Institute for Research in Immunology and Cancer

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.

The BioGRID (Biological General Repository for Interaction Datasets, thebiogrid.org) is an open-access database resource that houses manually curated protein and genetic interactions from multiple species including yeast, worm, fly, mouse, and human. The ~1.93 million curated interactions in BioGRID can be used to build complex networks to facilitate biomedical discoveries, particularly as related to human health and disease. All BioGRID content is curated from primary experimental evidence in the biomedical literature, and includes both focused low-throughput studies and large high-throughput datasets. BioGRID also captures protein post-translational modifications and protein or gene interactions with bioactive small molecules including many known drugs. A built-in network visualization tool combines all annotations and allows users to generate network graphs of protein, genetic and chemical interactions. In addition to general curation across species, BioGRID undertakes themed curation projects in specific aspects of cellular regulation, for example the ubiquitin-proteasome system, as well as specific disease areas, such as for the SARS-CoV-2 virus that causes COVID-19 severe acute respiratory syndrome. A recent extension of BioGRID, named the Open Repository of CRISPR Screens (ORCS, orcs.thebiogrid.org), captures single mutant phenotypes and genetic interactions from published high throughput genome-wide CRISPR/Cas9-based genetic screens. BioGRID-ORCS contains datasets for over 1,042 CRISPR screens carried out to date in human, mouse and fly cell lines. The biomedical research community can freely access all BioGRID data through the web interface, standardized file downloads, or via model organism databases and partner meta-databases.

The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the curation and archival storage of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2018 (build 3.4.164), BioGRID contains records for 1 598 688 biological interactions manually annotated from 55 809 publications for 71 species, as classified by an updated set of controlled vocabularies for experimental detection methods. BioGRID also houses records for >700 000 post-translational modification sites. BioGRID now captures chemical interaction data, including chemical-protein interactions for human drug targets drawn from the DrugBank database and manually curated bioactive compounds reported in the literature. A new dedicated aspect of BioGRID annotates genome-wide CRISPR/Cas9-based screens that report gene-phenotype and gene-gene relationships. An extension of the BioGRID resource called the Open Repository for CRISPR Screens (ORCS) database (https://orcs.thebiogrid.org) currently contains over 500 genome-wide screens carried out in human or mouse cell lines. All data in BioGRID is made freely available without restriction, is directly downloadable in standard formats and can be readily incorporated into existing applications via our web service platforms. BioGRID data are also freely distributed through partner model organism databases and meta-databases.

The Biological General Repository for Interaction Datasets (BioGRID: http//thebiogrid.org) is an open access archive of genetic and protein interactions that are curated from the primary biomedical literature for all major model organism species. As of September 2012, BioGRID houses more than 500 000 manually annotated interactions from more than 30 model organisms. BioGRID maintains complete curation coverage of the literature for the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the model plant Arabidopsis thaliana. A number of themed curation projects in areas of biomedical importance are also supported. BioGRID has established collaborations and/or shares data records for the annotation of interactions and phenotypes with most major model organism databases, including Saccharomyces Genome Database, PomBase, WormBase, FlyBase and The Arabidopsis Information Resource. BioGRID also actively engages with the text-mining community to benchmark and deploy automated tools to expedite curation workflows. BioGRID data are freely accessible through both a user-defined interactive interface and in batch downloads in a wide variety of formats, including PSI-MI2.5 and tab-delimited files. BioGRID records can also be interrogated and analyzed with a series of new bioinformatics tools, which include a post-translational modification viewer, a graphical viewer, a REST service and a Cytoscape plugin.

The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the annotation and archival of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2016 (build 3.4.140), the BioGRID contains 1 072 173 genetic and protein interactions, and 38 559 post-translational modifications, as manually annotated from 48 114 publications. This dataset represents interaction records for 66 model organisms and represents a 30% increase compared to the previous 2015 BioGRID update. BioGRID curates the biomedical literature for major model organism species, including humans, with a recent emphasis on central biological processes and specific human diseases. To facilitate network-based approaches to drug discovery, BioGRID now incorporates 27 501 chemical-protein interactions for human drug targets, as drawn from the DrugBank database. A new dynamic interaction network viewer allows the easy navigation and filtering of all genetic and protein interaction data, as well as for bioactive compounds and their established targets. BioGRID data are directly downloadable without restriction in a variety of standardized formats and are freely distributed through partner model organism databases and meta-databases.

The Biological General Repository for Interaction Datasets (BioGRID: http://thebiogrid.org) is an open access database that houses genetic and protein interactions curated from the primary biomedical literature for all major model organism species and humans. As of September 2014, the BioGRID contains 749,912 interactions as drawn from 43,149 publications that represent 30 model organisms. This interaction count represents a 50% increase compared to our previous 2013 BioGRID update. BioGRID data are freely distributed through partner model organism databases and meta-databases and are directly downloadable in a variety of formats. In addition to general curation of the published literature for the major model species, BioGRID undertakes themed curation projects in areas of particular relevance for biomedical sciences, such as the ubiquitin-proteasome system and various human disease-associated interaction networks. BioGRID curation is coordinated through an Interaction Management System (IMS) that facilitates the compilation interaction records through structured evidence codes, phenotype ontologies, and gene annotation. The BioGRID architecture has been improved in order to support a broader range of interaction and post-translational modification types, to allow the representation of more complex multi-gene/protein interactions, to account for cellular phenotypes through structured ontologies, to expedite curation through semi-automated text-mining approaches, and to enhance curation quality control.

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycin-resistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.

Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser235/236 in vitro and in vivo using an mTOR-independent mechanism. Mutation of rpS6 at Ser235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTOR-independent pathway linking the Ras/ERK signaling cascade to the translational machinery. Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser235/236 in vitro and in vivo using an mTOR-independent mechanism. Mutation of rpS6 at Ser235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTOR-independent pathway linking the Ras/ERK signaling cascade to the translational machinery. In eukaryotic cells, the main rate-limiting step of translation is initiation, which is controlled by an array of proteins that respond to signaling cascades activated by extracellular signals (reviewed in Refs. 1Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 2Clemens M.J. Oncogene. 2004; 23: 3180-3188Crossref PubMed Scopus (187) Google Scholar, 3Richter J.D. Sonenberg N. Nature. 2005; 433: 477-480Crossref PubMed Scopus (745) Google Scholar). The mammalian target of rapamycin, mTOR, 4The abbreviations used are: mTOR, mammalian target of rapamycin; RSK, p90 ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; S6K1, ribosomal S6 kinase 1; 4E-BP, 4E-binding proteins; rpS6, ribosomal protein S6; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; siRNA, short interfering RNA; PMA, phorbol 12-myristate 13-acetate; fmk, fluoromethylketone; GST, glutathione S-transferase; wt, wild type; EGF, epidermal growth factor; HA, hemagglutinin. 4The abbreviations used are: mTOR, mammalian target of rapamycin; RSK, p90 ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; S6K1, ribosomal S6 kinase 1; 4E-BP, 4E-binding proteins; rpS6, ribosomal protein S6; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; siRNA, short interfering RNA; PMA, phorbol 12-myristate 13-acetate; fmk, fluoromethylketone; GST, glutathione S-transferase; wt, wild type; EGF, epidermal growth factor; HA, hemagglutinin. is a conserved serine/threonine kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate mammalian cell growth (reviewed in Refs. 4Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3377) Google Scholar, 5Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1036) Google Scholar, 6Richardson C.J. Schalm S.S. Blenis J. Semin. Cell Dev. Biol. 2004; 15: 147-159Crossref PubMed Scopus (115) Google Scholar, 7Gingras A.C. Raught B. Sonenberg N. Curr. Top Microbiol. Immunol. 2004; 279: 169-197Crossref PubMed Google Scholar, 8Averous J. Proud C.G. Oncogene. 2006; 25: 6423-6435Crossref PubMed Scopus (164) Google Scholar). Under conditions of nutrient and energy sufficiency and insulin or mitogen stimulation, mTOR stimulates two important translational regulators, the ribosomal S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E). eIF4E is crucial for ribosome recruitment as it binds to the 7-methylguanosine cap structure (m7GpppN, where N is any nucleotide) at the 5′-end of nearly all transcribed mRNAs to initiate cap-dependent translation (reviewed in Ref. 7Gingras A.C. Raught B. Sonenberg N. Curr. Top Microbiol. Immunol. 2004; 279: 169-197Crossref PubMed Google Scholar). When mTOR is active, eIF4E nucleates the assembly of the translation preinitiation complex through recruitment of numerous initiation factors, resulting in association of the ribosomal subunits to the mRNA. S6K1 and S6K2 are serine/threonine kinases directly stimulated by mTOR which in turn, phosphorylate substrates involved in cell and body size (5Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1036) Google Scholar, 6Richardson C.J. Schalm S.S. Blenis J. Semin. Cell Dev. Biol. 2004; 15: 147-159Crossref PubMed Scopus (115) Google Scholar). S6K1 phosphorylates several substrates located in the cytoplasm and the nucleus, including the ribosomal protein (rp) S6 (reviewed in Ref. 9Ruvinsky I. Meyuhas O. Trends Biochem. Sci. 2006; 31: 342-348Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). Ribosomal protein S6 is one of 33 proteins that comprise the 40 S ribosomal subunit and represents the most extensively studied substrate of S6K1 (10Thomas G. Siegmann M. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3952-3956Crossref PubMed Scopus (107) Google Scholar). Because the initial discovery that liver-derived rpS6 was phosphorylated (11Gressner A.M. Wool I.G. J. Biol. Chem. 1974; 249: 6917-6925Abstract Full Text PDF PubMed Google Scholar), mitogenic stimulation of cells was found to correlate with phosphorylation of rpS6 on serines, which suggested that rpS6 may control mRNA translation in dividing cells (12Bandi H.R. Ferrari S. Krieg J. Meyer H.E. Thomas G. J. Biol. Chem. 1993; 268: 4530-4533Abstract Full Text PDF PubMed Google Scholar). rpS6 phosphorylation sites have been mapped to five clustered residues that are conserved in metazoans, consisting of Ser235, Ser236, Ser240, Ser244, and Ser247, located at the C-terminal part of the protein (13Krieg J. Hofsteenge J. Thomas G. J. Biol. Chem. 1988; 263: 11473-11477Abstract Full Text PDF PubMed Google Scholar). Two classes of protein kinases were found to phosphorylate rpS6 in vitro, the S6K1/2 and the p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases (reviewed in Refs. 14Roux P.P. Blenis J. Microbiol. Mol. Biol. Rev. 2004; 68: 320-344Crossref PubMed Scopus (1880) Google Scholar and 15Hauge C. Frodin M. J. Cell Sci. 2006; 119: 3021-3023Crossref PubMed Scopus (151) Google Scholar). Subsequent studies determined that rpS6 phosphorylation was largely sensitive to the mTOR inhibitor rapamycin, indicating that S6K1/2 were the main physiological rpS6 kinases operating in somatic cells (16Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1012) Google Scholar, 17Blenis J. Chung J. Erikson E. Alcorta D.A. Erikson R.L. Cell Growth Differ. 1991; 2: 279-285PubMed Google Scholar, 18Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (117) Google Scholar). The RSK family members, in contrast, are not affected by rapamycin as they are activated via the classical mitogen-activated protein kinase (MAPK) signaling pathway. The contribution of S6K1/2 in rpS6 phosphorylation was recently addressed using S6K1/S6K2 double knock-out animals, which were found to display no phosphorylation of rpS6 at Ser240/244, but persistent phosphorylation at Ser235/236 (19Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar). Phosphorylation of Ser235/236 was found to require extracellular signal-regulated kinase (ERK) signaling, suggesting that RSK or other kinases downstream of ERK, such as the mitogen- and stress-activated kinases (MSK1/2), contribute to rpS6 phosphorylation upon mitogen stimulation. The functional importance of rpS6 in animals was underscored by conditional ablation of rpS6 in the liver (20Volarevic S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (312) Google Scholar). In these mice, hepatocytes failed to proliferate after partial hepatectomy due to a blockage in ribosome biogenesis and cell cycle progression. In vivo and in vitro studies have suggested that rpS6 phosphorylation exerts an effect on translation at the level of mRNA binding; initial chemical protection studies and cross-linking experiments localized rpS6 to the mRNA/tRNA binding site junction between the small and large ribosomal subunits (21Nygard O. Nilsson L. J. Biol. Chem. 1990; 265: 6030-6034Abstract Full Text PDF PubMed Google Scholar). Consistent with this finding, highly phosphorylated ribosomes were found to bind and utilize both synthetic and natural mRNA more efficiently in vitro than unphosphorylated counterparts (22Burkhard S.J. Traugh J.A. J. Biol. Chem. 1983; 258: 14003-14008Abstract Full Text PDF PubMed Google Scholar). More recently, the role of rpS6 phosphorylation was addressed through the generation