Adiponectin, an adipokine predominantly derived from adipose tissue, exhibits potent antitumor properties in breast cancer cells. However, its mechanisms of action remain elusive. Inflammasomes—intracellular multimeric protein complexes—modulate cancer cell growth in a complicated manner, as well as playing a role in the innate immune system. Herein, we examined the potential role of inflammasomes in the antitumor activity of adiponectin and found that globular adiponectin (gAcrp) significantly suppressed inflammasomes activation in breast cancer cells both in vitro and in vivo conditions, as determined by decreased expression of inflammasomes components, including NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and the apoptosis-associated speck-like protein containing a CARD (ASC), and inhibition of interleukin-1β and caspase-1 activation. Treatment with pharmacological inhibitors of inflammasomes caused decrease in cell viability, apoptosis induction, and G0/G1 cell cycle arrest, suggesting that inflammasomes activation is implicated in the growth of breast cancer cells. In addition, treatment with gAcrp generated essentially similar results to those of inflammasomes inhibitors, further indicating that suppression of breast cancer cell growth by gAcrp is mediated via modulation of inflammasomes. Mechanistically, gAcrp suppressed inflammasomes activation through sestrin2 (SESN2) induction, liver kinase B1 (LKB-1)-dependent AMP-activated protein kinase (AMPK) phosphorylation, and alleviation of endoplasmic reticulum (ER) stress. Taken together, these results demonstrate that gAcrp inhibits growth of breast cancer cells by suppressing inflammasomes activation, at least in part, via SESN2 induction and AMPK activation-dependent mechanisms.

Background AMP-activated protein kinase (AMPK) is a metabolic sensor that maintains energy homeostasis. AMPK functions as a tumor suppressor in different cancers; however, its role in regulating antitumor immunity, particularly the function of regulatory T cells (Tregs), is poorly defined. Methods AMPKα1fl/flFoxp3YFP-Cre, Foxp3YFP-Cre, Rag1−/−, and C57BL/6 J mice were used for our research. Flow cytometry and cell sorting, western blotting, immuno-precipitation, immuno-fluorescence, glycolysis assay, and qRT-PCR were used to investigate the role of AMPK in suppressing programmed cell death 1 (PD-1) expression and for mechanistic investigation. Results The deletion of the AMPKα1 subunit in Tregs accelerates tumor growth by increasing the expression of PD-1. Metabolically, loss of AMPK in Tregs promotes glycolysis and the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme of the mevalonate pathway. Mechanistically, AMPK activates the p38 mitogen-activated protein kinase (MAPK) that phosphorylates glycogen synthase kinase-3β (GSK-3β), inhibiting the expression of PD-1 in Tregs. Conclusion Our study identified an AMPK regulatory mechanism of PD-1 expression via the HMGCR/p38 MAPK/GSK3β signaling pathway. We propose that the AMPK activator can display synergic antitumor effect in murine tumor models, supporting their potential clinical use when combined with anti-PD-1 antibody, anti-CTLA-4 antibody, or a HMGCR inhibitor.

Blocking the mevalonate pathway for cholesterol reduction by using statin may have adverse effects including statin‐induced colitis. Moreover, one of the predisposing factors for colitis is an imbalanced CD4+ T cell, which can be observed on the complete deletion of HMG‐CoA reductase (HMGCR), a target of statins. In this study, we inquired geranylgeranyl pyrophosphate (GGPP) is responsible for maintaining the T‐cell homeostasis. Following dextran sulfate sodium (DSS)‐induced colitis, simvastatin increased the severity of disease, while cotreatment with GGPP, but not with cholesterol, reversed the disease magnitude. GGPP ameliorated DSS‐induced colitis by increasing Treg cells. GGPP amplified Treg differentiation through increased IL‐2/STAT 5 signaling. GGPP prenylated Ras protein, a prerequisite for extracellular signal‐regulated kinase (ERK) pathway activation, leading to increased IL‐2 production. Higher simvastatin dose increased the severity of colitis. GGPP ameliorated simvastatin‐increased colitis by increasing Treg cells. Treg cells, which have the capacity to suppress inflammatory T cells and were generated through IL‐2/STAT5 signaling, increased IL‐2 production through prenylation and activation of the Ras/ERK pathway.

Programmed cell death protein 1 (PD-1), expressed on tumor-infiltrating T cells, is a T cell exhaustion marker. The mechanisms underlying PD-1 upregulation in CD4 T cells remain unknown. Here we develop nutrient-deprived media and a conditional knockout female mouse model to study the mechanism underlying PD-1 upregulation. Reduced methionine increases PD-1 expression on CD4 T cells. The genetic ablation of SLC43A2 in cancer cells restores methionine metabolism in CD4 T cells, increasing the intracellular levels of S-adenosylmethionine and yielding H3K79me2. Reduced H3K79me2 due to methionine deprivation downregulates AMPK, upregulates PD-1 expression and impairs antitumor immunity in CD4 T cells. Methionine supplementation restores H3K79 methylation and AMPK expression, lowering PD-1 levels. AMPK-deficient CD4 T cells exhibit increased endoplasmic reticulum stress and Xbp1s transcript levels. Our results demonstrate that AMPK is a methionine-dependent regulator of the epigenetic control of PD-1 expression in CD4 T cells, a metabolic checkpoint for CD4 T cell exhaustion.

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Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to nonspecific systemic immunosuppression results in immunodeficiency and has toxic effects on nonimmune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin-releasing poly(lactic- co -glycolic acid) microparticles (RAP-MPs) to prevent immune rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell-particle mixture in methylcellulose solution while maintaining high cell viability. RAP-MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ~3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP-MPs (200- to 4000-ng RAP per recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced programmed death-ligand 1 expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.

The development of an optimal treatment modality to improve the therapeutic outcome of breast cancer patients is still difficult. Poor antigen presentation to T cells is a major challenge in cancer immunotherapy. In this study, a synergistic immunotherapy strategy for breast cancer incorporating immune cell infiltration, immunogenic cell death (ICD), and dendritic cell (DC) maturation through a reactive oxygen species (ROS)‐responsive dual‐targeted smart nanosystem (anti‐PD‐L1‐TKNP) for the simultaneous release of DOX, R848, and MIP‐3α in the tumor microenvironment is reported. Following local injection, anti‐PD‐L1‐DOX‐R848‐MIP‐3α/thioketal nanoparticle (TKNP) converts tumor cells to a vaccine owing to the combinatorial effect of DOX‐induced ICD, R848‐mediated immunostimulatory properties, and MIP‐3α‐induced immune cell recruitment in the tumor microenvironment. Intratumoral injection of anti‐PD‐L1‐DOX‐R848‐MIP‐3α/TKNP caused significant regression of breast cancer. Mechanistic studies reveal that anti‐PD‐L1‐DOX‐R848‐MIP‐3α/TKNP specifically targets tumor tissue, resulting in maximum exposure of calreticulin (CRT) and HMGB1 in tumors, and significantly enhances intratumoral infiltration of CD4+ and CD8+ T cells in tumors. Therefore, a combined strategy using dual‐targeted ROS‐responsive TKNP highlights the significant application of nanoparticles in modulating the tumor microenvironment and could be a clinical treatment strategy for effective breast cancer management.

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