Abstract
Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide, yet its molecular drivers are not fully defined. Emerging evidence highlights the importance of tumor-stroma interactions mediated by secreted glycoproteins. However, the mechanisms by which cancer cells regulate the secretion of these protumorigenic proteins remain largely unknown. Endoplasmic reticulum–resident (ER-resident) N-glycan–processing enzymes regulate proper protein folding, a prerequisite for glycoproteins to exit the ER and undergo secretion. By evaluating their prognostic significance in lung tumors and conducting functional screening in lung cancer cells, we identify α-glucosidase II (α-Glc II) as a key regulator of NSCLC progression. α-Glc II promotes tumor growth and dissemination in a glucosidase activity–dependent manner in orthotopic mouse lung tumor model. Genetic disruption of α-Glc II induced ER stress and reduced cell proliferation and motility. Mechanistically, α-Glc II–mediated N-glycan modification regulated the ER-to-Golgi trafficking and secretion of specific oncogenic glycoproteins, including lysyl hydroxylase 2 (LH2), Tissue Inhibitor of Metalloproteinase 1 (TIMP1), and TGF-β, which are known to be associated with extracellular matrix remodeling. These findings uncover a role for ER glycosylation machinery in shaping the NSCLC secretome and highlight α-Glc II as a potential therapeutic target.
Authors
Shike Wang, Na Ding, Angelo Chen, Derrick Cardin, Yuting Xu, Kate Grimley, William K. Russell, Jun Xu, Jonathan M. Kurie, Guan-Yu Xiao, Xiaochao Tan
Abstract
Transcriptional reprogramming has an important role in kidney glomerular disease. Using in vivo murine models of podocyte injury, we studied the roles of the FOXC2 and WT1 transcription factors (TFs) in podocyte injury. Podocytes are a crucial cell type of glomeruli, the filtration units of each nephron. Podocyte injury is often the incipient event leading to chronic kidney disease. It is well established that the TFs FOXC2 and WT1 are required in podocytes to maintain the glomerular filtration barrier. Their role in the response to injury is less well understood. Here, we tested the hypothesis that FOXC2 and WT1 act together to mediate transcriptional reprogramming in response to podocyte injury. Similarly to that of WT1, genome-wide FOXC2 binding to target genes is dynamic during the course of injury, initially increasing, but late in injury there is a dramatic decrease in FOXC2 expression and in its binding to target genes. Podocyte-specific inactivation of FoxC2 or Wt1 in adult mice limits the transcriptional response to injury. Correlating FOXC2 and WT1 ChIP-seq analyses demonstrated that they co-bind many genes expressed in podocytes. Thus, reprogramming the transcriptome involves dynamic changes in the binding of FOXC2 and WT1 to their target genes during a reparative injury response.
Authors
Sandrine Ettou, Anya Greenberg, Sangyoon Lee, Arjun Rajesh, Liang Sun, Nahid Tabibzadeh, Haruka Oishi, Ran Konoe, Phillip J. McCown, Sean Eddy, Victoria Driscoll, Tomoya Miyoshi, Ken Hiratsuka, Jason Lam, R. Sathish Srinivasan, Youngsook L. Jung, Biju Isaac, Mingwei Sun, Mary E. Taglienti, Keith Keller, Hong Chen, Matthias Kretzler, Astrid Weins, Ryuji Morizane, Shira Rockowitz, Valerie A. Schumacher, Dongwon Lee, Jordan A. Kreidberg
Abstract
BK polyomavirus–associated nephropathy (BKVN) adversely impacts kidney allograft survival and often mimics acute T cell–mediated rejection (TCMR), confounding diagnosis and management. To address this conundrum, we performed unbiased RNA sequencing of urinary cells matched to biopsies classified as BKVN with intragraft inflammation (BKVN-P), BKVN without inflammation (BKVN-N), TCMR, or no rejection (NR). BKVN-N displayed dominant host DNA replication, cell cycle, and repair programs, while BKVN-P samples exhibited expansive innate immune activation, antigen presentation, chemokine upregulation, and epithelial injury. Both BKVN subtypes shared signatures of T cell exhaustion and mature and tolerogenic dendritic cell activation but differed in immune orientation — Th1 predominance in BKVN-N versus Treg and CD8 enrichment in BKVN-P. Compared with TCMR samples, BKVN-P lacked robust TCR/CD28 signaling and was enriched for viral and innate modules; BKVN-N lacked alloimmune activation. B cell exhaustion characterized BKVN-N, while BKVN-P displayed robust B cell activation with metabolic downregulation. A ratiometric urinary cell biomarker, CXCL10 mRNA/CD3E mRNA, distinguished both BKVN subtypes from TCMR with diagnostic accuracy, replicated by quantitative reverse transcription PCR for clinical translation, and confirmed in an independent cohort. These findings demonstrate the utility of urinary cell transcriptomics for resolving viral injury from alloimmunity, enabling precision diagnostics and targeted immunomodulation in kidney transplantation.