of viable and fertile knock-in mice containing alanine substitutions of all five phosphorylatable serine residues in rpS6 (rpS6P–/–) (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google Scholar). These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose an of protein synthesis and cell from these animals are than the size of was not upon rapamycin that rpS6 is a mTOR regulating cell size (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google Scholar). is in to data from S6K1 and S6K2 which that S6K2 rpS6 phosphorylation are involved in the growth of or cells (19Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar, S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (312) Google Scholar). it rpS6 phosphorylation a role in cell In the of all regulating rpS6 and this regulated to protein In this we demonstrate that agonists of the signaling pathway rpS6 phosphorylation using an mTOR-independent pathway that RSK We found that activation of all RSK stimulates cap-dependent indicating that RSK provides an and linking the signaling pathway to the of translation initiation. of phosphorylation of rpS6 that Ras/ERK signaling promotes translation initiation by assembly of the preinitiation complex. S6K1, and were P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). rpS6 was in with a The was by and been M. C. F. Mol. Cell Biol. PubMed Scopus Google Scholar). The was from and in with a protein was P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar). The used in this were using the Cell and and cells were in with and and using P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google or the were for and of where for cells were with rapamycin or a inhibitor C. J. Science. 2005; PubMed Scopus Google Scholar), and stimulated with insulin epidermal growth factor or the small interfering with two were from The which for S6K1, and control were were P.P. Blenis J. Curr. Biol. 2005; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, P.P. Mieulet V. Cohen Raught B. J. Blenis J. M. Sonenberg N. J. 2006; 25: PubMed Scopus Google Scholar). cells were using and was determined to than using a cells were for and stimulated with or The inhibitor was as C. J. Science. 2005; PubMed Scopus Google Scholar). and were using 1 1 1 of of as P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar). were with the for by with protein or protein for 1 cap binding were with for were in and with cell were to and as P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar, P.P. Blenis J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). were from were by of was A. A. Blenis J. Mol. Cell Biol. 1999; 19: PubMed Google Scholar). were by were from Cell with the of the RSK which was from and were P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar). and were from and from were in and in kinase were with as substrate and all were to of or was determined by or using a with or cells were with a M. C. F. Mol. Cell Biol. PubMed Scopus Google Scholar), which cap-dependent translation of the and translation of the was with the and were using a and a or were in and are as from the was to the from the ribosome stimulation of was to the were in with and in and were on for and at for at The resulting was on a and in a at for at the was and using a to a Ras/ERK rpS6 Phosphorylation on Ser235/236 an mTOR-independent but mTOR-independent pathways to rpS6 cells were stimulated with a Phosphorylation of rpS6 was using two that rpS6 phosphorylated on Ser235/236 or We of stimulation, rpS6 is phosphorylated at all sites phosphorylation at of stimulation The of rpS6 phosphorylation was between the sites with phosphorylation of Ser235/236 with than Ser240/244, suggesting that phosphorylation of these sites is regulated by signaling When cells were with rapamycin, which S6K1 and phosphorylation at its not rpS6 phosphorylation at Ser235/236 phosphorylated in cells with rapamycin, indicating the of an mTOR-independent pathway to rpS6 phosphorylation at these the signaling pathway was to Ser235/236 cells were with the inhibitor stimulation with phosphorylation of was not affected by at a that efficiently phosphorylation and RSK we found that Ser235/236 phosphorylation was by the in a of the of Ser235/236 phosphorylation suggesting a between signaling and the initial of rpS6 phosphorylation upon stimulation. rapamycin and were rpS6 phosphorylation was indicating that Ser235/236 phosphorylation through and phosphorylation is regulated via an mechanism. the role of signaling in rpS6 we the of rpS6 phosphorylation in cells with of the Ras/ERK signaling cascade. of cells with the phorbol PMA, which stimulates Ras/ERK but not signaling in cells, in a in and RSK and rpS6 phosphorylation Ser235/236 phosphorylation was by and by rapamycin, the more important contribution by the signaling cascade these we determined oncogenic Ras rpS6 phosphorylation at We found that oncogenic Ras but not a rpS6 phosphorylation at Ser235/236 stimulation was by indicating that signaling is required for rpS6 phosphorylation mTOR-independent Growth and Ribosomal S6 Phosphorylation via data that a kinase activated by Ras/ERK signaling regulates rpS6 phosphorylation at and the kinases of the RSK and the of RSK in rpS6 we used and the RSK in and of S6K1 rpS6 phosphorylation at all sites upon stimulation we found that of or rpS6 phosphorylation at and to a and was both RSK were indicating that and are involved in rpS6 phosphorylation upon and growth factor stimulation. inhibitor was recently and as an kinase inhibitor of RSK C. J. Science. 2005; PubMed Scopus Google Scholar). We the of this inhibitor and found that of cells and by while S6K1 and not to the data using of RSK using rpS6 phosphorylation at but not phosphorylation RSK was more at of stimulation which with the of RSK and as by phosphorylation at and These the RSK in the of rpS6 phosphorylation at as suggested by the of the inhibitor RSK is required for rpS6 phosphorylation by other mitogenic cells were with stimulation with serum, PMA, EGF, or insulin of RSK rpS6 phosphorylation at Ser235/236 in to all Ras/ERK pathway Consistent with the for Ras/ERK signaling, with no effect on rpS6 phosphorylation by insulin, which is not a of RSK in these cells P.P. Blenis J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and S6K1 rpS6 in and in with phosphorylation sites on rpS6 are located a highly C-terminal of the protein The of residues at the and and these phosphorylation for both RSK family and S6K1 Cohen Cohen P. PubMed Scopus Google Scholar). and are not by such residues indicating that they are not RSK or S6K1 phosphorylation the of and S6K1 rpS6, of these kinases were to in vitro kinase with a protein containing the C-terminal of rpS6 as of kinase were as and S6K1 were to phosphorylate rpS6 on Ser235/236 as with we found that S6K1, but not or was of rpS6 on while and stimulated in to levels by S6K1, of this was found to These that and have a for residues at the and and that the RSK not directly phosphorylate in the kinase of RSK rpS6 in cells were with wt, and kinase of cells were for and for rpS6 phosphorylation at Ser235/236 and of or a not rpS6 phosphorylation in of rpS6 phosphorylation at The level of Ser235/236 phosphorylation was to that in cells, but phosphorylation at was not by We have found that RSK modulate mTOR signaling through the phosphorylation and of P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). we found that activated rpS6 phosphorylation was to rapamycin indicating that RSK regulates rpS6 phosphorylation using mTOR-independent activated stimulated rpS6 phosphorylation cells were with or rapamycin for and for rpS6 phosphorylation We have that activation of is to these Blenis J. Mol. Cell Biol. PubMed Scopus Google and as rpS6 phosphorylation