Europelta carbonensis n. gen., n. sp. is a nodosaur and is the sister taxon to the Late Cretaceous nodosaurids Anoplosaurus, Hungarosaurus, and Struthiosaurus, defining a monophyletic clade of European nodosaurids– the Struthiosaurinae.Nodosaurids are poorly known from the Lower Cretaceous of Europe. Two associated ankylosaur skeletons excavated from the lower Albian carbonaceous member of the Escucha Formation near Ariño in northeastern Teruel, Spain reveal nearly all the diagnostic recognized character that define nodosaurid ankylosaurs. These new specimens comprise a new genus and species of nodosaurid ankylosaur and represent the single most complete taxon of ankylosaur from the Cretaceous of Europe. These two specimens were examined and compared to all other known ankylosaurs. Comparisons of these specimens document that  Hylaeosaurus armatus  described in 1833 Hylaeosaurus is one of the three dinosaurs on which the Dinosauria were defined th 1865 issue of the “The Illustrated London News” by Sir Richard Owen Polacanthus was not described formally as Polacanthus foxii by Hulke until 1882 Acanthopholis from the base of the Upper Cretaceous (Cenomanian) Anoplosaurus curtonotusPolacanthusAnkylosaurs were first described from the Lower Cretaceous of England with Struthiosaurus austriacus described from the Campanian of Austria in 1871 Struthiosaurus transylvanicusStruthiosaurusStruthiosaurus languedocensis from the Campanian of southern France Hungarosaurus tormaiOnly nodosaurids have been described from the Upper Cretaceous of Europe with Fragmentary ankylosaur remains are also known from a number of localities from the Middle to Upper Jurassic strata of Europe, but have been relatively uninformative as specimens are based largely on isolated skeletal elements PolacanthusStruthiosaurusNortheastern Spain has contributed many dinosaur discoveries from both Lower and Upper Cretaceous strata in recent years Proa valdearinnoensisEuropelta carbonensis n. gen., n. sp. This new taxon is the most completely known ankylosaur in Europe and adds considerable new information about Early Cretaceous ankylosaurian phylogeny and biogeography.The earliest reported dinosaur remains from Spain were found in the Escucha Formation, few significant vertebrate fossils had been recovered from these rocks in the 140 intervening years Formación Lignitos de Escucha and overlying Formación Arenas de Utrillas were initially described in 1971 Counterclockwise rotation of the Iberian Plate toward the end of the Early Cretaceous resulted in the development of a series of syndepositional sub-basins bounded by active faults within Ebro Basin south of the Pyrenean ranges, northeast of the Iberian Range, and northwest of the Catalan/Coastal Range The geologic age of the Escucha Formation has been considered to be early to middle Albian. It overlies Aptian strata in central basinal settings and is, in turn, overlain by the upper Albian Utrillas Formation A sample of the matrix from the bonebed was processed for both palynomorphs and calcareous microfossils. The palynomorphs were exclusively of terrestrial origin and indicated an Albian age . The microfossils consisted exclusively of freshwater ostracods and charophytes. The ostracods represent new species and the charophytes are also reported from the Albian of Tunisia The bonebed is located immediately below the lowest mineable coal seam in the Santa María coal mine , in a daThe bonebed was located many tens of meters underground prior to strip mining operations in the Santa María coal mine. As mining operations proceed, more of the plant debris stratum containing the bonebed is exposed as simultaneous reclamation covers the previously exposed surface. Thus, with the help of mine managers, efficient methodologies for the documentation and extraction of significant fossils have been established The bonebed designated AR-1 contains more than 5000 identifiable vertebrate specimens recovered from isolated skeletal remains and associated individual animals. All fossils receive a consecutive number from the site, each association is numbered as well. Thus:AR-1-#fossil identifies each fossil found at the Ariño site (the ID written on each fossil);AR-1/#concentration identifies a collection of bones belonging to a single skeleton;AR-1-#fossil/#concentration identifies a fossil from a bone concentration # belonging or not belonging to a single skeleton.The two associated ankylosaur skeletons described herein were separated by 200 meters. The location of the holotype AR-1/10  was stilAll of the specimens described in this paper (AR-1/10 and AR-1/31) are reposited in the collections of the Fundación Conjunto Paleontológico de Teruel-Dinópolis/Museo Aragonés de Paleontología (FCPTD/MAP). Locality information is available from the registrar of the museum as per museum policy. All necessary permits were obtained for the described study, which complied with all relevant regulations. All of these specimens were collected under permits obtained from the Sociedad Anónima Minera Catalano-Aragonesa.http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:9246FFA7-6271-4734-8E01-5590BE4A80C2. The LSID for Europelta carbonensis is: urn:lsid:zoobank.org:act:089040A3-1BCF-42D1-B99F-94840E2BB96D. The electronic edition of this work was published in a journal with an ISSN (1932-6203), and has been archived and is available from the following digital repositories: LOCKSS (http://www.lockss.org); PubMed Central (http://www.ncbi.nlm.nih.gov/pmc).The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “Gargoyleosaurus, Mymoorapelta, Hylaeosaurus, Polacanthus, Hoplitosaurus, and Gastonia). The most recent analysis of polacanthids as a monophylogenetic subfamily of nodosaurids was by Yang and others Polacanthus foxii but not Ankylosaurus magniventris or Panoplosaurus mirus.We do not refer to the “armor” on the skull roof as caputegulae, as we consider these patterns in the Nodosauridae to reflect impressions of scale boundaries on the skull roof as opposed to thickened remodeled cranial bone. We use the term caudal rib instead of caudal transverse process. We employ the monophyletic clade Polacanthidae of