Polymorphonuclear neutrophils (PMNs) are increasingly recognized to influence solid tumor development, but why their effects are so context-dependent and even frequently divergent remains poorly understood. Using an autochthonous mouse model of uterine cancer and the administration of respiratory hyperoxia as a means to improve tumor oxygenation, we provide in vivo evidence that hypoxia is a potent determinant of tumor-associated PMN phenotypes and direct PMN-tumor cell interactions. Upon relief of tumor hypoxia, PMNs were recruited less intensely to the tumor-bearing uterus but the recruited cells much more effectively killed tumor cells, an activity our data moreover suggested was mediated via their production of NADPH oxidase-derived reactive oxygen species and MMP-9. Simultaneously, their ability to promote tumor cell proliferation, which appeared mediated via their production of neutrophil elastase, was rendered less effective. Relieving tumor hypoxia thus greatly improved net PMN-dependent tumor control, leading to a massive reduction in tumor burden. Remarkably, this outcome was T cell-independent. Together, these findings identify key hypoxia-regulated molecular mechanisms through which PMNs directly induce tumor cell death and proliferation in vivo and suggest that the contrasting properties of PMNs in different tumor settings may in part reflect the effects of hypoxia on direct PMN-tumor cell interactions.
Karim Mahiddine, Adam Blaisdell, Stephany Ma, Amandine Créquer-Grandhomme, Clifford A. Lowell, Adrian Erlebacher
Patients with bladder cancer (BCa) with clinical lymph node (LN) metastasis have extremely poor prognosis. VEGF-C has been demonstrated to play vital roles in LN metastasis in BCa. However, approximately 20% of BCa with LN metastasis exhibits low VEGF-C expression, suggesting a VEGF-C-independent mechanism for LN metastasis of BCa. Herein, we demonstrated that BCa cell-secreted exosomes-mediated lymphangiogenesis promoted LN metastasis in BCa, which was in a VEGF-C-independent manner. We identified an exosomal long noncoding RNA (lncRNA), termed lymph node metastasis-associated transcript 2 (LNMAT2), stimulated HLEC tube formation and migration in vitro and enhanced tumor lymphangiogenesis and LN metastasis in vivo. Mechanistically, LNMAT2 was loaded to BCa cell-secreted exosomes by directly interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). Subsequently, exosomal LNMAT2 was internalized by HLECs and epigenetically upregulated prospero homeobox 1 (PROX1) expression by recruitment of hnRNPA2B1 and increasing the H3K4 trimethylation level in the PROX1 promoter, ultimately resulting in lymphangiogenesis and lymphatic metastasis. Therefore, our findings highlight a VEGF-C-independent mechanism of exosomal lncRNA-mediated LN metastasis and identify LNMAT2 as a therapeutic target for LN metastasis in BCa.
Changhao Chen, Yuming Luo, Wang He, Yue Zhao, Yao Kong, Hongwei Liu, Guangzheng Zhong, Yuting Li, Jun Li, Jian Huang, Rufu Chen, Tianxin Lin
Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly-identified highly-selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted-radiotherapy on human orthotopic lung tumors without influencing acute DNA-damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and toxicity of a parenterally-administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.
Catherine E. Willoughby, Yanyan Jiang, Huw D. Thomas, Elaine Willmore, Suzanne Kyle, Anita Wittner, Nicole Phillips, Yan Zhao, Susan J. Tudhope, Lisa Prendergast, Gesa Junge, Luiza Madia Lourenco, M. Raymond V. Finlay, Paul Turner, Joanne M. Munck, Roger J. Griffin, Tommy Rennison, James Pickles, Celine Cano, David R. Newell, Helen L. Reeves, Anderson J. Ryan, Stephen R. Wedge
Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40% to 50% of adults with CF. The age-at-onset of CF-related diabetes (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age-at-onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that two common DNA haplotypes formed by the risk variants account for the association with diabetes (high risk, P-value: 4.34E-3; low risk, P-value: 1.14E-3). Single-cell RNA (scRNA) sequencing indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells, and frequently co-expressed with CFTR along with transcription factors that have binding sites 5′ of SLC26A9. These findings replicated upon re-analysis of scRNA data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9 bearing variants from the low risk haplotype generated 12% to 20% higher levels of expression in PANC-1 and CFPAC-1 cells compared to the high risk haplotype (P-values: 2.00E-3 to 5.15E-9). Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age-at-onset of diabetes, thereby suggesting a CFTR-agnostic treatment for a major complication of CF.
Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Although CD4 T cells are implicated in MS pathogenesis and have been the main focus of MS research using the animal model experimental autoimmune encephalomyelitis (EAE), substantial evidence from patients with MS points to a role for CD8 T cells in disease pathogenesis. We previously showed that an MHC class I-restricted epitope of myelin basic protein (MBP) is presented in the CNS during CD4 T cell-initiated EAE. Here, we investigated whether naïve MBP-specific CD8 T cells recruited to the CNS during CD4 T cell-initiated EAE engaged in determinant-spreading and influenced disease. We found that the MBP-specific CD8 T cells exacerbated brain but not spinal cord inflammation. We show that a higher frequency of monocytes and monocyte-derived cells presented the MHC class I-restricted MBP ligand in the brain compared to the spinal cord. Infiltration of MBP-specific CD8 T cells enhanced ROS production in the brain only in these cell-types and only when the MBP-specific CD8 T cells expressed Fas ligand (FasL). These results suggest that myelin-specific CD8 T cells may contribute to disease pathogenesis via a FasL-dependent mechanism that preferentially promotes lesion formation in the brain.
Catriona A. Wagner, Pamela J. Roqué, Trevor R. Mileur, Denny Liggitt, Joan M. Goverman
Brown adipose tissue (BAT), as the main site of adaptive thermogenesis, exerts beneficial metabolic effects on obesity and insulin resistance. BAT has been previously assumed to contain a homogeneous population of brown adipocytes. Utilizing multiple mouse models capable of genetically labeling different cellular populations, as well as single-cell RNA sequencing, and 3D tissue profiling, we discovered a new brown adipocyte subpopulation with low thermogenic activity co-existing with the classical high thermogenic brown adipocytes within the BAT. These low thermogenic brown adipocytes had significantly lower Ucp1 and Adipoq expression, larger lipid droplets, and lower mitochondrial content. Functional analyses showed that the low thermogenic brown adipocytes have significant lower basal mitochondrial respiration, and they are specialized in fatty acid uptake. Upon changes in environmental temperature, the two brown adipocyte subpopulations underwent dynamic inter-conversions. Cold exposure converted low thermogenic brown adipocytes into high thermogenic cells, and a thermoneutral environment had the opposite effect. This recruitment of high thermogenic brown adipocytes by cold stimulation is not affected by high fat diet feeding, but significantly declined with age. Our results revealed a high degree of functional heterogeneity of brown adipocytes.
Anying Song, Wenting Dai, Min Jee Jang, Leonard Medrano, Zhuo Li, Hu Zhao, Mengle Shao, Jiayi Tan, Aimin Li, Tinglu Ning, Marcia M. Miller, Brian Armstrong, Janice M. Huss, Yi Zhu, Yong Liu, Viviana Gradinaru, Xiwei Wu, Lei Jiang, Philipp E. Scherer, Qiong A. Wang
Catecholamines released by sympathetic nerves can activate adrenergic receptors present on nearly every cell type, including myeloid derived suppressor cells (MDSCs). Using in vitro systems and murine tumor models, in wild-type mice and genetically modified (β2-AR–/–) mice, as well adoptive transfer approaches, we found that the degree of β2-AR signaling significantly influences MDSC frequency and survival in tumors and other tissues, modulates their expression of immunosuppressive molecules such as arginase-I and PDL-1 and alters their ability to suppress the proliferation of T cells. The regulatory functions of β-AR signaling in MDSCs were found to be dependent upon STAT3 phosphorylation. Moreover, we observed that the β2-AR-mediated increase in survival of MDSCs is dependent upon Fas-FasL interactions, and this is consistent with gene expression analyses which reveal a greater expression of apoptosis-related genes in β2-AR–/– MDSCs. Our data reveals the potential of β2-AR signaling to increase the generation of MDSCs from both murine and human peripheral blood cells and that the immunosuppressive function of MDSCs could be mitigated by treatment with β-AR antagonists, or enhanced by β-AR agonists, strongly supporting the possibility that reducing stress-induced activation of β2-ARs could help to overcome immune suppression and enhance the efficacy of immunotherapy and other cancer therapies.