stimulated by was not affected by of mTOR, or These data that the RSK directly target the translational by promoting rpS6 phosphorylation at Ser235/236 of mTOR Phosphorylation of rpS6 to the the function of rpS6 phosphorylation at we rpS6 with alanine or substitutions at and These were in cells and cell were for and rpS6 phosphorylation the while rpS6 phosphorylation was stimulated by phosphorylation of Ser235/236 was in the and phosphorylation of rpS6 its recruitment to the mRNA cap complex, we the of the rpS6 to bind 7-methylguanosine cap We found both in the and of serum, binding of the rpS6 was impaired with protein binding of the rpS6 to cap was more than protein in the of serum, indicating that phosphorylation of Ser235/236 promotes rpS6 binding to the 7-methylguanosine cap complex. Under conditions where rpS6 is phosphorylated in to serum, both and the rpS6 were found to to cap with we determined signaling agonists require phosphorylation of Ser235/236 to rpS6 to the 7-methylguanosine complex. rpS6 was to the 7-methylguanosine cap complex in a we found that of Ser235/236 to alanine residues impaired binding of rpS6 to cap Phosphorylation of Ser235/236 to important for recruitment of rpS6, as the of rpS6 to the mRNA cap complex in the of stimulation. These that rpS6 phosphorylation at Ser235/236 rpS6 recruitment to the mRNA complex, indicating that phosphorylation of rpS6 may assembly of the translation initiation complex. The RSK and the RSK are required for translation initiation, we used a that the between cap-dependent and translation initiation M. C. F. Mol. Cell Biol. PubMed Scopus Google Scholar, Blenis J. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). this we that and both cap-dependent translation and which was by rapamycin of the of eIF4E function RSK is required for cap-dependent or cells were in and for with of the inhibitor Under conditions that RSK we found that cap-dependent translation was in a with to a in translation initiation level was to the effect of rapamycin suggesting that RSK is important for translation initiation. this we the effect of RSK on cap-dependent in of or cap-dependent translation or of cap-dependent that kinase is to cap-dependent all RSK stimulated cap-dependent were in the we found that all RSK stimulated cap-dependent translation in cells translation initiation was found to sensitive to rapamycin, which is with the that mTOR is required for eIF4E and of the translation initiation complex. these data demonstrate that RSK to cap-dependent directly the for RSK in ribosomal recruitment to we the of ribosomes from cells with the RSK The at was the resulting to which two by the of and in stimulation in with cells that were of both rapamycin and for 1 the level of by stimulation indicating that mTOR and RSK are important to these data that RSK provides a between the Ras/ERK signaling cascade and the translational by promoting recruitment of rpS6 and ribosomal subunits to the translation preinitiation complex We have an important between the Ras/ERK signaling cascade and the translational which of the mechanisms by which of this cascade protein We found that serum, growth factors, oncogenic and phorbol rpS6 phosphorylation at Ser235/236 using an mTOR-independent pathway that RSK Phosphorylation of Ser235/236 was found to regulate the of rpS6 for the 7-methylguanosine cap complex, indicating that RSK signaling to the assembly of the translation initiation complex. We that all RSK cap-dependent translation in cells to and phorbol and that RSK was required for recruitment of ribosomes to indicating that RSK family are of translation initiation. the RSK of Ras signaling, and the of mTOR and signaling, modulate protein was in as a serine kinase that phosphorylated ribosomal protein S6 in vitro E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). S6K1 and S6K2 were found to the rpS6 kinases operating in somatic cells (16Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1012) Google Scholar, H. Thomas G. Nature. 1991; PubMed Scopus Google Scholar), RSK family were no to involved in rpS6 phosphorylation and translation initiation. in that S6K1 and S6K2 were the rpS6 but that a kinase phosphorylated rpS6 at Ser235/236 (19Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar). Consistent with these we found that all RSK phosphorylated rpS6 at Ser235/236 but not in vitro and in with the that S6K1/2 and the RSK on rpS6 to modulate mRNA We have that phosphorylate and the tumor a of mTOR signaling P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). was found to regulate L. H. P. P.P. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), indicating that Ras/ERK signaling modulate rpS6 phosphorylation through the of mTOR Here, we that rapamycin not rpS6 phosphorylation at indicating that RSK rpS6 phosphorylation using and by Meyuhas and (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google a knock-in (rpS6P–/–) with alanine substitutions of all five phosphorylatable the of These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose indicating that rpS6 phosphorylation is required for the synthesis or function of proteins that (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google Scholar). association and protein synthesis were found to in and from these mice, indicating that rpS6 phosphorylation regulates mRNA The of protein synthesis and were found to in but this from in protein to In are than wild indicating that rpS6 phosphorylation to the of cell size via an mechanism. on rpS6 phosphorylation to regulated by at two of the RSK and We found that the RSK phosphorylate Ser235/236 but not Ser240/244, suggesting that the sites of rpS6 phosphorylation may molecular The of the may the of a of molecular by phosphorylation of We that rpS6 is to the 7-methylguanosine cap binding complex more efficiently in cells with activated Ras/ERK signaling, suggesting a by which phosphorylation of rpS6 by RSK proteins promotes its recruitment to the translation preinitiation complex. These are in with that phosphorylated ribosomes with both synthetic and natural mRNA more efficiently than unphosphorylated counterparts (22Burkhard S.J. Traugh J.A. J. Biol. Chem. 1983; 258: 14003-14008Abstract Full Text PDF PubMed Google Scholar). that RSK is important for and a for the between the and Ras/ERK signaling cascades in translation initiation. Because these pathways have activation may provide cells with a for of molecular between these pathways was to contribute to by the recruitment of mRNAs to ribosomes A. M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the mRNAs most affected are proteins that regulate growth and suggesting a by which Ras/ERK and signaling to by the of mRNAs with of both pathways in mRNA translation been in of A. 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Cancer drug resistance continues to be a major impediment in medical oncology. Clinically, resistance can arise prior to or as a result of cancer therapy. In this review, we discuss different mechanisms adapted by cancerous cells to resist treatment, including alteration in drug transport and metabolism, mutation and amplification of drug targets, as well as genetic rewiring which can lead to impaired apoptosis. Tumor heterogeneity may also contribute to resistance, where small subpopulations of cells may acquire or stochastically already possess some of the features enabling them to emerge under selective drug pressure. Making the problem even more challenging, some of these resistance pathways lead to multidrug resistance, generating an even more difficult clinical problem to overcome. We provide examples of these mechanisms and some insights into how understanding these processes can influence the next generation of cancer therapies.

