Notch signaling can have either an oncogenic or tumor suppressive function in cancer depending on the cancer type and cellular context. While Notch can be oncogenic in early prostate cancer, we identified significant downregulation of the Notch pathway during prostate cancer progression from adenocarcinoma to neuroendocrine prostate cancer where it functions as a tumor suppressor. Activation of Notch in neuroendocrine and Rb1/Trp53-deficient prostate cancer models led to phenotypic conversion towards a more indolent non-neuroendocrine state with glandular features and expression of luminal lineage markers. This was accompanied by up-regulation of MHC and type I interferon and immune cell infiltration. Overall, these data support Notch signaling as a suppressor of neuroendocrine differentiation in advanced prostate cancer and provides insights into how Notch signaling influences lineage plasticity and the tumor microenvironment.
Sheng-Yu Ku, Yanqing Wang, Maria Mica Garcia, Yasutaka Yamada, Kei Mizuno, Mark D. Long, Spencer Rosario, Meenalakshmi Chinnam, Majd Al Assaad, Loredana Puca, Min Jin Kim, Martin K. Bakht, Varadha Balaji Venkadakrishnan, Brian D. Robinson, Andrés M. Acosta, Kristine M. Wadosky, Juan Miguel Mosquera, David W. Goodrich, Himisha Beltran
Intestinal fibrosis, a severe complication of Crohn’s disease (CD), is characterized by excessive extracellular matrix (ECM) deposition and induces intestinal strictures, but there are no effective anti-fibrosis drugs available for clinical application. We performed single-cell RNA sequencing (scRNA-seq) of fibrotic and non-fibrotic ileal tissues from CD patients with intestinal obstruction. Analysis revealed mesenchymal stromal cells (MSCs) as the major producers of ECM and the increased infiltration of its subset FAP+ fibroblasts in fibrotic sites, which was confirmed by immunofluorescence and flow cytometry. Single cell transcriptomic profiling of chronic Dextran Sulfate Sodium Salt (DSS) murine colitis model revealed Cd81+Pi16– fibroblasts exhibited transcriptomic and functional similarities to human FAP+ fibroblasts. Consistently, FAP+ fibroblasts were identified as the key subtype with the highest level of ECM production in fibrotic intestines. Furthermore, specific knockout or pharmacological inhibition of TWIST1, which was highly expressed by FAP+ fibroblasts, could significantly ameliorate fibrosis in mice. In addition, TWIST1 expression was induced by CXCL9+ macrophages enriched in fibrotic tissues via IL-1β and TGF-β signal. These findings suggest the inhibition of TWIST1 as a promising strategy for CD fibrosis treatment.
Yao Zhang, Jiaxin Wang, Hongxiang Sun, Zhenzhen Xun, Zirui He, Yizhou Zhao, Jingjing Qi, Sishen Sun, Qidi Yang, Yubei Gu, Ling Zhang, Chunhua Zhou, Youqiong Ye, Ningbo Wu, Duowu Zou, Bing Su
Neovascular age-related macular degeneration (nAMD) remains a major cause of visual impairment and puts considerable burden on patients and health care systems. L-DOPA-treated Parkinson Disease (PD) patients have been shown to be partially protected from nAMD, but the mechanism remains unknown. Using murine models, combining 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD and laser-induced nAMD, standard PD treatment of L-DOPA/DOPA-decarboxylase inhibitor, or specific dopamine receptor inhibitors, we here demonstrate that L-DOPA treatment-induced increase of dopamine mediated dopamine receptor D2 (DRD2) signaling inhibits choroidal neovascularization independently of MPTP-associated nigrostriatal pathway lesion. Analyzing a retrospective cohort of more than two hundred thousand nAMD patients receiving anti-VEGF treatment from the French nationwide insurance database, we show that DRD2-agonist treated (PD) patients have a significantly delayed age of onset for nAMD (81.4 (±7.0) vs 79.4 (±8.1) years old, respectively, p<0.0001) and reduced need for anti-VEGF therapies (-0.6 injections per 100 mg/day daily dose of DRD2 agonists the second year of treatment), similar to the L-DOPA treatment. While providing a mechanistic explanation for an intriguing epidemiological observation, our findings suggest that systemic DRD2 agonists might constitute an adjuvant therapy to delay and reduce the need for anti-VEGF therapy in nAMD patients.
