The origin of breast cancer, whether primary or recurrent, is unknown. Here, we show that invasive breast cancer cells exposed to hypoxia release small extracellular vesicles (sEV) that disrupt the differentiation of normal mammary epithelia, expand stem and luminal progenitor cells, and induce atypical ductal hyperplasia and intraepithelial neoplasia. This was accompanied by systemic immunosuppression with increased myeloid cell release of the “alarmin”, S100A9, and oncogenic traits of EMT, angiogenesis, and local and disseminated luminal cell invasion, in vivo. In the presence of a mammary gland driver oncogene (MMTV-PyMT), hypoxic sEV accelerated bilateral breast cancer onset and progression. Mechanistically, genetic or pharmacologic targeting of hypoxia-inducible factor-1α (HIF1α) packaged in hypoxic sEV, or homozygous deletion of S100A9 normalized mammary gland differentiation, restored T cell function and prevented atypical hyperplasia. The transcriptome of sEV-induced mammary gland lesions resembled luminal breast cancer, and detection of HIF1α in plasma circulating sEV from luminal breast cancer patients correlated with disease recurrence. Therefore, sEV-HIF1α signaling drives both local and systemic mechanisms of mammary gland transformation at high risk for evolution to multifocal breast cancer. This pathway may provide a readily accessible biomarker of luminal breast cancer progression.
Irene Bertolini, Michela Perego, Yulia Nefedova, Cindy Lin, Andrew T. Milcarek, Peter Vogel, Jagadish C. Ghosh, Andrew V. Kossenkov, Dario C. Altieri
Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared to matched non-invasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified three distinct transcriptomic groups in invasive organoids, two of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGFB1, IL6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify unique molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.
Yea Ji Jeong, Hildur Knutsdottir, Fatemeh Shojaeian, Michael G. Lerner, Maria F. Wissler, Elodie Henriet, Tammy Ng, Shalini Datta, Bernat Navarro-Serer, Peter Chianchiano, Benedict Kinny-Köster, Jacquelyn W. Zimmerman, Genevieve Stein-O'Brien, Matthias M. Gaida, James R. Eshleman, Ming-Tseh Lin, Elana J. Fertig, Andrew J. Ewald, Joel S. Bader, Laura D. Wood
IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin and relevance for fracture repair of these T cells are unknown. Here we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxa segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improvement of fracture repair. Mechanistically, fractures increased the S1P-receptor-1 (S1PR1) mediated egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20 mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics, blockade of Th17 cell egress from the gut or antibody neutralization of Th17 cell influx into the callus. These findings demonstrated the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum antibiotics may represent novel therapeutic strategies to optimize fracture healing.
Hamid Y. Dar, Daniel S. Perrien, Subhashis Pal, Andreea Stoica, Sasidhar Uppuganti, Jeffry S. Nyman, Rheinallt M. Jones, M. Neale Weitzmann, Roberto Pacifici
Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.
Nafisa Nuzhat, Kristof Van Schil, Sandra Liakopoulos, Miriam Bauwens, Alfredo Dueñas Rey, Stephan Käseberg, Melanie Jäger, Jason R. Willer, Jennifer Winter, Hanh M. Truong, Nuria Gruartmoner, Mattias Van Heetvelde, Joachim C. Wolf, Robert Merget, Sabine Grasshoff-Derr, Jo Van Dorpe, Anne Hoorens, Heidi Stöhr, Luke Mansard, Anne-Françoise Roux, Thomas Langmann, Katharina Dannhausen, David Rosenkranz, Karl M. Wissing, Michel Van Lint, Heidi Rossmann, Friederike Häuser, Peter Nürnberg, Holger Thiele, Ulrich Zechner, Jillian N. Pearring, Elfride De Baere, Hanno J. Bolz
The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here we demonstrated that Salt Inducible Kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole tissue and single cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Lastly, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful to stimulate 1,25-vitamin D production in CKD-MBD.
Sung-Hee Yoon, Mark B. Meyer, Carlos Arevalo Rivas, Murat Tekguc, Chengcheng Zhang, Jialiang S. Wang, Christian D. Castro Andrade, Katelyn E. Strauss, Tadatoshi Sato, Nancy Benkusky, Seong Min Lee, Rebecca Berdeaux, Marc Foretz, Thomas B. Sundberg, Ramnik J. Xavier, Charles H. Adelmann, Daniel J. Brooks, Anthony Anselmo, Ruslan I. Sadreyev, Ivy A. Rosales, David E. Fisher, Navin Gupta, Ryuji Morizane, Anna Greka, J. Wesley Pike, Michael Mannstadt, Marc N. Wein
Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. Todecipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysisperformed on control mice and mice with inducible deletion of the circadian clock regulator Bmal1 in the renal tubule (cKOt). With this unique resource, we demonstrated that ~30% RNAs, ~20% proteins and ~20% metabolites are rhythmic in kidneys of control mice. Several key metabolic pathways including NAD+ biosynthesis, fatty acid transport, carnitine shuttle,and b-oxidation displayed impairments in kidneys of cKOt, resulting in a perturbedmitochondrial activity. Carnitine reabsorption from the primary urine was one of the mostimpacted processes with a ~50% reduction in plasma carnitine levels and a parallel systemicdecrease in tissues carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology.