The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1–3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17–92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17–92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3′-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17–92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17–92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17–92 cluster. Altogether, these results suggest that the autoregulation between E2F1–3 and miR-20a is important for preventing an abnormal accumulation of E2F1–3 and may play a role in the regulation of cellular proliferation and apoptosis. The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1–3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17–92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17–92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3′-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17–92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17–92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17–92 cluster. Altogether, these results suggest that the autoregulation between E2F1–3 and miR-20a is important for preventing an abnormal accumulation of E2F1–3 and may play a role in the regulation of cellular proliferation and apoptosis. The proper regulation of cellular proliferation and cell cycle progression is critical for the normal development of organisms and the prevention of cancer. Among the numerous factors involved in these processes, the E2F transcription factors play an essential role (1DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar, 2Attwooll C. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4709-4716Crossref PubMed Scopus (419) Google Scholar, 3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar). E2F1, along with E2F2 and E2F3, are activators of cell cycle progression and promote the entry of quiescent cells into S phase (4DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (607) Google Scholar, 5Johnson D.G. Schwarz J.K. Cress W.D. Nevins J.R. Nature. 1993; 365: 349-352Crossref PubMed Scopus (834) Google Scholar). During G1, E2F1–3 are inhibited by their association with members of the retinoblastoma protein family (pRb, 5The abbreviations used are: Rb, retinoblastoma; miRNA, micro-RNA; UTR, untranslated region; ASO, antisense oligoribonucleotides; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; GFP, green fluorescent protein; ER, estrogen receptor. p107, and p130) (3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar). In mid to late G1, hyperphosphorylation of pRb by the cyclinD/cdk4-cdk6 complexes leads to the release of E2F1–3, which bind to specific E2F-responsive promoters, stimulating the transcription of genes involved in G1/S progression (6Stevaux O. Dyson N.J. Curr. Opin. Cell Biol. 2002; 14: 684-691Crossref PubMed Scopus (350) Google Scholar, 7Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (630) Google Scholar). Most cancer cells contain mutations that deregulate the pRb/E2F pathway, which highlights its importance in the control of cellular proliferation. Deregulation of Rb/E2F control can also result in the activation of E2F1-induced apoptosis. Indeed, inactivation of pRb or overexpression of E2F1 promote apoptosis in several cell lines. E2F3 has also been shown to stimulate apoptosis but in a E2F1-dependent pathway (8Lazzerini Denchi E. Helin K. EMBO Rep. 2005; 6: 661-668Crossref PubMed Scopus (99) Google Scholar). E2F1-responsive sites have been found in the promoters of several caspases as well as in other pro-apoptotic targets of p53 (9Nahle Z. Polakoff J. Davuluri R.V. McCurrach M.E. Jacobson M.D. Narita M. Zhang M.Q. Lazebnik Y. Bar-Sagi D. Lowe S.W. Nat. Cell Biol. 2002; 4: 859-864Crossref PubMed Scopus (364) Google Scholar, 10Furukawa Y. Nishimura N. Furukawa Y. Satoh M. Endo H. Iwase S. Yamada H. Matsuda M. Kano Y. Nakamura M. J. Biol. Chem. 2002; 277: 39760-39768Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). E2F1 is also activated by the DNA damage signaling pathway (ATM/ATR) leading to the activation of both p53-dependent and independent, pro-apoptotic pathways (11Urist M. Tanaka T. Poyurovsky M.V. Prives C. Genes Dev. 2004; 18: 3041-3054Crossref PubMed Scopus (195) Google Scholar, 12Lin W.-C. Lin F.-T. Nevins J.R. Genes Dev. 2001; 15: 1833-1844PubMed Google Scholar). Therefore, E2F1 provides direct coupling of the cell cycle and apoptotic machinery, and it has been suggested that cycling cells are primed for apoptosis by E2F1 should proliferation be perceived as aberrant (9Nahle Z. Polakoff J. Davuluri R.V. McCurrach M.E. Jacobson M.D. Narita M. Zhang M.Q. Lazebnik Y. Bar-Sagi D. Lowe S.W. Nat. Cell Biol. 2002; 4: 859-864Crossref PubMed Scopus (364) Google Scholar). Besides the control of their activity by association with pRb, E2F1–3 are also regulated by phosphorylation (13Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin J.W.G. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (413) Google Scholar), acetylation (14Martinez-Balbas M.A. Bauer U.-M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (568) Google Scholar, 15Marzio G. Wagener C. Gutierrez M.I. Cartwright P. Helin K. Giacca M. J. Biol. Chem. 2000; 275: 10887-10892Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), and ubiquitin-dependent degradation (16Campanero M.R. Flemington E.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2221-2226Crossref PubMed Scopus (183) Google Scholar). E2F1–3 also regulate their own transcription through E2F-binding sites within their promoters (17Johnson D.G. Ohtani K. Nevins J.R. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (453) Google Scholar, 18Hsiao K.M. McMahon S.L. Farnham P.J. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (221) Google Scholar). Recently, a novel mechanism of regulation of E2F1 activity has been identified: micro-RNAs (miRNAs) have been found to be important modulators of E2F1 mRNA translation (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar). miRNAs are small 21–23 nucleotides non-coding RNAs that control the stability and/or translation of specific transcripts through the recruitment of the RNA-inducing silencing complex (reviewed in Ref. 20Zamore P.D. Haley B. Science. 2005; 309: 1519-1524Crossref PubMed Scopus (1130) Google Scholar). Recent evidence suggests that miRNAs can regulate the expression of numerous genes (21Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9882) Google Scholar) and several studies point to the role of some miRNAs in the development of cancer (reviewed in Ref. 22Esquela-Kerscher A. Slack F.J. Nat. Rev. Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6202) Google Scholar). Among them, miRNAs from the mir-17–92 cluster have been shown to have an oncogenic activity when overexpressed with c-myc in a mouse model of human B-cell lymphoma (23He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. Nature. 2005; 435: 828-833Crossref PubMed Scopus (3146) Google Scholar). Interestingly, this cluster is amplified in large-B cell lymphoma and in other malignant lymphomas (24Ota A. Tagawa H. Karnan S. Tsuzuki S. Karpas A. Kira S. Yoshida Y. Seto M. Cancer Res. 2004; 64: 3087-3095Crossref PubMed Scopus (631) Google Scholar). Moreover, miRNAs from this cluster are overexpressed in lung cancer cells and in colon, pancreas, and prostate tumors (25Hayashita Y. Osada H. Tatematsu Y. Yamada H. Yanagisawa K. Tomida S. Yatabe Y. Kawahara K. Sekido Y. Takahashi T. Cancer Res. 2005; 65: 9628-9632Crossref PubMed Scopus (1381) Google Scholar, 26Volinia S. Calin G.A. Liu C.