September3, 2013|vol. 110 | no. 36 | pp. 14505-14814 aad
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`

`

`Coverimage:Pictured is an aggregate of dolomite nanocrystals on the surface of a carboxylated
`polystyrene sphere. The mineral dolomite is abundantin the geologic record andcan be synthesized
`in the laboratory at high temperatures, but the mechanism of its formation at low temperatures
`remains unclear. Jennifer A. Roberts et al. demonstrate that carboxyl groups on the surfaces of
`polystyrene spheres can catalyze dolomite precipitation at 30°C from solutions that simulate
`seawater. The findings suggest that natural surfaces that possess a high density of carboxyl groups,
`such as microbial biomass, may be key to the formation of dolomite in low temperature sedimentary
`systems.See the article by Roberts et al. on pages 14540-14545. Image courtesy of David S. Moore.
`
`14610 Biological role of a polyglutamine tract
`
`From the Cover
`
`14540 Dolomite formation at low temperature
`
`14520 Neutralization of differentiation in gliomagenesis
`
`14528 Characterizing a bacteria-amoeba symbiosis
`
`14546 Modeling ancient atmospheric oxygen levels
`
`Charles Choi
` 14505
`
`In This Issue
`
`14507 Spinning silk from spiders
`
`> See Inaugural Article on page 14520
`
`
`14508 QnAs with Ronald DePinho
`Prashant Nair
`
`E3368 Lack of chronological support for stepwise prehuman
`extinctions of Australian megafauna
`Barry W. Brook, Corey J. A. Bradshaw, Alan Cooper,
`Christopher N. Johnson, Trevor H. Worthy, Michael Bird,
`Richard Gillespie, and Richard G. Roberts
`
`14510
`
`Changingthe fate of cancer, one splice at a time
`David L. Schonberg, Monica Venere, and Jeremy N. Rich
`> See companion article on page 14520
`14512 Small molecules mediate bacterial farming by
`social amoebae
`Robert A. Raguso
`> See companionarticle on page 14528
`14514 Acold, hard look at ancient oxygen
`Boswell A. Wing
`> See companion article on page 14546
`14516 A flexible polyglutamine hinge opens new doors for
`understanding huntingtin function
`Flaviano Giorgini
`> See companion article on page 14610
`
`E3369 Reply to Brooket al: No empirical evidence for human
`overkill of megafauna in Sahul
`Stephen Wroe, Judith H. Field, Michael Archer, Donald K.
`Grayson, Gilbert J. Price, Julien Louys, J. Tyler Faith,
`Gregory
`E. Webb, Iain Davidson, and Scott D. Moone
`‘
`oc
`E3370 Concerns overinterspecies transcriptional comparisons
`in mice and humansafter trauma
`Andrew R.Osterburg, Philip Hexley, Dorothy M. Supp,
`Chad T. Robinson, Greg Noel, Cora Ogle, Steven T. Boyce,
`Bruce J. Aronow, and George F. Babcock
`
`E3371 Reply to Osterburg et al.: To study humaninflammatory
`diseases in humans
`Ronald G. Tompkins, H. Shaw Warren, Michael N.
`
`Mindrinos, Wenzhong Xiao, and Ronald W. Davis
`
`|
`
`QE Free online through the PNASopenaccess option.
`
`14518 Significance Statements
`=> Brief statements written by the authors aboutthe significance
`of their papers.
`
`PNAS | September 3, 2013 | vol. 110 | no. 36 |
`
`iii-vii
`
`

`

`
`
`14592
`
`14520
`
`Neutralization of terminal differentiation
`in gliomagenesis
`Jian Hu, Allen L. Ho, Liang Yuan, Baoli Hu, Sujun Hua,
`Soyoon Sarah Hwang, Jianhua Zhang, Tianyi Hu, Hongwu
`Zheng, Boyi Gan, Gongxiong Wu, Yaoqi Alan Wang,
`Lynda Chin, and Ronald A. DePinho
`=> See QnAs on page 14508
`= See Commentary on page 14510
`
`
`
`£3445
`
`14528
`
`14604
`
`14658
`
`14534
`
`14540
`
`14546
`
`14551
`
`CHEMISTRY
`
`Stapled a—helical peptide drug development: A potent
`dualinhibitor of MDM2 and MDMxXfor p53-dependent
`cancer therapy
`Yong S. Chang, Bradford Graves, Vincent Guerlavais,
`Christian Tovar, Kathryn Packman, Kwong-Him To, Karen A.