Authors
Franco B. Mueller, Carol Li, Darshana M. Dadhania, Surya V. Seshan, Thalia Salinas, Vijay K. Sharma, Jenny Z. Xiang, Hans H. Hirsch, Thangamani Muthukumar, Manikkam Suthanthiran
Abstract
β-Arrestins are ubiquitously expressed cytosolic adaptor proteins that regulate GPCR-dependent and -independent pathways essential for numerous physiological functions. This study investigated the role of β-arrestin1/2 in embryonic lymphatic vessel development and survival by generating and characterizing mice with lymphatic tamoxifen-inducible loss of the genes encoding β-arrestin1/2 (Arrb1/2ΔiLEC). At E15.5, Arrb1/2ΔiLEC embryos exhibited profound hydrops fetalis and increased embryonic mortality compared with control Arrb1/2fl/fl embryos. Edematous Arrb1/2ΔiLEC embryos, which were more often represented by the female sex, showed growth restriction and decreased lymphatic endothelial cell (LEC) proliferation in the jugular lymphatic sac compared with controls. In vitro knockdown of β-arrestin1 in LECs increased proliferation and increased activation of AKT, while knockdown of β-arrestin2 decreased proliferation and decreased activation of both ERK and CREB. Arrb1/2ΔiLEC embryos also exhibited dilated dermal lymphatics with decreased continuous VE-cadherin adherens junctions compared with controls. These results were recapitulated in vitro in β-arrestin1/2 knockdown human LECs, which showed a decrease in membrane VE-cadherin and β-catenin levels, in addition to prevention of adrenomedullin-induced linearization of VE-cadherin at endothelial cell–cell junctions. Collectively, these results demonstrate that loss of β-arrestin1/2 in lymphatics causes hydrops fetalis, midgestational growth arrest, and embryonic demise associated with reduced LEC proliferation and disrupted VE-cadherin adherens junctions.
Authors
Yanna Tian, D. Stephen Serafin, Monserrat Avila-Zozaya, Alyssa M. Tauro, Natalie M. Torres-Valle, Bryan M. Kistner, Danielle M. Dy, Elizabeth S. Douglas, Kathleen M. Caron
Abstract
Pancreatic ductal adenocarcinoma (PDAC) shows profound resistance to immunotherapy due to its immunosuppressive tumor microenvironment. Here, we studied the relationship between T cell infiltration and innate immune signaling in PDAC, identifying TLR2 as a key regulator of T cell exclusion. TLR2 expression correlated with T cell infiltration in both human and mouse PDAC tumors. Using genetic KO models and adoptive T cell transfer experiments, we found that TLR2 expression in both T cells and non–T cells contributes to T cell exclusion in PDAC. Notably, successful infiltration of adoptively transferred tumor-specific T cells required TLR2 deletion in both the transferred cells and the recipient host. The therapeutic implications of these findings are demonstrated through both genetic deletion and pharmacological inhibition of TLR2 using AAV-mediated and antibody-based approaches in murine models, resulting in decreased tumor growth and extended survival. Collectively, these findings identify TLR2 as a key modulator of T cell trafficking and immune suppression within the PDAC microenvironment, suggesting its potential as a therapeutic target for improving treatment outcomes.