-G. Ambs S. Cimmino A. Petrocca F. Visone R. Iorio M. Roldo C. Ferracin M. Prueitt R.L. Yanaihara N. Lanza G. Scarpa A. Vecchione A. Negrini M. Harris C.C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2257-2261Crossref PubMed Scopus (4963) Google Scholar). Another group has also shown that the mir-17–92 cluster is directly regulated by MYC, and two miRNAs from this cluster, miR-17a and miR-20a, inhibited the translation of the E2F1 mRNA (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar). Altogether, these results suggest that miRNAs from the mir-17–92 cluster can act as oncogenic miRNAs or “oncomirs” when overexpressed, possibly by acting on key regulators of the cell cycle and apoptosis, like E2F1. Here, we that miR-20a, a member of the mir-17–92 cluster, E2F1 but also E2F2 and E2F3 via binding sites in the of their We also report that E2F1–3 directly bind the promoter of the mir-17–92 cluster its While overexpression of miR-20a decreased apoptosis in a prostate cancer cell line, inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin to a anti-apoptotic role for Altogether, these results suggest that the autoregulation between E2F1–3 and miR-20a is important for a between E2F in cellular proliferation and apoptosis. E2Fs amplified from the DNA of cells the E2F1, and E2F2, and E2F3, The and of E2F1, E2F2, and E2F3 into the of the the the to the of the miR-20a miRNA in by the the promoter a of from the cluster promoter E2F-binding sites amplified from DNA the and The between the and sites of the miR-20a, we the miRNA after from human DNA with the and The into the and cells at cells well in a The the or with the of the cluster the a of the promoter into cells at cells in in with or with of of a protein and The of at by the to the of The after for activity the of E2Fs of into cells cells well in with the the promoter the the promoter or the the of E2F1 with the antisense used in this miR-20a and The by DNA the at in after activity to activity for the on to a a and the in the The to and at and the for Cell cells at cells well in a The after the and doxorubicin after and with to the cell death The in miR-20a or in cells and with cells well in a from of the in with or doxorubicin cells and with of cells the of Cell or into cells cells well directly into with or doxorubicin and cells to for with and with in with several with of and which is to cell with of and of used for at in with a the of and in the cell cells well directly into with or doxorubicin and cells to for with with and the of cells and with PBS, and the in of and on for and the for The with of with PBS, two with and in and at The on a and to The used for mouse mouse after with to by of E2F1–3 by E2F1 expression regulated by factors binding to its we the of E2F1 to a We a in activity from cells when with a to the of (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar) that the E2F1 mRNA binding sites for miRNAs that regulated its we the two sites in the E2F1 mRNA both sites we an in activity to the control level Moreover, of the with miR-20a resulted in a in activity with a control these data the that the translation of the E2F1 mRNA is regulated by miRNAs from the mir-17–92 cluster, miR-20a, through sites in the E2F1 mRNA (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar). it is that other members of the E2F E2F2 and E2F3, of like and activation (3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar, 15Marzio G. Wagener C. Gutierrez M.I. Cartwright P. Helin K. Giacca M. J. Biol. Chem. 2000; 275: 10887-10892Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, D.G. Ohtani K. Nevins J.R. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (453) Google Scholar), we the that miR-20a also regulate the translation of the E2F2 and E2F3 mRNAs via binding sites in their Indeed, the of sites in the of the E2F2 mRNA and in the of the E2F3 mRNA B. A. T. C. Biol. 2004; PubMed Scopus Google Scholar) at the of the in the control and in cells to its the E2F2 that the two with the control in activity from the and the we have for the E2F1 we the binding sites of miR-20a in the and E2F3 shown in of the miR-20a sites increased activity in both mRNAs the level of the suggesting that binding of miR-20a in these We also the of miR-20a on the expression of endogenous shown in of cells with the miR-20a resulted in an increased level of E2F1 and E2F2, as by with we have been to E2F3 member of the miR-20a also increased both E2F1 and E2F2 with the control Altogether, these results suggest that like E2F1, the E2F2 and E2F3 mRNAs are also targets of of endogenous of E2F1 and E2F2 in cells with miR-20a, or to with of by E2F1–3 are transcription we the that may regulate the expression of the miR-20a We cells estrogen of E2F1, E2F2, and E2F3 E. H. Prosperini E. G. Cartwright P. Moroni M.C. Helin K. Cell Biol. 19: PubMed Scopus Google Scholar). of from these cells and the level of miR-20a by shown in overexpression of E2F1–3 to an increased level of miR-20a with the control suggesting that the E2F1, E2F2, and E2F3 transcription factors can the expression of We also cells with a of a which the but the Interestingly, of this E2F also to increased miR-20a studies have of the in cellular C. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4709-4716Crossref PubMed Scopus (419) Google Scholar), which suggests that the of a complex from a promoter by the may be to The miR-20a miRNA is of a cluster of the mir-17–92 cluster, which is on the (24Ota A. Tagawa H. Karnan S. Tsuzuki S. Karpas A. Kira S. Yoshida Y. Seto M. Cancer Res. 2004; 64: 3087-3095Crossref PubMed Scopus (631) Google Scholar). This cluster has been shown to be by the (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar) and amplified in lymphomas (23He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. Nature. 2005; 435: 828-833Crossref PubMed Scopus (3146) Google Scholar). the increased expression of miR-20a at the level of the mir-17–92 cluster we of a cell We used to the level of of shown in of the resulted in an increased suggesting that the cluster is by this cluster may be directly regulated by the E2F1–3 transcription we for E2F-binding sites within the of the promoter of the mir-17–92 cluster E2Fs are to bind to a S in the promoters of their genes Y. Cress W.D. J.M. Cell Biol. 1997; PubMed Scopus Google Scholar). We E2F-binding sites in the mir-17–92 promoter to this and the binding of these E2Fs to the we to the association of the endogenous E2F1, E2F2, and E2F3 transcription factors with the mir-17–92 promoter in cells In the mir-17–92 we which is to two E2F-binding sites and which two other E2F-binding sites and and which the sites (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar). used as a while E2F1 in the E2F1 promoter used as a shown in and amplified after with and E2F3 but with an the amplified with the and Interestingly, a of the with E2F3 with E2F2 or E2F1. may that E2F3 to this of the promoter or that the E2F3 is for that the mir-17–92 cluster is regulated by the we a of this the E2F-binding sites the and of the in the of this with of the resulted in an increased activation of the E2F1 a activation of this promoter by E2F3 