Carpenter AMNH, American Museum of Natural History, New York, New York, NHMUK, Natural History Museum, London, England, CEUM, Prehistoric Museum, Utah State University, Price, Utah, DMNH, Denver Museum of Nature and Science, Denver, Colorado, MPC, Geological Institute, Ulaan Bataar, Mongolia, FCPTD/MAP, Fundación Conjunto Paleontológico de Teruel-Dinópolis/Museo Aragonés de Paleontología, Teruel, Spain, FMNH, Field Museum of Natural History, Chicago, MPC, Institute of Geology, Mongolian Academy of Sciences, Ulaan Baatar, Mongolia; INBR, Victor Valley Museum, Apple Valley, California, IVPP, Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China, KUVP, Kansas Museum of Natural History, Lawrence, Kansas, MPC, Mongolian Paleontological Center, Ulaan Baatar, Mongolia; MNA, Museum of Northern Arizona, Flagstaff, Arizona, NMC, National Museum of Canada, Ottawa, Canada, NMW, National Museum of Wales, Cardiff, England, PIN, National Institute of Paleontology, Moscow, Russia, QM, Queensland Museum, Queensland, Australia, ROM, Royal Ontario Museum, Toronto, Canada, SDNHM, San Diego Natural History Museum, San Diego, California, SGDS, Saint George Dinosaur Discovery Site at Johnson Farm, St. George, Utah, SMP, State Museum of Pennsylvania, Harrisburg, Pennsylvania, SMU, Schuler Museum, Southern Methodist University, Dallas, Texas, USNM, National Museum of Natural History, Smithsonian Institution, Washington D.C.Scutellosaurus lawleri (MNA P1.175), the type material of Scelidosaurus harrisoni (NHMUK R 1111), and a large, exceptionally well-preserved, articulated Scelidosaurus specimen with intact armor, collected and owned by David Sole and currently exhibited at the University of Bristol. Also, a full cast of the left side of the skeleton (SGDS 1311) exhibited in southwestern Utah was examined.In addition to accessing the ever-expanding ankylosaur literature, the senior and third authors have had the opportunity to study firsthand much of the important ankylosaur material collected globally. From the basal thyreophorans: the type material of Mymoorapelta maysi housed at the Museum of Western Colorado, Gargoyleosaurus parkpinorum (DMNH 27726), and the dentary of Sarcolestes leedsi (NHMUK R 2682) were studied.In regards to Jurassic ankylosaurs: the extensive type and paratype material of Polacanthus foxii , Hylaeosaurus armatus (NHMUK R 3775), Hoplitosaurus marshi (USNM 4752), and the extensive material of Gastonia burgei material housed at the Prehistoric Museum , and cranial material from a minimum of six individuals at Brigham Young University's Earth Science Museum, together with the postcranial skeleton of an unnamed new species of polacanthine (BYU 245).Early Cretaceous polacanthine ankylosaur material examined includes Cedarpelta bilbyhallorum , Shamosaurus scutatus (PIN 3779/2), and a cast of the skull of Gobisaurus domoculus (IVPP 12563) housed at the Royal Tyrell Museum were studied.Among basal shamosaurine-grade ankylosaurids, Nodocephalosaurus kirtlandensis (SMP-VP-900), Ankylosaurus magniventris , Anadontosaurus lambei (NMC 8530), Dyoplosaurus acutosquameus (ROM 784), Scolosaurus cutleri , and several important examples of Euoplocephalus tutus,  were examined.Among derived North American ankylosaurs, Tsagantegia longicranialis (MPC 100/1306), China, Pinacosaurus grangeri (AMNH 6523) and three undescribed skulls personally excavated by JIK from the Djadokhta Formation, Shabarakh Usu  and housed at MAS, Talarurus plicatospineus , cast skull of Saichania chulsanensis (PIN 3141/251), a relatively complete specimen referred to Saichania with in situ armor but lacking its skull (MPC 100/1305), Tarchia gigantea (PIN 3142/250), a cast skull of Minotaurasaurus ramachandrani (INBR 21004), and a cast skeleton of Crichtonsaurus benxiensis housed in the Museum at the Chaoyang Bird National Geopark, Liaoning.Asian ankylosaur material researched include an adult skull of Sauropelta edwardsi , Peloroplites cedrimontanus , and Pawpawsaurus campbelli . The early Late Cretaceous nodosaurids reviewed include Animantarx ramaljonesi (CEUM 6228), Silvisaurus condrayi (KUVP 10296), Nodosaurus textilis (YPM 1815), and Stegopelta landerensis(FMNH UR88) and the Late Cretaceous nodosaurids Panoplosaurus mirus (NMC 2759), Edmontonia rugosidens , Edmontonia longiceps (NMC 8531), Denversaurus schlessmani (DMNH 468), casts of Struthiosaurus austriacus at the Carnegie Museum (PIUW 2349) and Struthiosaurus transylvanicus (NHMUK R 4966).Numerous nodososaurids were examined, including the Early Cretaceous nodosaurids Minmi paravertebrata (QM F18101), the skeleton of Liaoningosaurus paradoxus (IVPP V12560), and Aletopelta coombsi (SDNHM 33909) were also examined.Enigmatic taxa such as the skull of Dinosauria Owen, 1842 Ornithischia Seeley, 1887 Thyreophora Nopcsa, 1915 Ankylosauria Osborn, 1908 Nodosauridae Marsh, 1890 Struthiosaurinae Nopcsa, 1923 Struthiosaurinae is defined as the most inclusive clade containing Europelta but not Cedarpelta, Peloroplites, Sauropelta or Edmontonia.Nodosaurid ankylosaurs that share a combination of characters including: narrow predentaries; a nearly horizontal, unfused quadrates that are oriented less than 30° from the skull roof, and condyles that are 3 times transversely wider than long; premaxillary teeth and dentary teeth that are near the predentary symphysis; dorsally arched sacra; an acromion process dorsal to midpoint of the scapula-coracoid suture; straight ischia, with a straight dorsal margin; relatively long slender limbs; a sacral shield of armor; and erect sacral armor with flat bases. Europelta Kirkland, Alcalá, Loewen, Espílez, Mampel, and Wiersma 2013 gen. nov.urn:lsid:zoobank.org:act:62808E3D-85BE-4AE3-B771-9CFF2C6AC054“Euro” as a contraction for Europe in regard to its origin and “pelta” Greek for shield, a common root for ankylosaurian genera; “Europe's shield”.Same as for the only known species below.Europelta carbonensis Kirkland, Alcalá, Loewen, Espílez, Mampel, and Wiersma 2013 gen. et sp. nov.urn:lsid:zoobank.org:act:089040A3-1BCF-42D1-B99F-94840E2BB96Dcarbonensis” from the coal, is in honor of access to the fossil locality in the Santa María coal mine provided by Sociedad