Cell biology
Abstract
Canonically PD-L1 functions as the inhibitory immune checkpoint on cell surface. Recent studies have observed PD-L1 expression in the nucleus of cancer cells. But the biological function of nuclear PD-L1 (nPD-L1) in tumor growth and antitumor immunity is unclear. Here we enforced nPD-L1 expression and established stable cells. nPD-L1 suppressed tumorigenesis and aggressiveness in vitro and in vivo. Compared with PD-L1 deletion, nPD-L1 expression repressed tumor growth and improved survival more markedly in immunocompetent mice. Phosphorylated AMPKα (p-AMPKα) facilitated nuclear PD-L1 compartmentalization and then cooperated with it to directly phosphorylate S146 of histone variant macroH2A1 (mH2A1) to epigenetically activate expression of genes of cellular senescence, JAK/STAT, and Hippo signaling pathways. Lipoic acid (LA) that induced nuclear PD-L1 translocation suppressed tumorigenesis and boosted antitumor immunity. Importantly, LA treatment synergized with PD-1 antibody and overcame immune checkpoint blockade (ICB) resistance, which likely resulted from nPD-L1–increased MHC-I expression and sensitivity of tumor cells to interferon-γ. These findings offer a conceptual advance for PD-L1 function and suggest LA as a promising therapeutic option for overcoming ICB resistance.
Authors
Yong Liu, Zhi Yang, Shuanglian Wang, Rui Miao, Chiung-Wen Mary Chang, Jingyu Zhang, Xin Zhang, Mien-Chie Hung, Junwei Hou
Abstract
The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergizes with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor-derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress-targeting strategy for treating a broad range of cancers.
Authors
Ting-Yu Chang, Yan Yan, Zih-Yao Yu, Moeez Rathore, Nian-Zhe Lee, Hui-Ju Tseng, Li-Hsin Cheng, Wei-Jan Huang, Wei Zhang, Ernest R. Chan, Yulan Qing, Ming-Lun Kang, Rui Wang, Kelvin K. Tsai, John J. Pink, William E. Harte, Stanton L. Gerson, Sung-Bau Lee
Abstract
The most common mutation in surfactant protein C gene (SFTPC), SFTPCI73T, causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPCI73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity purification-mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including Valosin Containing Protein (VCP) and its interactors. Treatment of SftpcI73T knock-in mice and SFTPCI73T expressing iAT2 cells derived from SFTPCI73T patient-specific iPSCs with the specific VCP inhibitor CB5083 restored alveolar structure and SFTPCI73T trafficking respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPCI73T mutant, providing novel therapeutical targets for SFTPCI73T-associated interstitial lung disease.
Authors
Xiaofang Tang, Wei Wei, Yuqing Sun, Timothy E. Weaver, Ernesto S. Nakayasu, Geremy Clair, John M. Snowball, Cheng-Lun Na, Karen S. Apsley, Emily P. Martin, Darrell N. Kotton, Konstantinos-Dionysios Alysandratos, Jiuzhou Huo, Jeffery D. Molkentin, William A. Gower, Xinhua Lin, Jeffrey A. Whitsett
Abstract
The high rate of recurrence after radiation therapy in triple-negative breast cancer (TNBC) indicates that novel approaches and targets are needed to enhance radiosensitivity. Here, we report that neuropilin-2 (NRP2), a receptor for vascular endothelial growth factor (VEGF) that is enriched on sub-populations of TNBC cells with stem cell properties, is an effective therapeutic target for sensitizing TNBC to radiotherapy. Specifically, VEGF/NRP2 signaling induces nitric oxide synthase 2 (NOS2) transcription by a mechanism dependent on Gli1. NRP2-expressing tumor cells serve as a hub to produce nitric oxide (NO), an autocrine and paracrine signaling metabolite, which promotes cysteine-nitrosylation of Kelch-like ECH-asssociated protein 1 (KEAP1) and, consequently, nuclear factor erythroid 2-related factor 2 (NFE2L2)-mediated transcription of antioxidant response genes. Inhibiting VEGF binding to NRP2, using a humanized monoclonal antibody (mAb), results in NFE2L2 degradation via KEAP1 rendering cell lines and organoids vulnerable to irradiation. Importantly, treatment of patient-derived xenografts with the NRP2 mAb and radiation resulted in significant tumor necrosis and regression compared to radiation alone. Together, these findings reveal a targetable mechanism of radioresistance and they support the use of NRP2 mAb as an effective radiosensitizer in TNBC.