Hemn Mohammadpour, Cameron R. MacDonald, Guanxi Qiao, Minhui Chen, Bowen Dong, Bonnie L. Hylander, Philip L. McCarthy, Scott I. Abrams, Elizabeth A. Repasky
Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly-inherited diseases—where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function (GoF) allele—is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapeutic for Charcot-Marie-Tooth type 2D (CMT2D), caused by dominant mutations in glycyl tRNA-synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks-of-age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least one year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof-of-concept for gene therapy approaches for dominant neuromuscular diseases.
Kathryn H. Morelli, Laurie B. Griffin, Nettie K. Pyne, Lindsay M. Wallace, Allison M. Fowler, Stephanie N. Oprescu, Ryuichi Takase, Na Wei, Rebecca Meyer-Schuman, Dattatreya Mellacheruvu, Jacob O. Kitzman, Samuel G. Kocen, Timothy J. Hines, Emily L. Spaulding, James R. Lupski, Alexey Nesvizhskii, Pedro Mancias, Ian J. Butler, Xiang-Lei Yang, Ya-Ming Hou, Anthony Antonellis, Scott Q. Harper, Robert W. Burgess
Arcuate nucleus agouti-related peptide (AgRP) neurons play a central role in feeding and are under complex regulation by both homeostatic hormonal and nutrient signals and hypothalamic neuronal pathways. Feeding may also be influenced by environmental cues, sensory inputs and other behaviors implying the involvement of higher brain regions. However, whether such pathways modulate feeding through direct synaptic control of AgRP neuron activity is unknown. Here we show that nociceptin-expressing neurons in the anterior bed nuclei of the stria terminalis (aBNST) make direct GABAergic inputs onto AgRP neurons. We found that activation of these neurons inhibited AgRP neurons and feeding. Activity of these neurons increased upon food availability and their ablation resulted in obesity. Furthermore, these neurons received afferent inputs from a range of upstream brain regions as well as hypothalamic nuclei. Therefore, aBNST nociceptin/GABAergic neurons may act as a gateway to feeding behavior by connecting AgRP neurons to both homeostatic and non-homeostatic neuronal inputs.
Mark A. Smith, Agharul I. Choudhury, Justyna A. Glegola, Paulius Viskaitis, Elaine E. Irvine, Pedro Caldas Custodio de Campos Silva, Sanjay Khadayate, Hanns Ulrich Zeilhofer, Dominic J. Withers
Currently, an effective targeted therapy for pancreatitis is still lacking. Hereditary pancreatitis (HP) is a heritable, autosomal-dominant disorder with recurrent acute pancreatitis (AP) progressing to chronic pancreatitis (CP) and a markedly increased risk of pancreatic cancer. In 1996, mutations in PRSS1 were linked to the development of HP. Here, we developed a mouse model by inserting a full-length human PRSS1R122Hgene, the most commonly mutated gene in human HP, into mice. Expression of PRSS1R122H protein in the pancreas markedly increased stress signaling pathways and exacerbated AP. After the attack of AP, all PRSS1R122H mice had disease progression to CP, with similar histologic features as those observed in human HP. By comparing PRSS1R122H mice with PRSS1WT mice as well as enzymatically inactivated Dead-PRSS1R122H mice, we unraveled that increased trypsin activity is the mechanism for R122H mutation to sensitize mice to the development of pancreatitis. We further discovered that trypsin inhibition, in combination with anti-coagulation therapy, synergistically prevented the progression to CP in PRSS1R122H mice. These animal models help us better understand the complex nature of this disease and provide powerful tools for developing and testing novel therapeutics for human pancreatitis.
Fu Gui, Yuebo Zhang, Jianhua Wan, Xianbao Zhan, Yao Yao, Yinghua Li, Ashley N. Haddock, Ji Shi, Jia Guo, Jiaxiang Chen, Xiaohui Zhu, Brandy H. Edenfield, Lu Zhuang, Cheng Hu, Ying Wang, Debabrata Mukhopadhyay, Evette S. Radisky, Lizhi Zhang, Aurelia Lugea, Stephen J. Pandol, Yan Bi, Baoan Ji
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