Yohan Bignon, Leonore Wigger, Camille Ansermet, Benjamin D. Weger, Sylviane Lagarrigue, Gabriel Centeno, Fanny Durussel, Lou Götz, Mark Ibberson, Sylvain Pradervand, Manfredo Quadroni, Meltem Weger, Francesca Amati, Frédéric Gachon, Dmitri Firsov
The rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identified S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) that was derived from an individual previously infected with wildtype SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrated broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1/XBB). Furthermore, S728-1157 protected hamsters against in vivo challenges with wildtype, Delta, and BA.1 viruses. Structural analysis showed that this antibody targets a class 1/RBS-A epitope in the receptor binding domain (RBD) via multiple hydrophobic and polar interactions with its heavy chain complementarity determining region region 3 (CDR-H3), in addition to common motifs in CDR-H1/CDR-H2 of class 1/RBS-A antibodies. Importantly, this epitope was more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.
Siriruk Changrob, Peter J. Halfmann, Hejun Liu, Jonathan L. Torres, Joshua J.C. McGrath, Gabriel Ozorowski, Lei Li, G. Dewey Wilbanks, Makoto Kuroda, Tadashi Maemura, Min Huang, Nai-Ying Zheng, Hannah L. Turner, Steven A. Erickson, Yanbin Fu, Atsuhiro Yasuhara, Gagandeep Singh, Brian Monahan, Jacob Mauldin, Komal Srivastava, Viviana Simon, Florian Krammer, D. Noah Sather, Andrew B Ward, Ian A. Wilson, Yoshihiro Kawaoka, Patrick C. Wilson
Genetic defects of GNAS, the imprinted gene encoding the stimulatory G protein α-subunit, are responsible for multiple diseases. Abnormal GNAS imprinting causes pseudohypoparathyroidism type 1B (PHP1B), a prototype of mammalian end-organ hormone resistance. Hypomethylation at the maternally methylated GNAS A/B region is the only shared defect in PHP1B patients. In autosomal dominant (AD) PHP1B kindreds, A/B hypomethylation is associated with maternal microdeletions at either the GNAS NESP55 differentially methylated region or the STX16 gene located ~170 kb upstream. Functional evidence is meager regarding the causality of these microdeletions. Moreover, the mechanisms linking A/B methylation and these putative imprinting control regions (ICRs), NESP-ICR and STX16-ICR, remain unknown. Here, we generated a human embryonic stem cell model of AD-PHP1B by introducing ICR deletions using CRISPR/Cas9. Using this model, we showed that NESP-ICR is required for methylation and transcriptional silencing of A/B on the maternal allele. We also found that SXT16-ICR is a long-range enhancer of NESP55 transcription, which originates from maternal NESP-ICR. Furthermore, we demonstrated that STX16-ICR is an embryonic stage-specific enhancer enabled by the direct binding of pluripotency factors. Our findings uncover an essential GNAS imprinting control mechanism and advance the molecular understanding of the PHP1B pathogenesis.
Yorihiro Iwasaki, Cagri Aksu, Monica Reyes, Birol Ay, Qing He, Murat Bastepe
Multiple Sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specifically causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin (ETX)-producing strains of C. perfringens within their gut microbiomes compared to healthy controls (HC). MS patient-derived isolates produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.
Yinghua Ma, David Sannino, Jennifer R. Linden, Sylvia Haigh, Baohua Zhao, John B. Grigg, Paul Zumbo, Friederike Dündar, Daniel J. Butler, Caterina P. Profaci, Kiel M. Telesford, Paige N. Winokur, Kareem R. Rumah, Susan A. Gauthier, Vincent A. Fischetti, Bruce A. McClane, Francisco A. Uzal, Lily Zexter, Michael Mazzucco, Richard Rudick, David Danko, Evan Balmuth, Nancy Nealon, Jai Perumal, Ulrike W. Kaunzner, Ilana L. Brito, Zhengming Chen, Jenny Z. Xiang, Doron Betel, Richard Daneman, Gregory F. Sonnenberg, Christopher E. Mason, Timothy Vartanian
CD8+ exhausted T-cells (Tex) are heterogeneous. PD-1 inhibitors reinvigorate progenitor Tex, which subsequently differentiate into irresponsive terminal Tex. Maintaining durable proliferative capacity of progenitor Tex is important but remains unclear. Here, we showed that low-dose DNA demethylating agent decitabine-pretreated CD8+ progenitor Tex had enhanced proliferation and effector function against tumors after anti-PD-1 treatment in vitro. Decitabine-plus-anti-PD-1 treatment promoted the activation and expansion of tumor-infiltrated CD8+ progenitor Tex and efficiently suppressed tumor growth in multiple tumor models. Transcriptional and epigenetic profiling of tumor-infiltrated T cells demonstrated that decitabine-plus-anti-PD-1 combination markedly elevated the clonally expansion and cytolytic activity of progenitor Tex compared with anti-PD-1 monotherapy and restrained CD8+ T-cell terminal differentiation. Strikingly, decitabine-plus-anti-PD-1 sustained the expression and activity of AP-1 transcription factor JunD, which was reduced following PD-1 blockade therapy. Downregulation of JunD repressed T cell proliferation and activating JNK/AP-1 signaling in CD8+ T-cells enhanced the antitumor capacity of PD-1 inhibitors. Together, epigenetic agent remodels CD8+ progenitor Tex and improves responsiveness to anti-PD-1 therapy.
Xiang Li, Yaru Li, Liang Dong, Yixin Chang, Xingying Zhang, Chunmeng Wang, Meixia Chen, Xiaochen Bo, Hebing Chen, Weidong Han, Jing Nie
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