Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

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Abstract

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

Authors

Lindsey N. Kent, Sooin Bae, Shih-Yin Tsai, Xing Tang, Arunima Srivastava, Christopher Koivisto, Chelsea K. Martin, Elisa Ridolfi, Grace C. Miller, Sarah M. Zorko, Emilia Plevris, Yannis Hadjiyannis, Miguel Perez, Eric Nolan, Raleigh Kladney, Bart Westendorp, Alain de Bruin, Soledad Fernandez, Thomas J. Rosol, Kamal S. Pohar, James M. Pipas, Gustavo Leone

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis

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Abstract

Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction–associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.

Authors

Chuan-Jin Wu, Xu Feng, Michael Lu, Sohshi Morimura, Mark C. Udey

Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

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Abstract

Aminoglycosides (AGs) are broad-spectrum antibiotics that are associated with kidney damage, balance disorders, and permanent hearing loss. This damage occurs primarily by killing of proximal tubule kidney cells and mechanosensory hair cells, though the mechanisms underlying cell death are not clear. Imaging molecules of interest in living cells can elucidate how molecules enter cells, traverse intracellular compartments, and interact with sites of activity. Here, we have imaged fluorescently labeled AGs in live zebrafish mechanosensory hair cells. We determined that AGs enter hair cells via both nonendocytic and endocytic pathways. Both routes deliver AGs from the extracellular space to lysosomes, and structural differences between AGs alter the efficiency of this delivery. AGs with slower delivery to lysosomes were immediately toxic to hair cells, and impeding lysosome delivery increased AG-induced death. Therefore, pro-death cascades induced at early time points of AG exposure do not appear to derive from the lysosome. Our findings help clarify how AGs induce hair cell death and reveal properties that predict toxicity. Establishing signatures for AG toxicity may enable more efficient evaluation of AG treatment paradigms and structural modifications to reduce hair cell damage. Further, this work demonstrates how following fluorescently labeled drugs at high resolution in living cells can reveal important details about how drugs of interest behave.

Authors

Dale W. Hailey, Robert Esterberg, Tor H. Linbo, Edwin W. Rubel, David W. Raible

Dual modulation of MCL-1 and mTOR determines the response to sunitinib

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Abstract

Most patients who initially respond to treatment with the multi–tyrosine kinase inhibitor sunitinib eventually relapse. Therefore, developing a deeper understanding of the contribution of sunitinib’s numerous targets to the clinical response or to resistance is crucial. Here, we have shown that cancer cells respond to clinically relevant doses of sunitinib by enhancing the stability of the antiapoptotic protein MCL-1 and inducing mTORC1 signaling, thus evoking little cytotoxicity. Inhibition of MCL-1 or mTORC1 signaling sensitized cells to clinically relevant doses of sunitinib in vitro and was synergistic with sunitinib in impairing tumor growth in vivo, indicating that these responses are triggered as prosurvival mechanisms that enable cells to tolerate the cytotoxic effects of sunitinib. Furthermore, higher doses of sunitinib were cytotoxic, triggered a decline in MCL-1 levels, and inhibited mTORC1 signaling. Mechanistically, we determined that sunitinib modulates MCL-1 stability by affecting its proteasomal degradation. Dual modulation of MCL-1 stability at different dose ranges of sunitinib was due to differential effects on ERK and GSK3β activity, and the latter also accounted for dual modulation of mTORC1 activity. Finally, comparison of patient samples prior to and following sunitinib treatment suggested that increases in MCL-1 levels and mTORC1 activity correlate with resistance to sunitinib in patients.

Authors

Mohamed Elgendy, Amal Kamal Abdel-Aziz, Salvatore Lorenzo Renne, Viviana Bornaghi, Giuseppe Procopio, Maurizio Colecchia, Ravindran Kanesvaran, Chee Keong Toh, Daniela Bossi, Isabella Pallavicini, Jose Luis Perez-Gracia, Maria Dolores Lozano, Valeria Giandomenico, Ciro Mercurio, Luisa Lanfrancone, Nicola Fazio, Franco Nole, Bin Tean Teh, Giuseppe Renne, Saverio Minucci

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy

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Abstract

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217–mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217–mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.

Authors

Bernard Thienpont, Jan Magnus Aronsen, Emma Louise Robinson, Hanneke Okkenhaug, Elena Loche, Arianna Ferrini, Patrick Brien, Kanar Alkass, Antonio Tomasso, Asmita Agrawal, Olaf Bergmann, Ivar Sjaastad, Wolf Reik, Hywel Llewelyn Roderick

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

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Abstract

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3–dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.