Authors
Ayush Kumar, Hira Goel, Christi Wisniewski, Tao Wang, Yansong Geng, Mengdie Wang, Shivam Goel, Kai Hu, Rui Li, Lihua J. Zhu, Jennifer L. Clark, Lindsay M. Ferreira, Michael Brehm, Thomas J. Fitzgerald, Arthur M. Mercurio
Abstract
Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic CIT missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it does not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lose cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupt polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.
Authors
Gianmarco Pallavicini, Amanda Moccia, Giorgia Iegiani, Roberta Parolisi, Emily R. Peirent, Gaia Elena Berto, Martina Lorenzati, Rami Y. Tshuva, Alessia Ferraro, Fiorella Balzac, Emilia Turco, Shachi U. Salvi, Hedvig F. Myklebust, Sophia Wang, Julia Eisenberg, Maushmi Chitale, Navjit S. Girgla, Enrica Boda, Orly Reiner, Annalisa Buffo, Ferdinando Di Cunto, Stephanie L. Bielas
Abstract
Chronic low back pain (LBP) can severely affect daily physical activity. Aberrant osteoclast-mediated resorption leads to porous endplates for the sensory innervation to cause LBP. Here, we report that the expression of proton-activated chloride (PAC) channel is induced during osteoclast differentiation in the porous endplates via a RANKL-NFATc1 signaling pathway. Extracellular acidosis evokes robust PAC currents in osteoclasts. An acidic environment of porous endplates and elevated PAC activation-enhanced osteoclast fusion provoke LBP. Further, we find that genetic knockout of PAC gene Pacc1 significantly reduces endplate porosity and spinal pain in a mouse LBP model, but it does not affect bone development or homeostasis of bone mass in adult mice. Moreover, both osteoclast bone resorptive compartment environment and PAC traffic from the plasma membrane to endosomes to form an intracellular organelle Cl channel have low pH around 5.0. The low pH environment activates PAC channel to increase sialyltransferase St3gal1 expression and sialylation of TLR2 in initiation of osteoclast fusion. Aberrant osteoclast-mediated resorption is also found in most skeletal disorders, including osteoarthritis, ankylosing spondylitis, rheumatoid arthritis, heterotopic ossification, enthesopathy. Thus, elevated Pacc1 expression and PAC activity could be a potential therapeutic target for LBP and osteoclast-associated pain.
Authors
Peng Xue, Weixin Zhang, Mengxi Shen, Junhua Yang, Jiachen Chu, Shenyu Wang, Mei Wan, Junying Zheng, Zhaozhu Qiu, Xu Cao
Abstract
CD44 is associated with a high risk of metastasis, recurrence, and drug resistance in various cancers. Here we report that platelet endothelial aggregation receptor 1 (PEAR1) is a CD44 chaperone protein that protected CD44 from endocytosis-mediated degradation and enhances cleavage of the CD44 intracellular domain (CD44-ICD). Furthermore, we found that lysyl oxidase–like protein 2 (LOXL2), an endogenous ligand of PEAR1, bound to the PEAR1-EMI domain and facilitated the interaction between PEAR1 and CD44 by inducing PEAR1 Ser891 phosphorylation in a manner that was independent of its enzyme activity. Levels of PEAR1 protein and PEAR1 phosphorylation at Ser891 were increased in patients with triple-negative breast cancer (TNBC), were positively correlated with expression of LOXL2 and CD44, and were negatively correlated with overall survival. The level of PEAR1 Ser891 phosphorylation was identified as the best independent prognostic factor in TNBC patients. The prognostic efficacy of the combination of PEAR1 phosphorylation at Ser891 and CD44 expression was superior to that of PEAR1 phosphorylation at Ser891 alone. Blocking the interaction between LOXL2 and PEAR1 with monoclonal antibodies significantly inhibited TNBC metastasis, representing a promising therapeutic strategy for TNBC.