Anónima Minera Catalano-Aragonesa (SAMCA Group), which has been extracting coal in Ariño (Teruel) since 1919.The specific name “AR-1/10, a disarticulated partial skeleton reposited at Fundación Conjunto Paleontológico de Teruel-Dinópolis/Museo Aragonés de Paleontología (FCPTD/MAP). The holotype consists of: a mostly complete skull (AR-1-544), isolated left and right nasals , a dentary fragment (AR-1-362), 15 isolated teeth , an atlas (AR-1-649), five cervical vertebrae , two cervical ribs , AR-1-638 , seven more posterior dorsal vertebrae , a section of synsacrum (AR-1-154), three isolated dorsal ribs , seven dorsal rib fragments , three caudal vertebrae , four chevrons , a coracoid with a small portion of scapula (AR-1-657), a scapular blade fragment (AR-1-429), two xiphosternal plates , two partial humeri , a right ilium-ischium-pubis (AR-1-479), a left ischium-pubis (AR-1-129), and 70 osteoderms .AR-1/31, a partial skeleton deposited at Fundación Conjunto Paleontológico de Teruel-Dinópolis/Museo Aragonés de Paleontología (FCPTD/MAP). The paratype consists of a partial left jaw with dentary and surangular (AR-1-3698) and isolated angular (AR-1-2945), 10 teeth , five cervical vertebrae , nine dorsal vertebrae , three to four? dorsosacral vertebrae , a sacrum (AR-1-3446), a caudosacral vertebra (AR-1-3512), two sacral rib fragments , 14 caudal vertebrae , a right ilium (AR-1-3490), two left ilium fragments , two ischia with fused pubes , a right femur (AR-1-3244), a right tibia (AR-1-3237), a right fibula (AR-1-3238), a calcaneum (AR-1-3239), four metatarsals , eight phalanges , nine unguals , and 90 osteoderms .The type locality, Fundación Conjunto Paleontológico of Teruel-Dinópolis locality AR-1, is located east of Ariño, Teruel Province, Spain. The fossil horizon is below the lowest mineable coal seam at Sociedad Anónima Minera Catalano-Aragonesa Group's Ariño coal mine in a plant debris bed in the lower Escucha Formation Elsewhere, the Escucha Formation has been interpreted as late Aptian to early Albian in age based on nanofossils, planktonic foraminifera, dinoflagellates and palynomorphs The quadrate is shorter and mediolaterally wider than in any other ankylosaur. The posterior margin of the skull is concave in dorsal view. The sacrum is arched dorsally about 55° in lateral view. The pubis is fully and uniquely fused to the ischium with a slot-shaped foramen between the post-pubic process and the position of the pubic peduncle forming an ischiopubis. The tibia is longer relative to the length of the femur (90%) than in other ankylosaurs for which these proportions are known. Laterally compresed, flanged osteoderm with a flat plate-like base is present anteriorly on the pelvic shield.The skull (AR-1-544/10) was lying on its dorsal surface and is moderately well preserved although distorted through compaction . The palThe skull has a minimum length of 370.3 mm from the anterior end of the maxillae to the rear margin of the squamosals. The skull has a maximum width of 299.1 mm at the orbits and narrows to 203.7 mm at the posterior end of the skull at the squamosals, giving the skull the “pear-shaped” dorsal profile characteristic of derived nodosaurids EdmontoniaPawpawsaurus, Edmontonia, and PanoplosaurusSilvisaurusThe maxillae  are irreSilvisaurusNiobrarasaurusThe nasals  are relatively large and subrectangular, tapering somewhat anteriorly . Both naMymoorapelta, Gargoyleosaurus, and Gastonia where the suborbital horn is below the orbit and is formed exclusively by the jugal The orbits are somewhat crushed and the sutures of the bones surrounding them are obscured by fusion. The orbits are subrectangular in shape, are slightly more elongate anteoposteriorly and are directed anterolaterally. The prominent and evenly rounded suborbital horn is formed mostly from the quadratojugal posterior to the ventral margin of the orbit, as in most derived ankylosaurs PeloroplitesSilvisaurusStruthiosaurus transylvanicusEdmontonia (ROM 1215) Europelta. The lower temporal opening is completely obscured in lateral view in CedarpeltaShamosaurusGobisaurus, ZhongyuansaurusThe lateral wall of the skull extends posteriorly behind orbit with a dorsoventally wide posterior notch, such that the lower temporal opening is just visible in lateral view. There is no lateral wall of skull behind the orbits in polacanthids Although the palate is fragmented and crushed along the internal surface of the skull roof, the fragments of the vomer suggest it did not extend ventrally to the level of the tooth row. Additionally, the broad sheet-like pterygoids appear to have been flexed nearly dorsally against the anterior portion of the basicranium as in nodosaurids and not like the open transversely oriented pterygoids characteristic of ankylosaurids or polacanthids Mymoorapelta  and CedarpeltaThe posterolateral margin of the pterygoid is fully fused to the quadrate. There is a sutural contact between the straight, nearly vertical quadrates and the quadratojugal laterally. The quadrates are wide transversely and thin rostrocaudally as compared to the mediolaterally narrower quadrates of other ankylosaurs Gargoyleosaurus, Gastonia, and most other derived anklylosaurs, and unlike the narrow, highly arched occipital region of StruthiosaurusGargoyleosaurusGastoniaVertical compaction has obscured the posterior view of the skull, in particular the foramen magnum and the supraoccipital. However, even with compaction it is apparent that in occipital view the skull was subrectangular and wider than tall as in CedarpeltaCedarpeltaThe subspherical occipital condyle  has a wiCedarpelta, the nodosaurids Sauropelta and Peloroplites, and the shamosaurine-grade ankylosaurids Shamosaurus and GobisaurusEuropelta differs from these specimens in that some of the margins of the scale impressions on the skull roof are visible, as seen in Edmontonia, Panoplosaurus and StruthiosaurusPeloroplites, Cedarpelta, Shamosaurus, and GobisaurusThe skull roof  is roughStruthiosaurusIn dorsal view, the posterior margin of the skull is concave, whereas it is nearly straight or convex in all other nodosaurids. This reflects the posterior angulation of the