Authors

Xiaochao Tan, Priyam Banerjee, Hou-Fu Guo, Stephen Ireland, Daniela Pankova, Young-ho Ahn, Irodotos Michail Nikolaidis, Xin Liu, Yanbin Zhao, Yongming Xue, Alan R. Burns, Jonathon Roybal, Don L. Gibbons, Tomasz Zal, Chad J. Creighton, Daniel Ungar, Yanzhuang Wang, Jonathan M. Kurie

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2

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Abstract

Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.

Authors

Yoshinori Matsumoto, Jose La Rose, Oliver A. Kent, Melany J. Wagner, Masahiro Narimatsu, Aaron D. Levy, Mitchell H. Omar, Jiefei Tong, Jonathan R. Krieger, Emily Riggs, Yaryna Storozhuk, Julia Pasquale, Manuela Ventura, Behzad Yeganeh, Martin Post, Michael F. Moran, Marc D. Grynpas, Jeffrey L. Wrana, Giulio Superti-Furga, Anthony J. Koleske, Ann Marie Pendergast, Robert Rottapel

Store-operated Ca²⁺ entry regulates Ca²⁺-activated chloride channels and eccrine sweat gland function

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Abstract

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca²⁺ release–activated Ca²⁺ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca²⁺ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel–deficient patients and mice with ectodermal tissue–specific deletion of Orai1 (Orai1^K14Cre) or Stim1 and Stim2 (Stim1/2^K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1^K14Cre and Stim1/2^K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1^K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca²⁺-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca²⁺ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

Authors

Axel R. Concepcion, Martin Vaeth, Larry E. Wagner II, Miriam Eckstein, Lee Hecht, Jun Yang, David Crottes, Maximilian Seidl, Hyosup P. Shin, Carl Weidinger, Scott Cameron, Stuart E. Turvey, Thomas Issekutz, Isabelle Meyts, Rodrigo S. Lacruz, Mario Cuk, David I. Yule, Stefan Feske

Cancer-associated fibroblast-derived annexin A6⁺ extracellular vesicles support pancreatic cancer aggressiveness

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Abstract

The intratumoral microenvironment, or stroma, is of major importance in the pathobiology of pancreatic ductal adenocarcinoma (PDA), and specific conditions in the stroma may promote increased cancer aggressiveness. We hypothesized that this heterogeneous and evolving compartment drastically influences tumor cell abilities, which in turn influences PDA aggressiveness through crosstalk that is mediated by extracellular vesicles (EVs). Here, we have analyzed the PDA proteomic stromal signature and identified a contribution of the annexin A6/LDL receptor-related protein 1/thrombospondin 1 (ANXA6/LRP1/TSP1) complex in tumor cell crosstalk. Formation of the ANXA6/LRP1/TSP1 complex was restricted to cancer-associated fibroblasts (CAFs) and required physiopathologic culture conditions that improved tumor cell survival and migration. Increased PDA aggressiveness was dependent on tumor cell–mediated uptake of CAF-derived ANXA6⁺ EVs carrying the ANXA6/LRP1/TSP1 complex. Depletion of ANXA6 in CAFs impaired complex formation and subsequently impaired PDA and metastasis occurrence, while injection of CAF-derived ANXA6⁺ EVs enhanced tumorigenesis. We found that the presence of ANXA6⁺ EVs in serum was restricted to PDA patients and represents a potential biomarker for PDA grade. These findings suggest that CAF–tumor cell crosstalk supported by ANXA6⁺ EVs is predictive of PDA aggressiveness, highlighting a therapeutic target and potential biomarker for PDA.

Authors

Julie Leca, Sébastien Martinez, Sophie Lac, Jérémy Nigri, Véronique Secq, Marion Rubis, Christian Bressy, Arnauld Sergé, Marie-Noelle Lavaut, Nelson Dusetti, Céline Loncle, Julie Roques, Daniel Pietrasz, Corinne Bousquet, Stéphane Garcia, Samuel Granjeaud, Mehdi Ouaissi, Jean Baptiste Bachet, Christine Brun, Juan L. Iovanna, Pascale Zimmermann, Sophie Vasseur, Richard Tomasini

Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways

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Abstract

Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.

Authors

Seong M. Kim, Saurabh G. Roy, Bin Chen, Tiffany M. Nguyen, Ryan J. McMonigle, Alison N. McCracken, Yanling Zhang, Satoshi Kofuji, Jue Hou, Elizabeth Selwan, Brendan T. Finicle, Tricia T. Nguyen, Archna Ravi, Manuel U. Ramirez, Tim Wiher, Garret G. Guenther, Mari Kono, Atsuo T. Sasaki, Lois S. Weisman, Eric O. Potma, Bruce J. Tromberg, Robert A. Edwards, Stephen Hanessian, Aimee L. Edinger