The mitochondrial stress signaling tunes immunity from a view of systemic tumor microenvironment and ecosystem.

Mitochondria play important roles in cell fate, calcium signaling, mitophagy, and the signaling through reactive oxygen species (ROS). Recently, mitochondria are considered as a signaling organelle in the cell and communicate with other organelles to constitute the mitochondrial information processing system (MIPS) that transduce input-to-output biological information. The success in immunotherapy, a concept of systemic therapy, has been proved to be dependent on paracrine interactions within the tumor microenvironment (TME) and distant organs including microbiota and immune components. We will adopt a broader view from the concept of TME to tumor micro- and macroenvironment (TM 2 E) or tumor-organ ecosystem (TOE). In this review, we will discuss the role of mitochondrial signaling by mitochondrial ROS, calcium flux, metabolites, mtDNA, vesicle transportation, and mitochondria-derived peptide in the TME and TOE, in particular immune regulation and effective cancer immunotherapy.

Mitochondrial Lon-induced mitophagy benefits hypoxic resistance via Ca2+-dependent FUNDC1 phosphorylation at the ER-mitochondria interface.

During hypoxia, FUNDC1 acts as a mitophagy receptor and accumulates at the ER (endoplasmic reticulum)-mitochondria contact sites (EMC), also called mitochondria-associated membranes (MAM). In mitophagy, the ULK1 complex phosphorylates FUNDC1(S17) at the EMC site. However, how mitochondria sense the stress and send the signal from the inside to the outside of mitochondria to trigger mitophagy is still unclear. Mitochondrial Lon was reported to be localized at the EMC under stress although the function remained unknown. In this study, we explored the mechanism of how mitochondrial sensors of hypoxia trigger and stabilize the FUNDC1-ULK1 complex by Lon in the EMC for cell survival and cancer progression. We demonstrated that Lon is accumulated in the EMC and associated with FUNDC1-ULK1 complex to induce mitophagy via chaperone activity under hypoxia. Intriguingly, we found that Lon-induced mitophagy is through binding with mitochondrial Na+/Ca2+ exchanger (NCLX) to promote FUNDC1-ULK1-mediated mitophagy at the EMC site in vitro and in vivo. Accordingly, our findings highlight a novel mechanism responsible for mitophagy initiation under hypoxia by chaperone Lon in mitochondria through the interaction with FUNDC1-ULK1 complex at the EMC site. These findings provide a direct correlation between Lon and mitophagy on cell survival and cancer progression.

A Fc-VEGF chimeric fusion enhances PD-L1 immunotherapy via inducing immune reprogramming and infiltration in the immunosuppressive tumor microenvironment.

BACKGROUND: Immunotherapy is an emerging cancer therapy with potential great success; however, immune checkpoint inhibitor (e.g., anti-PD-1) has response rates of only 10-30% in solid tumor because of the immunosuppressive tumor microenvironment (TME). This affliction can be solved by vascular normalization and TME reprogramming. METHODS: By using the single-cell RNA sequencing (scRNAseq) approach, we tried to find out the reprogramming mechanism that the Fc-VEGF chimeric antibody drug (Fc-VFD) enhances immune cell infiltration in the TME. RESULTS: In this work, we showed that Fc-VEGF121-VEGF165 (Fc-VEGF chimeric antibody drug, Fc-VFD) arrests excess angiogenesis and tumor growth through vascular normalization using in vitro and in vivo studies. The results confirmed that the treatment of Fc-VFD increases immune cell infiltration including cytotoxic T, NK, and M1-macrophages cells. Indeed, Fc-VFD inhibits Lon-induced M2 macrophages polarization that induces angiogenesis. Furthermore, Fc-VFD inhibits the secretion of VEGF-A, IL-6, TGF-ß, or IL-10 from endothelial, cancer cells, and M2 macrophage, which reprograms immunosuppressive TME. Importantly, Fc-VFD enhances the synergistic effect on the combination immunotherapy with anti-PD-L1 in vivo. CONCLUSIONS: In short, Fc-VFD fusion normalizes intratumor vasculature to reprogram the immunosuppressive TME and enhance cancer immunotherapy.

Mitochondrial oxidative stress in the tumor microenvironment and cancer immunoescape: foe or friend?

The major concept of "oxidative stress" is an excess elevated level of reactive oxygen species (ROS) which are generated from vigorous metabolism and consumption of oxygen. The precise harmonization of oxidative stresses between mitochondria and other organelles in the cell is absolutely vital to cell survival. Under oxidative stress, ROS produced from mitochondria and are the major mediator for tumorigenesis in different aspects, such as proliferation, migration/invasion, angiogenesis, inflammation, and immunoescape to allow cancer cells to adapt to the rigorous environment. Accordingly, the dynamic balance of oxidative stresses not only orchestrate complex cell signaling events in cancer cells but also affect other components in the tumor microenvironment (TME). Immune cells, such as M2 macrophages, dendritic cells, and T cells are the major components of the immunosuppressive TME from the ROS-induced inflammation. Based on this notion, numerous strategies to mitigate oxidative stresses in tumors have been tested for cancer prevention or therapies; however, these manipulations are devised from different sources and mechanisms without established effectiveness. Herein, we integrate current progress regarding the impact of mitochondrial ROS in the TME, not only in cancer cells but also in immune cells, and discuss the combination of emerging ROS-modulating strategies with immunotherapies to achieve antitumor effects.

Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells.

Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca2+) via NCLX, a mitochondrial Na+/Ca2+ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.