E2F2 activation While E2F2 as as E2F1 and E2F3 to miR-20a it may bind to sites that are of the in the of an E2F in the results that this of the mir-17–92 cluster promoter is to the E2F1–3 transcription Altogether, these data suggest a where the translation of the E2F1–3 mRNAs is controlled by the miR-20a miRNA, which is regulated by the E2Fs at the of the of E2F1–3 by an of E2F1–3 miR-20a should expression and may act as an and/or as an anti-apoptotic these we the of the of miR-20a on the activity of endogenous we cells with genes the control of the or both promoters K. J. Leone G. Nevins J.R. Cell. Biol. PubMed Scopus Google Scholar, T. Nat. 2000; PubMed Scopus Google Scholar). The cells with a the endogenous miR-20a shown in inhibition of miR-20a resulted in a in the activation of the promoter and promoter control the miRNA result in the of these with the increased E2F1–3 expression when cells with the miR-20a and suggests an increased E2F1–3 activity in these the of an increased or decreased miR-20a we of a prostate cancer cell that used to apoptosis after with the DNA damage doxorubicin G. Cancer Res. 2005; 65: PubMed Scopus Google Scholar). In these an increased E2F activity with apoptosis after with doxorubicin G. Cancer Res. 2005; 65: PubMed Scopus Google Scholar). We an endogenous miR-20a activity in cells by a decreased activity from the with the with miR-20a sites the inhibition of miR-20a the level of apoptosis of with the or a and with Cell death by after in shown in of the miR-20a resulted in a in cell death after doxorubicin with the suggesting that inhibition of miR-20a these cells to apoptosis. We also overexpressed the miR-20a miRNA in cells by of a a miR-20a from cells with the an expression of the miR-20a miRNA of miR-20a resulted in a in the endogenous level of E2F1 and E2F2, as shown by The of these cells by the overexpression of miR-20a, suggesting that it may E2F1–3 expression to an to cell cycle after with cells the miR-20a miRNA a in cell death with the control cells cell after doxorubicin with a we a in cell of cells miR-20a with control cells with the We also cell accumulation in after doxorubicin of cells or when miR-20a overexpressed, we cells in to the control cells and suggest an increased level of cell after with a DNA damage when miR-20a is overexpressed in these the that miR-20a has an anti-apoptotic possibly through its regulation of E2F1 cells from cell on miR-20a miRNA from cells with the control or the of the of E2F1 and E2F2 in cells with the control or the to with cell death of cells with or control after with doxorubicin Cell death the of cells with or after with doxorubicin to the with of cells with or after with doxorubicin to the with The of which explain the of cell or their to have been as the of the of regulators that can or specific a family of small has to this of miRNAs can regulate expression both at and P.D. Haley B. Science. 2005; 309: 1519-1524Crossref PubMed Scopus (1130) Google Scholar). has been that can act as or as regulators of Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). miRNAs are by can be regulated by This that can be between genes for transcription factors and genes for We report that the mir-17–92 cluster is directly regulated by the E2F family of transcription several miRNAs in this cluster can E2F1–3 an autoregulatory feedback loop can be between E2Fs and the mir-17–92 cluster is well that E2F1–3 are involved in a autoregulatory loop stimulate their own genes (17Johnson D.G. Ohtani K. Nevins J.R. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (453) Google Scholar, 18Hsiao K.M. McMahon S.L. Farnham P.J. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (221) Google Scholar). We that a role of the miR-20a miRNA family is to the autoregulatory loop of E2F1–3 by a feedback loop to control the level of expression of these transcription We suggest that other transcription factors involved in autoregulatory may also be controlled by feedback miRNAs as may be the transcription which is involved in and which is to the transcription of its own S.J. R.L. H. Cell. Full Text PDF PubMed Scopus Google Scholar). Recently, and M. S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: PubMed Scopus Google Scholar) have shown that the transcription of the specific and Interestingly, the mRNA has a in its (21Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9882) Google Scholar), and it may be involved in a feedback loop that and In the feedback loop between E2F1–3 and miR-20a, of be to the activation of the mir-17–92 cluster by (19O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. Nature. 2005; 435: 839-843Crossref PubMed Scopus (2467) Google Scholar). E2F1–3 are to the transcription of K. H. R. M. Genes Dev. PubMed Scopus Google Scholar), and can the transcription of E2F1–3 G. J. R. Jakoi L. Nevins J.R. Nature. 1997; PubMed Scopus Google Scholar, M.R. R. F. Leone G. Nevins J.R. Cell. Biol. 2000; PubMed Scopus Google Scholar), and both transcription factors the mir-17–92 cluster, it suggest that this may a novel of the loop S. U. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), which can be as a loop of the feedback loop between E2Fs and the miR-20a family of miRNAs be to a mechanism to E2F E2F activity is for the it can to cell death or malignant on the cellular The of this may be to normal cell cycle where E2F1–3 can to the well that E2F1–3 regulate their own This also the that the E2Fs with miR-20a inhibition may from an increased E2Fs mRNA translation but may be in by a of the of the E2F1–3 genes by E2F Our that miR-20a targets the of the E2F1 mRNA with the mRNAs of E2F2 and E2F3 suggest that E2F1 are critical to cell possibly to the of E2F1. This regulation may also be to cell where the family is by hyperphosphorylation and E2F activity is to the cell cycle J. E. R. S. Cartwright P. S. Biol. Cell. 2005; PubMed Scopus Google Scholar). Interestingly, the mir-17–92 cluster is in mouse cells J.M. J. C.M. Hammond S.M. Nat. 2004; PubMed Scopus Google Scholar), which suggests the that in the of E2F1–3 activity may be controlled by this feedback The autoregulatory feedback loop may be also the of normal cells into E2F1 is to have both and oncogenic on the cellular the miRNA mechanism that E2Fs can promote or oncogenic role for the miR-20a family of miRNAs is with the anti-apoptotic role of this miRNA in this expression of the mir-17–92 cluster in a mouse (23He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. Nature. 2005; 435: 828-833Crossref PubMed Scopus (3146) Google Scholar). While the anti-apoptotic of miR-20a may explain this oncogenic activity and the in cell death in the mouse the mir-17–92 cluster, a has shown that apoptosis in the mouse is J. E. C. A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). it is that other members of the mir-17–92 cluster may other In the of miRNAs are and the in where expression is the other in other cell the miR-20a family may act as a by preventing the activity of In the mir-17–92 cluster found in a of and L. J. N. J. A. S. A. M.R. G. A. A. D. A. G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: PubMed Scopus Google Scholar). Our results to the of the complex pathways E2F activity and are for studies on cell cycle cell and In the between miRNAs and transcription factors may a We of for the and K. Helin for the E2F1–3 expression with