`Olson, Kamala Kesavan, Pranoti Gangurde, Aditi Mukherjee,
`Theresa Baker, Krzysztof Darlak, Carl Elkin, Zoran Filipovic,
`Farooq Z. Qureshi, Hongliang Cai, Pamela Berry, Eric
`Feyfant, Xiangguo E. Shi, James Horstick, D. Allen Annis,
`Anthony M. Manning, Nader Fotouhi, Huw Nash,
`Lyubomir T. Vassilev, and Tomi K. Sawyer
`
`A bacterial symbiont is converted from an inedible
`producerof beneficial molecules into food by a single
`mutation in the gacA gene
`Pierre Stallforth, Debra A. Brock, Alexandra M. Cantley,
`Xiangjun Tian, David C. Queller, Joan E. Strassmann,
`and Jon Clardy
`=> See Commentary on page 14512
`Pulsed hydrogen-deuterium exchange mass
`spectrometry probes conformational changes in
`amyloid beta (Ap) peptide aggregation
`Ying Zhang, Don L. Rempel, Jun Zhang, Anuj K. Sharma,
`Liviu M. Mirica, and Michael L. Gross
`
`Axial interactions in the mixed-valent Cu, active site and
`role of the axial methionine in electron transfer
`Ming-Li Tsai, Ryan G, Hadt, Nicholas M. Marshall,
`Tiffany D. Wilson, Yi Lu, and Edward I. Solomon
`
`COMPUTER SCIENCES
`
`Efficient discovery of overlapping communities in
`massive networks
`Prem K. Gopalan and David M.Blei
`
`EARTH, ATMOSPHERIC, AND PLANETARY SCIENCES
`Surface chemistry allows for abiotic precipitation of
`dolomite at low temperature
`Jennifer A. Roberts, Paul A. Kenward, David A.
`Fowle, Robert H. Goldstein, Luis A. Gonzalez,
`and David S. Moore
`
`Dynamic modelconstraints on oxygen-17 depletion in
`atmospheric O2 after a snowball Earth
`Xiaobin Cao and Huiming Bao
`> See Commentary on page 14514
`
`Synchronousinterhemispheric Holocene climate trends
`in the tropical Andes
`Pratigya J. Polissar, Mark B. Abbott, Alexander P. Wolfe,
`Mathias Vuille, and Maximiliano Bezada
`
`iv
`
`| www.pnas.org
`
`ENGINEERING
`
`Design and characterization of synthetic fungal-bacterial
`consortia for direct production of isobutanol from
`cellulosic biomass
`Jeremy J. Minty, Marc E. Singer, Scott A. Scholz,
`Chang-Hoon Bae, Jung-Ho Ahn,Clifton E. Foster,
`James C. Liao, and Xiaoxia Nina Lin
`
`STATISTICS
`
`14557
`
`14563
`
`On robust regression with high-dimensional predictors
`Noureddine El Karoui, Derek Bean, Peter J. Bickel,
`Chinghway Lim, and Bin Yu
`
`Optimal M-estimation in high-dimensional regression
`Derek Bean, Peter J. Bickel, Noureddine El Karoui,
`and Bin Yu
`
`
`
`14569
`
`14574
`
`14580
`
`14586
`
`ANTHROPOLOGY
`
`Environmental productivity predicts migration,
`demographic, and linguistic patterns in
`prehistoric California
`Brian F. Codding and Terry L. Jones
`
`PSYCHOLOGICAL AND COGNITIVE SCIENCES
`Neural mechanisms of communicative innovation
`Arjen Stolk, Lennart Verhagen, Jan-Mathijs Schoffelen,
`Robert Oostenveld, Mark Blokpoel, Peter Hagoort,
`Iris van Rooij, and Ivan Toni
`
`Sight over soundin the judgment of music performance
`Chia-Jung Tsay
`
`Ontogenyof prosocial behavior across diverse societies
`Bailey R. House, Joan B. Silk, Joseph Henrich, H. Clark
`Barrett, Brooke A. Scelza, Adam H.Boyette, Barry S.
