ABSTRACT
High infiltration of M2-polarized macrophages in the primary tumor indicates unfavorable prognosis and poor overall survival in the patients with triple-negative breast cancer (TNBC). Thus, reversing M2-polarized tumor-associated macrophages in the tumors has been considered as a potential therapeutic strategy for TNBC. Sphingomyelin synthase 2 (SMS2) is the key enzyme for sphingomyelin production, which plays an important role in plasma membrane integrity and function. In this study we investigated whether SMS2 inhibitor or SMS2 gene knockout could reduce macrophages M2 polarization and tumor progression in a mouse model of TNBC. We showed that SMS2 mRNA expression was linked to immunosuppressive tumor microenvironment and poor prognosis in TNBC patients. The knockout of SMS2 or application of 15w (a specific SMS2 inhibitor) markedly decreased the generation of M2-type macrophages in vitro, and reduced the tumor weight and lung metastatic niche formation in a 4T1-TNBC mouse model. We further demonstrated that the in vivo antitumor efficacy of 15w was accompanied by a multifaceted remodeling of tumor immune environment reflecting not only the suppression of M2-type macrophages but also diminished levels of regulatory T cells and myeloid-derived suppressor cells leading to a dramatically improved infiltration of antitumor CD8+ T lymphocytes. Collectively, our results reveal a novel and important role of SMS2 in the protumorigenic function and may offer a new strategy for macrophage-targeted anticancer therapy.
Subject(s)
Macrophages/metabolism , Transferases (Other Substituted Phosphate Groups)/metabolism , Triple Negative Breast Neoplasms/physiopathology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Disease Progression , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Gene Knockout Techniques , Humans , Immunity, Cellular/drug effects , Isoxazoles/pharmacology , Isoxazoles/therapeutic use , Macrophage Activation/drug effects , Macrophage Activation/physiology , Macrophages/drug effects , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Prognosis , Transferases (Other Substituted Phosphate Groups)/antagonists & inhibitors , Transferases (Other Substituted Phosphate Groups)/genetics , Triple Negative Breast Neoplasms/diagnosis , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/immunologyABSTRACT
The flavonoid quercetin exhibits significant anticancer activities with few side effects. In the current study, we characterized TL-2-8, a quercetin derivative, as a novel anticancer agent in vitro and in vivo. Cell proliferation and viability were assessed using Cell Counting Kit-8 and CellTiter-Blue assay, respectively. Cell death was examined using PI staining or a TUNEL assay. Mitophagy was determined by measuring autophagic flux and by confocal imaging. Protein expression was examined by Western blotting. We found that TL-2-8 selectively inhibited the proliferation and decreased the viability of various cancer cells (the anti-proliferation IC50 values in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells at 72 h were 8.28, 8.56, and 9.58 µmol/L, respectively), and it displayed only slight cytotoxicity against normal MCF-10A and HEK-293 cells. In MDA-MB-231 and MDA-MB-468 breast cancer cells, TL-2-8 treatment induced the degradation of multiple Hsp90 client proteins without inducing Hsp70. TL-2-8 (3, 6, 12 µmol/L) dose-dependently inhibited the expression of AHA1, an activator of Hsp90 ATPase, and decreased Hsp90-AHA1 complex formation, leading to decreased Hsp90 chaperone function and reduced polo-like kinase 1 (PLK1) signaling. Consequently, impaired mitophagy was induced via the downregulation of lysosomal-associated membrane protein 2 (LAMP2). The in vivo anticancer effects of TL-2-8 were evaluated in an MDA-MB-231 breast cancer xenograft model, which was treated with TL-2-8 (25, 50, 100 mg·kg-1·d-1, po). Administration of TL-2-8 resulted in tumor growth inhibition rates of 37.9%, 58.9% and 70.9%, respectively, whereas quercetin treatment (100 mg·kg-1·d-1, po) produced only a lower tumor growth inhibition rate (49.5%). Furthermore, TL-2-8 treatment significantly extended the lifespan of mice bearing MDA-MB-231 breast cancer cell xenografts. Our results demonstrate that TL-2-8 induces significant cell death and immature mitophagy in breast cancer cells in vitro and in vivo via AHA1 abrogation.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Flavonoids/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Breast Neoplasms/pathology , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Down-Regulation , Female , HEK293 Cells , HSP90 Heat-Shock Proteins/metabolism , Humans , In Situ Nick-End Labeling , Inhibitory Concentration 50 , MCF-7 Cells , Mice , Mice, Inbred BALB C , Mice, Nude , Mitophagy/drug effects , Molecular Chaperones/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value of 5.2 µM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC50 value of 2.1 µM in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors.
