Background: Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is frequently upregulated in many cancers and is closely implicated in tumorigenesis. However, the mechanism underling Drp1 to influence tumor progression is largely unknown, especially in esophageal squamous cell carcinoma (ESCC). Methods: Immunohistochemistry was used to examine Drp1 and LC3B expression in tissues of ESCC patients. Autophagic vesicles were investigated by transmission electron microscopy. Fluorescent LC3B puncta and mitochondrial nucleoid were observed by fluorescent and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP levels. Xenograft tumor model was performed in BALB/c nude mice to analyze the role of Drp1 on ESCC progression. Results: We found that Drp1 high expression is correlated with poor overall survival of ESCC patients. Drp1 overexpression promotes cell proliferation and xenograft ESCC tumor growth by triggering autophagy. Furthermore, we demonstrated that Drp1 overexpression disturbs mitochondrial function and subsequent induces mitochondrial DNA (mtDNA) released into the cytosol thereby inducing cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA activates the cGAS-STING pathway and facilitates autophagy, which promotes ESCC cancer growth. Moreover, mtDNA digestion with DNase I and autophagy inhibition with chloroquine attenuates the cGAS-STING pathway activation and ESCC cancer growth. Conclusions: Our finding reveals that Drp1 overexpression induces mitochondrial dysfunction and cytosolic mtDNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression.

The systemic spread of malignancies and the risk of cancer-associated thrombosis are major clinical challenges in cancer therapy worldwide. As an important post-translational modification enzyme, peptidyl arginine deiminase 4 (PAD4) could mediate the citrullination of protein in different components (including nucleus and cytoplasm, etc.) of a variety of cells (tumor cells, neutrophils, macrophages, etc.), thus participating in gene regulation, neutrophil extracellular trap (NET) and macrophage extracellular trap (MET). Thereby, PAD4 plays an important role in enhancing the growth of primary tumors and facilitating the distant metastasis of cancer cells. In addition, it is related to the formation of cancer-associated thrombosis. Therefore, the development of PAD4-specific inhibitors may be a promising strategy for treating cancer, and it may improve patient prognosis. In this review, we describe PAD4 involvement in gene regulation, protein citrullination, and NET formation. We also discuss its potential role in cancer and cancer-associated thrombosis, and we summarize the development and application of PAD4 inhibitors.

Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttranslational modification enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hypercitrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps (NET). NETs were first described as antimicrobial fibers that bind and kill bacteria. However, it is not known whether PAD4 can mediate the release of chromatin DNA into the extracellular space of cancer cells. Here, we report that murine breast cancer 4T1 cells expressing high levels of PADI4 can release cancer extracellular chromatin networks (CECN) in vitro and in vivo. Deletion of Padi4 using CRISPR/Cas9 abolished CECN formation in 4T1 cells. Padi4 deletion from 4T1 cells also reduced the rate of tumor growth in an allograft model, and decreased lung metastasis by 4T1 breast cancers. DNase I treatment, which degrades extracellular DNA including CECNs, also reduced breast to lung metastasis of Padi4 wild-type 4T1 cells in allograft experiments in the Padi4-knockout mice. We further demonstrated that DNase I treatment in this mouse model did not alter circulating tumor cells but decreased metastasis through steps after intravasation. Taken together, our genetic studies show that PAD4 plays a cell autonomous role in cancer metastasis, thus revealing a novel strategy for preventing cancer metastasis by inhibiting cancer cell endogenous PAD4. Implications: This study shows that PADI4 can mediate the formation of CECNs in 4T1 cells, and that endogenous PADI4 plays an essential role in breast cancer lung metastasis. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/18/5/735/F1.large.jpg. Visual Overview

Exhaustion of chimeric antigen receptor (CAR) T cells is one of the limitations for CAR T efficacy in solid tumors and for tumor recurrence after initial CAR T treatment. Tumor treatment with a combination of programmed cell death receptor-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blockage and CD28-based CAR T cells has been intensively studied. However, it remains largely unclear whether autocrine single-chain variable fragments (scFv) PD-L1 antibody can improve 4-1BB-based CAR T cell anti-tumor activity and revert CAR T cell exhaustion. Here, we studied T cells engineered with autocrine PD-L1 scFv and 4-1BB-containing CAR. The antitumor activity and exhaustion of CAR T cells were investigated in vitro and in a xenograft cancer model using NCG mice. CAR T cells with autocrine PD-L1 scFv antibody demonstrate enhanced anti-tumor activity in solid tumors and hematologic malignancies by blocking the PD-1/PD-L1 signaling. Importantly, we found that CAR T exhaustion was largely diminished by autocrine PD-L1 scFv antibody in vivo. As such, 4-1BB CAR T with autocrine PD-L1 scFv antibody combined the power of CAR T cells and the immune checkpoint inhibitor, thereby increasing the anti-tumor immune function and CAR T persistence, providing a cell therapy solution for a better clinical outcome.

Protein arginine deiminase 4 (PAD4) plays an important role in cancer progression by participating in gene regulation, protein modification, and neutrophil extracellular trap (NET) formation. Many reversible and irreversible PAD4 inhibitors have been reported recently. In this review, we summarize the structure–activity relationships of newly investigated PAD4 inhibitors to bring researchers up to speed by guiding and describing new scaffolds as optimization and development leads for new effective, safe, and selective cancer treatments. In addition, some recent reports have shown evidence that PAD4 inhibitors are expected to trigger antitumor immune responses, regulate immune cells and related immune factors, enhance the effects of immune checkpoint inhibitors, and enhance their antitumor efficacy. Therefore, PAD4 inhibitors may potentially change tumor immunotherapy and provide an excellent direction for the development and clinical application of immunotherapy strategies for related diseases.