paraoccipital processes and the squamosal horns. Interestingly, the occipital condyle is barely visible, though not completely obscured in dorsal view. There is no evidence of any distinct nuchal sculpturing. The skull roof is relatively flat but a slight dome may have been present prior to crushing. However, it is clear that the skull roof is not as highly domed as in many other nodosaurids, such as Attempts were made to image the skull using X-ray photography and CT scanning. The abundance of pyrite present in the skull  presentsA small dentary fragment extending for only four complete alveolae (AR-1-133/10) was preserved from the holotype skeleton . HoweverSarcolestesGargoyleosaurus, SilvisaurusAnimantarxSauropeltaAnoplosaurusHungarosaurusStruthiosaurusEuropelta is robust and dorsoventrally deeper (45.0 mm deep and 29.00 mm across) than in ankylosaurs HungarosaurusHungarosaurus is deeper dorsoventrally than that of EuropeltaThe dentary is 184.7 mm long with a minimum of 21 tooth positions, with no possibility of more than two unpreserved alveoli as determined by the position of the suture with the angular and surangular. As with the maxillary teeth, the alveoli are more than twice as large posteriorly. There is only 1.5 cm between the anteriormost alveoli and the symphysis, suggesting that there may have been premaxillary teeth as at least nine anterior teeth would have been positioned to oppose the premaxilla. The primitive ankylosaurs The splenial  is a thiEuoplocephalusMinataurasaurusGargoyleosaurusSauropeltaPanoplosaurusThe angular  has a maA large number of teeth are preserved from both the holotype AR-1/10 (20+) and the paratype AR-1/31 (15+) although many have drifted away from the alvaeolae. We assume that the teeth associated with the holotype pertain to the maxilla  and those of the paratype pertain to the dentary . In general, the cutting surfaces of the teeth are not well preserved, but a few exceptions exist. Wear facets were not observed on any of the teeth. The roots for both dentary and maxillary teeth are swollen lingually, are three to four times the length of the crowns, and are subquadrate in cross-section. One small tooth (AR-1-343/10) is more highly asymmetrical mesiodistally and may represent a premaxillary tooth .The isolated maxillary teeth  have a wThe isolated dentary teeth  are idenEuropelta are most comparable to those of other nodosaurids Cedarpelta, SauropeltaEdmontonia and PanoplosaurusSarcolestes and PriodontognathusGargoyleosaurusMymoorapelta , the nodosaurids PeloroplitesHungarosaurusGobisaurus are more inflated labiolingually than in Europelta and other ankylosaurs. The teeth of Gastonia and putative Polacanthus teeth are also inflated, but are smaller proportionally Europelta differ from an isolated tooth from the Cenomanian of France which is about half the size, and proportionally is longer mesiodistally with more deeply divided denticles forming ridges on the labiolingual surfaces of the tooth Acanthopholis” have more deeply divided denticles in what is a proportionally taller tooth Struthiosaurus languedocensisWith their relatively large size and well-developed cingula, the teeth of There are numerous ribs and vertebrae preserved from the holotype (AR-1/10) and the paratype specimen (AR-1/31). Vertebral measurements are presented in The complete atlas (AR-1-649/10) from the holotype has a total width of 195.6 mm . The neuAnimantarxThere are five post-axis cervical vertebrae  preserved from the holotype skeleton  and fiveSaichaniaAnkylosaurusSilvisaurusThere are two complete cervical ribs preserved for the holotype. AR-1-450/10 is a relatively anterior cervical rib  and AR-1PolacanthusSauropeltaSauropeltaShamosaurusSeveral amphiplatan to amphicoelus dorsal vertebra are preserved: eight for the holotype AR-1/10 and nine for the paratype AR-1/31. The diapophyses originate at the level of the post-zygopophyses at the dorsal extent of the neural canal. The more anterior vertebrae have large cylindrical amphiplatan centra which lack a constricted ventral keel with circular neural canals and fused ribs . The broad transverse processes are T-shaped in cross-section and angled dorsally, unlike the laterally directed transverse processes in There are a number of rib fragments preserved with AR-1/10, but there are only three  relatively complete ribs . As withEuoplocephalusSaichaniaThe sacrum is not preserved in AR-1/10 other than an anteriormost centrum (AR-1-154/10) of the synscacrum . HoweverStruthiosaurus languedocensisEuropelta. Similarly, the sacrum of Hungarosaurus, as exhibited at the Hungarian Natural History Museum, appears to be moderately arched. The moderate angulation of the faces of the sacral centra  in AnoplosaurusSilvisaurus, which appears to be restricted to the posterior part of the sacrum and two sacrocaudals. In other ankylosaurs, the downward flexure of the tail from the hips is taken up in the proximal caudal vertebrae as in MymoorapeltaEuoplocephalusThe sacrum of GargoyleosaurusCedarpeltaEdmontoniaHungarosaurusEuoplocephalusMymoorapeltaGastoniaPolacanthusSauropeltaSilvisaurusEuropelta originate high on the sides of the centrum and angle ventrally proximal to flexing laterally, giving them a dorsally concave profile in anterior view like Hungarosaurus, Struthiosaurus, and Peloroplites, and unlike the ventrally flexed caudal ribs of many polacanthids Acanthopholis” GargoyleosaurusCedarpelta, PeloroplitesEdmontoniaHylaeosaurus differ in being swept back posteriorly PeloroplitesStruthiosaurus, which actually appear to bifurcate Only three proximal caudal vertebrae  are present . The proPinacosaurus and SaichaniaAnkylosaurusEdmontonia (ROM 1215) Additionally, there are four chevrons preserved from about the same region of the tail  of which three are illustrated . The proNodosaurusMymoorapeltaSauropeltaSeveral more distal caudal vertebrae are preserved in the paratype. The two most proximal of these  have centra of nearly equal height, width, and length, with a ventral groove, and caudal ribs shorter than the diameter of the centrum that extend laterally and angle posteriorly . The cheParts of the right scapulocoracoid are preserved. A portion of