Authors
Yingzhi Shen, Jie Yan, Lin Li, Huiyan Sun, Lin Zhang, Guoming Li, Xinxia Wang, Ruoyan Liu, Xuefeng Wu, Baosan Han, Xueqing Sun, Junling Liu, Xuemei Fan
Abstract
Variants of the G protein-coupled receptor 75 (GPR75) are associated with lower BMI in large-scale human exome sequencing studies. However, how GPR75 regulates body weight remains poorly understood. Using random germline mutagenesis in mice, we identified a missense allele (Thinner) of Gpr75 that resulted in a lean phenotype and verified the decreased body weight and fat weight in Gpr75 knockout (Gpr75–/–) mice. Gpr75–/– mice displayed reduced food intake under a high-fat diet (HFD), and pair-feeding normalized their body weight. The endogenous GPR75 protein was exclusively expressed in the brains of 3xFlag tagged Gpr75 knock-in (3xFlag-Gpr75) mice, with consistent expression across different brain regions. GPR75 interacted with Gαq to activate various signaling pathways after HFD feeding. Additionally, GPR75 was localized in the primary cilia of hypothalamic cells, whereas the Thinner mutation (L144P) and human GPR75 variants with lower BMI failed to localize in the cilia. Loss of GPR75 selectively inhibited weight gain in HFD-fed mice but failed to suppress the development of obesity in Leptin ob mice and Adenylate cyclase 3 (Adcy3) mutant mice on a chow diet. Our data reveal that GPR75 is a ciliary protein expressed in the brain and plays an important role in regulating food intake.
Authors
Yiao Jiang, Yu Xun, Zhao Zhang
Abstract
Cell cycle regulation is largely abnormal in cancers. Molecular understanding and therapeutic targeting of the aberrant cell cycle are essentially meaningful. Here, we identified an under-appreciated Serine/Threonine kinase, CDKL3 (Cyclin-dependent kinase like 3), crucially drives the rapid cell cycle progression and cell growth in cancers. Mechanism-wise, CDKL3 localizes in the nucleus and associates with specific cyclin to directly phosphorylate Retinoblastoma (Rb) for quiescence exit. In parallel, CDKL3 prevents the ubiquitin-proteasomal degradation of CDK4 by direct phosphorylation on T172 to sustain G1 phase advancement. The crucial function of CDKL3 in cancers was demonstrated both in vitro and in vivo. We also designed, synthesized and characterized a first-in-class CDKL3-specific inhibitor, HZ1. HZ1 exhibits greater potency than CDK4/6 (Cyclin-dependent kinase 4/6) inhibitor in pan-cancer treatment by causing cell cycle arrest and overcomes the acquired resistance of the latter. In particular, CDKL3 has significant clinical relevance in colon cancer, and the effectiveness of HZ1 was demonstrated by murine and patient-derived cancer models. Collectively, this work presented an integrated paradigm of cancer cell cycle regulation and suggested CDKL3-targeting as a feasible approach in cancer treatment.
Authors
Haijiao Zhang, Jiahui Lin, Shaoqin Zheng, Lanjing Ma, Zhongqiu Pang, Hongyi Yin, Chengcheng Meng, Yinuo Wang, Qing Han, Xi Zhang, Zexu Li, Liu Cao, Lijun Liu, Teng Fei, Daming Gao, Liang Yang, Xueqiang Peng, Chen Ding, Shixue Wang, Ren Sheng
Abstract
Mitochondria-related neurodegenerative diseases have been implicated in the disruption of primary cilia function. Mutation in an intrinsic mitochondrial complex I component NDUFAF2 has been identified in Leigh syndrome, a severe inherited mitochondriopathy. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye. Here, we report a mechanistic link between mitochondria metabolism and primary cilia signaling. We discovered that loss of NDUFAF2 caused both mitochondrial and ciliary defects in vitro and in vivo and identified NDUFAF2 as a binding partner for ARMC9. We also found that NDUFAF2 was both necessary and sufficient for cilia formation and that exogenous expression of NDUFAF2 rescued the ciliary and mitochondrial defects observed in cells from patients with known ARMC9 deficiency. NAD+ supplementation restored mitochondrial and ciliary dysfunction in ARMC9-deficient cells and zebrafish and ameliorated the ocular motility and motor deficits of a patient with ARMC9 deficiency. The present results provide a compelling mechanistic link, supported by evidence from human studies, between primary cilia and mitochondrial signaling. Importantly, our findings have significant implications for the development of therapeutic approaches targeting ciliopathies.
Authors
Chien-Hui Lo, Zhiquan Liu, Siyu Chen, Frank Lin, Andrew R. Berneshawi, Charles Q. Yu, Euna B. Koo, Tia J. Kowal, Ke Ning, Yang Hu, Won-Jing Wang, Y. Joyce Liao, Yang Sun
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ISSN: 0021-9738 (print), 1558-8238 (online)