Authors
Jacqueline Plesset, Meredith L. Stone, John C. McVey, Heather Coho, Kelly Markowitz, Kayjana Coho, Jesse Lee, Anna S. Thickens, Devora Delman, Gregory L. Beatty
Abstract
Cytotoxic chemotherapy primarily targets rapidly proliferating cancer cells but also depletes normal myeloid cells. The resulting cell loss triggers reactive myelopoiesis, a compensatory process in which hematopoietic stem and progenitor cells in the bone marrow (BM) regenerate myeloid lineages. We previously showed that the alkylating agent cyclophosphamide (CTX) induces myelopoiesis, leading to the expansion of immunosuppressive monocytes in mice. However, the molecular features and clinical relevance of these cells remain poorly understood. Here, we report the emergence of immunosuppressive monocytes in the peripheral blood of lymphoma patients receiving CTX-containing chemotherapy. To gain mechanistic insight into CTX-induced myelopoiesis, we performed single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) on BM monocytes from CTX-treated mice. These analyses revealed a heterogeneous monocyte population and demonstrated that CTX skews myelopoiesis toward the generation of neutrophil-like monocytes (NeuMo). Moreover, CTX-induced NeuMo cells, enriched within the CXCR4+CX3CR1– monocyte subset, exhibited potent T cell–suppressive activity. Using the NeuMo gene signature, reanalysis of public scRNA-seq datasets identified a transcriptionally similar monocyte subset in chemotherapy-treated cancer patients. Collectively, our findings suggest that the expansion of NeuMo cells following chemotherapy represents a conserved immunoregulatory feedback mechanism with potential impact on tumor response to chemoimmunotherapy.
Authors
Huidong Shi, Zhi-Chun Ding, Ogacheko D. Okoko, Xin Wang, George Zhou, Yan Ye, Md Yeashin Gazi, Caitlin Brandle, Lirong Pei, Rafal Pacholczyk, Catherine C. Hedrick, Locke J. Bryan, Gang Zhou
Abstract
Hypoxia critically restricts the effectiveness of immunotherapy in triple-negative breast cancer (TNBC). Comprehensive bioinformatics analyses have demonstrated that highly hypoxic TNBC tumors exhibited elevated T cell exhaustion, increased immune checkpoint molecule expression, and diminished responsiveness to immune checkpoint blockade (ICB). Consequently, strategies aimed at alleviating tumor hypoxia may effectively augment ICB therapy. Although ultrasound-targeted microbubble cavitation (UTMC) has been shown to reduce tumor hypoxia, the precise molecular mechanisms remain unclear. Here, we provide evidence that UTMC activated endothelial nitric oxide synthase (eNOS) through G protein–coupled signaling, resembling pathways induced by fluid shear stress. UTMC-induced eNOS activation was largely Ca2+ dependent and resulted in increased nitric oxide production. Enhanced nitric oxide generation was associated with improved tumor perfusion and reduced hypoxia. Combining UTMC with anti–PD-L1 therapy markedly improved the tumor immune microenvironment, characterized by increased CD8+ T cell infiltration, reduced T cell exhaustion, diminished regulatory T cell infiltration, increased macrophage polarization from an M2 to M1 phenotype, and elevated production of proinflammatory cytokines. Collectively, our findings identified UTMC as a promising adjunctive therapeutic approach to mitigate hypoxia and enhance the efficacy of anti–PD-L1 immunotherapy in TNBC. These results support further translational evaluation of UTMC-based combination strategies in hypoxic TNBC.