`Hewlett, Richard McElreath, and Stephen Laurence
`
`
`
`14569
`
`14586
`
`14592
`
`ANTHROPOLOGY
`
`Environmental productivity predicts migration,
`demographic, and linguistic patternsin
`prehistoric California
`Brian F. Codding and Terry L. Jones
`
`Ontogenyof prosocial behavior across diverse societies
`Bailey R. House, Joan B.Silk, Joseph Henrich, H. Clark
`Barrett, Brooke A. Scelza, Adam H. Boyette, Barry S.
`Hewlett, Richard McElreath, and Stephen Laurence
`
`APPLIED BIOLOGICAL SCIENCES
`
`;
`
`Design and characterization of synthetic fungal-bacterial
`consortia for direct production of isobutanol from
`cellulosic biomass
`Jeremy J. Minty, Mare E. Singer, Scott A. Scholz,
`Chang-Hoon Bae, Jung-Ho Ahn, Clifton E. Foster,
`James C. Liao, and Xiaoxia Nina Lin
`
`

`

`14598
`
`14604
`
`14610
`
`14616
`
`14622
`
`14628
`
`14634
`
`14640
`
`14646
`
`14652
`
`E3372
`
`E3381
`
`Engineered knottin peptide enables noninvasive optical
`imaging ofintracranial medulloblastoma
`Sarah J. Moore, Melanie G. Hayden Gephart, Jamie M.
`Bergen, YouRongS. Su, Helen Rayburn, Matthew P. Scott,
`and Jennifer R. Cochran
`
`BIOCHEMISTRY
`
`Pulsed hydrogen-deuterium exchange mass
`spectrometry probes conformational changes in
`amyloid beta (AB) peptide aggregation
`Ying Zhang, Don L. Rempel, Jun Zhang, Anuj K. Sharma,
`Liviu M. Mirica, and Michael L. Gross
`
`Polyglutamine domainflexibility mediates the proximity
`between flanking sequencesin huntingtin
`Nicholas Stephane Caron, Carly Robyn Desmond,
`Jianrun Xia, and Ray Truant
`=> See Commentary on page 14516
`
`Glucocorticoid receptor represses proinflammatory
`genes at distinct steps of the transcription cycle
`Rebecca Gupte, Ginger W. Muse, Yurii Chinenov,
`Karen Adelman, and Inez Rogatsky
`
`Analysis of Chlamydomonas thiamin metabolism in vivo
`reveals riboswitch plasticity
`Michael Moulin, Ginnie T. D. T. Nguyen, Mark A.Scaife,
`Alison G. Smith, and Teresa B. Fitzpatrick
`
`Human a-t-iduronidase uses its own N-glycan as
`a substrate-binding and catalytic module
`Nobuo Maita, Takahiro Tsukimura, Takako Taniguchi, Seiji
`Saito, Kazuki Ohno,Hisaaki Taniguchi, and Hitoshi Sakuraba
`
`Insights into Eph receptor tyrosine kinase activation
`from crystal structures of the EphA4 ectodomain and its
`complex with ephrin-A5
`Kai Xu, Dorothea Tzvetkova-Robev, Yan Xu, Yehuda
`Goldgur, Yee-Peng Chan, Juha P. Himanen,
`and Dimitar B. Nikolov
`
`Cotranslational folding of membrane proteins probed by
`arrest-peptide-mediated force measurements
`Florian Cymer and Gunnar von Heijne
`
`Glycosylated linkers in multimodular
`lignocellulose-degrading enzymes dynamically bind
`to cellulose
`Christina M. Payne, Michael G. Resch, Liqun Chen,
`Michael F. Crowley, Michael E. Himmel, Larry E. Taylor II,
`Mats Sandgren,Jerry Stahlberg, Ingeborg Stals, Zhongping
`Tan, and Gregg T. Beckham
`
`Implications for the evolution of eukaryotic
`amino-terminal acetyltransferase (NAT) enzymes
`from the structure of an archaeal ortholog
`Glen Liszezak and Ronen Marmorstein
`
`14658