the distal scapular blade (AR-1-429/10) is preserved with a portion of the distal ventral margin missing with a curved section broken away. There is no evidence of any distal expansion of the scapular blade as in many nodosaurids PeleroplitesTexasitesAnimantarxShamosaurusThe coracoid (AR-1-657/10) is preserved with only the most proximal portion of the scapula fused on . It appeEuropelta, whereas similar in overall shape to other nodosaurid xiphisterna, are not fenestrate or scalloped along their margins as in North American nodosaurids for which they are known Both xiphisternal plates are preserved . The besSauropeltaParts of both humeri are preserved. The right humerus (AR-1-655/10) is represented by the proximal end . It is 2Mymoorapelta  compared to the longer deltopectoral crests of ankylosaurids Europelta is similar in proportions to NiobrarasaurusStruthiosaurus is similar to that of Europelta, whereas the humerus of co-occuring Hungarosaurusis is more slender proportionally.The left humerus (AR-1-327/10) is represented by a midshaft for which both the proximal and distal ends appear to have rotted off and the core of the shaft has rotted away . The shaAmong the nine unguals preserved for AR-1/31, one specimen (AR-1-3711/31) may represent a manual ungual. It is more equidimensuional than the other eight more elongate unguals.Mymoorapelta is known to retain an open acetabulum Scelidosaurus and stegosaurs The right ilium of AR-1/10 is fused with its ischium and pubis (AR-1-479/10) which are flexed medially due to compaction . The aceCedarpeltaStruthiosaurusHungarosaurusBeyond some relatively uninformative fragments of the ilium , AR-1/31Polacanthus and nodosaurids, a derived character Mymoorapelta) is bent, a trait that is also observed in some stegosaurs such as KentrosaurusMymoorapelta (Kirkland pers. obs.) and GastoniaEuropelta is the oldest known ankylosaur preserving a straight ischium. The slight kink in the distal end of the ischium of Europelta suggests the straight ischium in European nodosaurids and ankylosaurids is achieved by shortening the ischium distal to the bend.A straight ischium has been considered to be the primitive character state for ankylosaurs, with the bent ischium of Europelta is expanded such that it overhangs the femoral shaft both anteriorly and posteriorly. The greater trochanter is well demarcated from the femoral head by a constriction across the proximal end of the femur, and the anterior trochanter forms a ridge ventral to the greater trochanter that is fully fused to the femur. The robust fourth trochanter overlaps the midpoint of the femoral shaft and its midpoint is located proximal at the midpoint of the femur. Polacanthids and nodosaurid ankylosaurs have this configuration, whereas in ankylosaurids the fourth trochanter is distal to the middle of the shaft The right femur, tibia, and fibula were closely associated . The robMymoorapeltaThe right tibia (AR-1-3237/31) and fibula (AR-1-3238/31) were closely associated  and postCedarpeltaMymoorapeltaGastoniaPolacanthusSauropeltaPeloroplitesZhejiangosaurusSaichaniaMymoorapeltaGastoniaHylaeosaurusPeloroplitesThe tibia is 458.8 mm long and robust for its entire length  as in CeEuropelta has the proportionally longest tibia of any ankylosaur for which this ratio is known. Both Cedarpelta and Peloroplites have relatively longer tibiae than other ankylosaurs Peloroplites differs in its proportionally more narrowly waisted tibial shaft.Generally, ankylosaurids have tibiae that are less than two-thirds the length of their femora, as opposed to nodosaurids which have proportionally longer lower leg elements The fibula is 395.5 mm long  and lateAnodontosaurusNiobrarasaurus coleii preserves an articulated lower hind limb, with an astragalus fully fused with the tibia and possessing an articulation with the distal end of the fibula and an unfused calcaneum of similar morphology to that of EuropeltaSaichaniaA calcaneum (AR-1-3289/31) was identified in association with the lower right leg of AR-1/31. It is laterally compressed, convex laterally and concave medially . Its dorEuropelta possesses four pedal phalanges as in most other nodosaurids Liaoningosaurus has three digits on the pes. The eight similar unguals are interpreted as pedal unguals and the smallest ungual , possibly from the cervical or pectoral region, is recognized from the holotype . It appeMymoorapeltaGargoyleosaurusGastoniaPolacanthusMinmiEuropelta are mediolaterally shorter and anteroposteriorly longer with a more posteriorly swept apices. Two pairs of similar plates are known for the holotype of Sauropelta (AMNH 3032), with one of the larger plates being illustrated Sauropelta has a unique double apex (YPM 5490). Given the rarity of Type B armor in Sauropelta and Europelta we hypothesize that caudal plates in these nodosaurids ran down the sides of the tail but decreased in size more rapidly, such that long-keeled osteoderms of Type E morphology made up the lateral armor down most of the length of the tail. It is also possible that these large plate-like osteoderms were on the lateral margin of the sacrum as has been documented by Carpenter and others Saichania. Struthiosaurus preserves several osteoderms of this morphology that have been reconstructed as in Polacanthus as being medial, dorsally-projecting caudal osteoderms Dorsoventrally compressed, hollow, asymmetric-based plate-like osteoderms with sharp anterior and posterior edges and lateroposteriorly directed apices are identified for AR-1/10  and AR-1StegopeltaNiobrarasaurusPanoplosaurusEdmontoniaBoth AR-1/10  and AR-1Both AR-1/10 and AR-1/31 preserve large (∼10-20 cm long) asymmetric, diamond  to tear-SauropeltaPanoplosaurusEdmontonia. Gastonia is documented to have similar armor Tarchia by Arbour and others Tarchia most of the intermediate scales lacked ossified cores. Similar armor is known from the lateral sides of the legs in some ankylosaurs such as SaichaniaType D Armor is widely known in the nodosaurids such as SauropeltaTexasetesScelidosurus and SaichaniaBoth AR-1/10 and AR-1/31 preserve large (10-15 cm long) moderately asymmetric osteoderms more than twice as long as wide with a