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".

There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1-4 (RXFP1-4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.

Tumor-specific antigens (TSAs) represent ideal targets for cancer immunotherapy, but few have been identified thus far. We therefore developed a proteogenomic approach to enable the high-throughput discovery of TSAs coded by potentially all genomic regions. In two murine cancer cell lines and seven human primary tumors, we identified a total of 40 TSAs, about 90% of which derived from allegedly noncoding regions and would have been missed by standard exome-based approaches. Moreover, most of these TSAs derived from nonmutated yet aberrantly expressed transcripts (such as endogenous retroelements) that could be shared by multiple tumor types. Last, we demonstrated that, in mice, the strength of antitumor responses after TSA vaccination was influenced by two parameters that can be estimated in humans and could serve for TSA prioritization in clinical studies: TSA expression and the frequency of TSA-responsive T cells in the preimmune repertoire. In conclusion, the strategy reported herein could considerably facilitate the identification and prioritization of actionable human TSAs.

Comparison of constitutional and tumor genotypes at chromosomal loci defined by restriction fragment length alleles has proven useful in determining the genomic position and tissue specificity of recessive mutations that predispose to cancer (Hansen, M.F., and Cavenee, W.K. Cancer Res., 47:5518-5527, 1987). Here we have applied this approach to 53 unrelated patients with glial tumors of varying histological malignancy grade. Loss of constitutional heterozygosity for loci on chromosome 10 was observed in 28 of 29 tumors histologically classified as glioblastoma (malignancy grade IV) whereas no similar losses were observed in any of 22 gliomas of lower malignancy grade. Examination of restriction fragment length alleles on other chromosomes revealed that loss of sequences on chromosomes 13, 17, or 22 had occurred at nonrandom frequencies and in at least one instance of each malignancy grade of adult glioma. The tumors in which loss of constitutional heterozygosity was observed were composed of one or a mixture of glial cell subtypes displaying astrocytic, oligodendrocytic, and/or ependymal differentiation. These results demonstrate a close association of the loss of chromosome 10 sequences with the most malignant histological stage of glioma and that glioblastoma arises as the clonal expansion of an earlier staged precursor. Furthermore they suggest that glioblastoma is a common phenotypic and malignancy terminus for glial tumors of various cellular subtypes which is reached through a common molecular pathway. This approach which involves the identification of malignancy stage specific somatic losses of heterozygosity provides a genotypic, rather than phenotypic, analysis of tumor progression.

Translational regulation plays a critical role in the control of cell growth and proliferation. A key player in translational control is eIF4E, the mRNA 5' cap-binding protein. Aberrant expression of eIF4E promotes tumorigenesis and has been implicated in cancer development and progression. The activity of eIF4E is dysregulated in cancer. Regulation of eIF4E is partly achieved through phosphorylation. However, the physiological significance of eIF4E phosphorylation in mammals is not clear. Here, we show that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide analysis of translated mRNAs, we show that the phosphorylation of eIF4E is required for translational up-regulation of several proteins implicated in tumorigenesis. Accordingly, increased phospho-eIF4E levels correlate with disease progression in patients with prostate cancer. Our findings establish eIF4E phosphorylation as a critical event in tumorigenesis. These findings raise the possibility that chemical compounds that prevent the phosphorylation of eIF4E could act as anticancer drugs.

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3 alpha/beta (GSK3 alpha/beta) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.