`
`14664
`
`14670
`
`E3388
`
`14520
`
`14676
`
`Axial interactions in the mixed-valent Cu, active site and
`role of the axial methionine in electron transfer
`Ming-Li Tsai, Ryan G. Hadt, Nicholas M. Marshall,
`Tiffany D. Wilson, Yi Lu, and Edward I. Solomon
`
`Structure of the YajR transporter suggests a transport
`mechanism based on the conserved motif A
`Daohua Jiang, Yan Zhao, Xianping Wang, Junping Fan,
`Jie Heng, Xuehui Liu, Wei Feng, Xusheng Kang, Bo
`Huang, Jianfeng Liu, and Xuejun Cai Zhang
`
`The growth speed of microtubules with
`XMAP215-coated beads coupled to their ends
`is increased by tensile force
`Anastasiya Trushko, Erik Schaffer, and Jonathon Howard
`
`CELL BIOLOGY
`
`Transcriptional responseto stress in the dynamic
`chromatin environmentof cycling and mitotic cells
`Anniina Vihervaara, Christian Sergelius, Jenni Vasara,
`Malin A. H. Blom, Alexandra N. Elsing, Pia Roos-Mattjus,
`and Lea Sistonen
`
`Neutralization of terminal differentiation
`in gliomagenesis
`Jian Hu, Allen L. Ho, Liang Yuan, Baoli Hu, Sujun Hua,
`Soyoon Sarah Hwang, Jianhua Zhang, Tianyi Hu, Hongwu
`Zheng, Boyi Gan, Gongxiong Wu, Yaoqi Alan Wang,
`Lynda Chin, and Ronald A. DePinho
`=~» See QnAs on page 14508
`> See Commentary on page 14510
`
`NAF-1 and mitoNEETare central to human breast cancer
`proliferation by maintaining mitochondrial homeostasis
`and promoting tumor growth
`Yang-Sung Sohn, Sagi Tamir, Luhua Song, Dorit Michaeli,
`Imad Matouk, Andrea R. Conlan, Yael Harir, Sarah H. Holt,
`Vladimir Shulaev, Mark L. Paddock, Abraham Hochberg,
`Ioav Z. Cabanchick, José N. Onuchic, Patricia A. Jennings,
`Rachel Nechushtai, and Ron Mittler
`
`14682
`
`14688
`
`DEVELOPMENTAL BIOLOGY
`
`Alteration of genic 5-hydroxymethylcytosine patterning
`in olfactory neurons correlates with changes in gene
`expression andcell identity
`Bradley M. Colquitt, William E. Allen, Gilad Barnea,
`and Stavros Lomvardas
`
`Torso-like functions independently of Torso to regulate
`Drosophila growth and developmental timing
`Travis K. Johnson, Tova Crossman, Karyn A. Foote,
`Michelle A. Henstridge, Melissa J. Saligari, Lauren
`Forbes Beadle, Anabel Herr, James C. Whisstock,
`and Coral G. Warr
`
`BIOPHYSICS AND COMPUTATIONAL BIOLOGY
`
`14528
`
`Two-state dynamics of the SH3-SH2 tandem of Abl
`kinase and theallosteric role of the N-cap
`Carles Corbi-Verge, Fabrizio Marinelli, Ana Zafra-Ruano,
`Javier Ruiz-Sanz, Irene Luque, and José D. Faraldo-Gémez
`
`Consequences of domain insertion on
`sequence-structure divergence in a superfold
`Chetanya Pandya, Shoshana Brown, Ursula Pieper, Andrej
`Sali, Debra Dunaway-Mariano, Patricia C. Babbitt, Yu Xia,
`and Karen N. Allen
`
`ECOLOGY
`
`A bacterial symbiont is converted from an inedible
`producer of beneficial molecules into food by a single
`mutation in the gacA gene
`Pierre Stallforth, Debra A. Brock, Alexandra M. Cantley,
`Xiangjun Tian, David C. Queller, Joan E. Strassmann,
`and Jon Clardy
`=> See Commentary on page 14512
`
`14693
`
`A physical explanation of the temperature dependence