long keel higher on the assumed posterior end . These oSauropelta anterior to the sacrum in AMNH 3036 PanoplosaurusEdmontonia  and the polacanthids Sauropelta (AMNH 3036). They may also lie on the tail between the Type B caudal plate-like osteoderms, or could be arranged along the lateral side of the limbs as in SaichaniaMedium to large (∼5-15 cm long) oval to circular osteoderms of low profile with a median keel extending into an apex near or overhanging the posterior margin of the osteoderm are represented in both AR-1/10  and AR-1Sauropelta specimen AMNH 3032 was curated with a note from the collector, Barnum Brown, stating that these distinct osteoderms were associated with the forelimbs. Therefore, we suggest a similar position for Type G armor in Europelta.One piece (AR-1-192/10) of flat, oval to subtriangular armor (AR-1-192/10) from AR-1/10 is about 12 cm long and 7 cm wide and is about 0.5 cm thick throughout . A pair SauropeltadmontoniaGastoniaTarchiaGastonia ossicles is smoother than those of Europelta.Small (∼1-4 cm long) solid ossicles are abundant, with 71 examples from both AR-1/10  and AR-1StegopeltaNodosaurusAletopeltaGlyptodontopeltaAletopeltaPolacanthus and GastoniaArmor is present on the posterior margin of the ilium AR-1-479/10. It is composed of large, subequal-sized (7-10 cm) osteoderms that are tightly sutured together  as in thHungarosaurus and interpreted to be present in StruthiosaurusAdditionally, there is a unique osteoderm AR-1-653/10 that has a large, posteriorly-curved, plate-like keel extending out from the surface that, considered in isolation, is comparable in size and morphology to Type B armor . The basSome irregularly shaped armor specimens are not represented by more than one element among this material or in the armor from other taxa. At this time, we can offer no positional interpretation of this armor. AR-1-447/10 is an irregular mass of what we interpret as an osteoderm, although it could be sacral armor . AR-1-43Europelta  ankylosaurids and was the first shamosaurine-grade ankylosaurid documented to not have a tail club. However, its distal tail is modified into a stiffened structure of the same morphology as the “handle” of the tail club in more derived ankylosaurids Zhejiangosaurus was assigned to the nodosaurids based on characteristics of the femur and sacrum, together with the lack of a tail club Zhejiangosaurus have raised ridges on the sides of the centra as in the distal vertebrae of polacanthids and nodosaurids. Additionally, whereas the position of its most proximal preserved caudal vertebrae is not known, morphologically, they do not appear to represent the most proximal caudal vertebrae. Thus, while Zhejiangosaurus' 13 preserved caudal vertebra are more than the number of free caudals preserved in most ankylosaurs with tail clubs  and Struthiosaurus might represent a separate lineage of European nodosaurids. Unlike Hylaeosaurus (in which he included Polacanthus), these taxa had a well-developed supraspinus fossa developed anteriorly on the scapula as did all North American nodosaurids. This European lineage was hypothesized based on their small body size, presence of premaxillary teeth, and their possessing an unfused scapula and corocoid. Although, none of the characters are valid in defining such a group, our research on Europelta has resulted in supporting the taxonomic hypothesis of Coombs In his seminal paper defining a bipartite division of the Ankylosauria into Ankylosauridae and Nodosauridae, Coombs Acanthopholis ( =  Anoplosaurus), Polacanthus, Stegopelta, Stegoceras, and Struthiosaurus. In 1923, he divided the Acanthopholidae into an Acanthopholinae and a Struthiosaurinae without comment Ankylosaurus and restricted the Acanthopholinae to Acanthopholis, Hylaeosaurus, Rhodanosaurus, Struthiosaurus, TroodonAcanthopholis, now considered a nomen dubiumEuropelta but not Cedarpelta, Peloroplites, Sauropelta or Edmontonia.We use Struthiosaurinae to define the clade of European nodosaurs. Nopcsa Europelta, it was found that previous cladistic analyses In order to determine the systematic position of Europelta with the European nodosaurids Anoplosaurus, Hungarosaurus and all species assigned to Struthiosaurus. This clade of European nodosaurids has not been previously recognized. Europelta represents the earliest member of the European clade Struthiosaurinae.We present a character based definition of Struthiosaurinae as: nodosaurid ankylosaurs that share a combination of characters including: narrow predentary; a nearly horizontal, unfused quadrate that is oriented less than 30° from the skull roof, and mandibular condyles that are 3 times transversely wider than long; premaxillary teeth and dentary teeth that are near the predentary symphysis; dorsally arched sacrum; an acromion process dorsal to midpoint of the scapula-coracoid suture; straight ischium, with a straight dorsal margin; relatively long slender limbs; a sacral shield of armor; and erect pelvic osteoderms with flat bases. This suite of characters unites Europelta is the oldest nodosaurid known in Europe, it derived from strata in the lower Escucha Formation that is dated to early Albian. The oldest nodosaurid from western North America is Sauropelta, which in the lower part of its range is in the lower Albian Little Sheep Mudstone Member  of the Cloverly Formation in northern Wyoming and southern Montana Priconodon crassus are known from the Arundel Clay of the Potomac Group Propanoplosaurus is from the base of the underlying Patuxent Formation of the Potomac Group of Maryland, which has been dated as late Aptian Propanoplosaurus the oldest known nodosaurid. Polacanthid ankylosaurs characterize pre-Aptian faunas in both Europe Polacanthus in the Lower Aptian Vectis Formation of the Wealden Group, although Polacanthus has been reported to occur in those strata Sauropelta as reported by Carpenter and others HoplitosaurusSauropelta (DMNS 49764) has been recovered from the overlying Ruby Ranch Member about 20 m up section in the same region Tenontosaurus at this time, while in Europe the lower Albian more derived iguanodont Proa is phylogenetically close to IguanodonThe near simultaneous appearance of nodosaurids in both North America and Europe is worthy of consideration . EuropelAdditionally, whereas there is no definitive evidence for nodosaurids in Asia, apparently polacanthids entered Asia in the later portion of the Early Cretaceous and survived there in isolation into the early Late Cretaceous.Europelta carbonensis, a new nodosaurid ankylosaur from the lower Albian Escucha Formation in Spain represents the earliest member of a European clade of nodosaurs defined as the Struthiosaurinae. Other members of this Late Cretaceous clade include: Anoplosaurus, Hungarosaurus, and Struthiosaurus. This clade of nodosaurs replaced the polacanthids in Europe during the Albian, similar to the Albian replacement of polacanthids by nodosaurids in North America.