Mitochondrial Lon-induced mtDNA leakage contributes to PD-L1-mediated immunoescape via STING-IFN signaling and extracellular vesicles.

BACKGROUND: Mitochondrial Lon is a chaperone and DNA-binding protein that functions in protein quality control and stress response pathways. The level of Lon regulates mitochondrial DNA (mtDNA) metabolism and the production of mitochondrial reactive oxygen species (ROS). However, there is little information in detail on how mitochondrial Lon regulates ROS-dependent cancer immunoescape through mtDNA metabolism in the tumor microenvironment (TME). METHODS: We explored the understanding of the intricate interplay between mitochondria and the innate immune response in the inflammatory TME. RESULTS: We found that oxidized mtDNA is released into the cytosol when Lon is overexpressed and then it induces interferon (IFN) signaling via cGAS-STING-TBK1, which upregulates PD-L1 and IDO-1 expression to inhibit T-cell activation. Unexpectedly, upregulation of Lon also induces the secretion of extracellular vehicles (EVs), which carry mtDNA and PD-L1. Lon-induced EVs further induce the production of IFN and IL-6 from macrophages, which attenuates T-cell immunity in the TME. CONCLUSIONS: The levels of mtDNA and PD-L1 in EVs in patients with oral cancer function as a potential diagnostic biomarker for anti-PD-L1 immunotherapy. Our studies provide an insight into the immunosuppression on mitochondrial stress and suggest a therapeutic synergy between anti-inflammation therapy and immunotherapy in cancer.

Mitochondrial oxidative stress by Lon-PYCR1 maintains an immunosuppressive tumor microenvironment that promotes cancer progression and metastasis.

Mitochondrial Lon is a chaperone protein whose upregulation increases the production of mitochondrial reactive oxygen species (ROS). However, there is a lack of information in detail on how mitochondrial Lon regulates cancer metastasis through ROS production in the tumor microenvironment (TME). Our results show that elevated Lon promotes epithelial-mesenchymal transition (EMT) via ROS-dependent p38 and NF-κB-signaling. We further identified pyrroline-5-carboxylate reductase 1 (PYCR1) as a client of chaperone Lon, which induces mitochondrial ROS and EMT by Lon. Mitochondrial Lon induces ROS-dependent production of inflammatory cytokines, such as TGF-ß, IL-6, IL-13, and VEGF-A, which consequently activates EMT, angiogenesis, and M2 macrophage polarization. In addition, Lon expression is induced upon the activation and M2 polarization of macrophages, which further promotes M2 macrophages to enhance the immunosuppressive microenvironment and metastatic behaviors in the TME. This raises the possibility that manipulation of the mitochondrial redox balance in the TME may serve as a therapeutic strategy to improve T cell function in cancer immunotherapy.

Mitochondrial Lon sequesters and stabilizes p53 in the matrix to restrain apoptosis under oxidative stress via its chaperone activity.

Mitochondrial Lon is a multi-function matrix protease with chaperone activity. However, little literature has been undertaken into detailed investigations on how Lon regulates apoptosis through its chaperone activity. Accumulating evidences indicate that various stresses induce transportation of p53 to mitochondria and activate apoptosis in a transcription-independent manner. Here we found that increased Lon interacts with p53 in mitochondrial matrix and restrains the apoptosis induced by p53 under oxidative stress by rescuing the loss of mitochondrial membrane potential (Δψm) and the release of cytochrome C and SMAC/Diablo. Increased chaperone Lon hampers the transcription-dependent apoptotic function of p53 by reducing the mRNA expression of p53 target genes. The ATPase mutant (K529R) of chaperone Lon decreases the interaction with p53 and fails to inhibit apoptosis. Furthermore, the chaperone activity of Lon is important for mitochondrial p53 accumulation in an mtHsp70-dependent manner, which is also important to prevent the cytosolic distribution of p53 from proteasome-dependent degradation. These results indicate that the chaperone activity of Lon is important to bind with mitochondrial p53 by which increased Lon suppresses the apoptotic function of p53 under oxidative stress. Furthermore, mitochondrial Lon-mtHsp70 increases the stability/level of p53 through trafficking and retaining p53 in mitochondrial matrix and preventing the pool of cytosolic p53 from proteasome-dependent degradation in vitro and in clinic.

Bufalin induced apoptosis in SCC­4 human tongue cancer cells by decreasing Bcl­2 and increasing Bax expression via the mitochondria­dependent pathway.

The aim of the present study was to investigate the cytotoxic effects of bufalin on SCC­4 human tongue cancer cells. Cell morphological changes and viability were examined using phase contrast microscopy and flow cytometry, respectively. The results indicated that bufalin induced morphological changes and reduced total viable cells. Apoptotic cell death was analyzed by DAPI staining and DNA gel electrophoresis; the results revealed that bufalin induced cell apoptosis. Levels of reactive oxygen species (ROS), Ca2+, nitric oxide (NO) and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry, and bufalin was observed to increase Ca2+ and NO production, decrease the ΔΨm and reduce ROS production in SCC­4 cells. In addition, western blotting was performed to detect apoptosis­associated protein expression. The results demonstrated that bufalin reduced the expression of the anti­apoptotic protein B­cell lymphoma 2 (Bcl­2) and increased the expression of the pro­apoptotic protein, Bcl­2­associated X protein. However, bufalin treatment also increased the expression of other apoptosis­associated proteins such as apoptosis­inducing factor and endonuclease G in SCC­4 cells. Based on these findings, bufalin may induce apoptotic cell death via mitochondria­dependent pathways in human tongue cancer SCC­4 cells.