Authors
Zhiyu Zhao, Li Ba, Siwei Li, Jianxin Wang, Yuzhou Luo, Sihan Wang, Yan Jin, Changjun Wu
Abstract
Chronic neuropathic pain is frequently comorbid with anxiety disorders, yet the neural circuits underlying this interaction remain poorly defined. The parafascicular nucleus (PF) of the thalamus integrates nociceptive and affective signals, but its specific regulatory mechanisms in pain-anxiety comorbidity are not well known. Using spared nerve injury (SNI) model mice, we combined viral neural tracing, chemogenetics, pharmacology, and electrophysiology to dissect the locus coeruleus (LC)–PF neural pathway. Viral tracing revealed monosynaptic projections from norepinephrinergic (NEergic) neurons in the dorsal LC to Ca2+/calmodulin–dependent protein kinase IIα–immunopositive (CaMKIIα+) neurons within the PF. Chemogenetic inhibition and activation of this pathway were performed in naive and SNI mice, alongside intra-PF microinjection of the α-2 adrenergic receptor (ADRA2) antagonist yohimbine. Behavioral tests assessed mechanical/thermal hypersensitivity and anxiety-like behaviors. Results showed that 92.1% of PF-projecting LC neurons were NEergic, with 70.1% localized dorsally. Chemogenetic inhibition of the LCNE-PFCaMKIIα neural pathway significantly alleviated both acute-phase mechanical hypersensitivity (<7 days after surgery) and chronic-phase anxiety-like behaviors in SNI mice, while activation of this pathway induced pain sensitization and anxiety-like behaviors in naive mice. Intra-PF yohimbine reversed SNI-induced allodynia and anxiety-like behaviors. Electrophysiology confirmed that yohimbine increased PF neuronal intrinsic excitability. These results suggest that the LCNE-PFCaMKIIα neural pathway promotes neuropathic pain and comorbid anxiety via ADRA2-mediated suppression of PF neuronal activity. Targeted inhibition of this circuit may represent a therapeutic strategy for pain-related affective disorders.
Authors
Zhong-Yi Liu, Fei Li, Li-Ming Liu, Yao-Hua Liu, Jia Li, Zi-Ang Li, Jin Cheng, Tian-Yu Zhao, Hui-Min Tian, Dong-Ning Li, Sha-Sha Tao, Hui Li, Fen-Sheng Huang, Yun-Qing Li
Abstract
X-linked myotubular myopathy (XLMTM) is a rare genetic disorder that typically presents at birth with progressive muscle weakness and respiratory difficulties and is caused by myotubularin 1 (MTM1) gene mutations. Here, we examine the role of phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2-β (PIK3C2B), a lipid kinase that interacts with MTM1, in XLMTM in various models. We examined the effect of BLU3797, a highly potent, selective, orally bioavailable PIK3C2B inhibitor, on survival, muscle development, myofiber phenotypes, and gene expression in MTM1–/y mice. PIK3C2B-deficient XLMTM animals demonstrated increased survival, restored muscle function, fewer myofibers with centralized nuclei, and normalization of disease-associated molecular markers. BLU3797 alleviated the XLMTM phenotype in a dose-dependent and reversible manner. Loss of functional PIK3C2B in XLMTM mice promoted a more differentiated, adult-like myofiber profile, which was strongly associated with normalization of disease surrogates and a reduction in markers of early muscle development and regeneration. BLU3797 treatment appears to modulate the expression of miRNAs associated with satellite cell activation and myofiber fusion. These findings indicate that PIK3C2B inhibition with BLU3797 effectively reverses the XLMTM disease phenotype by enhancing muscle function and promoting development toward a more mature state.
Authors
Andrew Shearer, Melissa L. Brooks, Maxine M. Chen, Thiwanka Samarakoon, John Hsieh, Gramoz Kondakci, Emanuele Perola, Jason Brubaker, Kristina Fetalvero, Stefanie Schalm, Joana Caetano-Lopes
Abstract
Huntington’s disease (HD) is a fatal neurodegenerative disease caused by an expanded polyglutamine (CAG) repeat in the N-terminal of the huntingtin protein (HTT). Microglial activation and elevated proinflammatory cytokines are observed in HD brains, but the mechanisms regulating neuroinflammation and microglial activation are poorly understood. Metformin-mediated neuroprotection has been demonstrated in experimental models of neurodegeneration, including HD. We found that metformin inhibits mitochondrial DNA (mtDNA) release and subsequent neuroinflammation in the cortex and striatum of a mouse model of HD. Moreover, elevated proinflammatory cytokines and microglial activation are inhibited by metformin in HD transgenic mouse brains. Metformin reduced pathological microglial clusters and shifted toward a quiescent, homeostatic phenotype. Metformin improved aberrant immunometabolism in HD mouse brains and primary microglia. Mechanistically, we found that metformin regulates mitochondrial fission, reprograms deregulated metabolism in HD microglia, and controls microglial activation and inflammation in HD transgenic mice.
Authors
Abhishek Jauhari, Adam C. Monek, Olena S. Abakumova, Tanisha Singh, Sukhman Singh, Xiaomin Wang, Carley S. Clise, Diane L. Carlisle, Robert M. Friedlander
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