Recent therapeutic advances for managing advanced prostate cancer include the successful targeting of the androgen-AR axis with several new drugs in castrate resistant prostate cancer including abiraterone acetate and enzalutamide (MDV3100). This translational progress from “bench to bed-side” has resulted in an enlarging repertoire of novel and traditional drug choices now available for use in advanced prostate cancer therapeutics, which has had a positive clinical impact in prolonging longevity and quality of life of advanced prostate cancer patients. In order to further the clinical utility of these drugs, development of predictive biomarkers guiding individual therapeutic choices remains an ongoing challenge. This paper will summarize the potential in developing predictive biomarkers based on the pathophysiology of the androgen-AR axis in tumor tissue from patients with advanced prostate cancer as well as inherited variation in the patient's genome. Specific examples of rational clinical trial designs incorporating potential predictive biomarkers from these pathways will illustrate several aspects of pharmacogenetic and pharmacogenomic predictive biomarker development in advanced prostate cancer therapeutics. Prostate cancer (PCa) is the second leading cause of cancer-related mortality in US men with an estimated 33,720 deaths in 2011 . ADT theThis paper will evaluate potential opportunities for androgen-AR axis-based biomarker development with a specific focus on somatic genomic alterations of the AR and components of the androgen-AR axis. Emerging evidence of germline variation in androgen-AR axis genes and their effects on clinical outcomes of ADT responses in advanced PCa will also be discussed. Finally, the paper will present potential clinical design models and scenarios that incorporate androgen-AR axis-based biomarkers into the design of PCa therapeutic trials that use novel and emerging agents targeting androgen-AR axis biology in combination with ADT. The ultimate goal of these trials would be to enhance the current efficacy of drugs used for treating advanced PCa.α-reductase in some, but not all, tissue types (depending on the presence of type I or II isoenzyme) [The androgen-AR axis regulates activity of the AR transcription factor, which is a master regulator of the prostate lineage. The lineage dependency hypothesis is an offshoot of the oncogene addiction hypothesis , statingoenzyme) . Both DHoenzyme) . The AR oenzyme) , 15 and oenzyme) .In the unliganded state, the AR resides primarily in the cytoplasmic compartment of prostate epithelial and stromal cells, in a stable complex bound to heat shock proteins (Hsps). DHT binding to the AR leads to higher AR stability, greater biological activity, and slower dissociation than testosterone. Upon ligand activation, changes occur in the composition and conformation of the AR-Hsp complex, leading to nuclear translocation . In the The clinical efficacy of ADT in advanced PCa is heterogeneous and ranges from a few weeks to several years, with an overall median time of 18 to 30 months –8. ApproKnowledge of somatic alterations in the PCa genome continues to expand at a rapid pace. Numerous early studies laid a broad foundation by assessing copy number gains and losses in tumor DNA using low-resolution comparative genomic hybridization (CGH) with spotted or manufactured arrays. A combined analysis of 41 published PCa CGH studies, which represented a total of 872 individual tumors, revealed multiple genomic regions that displayed frequent gain or loss in the PCa genome . SubsequThese large-scale genomic studies have also reinforced the importance of the AR gene in PCa, particularly when the disease has progressed to a CRPC phenotype. For example, direct alterations in the AR do not occur in primary PCa but occur in 58% of metastatic cases. Integration of exome sequence, genome copy number, and gene expression data demonstrated that the broader “AR pathway” is altered in 56% of primary PCa and 100% of metastases. Therefore, direct alterations in the AR and broader alterations affecting the androgen-AR axis are among the most frequent events that occur in PCa development and progression. in situ hybridization (FISH). These FISH-based studies have indicated that AR gene amplification occurs at a rate of 20–33% in CRPC [AR copy number > 4) was only observed in 5/14 (36%) subjects, which closely matches previous FISH-based estimates of AR amplification prevalence. This suggests that FISH may fail to identify AR amplicons that are focal in nature, which was demonstrated recently in a study that compared CGH-based AR gene copy number to AR FISH signals in cells derived from CRPC tissues [n = 33) had evidence of AR amplification in at least one of their CTCs, with considerable heterogeneity between CTCs originating from a single patient [One mechanism thought to increase AR protein expression is amplification of the AR gene. For example, an early study that analyzed the relationship between AR gene amplification and AR protein expression levels in matched androgen-dependent and CRPC tumors showed that 80% of tumors which acquired amplification of the AR gene also exhibited higher expression levels of AR protein . Histori in CRPC , 38–41 b in CRPC , 38. The in CRPC , 31, 36. in CRPC . However tissues . However patient . Overallbona fide drivers of resistance to AR-targeted