`of physiological processes mediated bycilia and flagella
`Stuart Humphries
`
`PNAS | September 3, 2013 | vol. 110 | no.36 | v
`
`

`

`EVOLUTION
`
`14735
`
`14699
`
`E3398
`
`E3408
`
`E3417
`
`14705
`
`14711
`
`E3425
`
`14717
`
`14723
`
`14729
`
`Segmenting the human genomebased onstates of
`neutral genetic divergence
`Prabhani Kuruppumullage Don, Guruprasad Ananda,
`Francesca Chiaromonte, and Kateryna D. Makova
`
`GENETICS
`
`Establishing a hematopoietic genetic network through
`locus-specific integration of chromatin regulators
`Andrew W. DeVilbiss, Meghan E. Boyer,
`and Emery H.Bresnick
`
`Inherited mutations in the helicase RTEL1 cause telomere
`dysfunction and Hoyeraal-Hreidarsson syndrome
`Zhong Deng, Galina Glousker, Aliah Molczan,
`Alan J. Fox, Noa Lamm, Jayaraju Dheekollu, Orr-El
`Weizman, Michael Schertzer, Zhuo Wang, Olga
`Vladimirova, Jonathan Schug, Memet Aker, Arturo
`Londono-Vallejo, Klaus H. Kaestner, Paul M. Lieberman,
`and Yehuda Tzfati
`
`Assembler for de novo assembly of large genomes
`Te-Chin Chu, Chen-Hua Lu, Tsunglin Liu, Greg C. Lee,
`Wen-Hsiung Li, and Arthur Chun-Chieh Shih
`
`Gene duplication in the majorinsecticide targetsite, Ral,
`in Drosophila melanogaster
`Emily J. Remnant, Robert T. Good, Joshua M. Schmidt,
`Christopher Lumb, Charles Robin, Phillip J. Daborn,
`and Philip Batterham
`
`2
`
`IMMUNOLOGY
`
`Blockade of A2, receptors potently suppresses the
`metastasis of CD73* tumors
`Paul A. Beavis, Upulie Divisekera, Christophe
`Paget, Melvyn T. Chow, Liza B. John, Christel
`Devaud, Karen Dwyer, John Stagg, Mark J. Smyth,
`and Phillip K. Darey
`
`MEDICAL SCIENCES
`
`Hypoxic retinal Miller cells promote vascular
`permeability by HIF-1-dependentup-regulation of
`angiopoietin-like 4
`Xiaoban Xin, Murilo Rodrigues, Mahaa Umapathi, Fabiana
`Kashiwabuchi, Tao Ma, Savalan Babapoor-Farrokhran,
`Shuang Wang, Jiadi Hu, Imran Bhutto, Derek S.
`Welsbie, Elia J. Duh, James T. Handa, Charles G.
`Eberhart, Gerard Lutty, Gregg L. Semenza,
`Silvia Montaner, and Akrit Sodhi
`
`A nanoparticle formulation that selectively transfects
`metastatic tumors in mice
`Jian Yang, William Hendricks, Guosheng Liu, J. Michael
`McCaffery, Kenneth W. Kinzler, David L. Huso,
`Bert Vogelstein, and Shibin Zhou
`
`Retinoblastoma tumor suppressorprotein in pancreatic
`progenitors controls a- and f-cell fate
`Erica P. Cai, Xiaohong Wu, Stephanie A. Schroer,
`Andrew J. Elia, M. Cristina Nostro, Eldad Zacksenhaus,
`and Minna Woo
`
`PINK1 regulates histone H3 trimethylation and gene
`expression by interaction with the polycomb
`protein EED/WAIT1
`Arnaud Berthier, Judit Jiménez-Sdinz, and Rafael Pulido
`
`14540
`
`14741
`
`14747
`
`14753
`
`E3435
`
`14574
`
`14759
`
`14765
`
`14771
`
`14777
`
`Protein kinase Cf as a therapeutic target stabilizing
`blood-brain barrier disruption in experimental
`autoimmuneencephalomyelitis
`Tobias V. Lanz, Simon Becker, Matthias Osswald, Stefan
`Bittner, Michael K. Schuhmann, Christiane A. Opitz,
`Sadanand Gaikwad, Benedikt Wiestler, Ulrike M.
