice. Tumor volume was measured by caliper 12 weeks after implantation, prior to sacrificing the mice. Data are represented as mean volume of six tumors for each condition, and error bars represent SEM. E, Tumor weight was measured at 12 weeks postinjection at the time of harvest. Data are represented as mean volume of six tumors for each condition, and error bars represent SEM. *, P < 0.05; **, P < 0.01. F, Oil Red O staining of control (clone #1) and Sigma1 shRNA (clone #4 and #5) xenografted C4-2 tumors.

LDs as buffers of DHT induced ROS and DHT promotes ROS homeostasis. A, Confocal micrograph showing HCS LipidTox stained LDs in LNCaP cells cultured in CSS containing medium for 3 days and treated with DMSO (vehicle) and 1 nmol/L DHT alone or combined with 2.5 mmol/L NAC for 3 days. B, LD quantification of LNCaP cells from A. Data represent LDs per cell and error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. C, Quantification of ROS, detected with CM-H2DCFDA in LNCaP treated as described above in A. Data are presented as mean ± SEM from at least three independent determinations. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Illustration of concept that DHT initially induces ROS to trigger proliferation (1 day of DHT), and subsequently decreases intracellular ROS levels. LD accumulation is observed as DHT decreases ROS levels. E, Quantification of ROS in control (#1) or Sigma1 shRNA (#5) transduced LNCaP cells treated as described above in A. Each datapoint represents mean CM-H2DCFDA signal per cell from three fields in three independent wells. F, Redox balance. Total GSH levels and ratio of GSH-to-GSSG measured in nonspecific control shRNA (ns) and in Sigma1 shRNA (#5) transduced LNCaP cells treated as described above in A.

Working Model. Sigma1 targeting disrupts LD-mediated redox homeostasis in prostate cancer cells. LD, lipid droplet; TG, triacylglycerol; DHT, dihydrotestosterone; ROS, reactive oxygen species.

Specificity of ARV7 and ARv567es antibodies.

There are two known subtypes of the so-called sigma receptors, Sigma1 and Sigma2. Sigma1 (encoded by the SIGMAR1 gene and also known as Sigma-1 receptor, S1R) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein that allosterically modulates the activity of its associated proteins. Sigma2, recently identified as transmembrane protein 97 (TMEM97), is an integral membrane protein implicated in cellular cholesterol homeostasis. A number of publications over the past two decades have suggested a role for both sigma proteins in tumor biology. Although there is currently no clinically used anti-cancer drug that targets Sigma1 or Sigma2/TMEM97, a growing body of evidence supports the potential of small-molecule compounds with affinity for these proteins, putative sigma ligands, as therapeutic agents to treat cancer. In preclinical models, these compounds have been reported to inhibit cancer cell proliferation, survival, adhesion, and migration; furthermore, they have been demonstrated to suppress tumor growth, to alleviate cancer-associated pain, and to exert immunomodulatory properties. Here, we will address the known knowns and the known unknowns of Sigma1 and Sigma2/TMEM97 ligand actions in the context of cancer. This review will highlight key discoveries and published evidence in support of a role for sigma proteins in cancer and will discuss several fundamental questions regarding the physiological roles of sigma proteins in cancer and sigma ligand mechanism of action.

There are two known subtypes of the so-called sigma receptors, Sigma1 and Sigma2. Sigma1 (encoded by the SIGMAR1 gene and also known as Sigma-1 receptor, S1R) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein that allosterically modulates the activity of its associated proteins. Sigma2, recently identified as transmembrane protein 97 (TMEM97), is an integral membrane protein implicated in cellular cholesterol homeostasis. A number of publications over the past two decades have suggested a role for both sigma proteins in tumor biology. Although there is currently no clinically used anti-cancer drug that targets Sigma1 or Sigma2/TMEM97, a growing body of evidence supports the potential of small-molecule compounds with affinity for these proteins, putative sigma ligands, as therapeutic agents to treat cancer. In preclinical models, these compounds have been reported to inhibit cancer cell proliferation, survival, adhesion, and migration; furthermore, they have been demonstrated to suppress tumor growth, to alleviate cancer-associated pain, and to exert immunomodulatory properties. Here, we will address the known knowns and the known unknowns of Sigma1 and Sigma2/TMEM97 ligand actions in the context of cancer. This review will highlight key discoveries and published evidence in support of a role for sigma proteins in cancer and will discuss several fundamental questions regarding the physiological roles of sigma proteins in cancer and sigma ligand mechanism of action.