Cell biology
Abstract
Estrogen receptor (ER)-negative breast cancer is thought to be more malignant and devastating than ER-positive breast cancer and exhibit elevated NF-κB activity. How abnormally high NF-κB activity is maintained in ER-negative breast cancer is poorly understood. The importance of linear ubiquitination, which is generated by the linear ubiquitin chain assembly complex (LUBAC), is increasingly appreciated in NF-κB signaling, which regulates cell activation and death. Here, we showed that epsin proteins, a family of ubiquitin-binding endocytic adaptors, interacted with LUBAC via its Ubiquitin-Interacting Motif (UIM) and bound LUBAC’s bona fide substrate NEMO via its N-terminal homolog (ENTH) domain. Furthermore, epsins promoted NF-κB essential modulator (NEMO) linear ubiquitination and served as scaffolds for recruiting other components of the IκB kinase (IKK) complex; thereby, resulting in the heightened IKK activation and sustained NF-κB signaling essential for the development of ER-negative breast cancer. Heightened epsin levels in ER-negative human breast cancer are associated with poor, relapse-free survival. We showed that transgenic and pharmacological approaches eliminating epsins potently impeded breast cancer development in both spontaneous and patient-derived xenograft breast cancer mouse models. Our findings established the pivotal role epsins played in promoting breast cancer. Thus, targeting epsins may represent a strategy to restrain NF-κB signaling, and provide an important perspective into ER-negative breast cancer treatment.
Authors
Kai Song, Xiaofeng Cai, Yunzhou Dong, Hao Wu, Yong Wei, Uma Shankavaram, Kui Cui, Yang Lee, Bo Zhu, Sudarshan Bhattacharjee, Beibei Wang, Kun Zhang, Aiyun Wen, Scott Wong, Lili Yu, Lijun Xia, Alana L Welm, Diane R. Bielenberg, Kevin Camphausen, Yibin Kang, Hong Chen
Abstract
Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kD (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45/myosin IIA-containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a-dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8+ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55/G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.
Authors
Xiaochao Tan, Lei Shi, Priyam Banerjee, Xin Liu, Hou-Fu Guo, Jiang Yu, Neus Bota-Rabassedas, B. Leticia Rodriguez, Don L. Gibbons, William K. Russell, Chad J. Creighton, Jonathan M. Kurie
Abstract
Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-κB signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/Beclin 1/LC3–independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated ATG9A+ vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9A-dependent, but ATG16L/Beclin 1/LC3–independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer.
Authors
Motao Zhu, Guangtong Deng, Peng Tan, Changsheng Xing, Cuiping Guan, Chongming Jiang, Yinlong Zhang, Bo Ning, Chaoran Li, Bingnan Yin, Kaifu Chen, Yuliang Zhao, Helen Y. Wang, Beth Levine, Guangjun Nie, Rong-Fu Wang
Abstract
Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell–derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.
Authors
Klitos Konstantinidis, Vassilios J. Bezzerides, Lo Lai, Holly M. Isbell, An-Chi Wei, Yuejin Wu, Meera C. Viswanathan, Ian D. Blum, Jonathan M. Granger, Danielle Heims-Waldron, Donghui Zhang, Elizabeth D. Luczak, Kevin R. Murphy, Fujian Lu, Daniel H. Gratz, Bruno Manta, Qiang Wang, Qinchuan Wang, Alex L. Kolodkin, Vadim N. Gladyshev, Thomas J. Hund, William T. Pu, Mark N. Wu, Anthony Cammarato, Mario A. Bianchet, Madeline A. Shea, Rodney L. Levine, Mark E. Anderson
Abstract
Store-operated calcium entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming puncta structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet Bin2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. These defects translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. These results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.
Authors
Julia Volz, Charly Kusch, Sarah Beck, Michael Popp, Timo Vögtle, Mara Meub, Inga Scheller, Hannah S. Heil, Julia Preu, Michael K. Schuhmann, Katherina Hemmen, Thomas Premsler, Albert Sickmann, Katrin G. Heinze, David Stegner, Guido Stoll, Attila Braun, Markus Sauer, Bernhard Nieswandt
Abstract
While cancer is commonly perceived as a disease of dedifferentiation, the hallmark of early stage prostate cancer is paradoxically the loss of more plastic basal cells and the abnormal proliferation of more differentiated secretory luminal cells. However, the mechanism of prostate cancer pro-luminal differentiation is largely unknown. Through integrating analysis of the transcription factors (TFs) from 806 human prostate cancers, we have identified that ERG highly correlated with prostate cancer luminal subtyping. ERG overexpression in luminal epithelial cells inhibits its normal plasticity to transdifferentiate into basal lineage and ERG supersedes PTEN-loss which favors basal differentiation. ERG knock-out disrupted prostate cell luminal differentiation, whereas AR knock-out had no such effects. Trp63 is a known master regulator of prostate basal lineage. Through analysis of 3D chromatin architecture, we found that ERG binds and inhibits the enhancer activity and chromatin looping of a Trp63 distal enhancer, thereby silencing its gene expression. Specific deletion of the distal ERG binding site resulted in the loss of ERG-mediated inhibition of basal differentiation. Thus, ERG orchestrates chromatin interactions and regulates prostate cell lineage toward pro-luminal program, as its fundamental role on lineage differentiation in prostate cancer initiation.
