The loss of contact inhibition is a key step during carcinogenesis. The Hippo–Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density–dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane–stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity–regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20–like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling.
Dongxue Su, Yuxi Li, Weiji Zhang, Huan Gao, Yao Cheng, Yongqiang Hou, Junhong Li, Yi Ye, Zhangjian Lai, Zhe Li, Haitao Huang, Jiaxin Li, Jinhuan Li, Mengyu Cheng, Cheng Nian, Na Wu, Zhien Zhou, Yunzhi Xing, Yu Zhao, He Liu, Jiayu Tang, Qinghua Chen, Lixin Hong, Wengang Li, Zhihai Peng, Bin Zhao, Randy L. Johnson, Pingguo Liu, Wanjin Hong, Lanfen Chen, Dawang Zhou
An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target which when inhibited improved radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting resulted in increased NF-kB-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 could improve radiotherapy even in tumours displaying immunosuppressive features.
Callum Beach, David MacLean, Dominika Majorova, Stavros Melemenidis, Dhanya K. Nambiar, Ryan K. Kim, Gabriel N. Valbuena, Silvia Guglietta, Carsten Krieg, Mahnaz Darvish-Damavandi, Tatsuya Suwa, Alistair Easton, Lily V.S. Hillson, Ashley K. McCulloch, Ross K. McMahon, Kathryn Pennel, Joanne Edwards, Sean M. O’Cathail, Campbell S. Roxburgh, Enric Domingo, Eui Jung Moon, Dadi Jiang, Yanyan Jiang, Qingyang Zhang, Albert C. Koong, Trent M. Woodruff, Edward E. Graves, Tim Maughan, Simon J.A. Buczacki, Manuel Stucki, Quynh Thu Le, Simon J. Leedham, Amato J. Giaccia, Monica M. Olcina
Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying pro-tumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform to identify potential therapeutic targets to disrupt the contribution of pro-tumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC, and inform the development of therapeutics that target ECM stiffness in EAC.
Ricardo Cruz-Acuña, Secunda W. Kariuki, Kensuke Sugiura, Spyros Karaiskos, Eleanor M. Plaster, Claudia Loebel, Gizem Efe, Tatiana A. Karakasheva, Joel T. Gabre, Jianhua Hu, Jason A. Burdick, Anil K. Rustgi
Antibody-drug conjugates(ADCs) are promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer(mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, conferred sensitivity and were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive(ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 wild-type ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 non-expressors governed response. Importantly, wild-type TP53 predicted non-responsiveness (7/8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.
Supreet Agarwal, Lei Fang, Kerry McGowen, JuanJuan Yin, Joel Bowman, Anson T. Ku, Aian Neil Alilin, Eva Corey, Martine P. Roudier, Lawrence D. True, Ruth F. Dumpit, Ilsa Coleman, John K. Lee, Peter S. Nelson, Brian J. Capaldo, Aida Mariani, Clare E. Hoover, Ilya S. Senatorov, Michael Beshiri, Adam G. Sowalsky, Elaine M. Hurt, Kathleen Kelly
Unabated activation of NLRP3 inflammasome activation is linked with the pathogenesis of various inflammatory disorders. PLK1 has been widely studied for its role in mitosis. Here, employing both pharmacological and genetic approaches, we demonstrated that PLK1 promoted NLRP3 inflammasome activation at cell interphase. Using an unbiased Bio-ID screen for PLK1 interactome in macrophages, we showed an enhanced proximal association of NLRP3 with PLK1 upon NLRP3 inflammasome activation. We further confirmed the interaction between PLK1 and NLRP3, and identified the interacting domains. Mechanistically, we showed that PLK1 orchestrated microtubule organizing center (MTOC) structure and NLRP3 subcellular positioning upon inflammasome activation. Treatment with a selective PLK1 kinase inhibitor suppressed IL1B production in in-vivo inflammatory models, including lipopolysaccharide-induced endotoxemia and monosodium urate-induced peritonitis in mice. Our results uncover an unprecedented role of PLK1 in regulating NLRP3 inflammasome activation during interphase, and identify pharmacological inhibition of PLK1 as a potential therapeutic strategy for inflammatory diseases with excessive NLRP3 inflammasome activation.