`Litzenburger, Felix Sahm, Martina Ott, Simeon Iwantscheff,
`Carl Grabitz, Michel Mittelbronn, Andreas von Deimling,
`Frank Winkler, Sven G. Meuth, Wolfgang Wick,
`and Michael Platten
`
`MICROBIOLOGY
`
`Surface chemistry allows for abiotic precipitation of
`dolomite at low temperature
`Jennifer A. Roberts, Paul A. Kenward, David A.
`Fowle, Robert H. Goldstein, Luis A. Gonzalez,
`and David S$. Moore
`
`Single point mutations in ATP synthase compensate for
`mitochondrial genomeloss in trypanosomes
`Samuel Dean, Matthew K. Gould, Caroline E. Dewar,
`and Achim C. Schnaufer
`
`Treatment with suboptimal doses of raltegravir leads to
`aberrant HIV-1 integrations
`Janani Varadarajan, Mary Jane McWilliams,
`and Stephen H. Hughes
`
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`| September 3, 2013 | vol. 110 | no. 36 | vii
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`This ‘material may beprotected by Copyrightlaw (Title 17 U.S. Code)
`
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`Blockade of Az, receptors potently suppresses the
`metastasis of CD73* tumors
`PaulA. Beavis”, Upulie Divisekera®”, Christophe Paget®”, Melvyn T. Chow”, Liza B. John”, Christel Devaud””,
`Karen Dwyer“, John Stagg®’, Mark J. Smyth®>-*9-1, and Phillip K. Darcy?
`Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia; °Sir Peter MacCallum Department of Oncology,
`The University of Melbourne,Parkville, VIC 3010, Australia; “St Vincent's Hospital, Immunology Research Centre, Fitzroy, VIC 3065, Australia; ‘Department
`of Medicine, University of Melbourne, Parkville, VIC 3010, Australia; “Centre de Recherche du Centre Hospitalier de I'Université de Montréal, Faculté de
`Pharmacie et Institut du Cancer de Montréal, Montréal, QC, Canada H2L 4M1; ‘immunology in Cancer and Infection Laboratory, Queensland Institute
`of Medical Research, Herston, QLD 4006, Australia; and °School of Medicine, University of Queensland, Herston, QLD 4006, Australia
`
`Edited by Steven A. Rosenberg, ‘National Institutes of Health/National Cancer Institute, Bethesda, MD, and accepted by the Editorial Board July 23, 2013
`(received for review May 5, 2013)
`
`CD73 inhibits antitumor immunity through the activation of aden-
`osine receptors expressed on multiple immune subsets. CD73 also
`enhances tumor metastasis, although the nature of the immune
`subsets and adenosine receptor subtypes involved in this process
`are largely unknown. In this study, we revealed that A2,/A2. re-
`ceptor antagonists were effective in reducing the metastasis of
`tumors expressing CD73 endogenously (411.2 breast tumors) and
`when CD73 wasectopically expressed (B16F10 melanoma). Azq~’~
`mice were strongly protected against tumor metastasis, indicating
`that host Aj, receptors enhanced tumor metastasis. Az, blockade
`enhanced natural killer (NK) cell maturation and cytotoxic function
`in vitro, reduced metastasis in a perforin-dependent manner, and
`enhanced NK cell expression of granzymeB in vivo, strongly sug-
`gesting that the antimetastatic effect of Az, blockade was due to
`enhanced NK cell function. Interestingly, Az, blockade had no
`effect on NK cell cytotoxicity, indicating that an NK cell-indepen-
`dent mechanism also contributed to the increased metastasis of
`CD73* tumors. Our results thus revealed that CD73 promotes tumor
`metastasis through multiple mechanisms, including suppression of
`NKcell function. Furthermore, our data strongly suggest that Az, or
`Azp antagonists may be useful for the treatment of metastatic dis-
`ease. Overall, our study has potential therapeutic implications
`given that A2,/A2, receptor antagonists have already entered clin-
`ical trials in other therapeutic settings.
`
`cancer metastasis | immunotherapy | tumor immunosuppression |
`innate immunity
`
`is now well-established that tumors use numerous immuno-
`t
`suppressive mechanismsto facilitate tumor growth (1, 2). Land-
`mark studies by Sitkovsky and colleagues established that one such
`mechanism is the catabolism of extracellular AMP into immuno-
`suppressive adenosine (3, 4). Extracellular adenosine is generated
`from adenosine monophosphate (AMP)by the ectoenzyme CD73
`and binds to four knowncell surface receptors (A;, Aza, Aop, and
`A3) that are expressed on multiple immunesubsets including T
`cells, natural killer (NK) cells, natural killer T cells, macrophages,
`dendritic cells, and myeloid-derived suppressorcells (MDSCs)(5).