Authors
Fei Li, Qiuyue Yuan, Wei Di, Xinyi Xia, Zhuang Liu, Ninghui Mao, Lin Li, Chunfeng Li, Juan He, Yunguang Li, Wangxin Guo, Xiaoyu Zhang, Yiqin Zhu, Rebiguli Aji, Shangqian Wang, Xinyuan Tong, Hongbin Ji, Ping Chi, Brett Carver, Yong Wang, Yu Chen, Dong Gao
Abstract
Utilizing the Nephrotic Syndrome Study Network Consortium and other publicly available transcriptomic datasets, we identified Retinoic acid receptor responder protein 1 (RARRES1) as a gene whose expression positively correlated with renal function decline in human glomerular disease. The glomerular expression of RARRES1, which is largely restricted to podocytes, increased in focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). Tumor necrosis factor-α (TNF-α) was a potent inducer of RARRES1 expression in cultured podocytes, and transcriptomic analysis showed the enrichment of cell death pathway genes with RARRES1 overexpression. The overexpression of RARRES1 indeed induced podocyte apoptosis in vitro. Notably, this effect was dependent on its cleavage in the extracellular domain, as the mutation of its cleavage site abolished the apoptotic effect. Mechanistically, the soluble RARRES1 is endocytosed and interacts with and inhibits RIO kinase 1 (RIOK1), resulting in p53 activation and podocyte apoptosis. In mice, podocyte-specific overexpression of RARRES1 resulted in marked glomerular injury and albuminuria, while the overexpression of RARRES1 cleavage mutant had no effect. Conversely, podocyte-specific knockdown of Rarres1 in mice ameliorated glomerular injury in the setting of Adriamycin-induced nephropathy. Together, our study demonstrates an important role and the mechanism of RARRES1 in podocyte injury in glomerular disease.
Authors
Anqun Chen, Ye Feng, Han Lai, Wenjun Ju, Zhengzhe Li, Yu Li, Andrew Wang, Quan Hong, Fang Zhong, Chengguo Wei, Jia Fu, Tian-Jun Guan, Bi-Cheng Liu, Matthias Kretzler, Kyung Lee, John Cijiang He
Abstract
TGFβ is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identify the nuclear lncRNA H19X as a master regulator of TGFβ-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGFβ, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X is an obligatory factor for the TGFβ-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of the TGFβ-induced ECM remodeling and fibrosis.
Authors
Elena Pachera, Shervin Assassi, Gloria A. Salazar, Mara Stellato, Florian Renoux, Adam Wunderlin, Przemyslaw Blyszczuk, Robert Lafyatis, Fina Kurreeman, Jeska de Vries-Bouwstra, Tobias Messemaker, Carol A. Feghali-Bostwick, Gerhard Rogler, Wouter T. van Haaften, Gerard Dijkstra, Fiona Oakley, Maurizio Calcagni, Janine Schniering, Britta Maurer, Jörg H.W. Distler, Gabriela Kania, Mojca Frank-Bertoncelj, Oliver Distler
Abstract
Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.
Authors
Lakshya Bajaj, Jaiprakash Sharma, Alberto di Ronza, Pengcheng Zhang, Aiden Eblimit, Rituraj Pal, Dany Roman, John R. Collette, Clarissa Booth, Kevin T. Chang, Richard N. Sifers, Sung Y. Jung, Jill M. Weimer, Rui Chen, Randy W. Schekman, Marco Sardiello
Abstract
Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.
Authors
Catherine R. Cochrane, Vijesh Vaghjiani, Anette Szczepny, W. Samantha N. Jayasekara, Alvaro Gonzalez-Rajal, Kazu Kikuchi, Geoffrey W. McCaughan, Andrew Burgess, Daniel J. Gough, D. Neil Watkins, Jason E. Cain
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Copyright © 2020 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)