Marta Baldrighi, Christian Doreth, Yang Li, Xiaohui Zhao, Emily F. Warner, Hannah Chenoweth, Kamal Kishore, Yagnesh Umrania, David-Paul Minde, Sarah Winkler, Xian Yu, Yuning Lu, Alice Knapton, James Harrison, Murray C.H. Clarke, Eicke Latz, Guillermo de Cárcer, Marcos Malumbres, Bernhard Ryffel, Clare E. Bryant, Jinping Liu, Kathryn S. Lilley, Ziad Mallat, Xuan Li
PCIF1 can mediate the methylation of N6,2′-O-dimethyladenosine (m6Am) in mRNA. Yet, the detailed interplay between PCIF1 and the potential cofactors and its pathological significance remains elusive. Here, we demonstrated that PCIF1-mediated cap mRNA m6Am modification promoted head and neck squamous cell carcinoma (HNSCC) progression both in vitro and in vivo. CTBP2 was identified as a cofactor of PCIF1 to catalyze m6Am deposition on mRNA. CLIP-seq data demonstrated CTBP2 bound to similar mRNAs as PCIF1. We then utilized m6Am-seq method to profile mRNA m6Am site at single-base resolution and found mRNA of TET2, a well-known tumor suppressor, was a major target substrate of PCIF1-CTBP2 complex. Mechanistically, knockout of CTBP2 reduced PCIF1 occupancy on TET2 mRNA and PCIF1-CTBP2 complex negatively regulated the translation of TET2 mRNA. Collectively, our study demonstrated the oncogenic function of the epitranscriptome regulator PCIF1-CTBP2 complex, highlighting the importance of the m6Am modification in tumor progression.
Kang Li, Jie Chen, Caihua Zhang, Maosheng Cheng, Shuang Chen, Wei Song, Chunlong Yang, Rongsong Ling, Zhi Chen, Xiaocheng Wang, Gan Xiong, Jieyi Ma, Yan Zhu, Quan Yuan, Qi Liu, Liang Peng, Qianming Chen, Demeng Chen
The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl– into the airway surface liquid through CFTR (cystic fibrosis transmembrane conductance regulator) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl– channels in their basolateral membrane. Disrupting barttin/Cl– channel function impaired liquid absorption, and overexpressing barttin/Cl– channels increased absorption. Together, apical CFTR and basolateral barttin/Cl– channels provide an electrically conductive pathway for Cl– flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.
Lei Lei, Soumba Traore, Guillermo S. Romano Ibarra, Philip H. Karp, Tayyab Rehman, David K. Meyerholz, Joseph Zabner, David A. Stoltz, Patrick L. Sinn, Michael J. Welsh, Paul B. McCray, Jr., Ian M. Thornell
Cystinosis is a lysosomal storage disease that is characterized by the accumulation of di-peptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7∆). We demonstrated that this mutant is retained in the endoplasmic reticulum (ER) and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the role of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7∆) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones, previously used for treating CFTR F508∆, and demonstrated that they could facilitate the folding and trafficking of cystinosin(7∆). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by ~70%. Conclusively, our study establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7∆).
Varsha Venkatarangan, Weichao Zhang, Xi Yang, Jess G. Thoene, Si H. Hahn, Ming Li
Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow–derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture–induced sepsis but not in CIRP–/– mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non–APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ–mediated hyper-NETosis.
Hui Jin, Monowar Aziz, Atsushi Murao, Molly Kobritz, Andrew J. Shih, Robert P. Adelson, Max Brenner, Ping Wang
RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS–mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.
Jian-Gang Ren, Bowen Xing, Kaosheng Lv, Rachel A. O’Keefe, Mengfang Wu, Ruoxing Wang, Kaylyn M. Bauer, Arevik Ghazaryan, George M. Burslem, Jing Zhang, Ryan M. O’Connell, Vinodh Pillai, Elizabeth O. Hexner, Mark R. Philips, Wei Tong
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