Thibaud Mathis, Florian Baudin, Anne-Sophie Mariet, Sébastien Augustin, Marion Bricout, Lauriane Przegralek, Christophe Roubeix, Éric Benzenine, Guillaume Blot, Caroline Nous, Laurent Kodjikian, Martine Mauget-Faÿsse, José-Alain Sahel, Robin Plevin, Christina Zeitz, Cécile Delarasse, Xavier Guillonneau, Catherine Creuzot-Garcher, Catherine Quantin, Stéphane Hunot, Florian Sennlaub
Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGFβ1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Biopsies from untreated patients showed higher fibroblast TGFβ1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGFβ1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzle-like receptor protein 2 (sFRP2), an unrecognized TGFβ1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision cut lung slices (PCLS) from non-diseased donors, we found TGFβ1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature Krt5+ basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGFβ1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify TGFβ1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.
Max L. Cohen, Alexis N. Brumwell, Tsung Che Ho, Kiana Garakani, Genevieve Montas, Darren Leong, Vivianne W. Ding, Jeffrey A. Golden, Binh N. Trinh, David M. Jablons, Michael A. Matthay, Kirk D. Jones, Paul J. Wolters, Ying Wei, Harold A. Chapman, Claude Jourdan Le Saux
Reelin (RELN) is a secreted glycoprotein essential for cerebral cortex development. In humans, recessive RELN variants cause cortical and cerebellar malformations, while heterozygous variants were associated to epilepsy, autism and mild cortical abnormalities. However, their functional effects remain unknown. We identified inherited and de novo RELN missense variants in heterozygous patients with neuronal migration disorders (NMDs) as diverse as pachygyria and polymicrogyria. We investigated in culture and in the developing mouse cerebral cortex how different variants impacted RELN function. Polymicrogyria-associated variants behaved as gain-of-function showing an enhanced ability to induce neuronal aggregation, while those linked to pachygyria as loss-of-function leading to defective neuronal aggregation/migration. The pachygyria-associated de novo heterozygous RELN variants acted as dominant-negative by preventing wild-type RELN secretion in culture, animal models and patients, thereby causing dominant NMDs. We demonstrated how mutant RELN proteins in vitro and in vivo predict cortical malformation phenotypes, providing valuable insights into the pathogenesis of such disorders.
Martina Riva, Sofia Ferreira, Kotaro Hayashi, Yoann Saillour, Vera P. Medvedeva, Takao Honda, Kanehiro Hayashi, Claire Altersitz, Shahad Albadri, Marion Rosello, Julie Dang, Malo Serafini, Frédéric Causeret, Olivia J. Henry, Charles-Joris Roux, Céline Bellesme, Elena Freri, Dragana Josifova, Elena Parrini, Renzo Guerrini, Filippo Del Bene, Kazunori Nakajima, Nadia Bahi-Buisson, Alessandra Pierani
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.
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
Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in immunocompromised individuals such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in immunosuppressed individuals have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in immunocompromised patients and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with non-controlled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hypo-responsiveness in individuals who fail to control chronic viral infection.
