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Cytotoxic Sigma-2 Ligands Trigger Cancer Cell Death via Cholesterol-Induced-ER-Stress

ABSTRACT: Sigma-2-ligands (S2L) are characterized by high-affinity interaction with their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes following chemical conjugation (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across pancreatic, ovarian, and synovial cancer cell lines. While C6 and C10 displayed distinct cytotoxicities (extended aliphatic side chains), C0 (no aliphatic side chain) and ISO1 (structurally unrelated) were essentially non-toxic, enabling us to investigate the unique mechanism of S2L cytotoxicity. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced an increase in intralysosomal free cholesterol, a corresponding increase in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking and processing, and cytotoxicity due to cholesterol excess, a phenomenon that contrasts the effects of inhibiting NPC1 function. RNA-sequencing revealed that gene clusters involved in cholesterol homeostasis and ER stress response were activated exclusively by cytotoxic S2L. ER stress markers were confirmed on qPCR, and ER stress modulators inhibited or enhanced cytotoxicity of C10 confirming the involvement of ER stress response in the cytotoxicity of S2L. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) which were also confirmed with qPCR and at the protein level and were found to be a pro-survival mechanism activated by ER stress response. Moreover, inhibiting the processes downstream this adaptive response with simvastatin resulted in synergistic treatment outcomes when combined with C10 in vitro and in vivo. Of note, the conjugates retained the S2L-mediated cholesterol phenotype of their parental ligands, which suggests that disrupting cholesterol homeostasis might contribute to the overall cytotoxicity of the drug conjugates. Furthermore, these phenomena were seen exclusively with cytotoxic S2L. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

ORGANISM(S): Homo sapiens

PROVIDER: GSE260557 | GEO | 2024/04/30

REPOSITORIES: GEO

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Project description:Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy. Yoon DJ1, Chen KY1, Lopes AM1, Pan AA1, Shiloach J2, Mason AB3, Kamei DT4. Author information 1 Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza, 5121 Engineering V, Los Angeles, CA 90095, USA. 2 Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892, USA. 3 Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA. 4 Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza, 5121 Engineering V, Los Angeles, CA 90095, USA. Electronic address: kamei@seas.ucla.edu. Abstract The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design. Copyright © 2017 Elsevier Ltd. All rights reserved.

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Cytotoxic Sigma-2 Ligands Trigger Cancer Cell Death via Cholesterol-Induced-ER-Stress

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