`The Asa and Asp receptor subtypes are essentially responsible
`for the immunosuppressive effects of adenosine. They share
`a commonsignaling pathway, both resulting in the activation of
`adenylate cyclase and the accumulation of intracellular cAMP.
`Extracellular AMP can be generated from ATP, an immune-
`activating molecule, by the ectoenzyme CD39. Thus, CD73 and
`CD39 acting in concert can catabolize ATP into adenosine and
`therefore tip the balance from an immunostimulatory to an im-
`munosuppressive microenvironment.
`Physiologically, CD73 is expressed on endothelial cells, epi-
`thelial cells, and subsets of the immune system, including Tregs
`(5). CD73 has been found to be up-regulated in several types of
`cancer and recently it has been shownto be a negative prognostic
`marker in triple negative breast cancer (5, 6). Several preclinical
`studies indicate that targeting the CD73-adenosine pathway may
`
`be beneficial. Proof-of-concept studies demonstrated that target-
`ing CD73 with an anti-CD73-blocking mAb could significantly
`delay tumor growth (7). This was due to enhanced antitumor
`immune T-cell responses as a result of attenuated A2,-mediated
`immunosuppression. Subsequently, both host and tumor-expressed
`CD73 have been shown to be important in the suppression of
`antitumor T-cell responses. Indeed, CD73-deficient mice are re-
`sistant to several transplantable tumors and de novo carcinogen-
`esis (8-11). On the other hand, specific targeting of CD73 on
`tumorcells using shRNAalso significantly reduced tumor growth
`and prolonged survival (7, 12).
`Furthermore,
`the knockdown of CD73 expression on tumor
`cells reduced their ability to metastasize (7). However, the mech-
`anism by which CD73 promotes tumor metastasis is unclear.
`Understanding the mechanism by which CD73 increases tumor
`metastasis is vital
`to developing new strategies that target
`metastasis because there are no anti-CD73 mAbscurrently in
`clinical development. Theclinical relevance of this pathwayis
`underlined by the correlation between CD73 expression and
`an increased risk of metastasis in breast cancer (13, 14). Thus,
`the CD73 pathway has been proposed as a novel target for
`immunotherapy(15).
`In this current study, we found that CD73 promoted metas-
`tasis through the activation of both Az4 and Agg receptors. Ao,
`blockade resulted in enhanced NK-cell activity in vitro and in
`vivo and reduced metastasis in a perforin-dependent manner.
`Thus, Az4 or Agp antagonists, which have already been used in
`clinicaltrials in other disease settings (16), are promising for the
`treatment of cancer metastasis for which there is a lack of ef-
`fective options available.
`Results
`
`Tumor-CD73 Expression Promotes in Vivo Metastasis. Previous inves-
`tigations in our laboratory suggested that the expression of CD73
`on tumorcells could positively regulate metastasis (7). To in-
`vestigate whether CD73 expression was sufficient to increase the
`migratory properties of tumorcells, AT-3 breast adenocarcinoma
`and B16F10 melanomacells (both CD73~) were transduced with
`retrovirus-encoding murine CD73 cDNA. CD73 expression on
`transduced AT-3 and B16FI10 cells was at a level equivalent to
`4T1.2 breast carcinomacells, a metastatic cell line expressing high
`levels of CD73 (Fig. S1A) (7). Ectopically expressed CD73 was
`
`Author contributions: P.A.B., J.S., MJ.S., and P.K.D. designed research; P.A.B., U.D., C.P.,
`N.T.C,, L.BJ., C.D., and P.K.D. performed research; K.D. and M.J.5. contributed new re-
`agents/analytic tools; P.A.B., J.S., M..S., and P.K.D. analyzed data; and P.A.B. wrote
`the paper.
`The authors declare no conflict of interest.
`This article is a PNAS Direct Submission. $.A.R. is a guest editor invited by the