Nils Mülling, Felix M. Behr, Graham A. Heieis, Kristina Boss, Suzanne van Duikeren, Floortje J. van Haften, Iris N. Pardieck, Esmé T.I. van der Gracht, Ward Vleeshouwers, Tetje C. van der Sluis, J. Fréderique de Graaf, Dominique M.B. Veerkamp, Kees L.M.C. Franken, Xin Lei, Lukas van de Sand, Sjoerd H. van der Burg, Marij J.P. Welters, Sebastiaan Heidt, Wesley Huisman, Simon P. Jochems, Martin Giera, Oliver Witzke, Aiko P.J. de Vries, Andreas Kribben, Bart Everts, Benjamin Wilde, Ramon Arens
Cystic fibrosis (CF) results from mutations in the CFTR anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The BKCa potassium channel is also critical for maintaining lung ASL volume. Here, we show the CFTR corrector, VX-445 (Elexacaftor), a component of Trikafta, induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121 – a corrector under clinical evaluation. Whole-cell patch-clamp recordings confirmed potentiated channel activity from CFTR correctors on the BKCa α-subunit, and excised patch-clamp recordings demonstrated a significant increase in open probability. In mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced action potential firing frequency in primary hippocampal and cortical neurons. VX-445 effects were observed at low micomolar concentrations (1-10 µM) – within the range reported in plasma and tissues from CF patients. We raise the possibilities that CFTR correctors gain additional clinical benefit by activation of BKCa in the lung, yet may lead to adverse events through BKCa activation, elsewhere.
Aaron Kolski-Andreaco, Stefanie Taiclet, Michael M. Myerburg, john sembrat, Robert J. Bridges, Adam C. Straub, Zachary P. Wills, Michael B. Butterworth, Daniel C. Devor
The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.
Segewkal Hawaze Heruye, Jered Myslinski, Chao Zeng, Amy Zollman, Shinichi Makino, Azuma Nanamatsu, Quoseena Mir, Sarath Chandra Janga, Emma H. Doud, Michael T. Eadon, Bernhard Maier, Michiaki Hamada, Tuan M. Tran, Pierre C. Dagher, Takashi Hato
Aortic aneurysm is a life-threatening disease with limited interventions, closely related to vascular smooth muscle cells (VSMCs) phenotypic switching. SLC44A2, a member of solute carrier series 44 (SLC44) family, remains under-characterized in the context of cardiovascular diseases. Venn diagram analysis based on microarray and single-cell RNA sequencing identified SLC44A2 as a major regulator of VSMCs phenotypic switching in aortic aneurysm. Screening for Slc44a2 amongst aortic cell lineages demonstrated its predominant location in VSMCs. Elevated levels of SLC44A2 were evidenced in the aorta of both abdominal aortic aneurysm patients and angiotensin II (Ang II)-infused Apoe–/– mice. In vitro, SLC44A2 silencing promoted VSMCs towards a synthetic phenotype, while SLC44A2 overexpression attenuated VSMCs phenotypic switching. VSMCs-specific SLC44A2 knockout mice were more susceptible to aortic aneurysm under Ang II infusion, while SLC44A2 overexpression showed protective effects. Mechanistically, SLC44A2 interaction with NRP1 and ITGB3 activates TGF-β/SMAD signaling, thereby promoting contractile genes expression. Elevated SLC44A2 in aortic aneurysm is associated with upregulated runt-related transcription factor 1 (RUNX1). Furthermore, low dose of lenalidomide (LEN) suppressed aortic aneurysm progression by enhancing SLC44A2 expression. These findings reveal SLC44A2/NRP1/ITGB3 complex is a major regulator of VSMCs phenotypic switching and provide potential therapeutic approach (LEN) for aortic aneurysm treatment.
Tianyu Song, Shuang Zhao, Shanshan Luo, Chuansheng Chen, Xingeng Liu, Xiaoqi Wu, Zhongxu Sun, Jiawei Cao, Ziyu Wang, Yineng Wang, Bo Yu, Zhiren Zhang, Xiaolong Du, Xiaoqiang Li, Zhijian Han, Hongshan Chen, Feng Chen, Liansheng Wang, Hong Wang, Kangyun Sun, Yi Han, Liping Xie, Yong Ji
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