therapy.AR mutation is a mechanism of therapy resistance operating in a subset of CRPC. This area of PCa biology has been reviewed extensively , 45, anddownstream of exon 3 [Altered splicing and synthesis of COOH-truncated, constitutively active AR variant proteins has emerged as an important mechanism of PCa resistance to AR-targeted therapy –72. Moref exon 3 , 73. Subf exon 3 . This xef exon 3 . Similarf exon 3 . Importaf exon 3 . upstream from an amplified AR locus [These types of focal, medium-scale rearrangements are not easily detected by low-resolution legacy techniques such as FISH or array CGH. However, high-resolution genome copy number analysis using platforms such as Affymetrix SNP6.0 would be expected to identify focal (<50 kb) copy number gains and/or losses when data are analyzed with an appropriate breakpoint-finding algorithm. Indeed, reanalysis of genomewide SNP6.0 data from metastatic CRPC and primary PCa demonstrated that complex patterns of copy number gain and/or loss occur along the length of the AR gene, exclusively in CRPC . This arAR locus . Thus faAR locus , 74. TheTMPRSS2 and members of the Ets transcription factor family [C15orf21 promoter and the Myc transcription factor was identified in a patient with aggressive PCa [MYC gene is frequently amplified in PCa [C15orf21 promoter could drive Myc expression to a similar high level as Myc gene amplification.The earliest somatic alterations that occur in the androgen-AR pathway are fusions between 5′ genomic regulatory elements such as r family . In thisr family . Moreover family . More resive PCa . The MYCd in PCa , and it HSD17B2 gene undergoes copy number loss, whereas the HSD17B3 gene undergoes copy number gain in CRPC specimens [HSD17B3 and/or loss of HSD17B2 would favor testosterone production in CRPC tissue, thereby increasing the level of AR activation. In addition, chromosome 8q13 displays broad copy number gain in 17% of PCa and amplification in 1.9% of primary PCa and 24.3–60% of metastatic CRPC [NCOA2, which encodes the AR coactivator SRC-2/TIF2. Mechanistically, NCOA2 amplification was linked to increased expression of SRC-2/TIF2 mRNA levels [NCOA2 gene has also been shown to harbor somatic point mutations that are predicted to alter protein sequence in clinical PCa specimens [NRIP1/RIP140 and the AR corepressor NCOR2/SMRT have been shown to harbor somatic mutations in PCa [In addition to gene fusions allowing Ets family members (and perhaps other factors such as Myc) to become new downstream components of the androgen-AR axis, traditional upstream regulators of AR transcriptional activity have also been shown to undergo somatic alterations in PCa cells. For example, the pecimens . These gpecimens . Gain oftic CRPC , 78. Whitic CRPC , the 8q1A levels . This isA levels . These sA levels , 81. Rempecimens . Similarpecimens . Overexppecimens , 83. Adds in PCa . While tTPMT and thiopurine toxicity, UGT1A1 and irinotecan toxicity, CYP2D6 and tamoxifen efficacy, and ERCC1 or GSTP1/GSTP1-I105V and oxaliplatin response and survival [CYP19A1, HSD3B1, HSD17B4 [TRMT11 [SLCO2B1 and SLCO1B3 [BNC2 (rs16934641) affected disease progression for patients treated with ADT for hormone sensitive disease, while variation in another estrogen receptor binding site related gene TACC2 (rs3763763) was associated with prostate cancer specific mortality, and variation in a third related gene ALPK1 (rs2051778) along with TACC2 (rs3763763) was associated with all-cause mortality in advanced stage disease [ARRDC3 (rs2939244); FLT1 (rs9508016). SKAP1 (rs6504145) in Han Chinese populations was detected to affect prostate cancer specific mortality in advanced prostate cancer populations, and variation in FBXO32 (rs7830622) and FLT1 (rs9508016) appeared to affect all-cause mortality in advanced prostate cancer [The availability of information from the Human Genome Project has raised interest in investigating a role for host genome variation in predicting response to chemotherapeutic medications. A nascent, but very promising class of pharmacogenetic predictive markers are host (germline) genetic variations and their association with response and toxicity to chemotherapeutic agents; some successful examples include survival , 85. In  [TRMT11 , as well SLCO1B3 . Interes disease . Similare cancer . On the e cancer . These iThe two broad challenges for achieving the long-term goal of personalizing treatments in advanced PCa stages included a current lack of an adequate and systematic prospective collection of cancer tissue and lack of availability of functional xenograft models from patient tumor samples. Meanwhile, the rapid advancement and availability of next-generation sequencing technologies has permitted quick and comprehensive characterization of genomic changes. Drug development and selection in oncology has thus focused on the identification of specific abnormalities in genes and gene pathways. These next-generation sequencing technologies could be particularly useful for analysis of the entire tumor genome for alterations in genes and gene pathways associated with response or resistance to standard hormonal and chemotherapy interventions but have yet to make a clinical impact. In the advanced PCa stage, a critical need exists for the identification of the heterogeneous somatic changes characterizing this stage that are “druggable,” as this disease stage is inevitably fatal. Identification, functional validation, and clinical application of pathways responsible for the malignant phenotype and drug response at this stage based on the genomic status of the patient's tumor are more likely to lead to promoting longevity and quality of life. However, this will involve high-throughput analyses of advanced PCa stage tumors via next-generation sequencing, gene expression (RNA-seq), and CpG methylation, followed by an integrated analysis of the molecular signatures associated with response to treatments. Assessing germline variants in androgen-AR axis genes is relatively straightforward because germline DNA can be obtained from blood. In contrast, assessing somatic alterations in androgen-AR axis genes is more challenging, because this requires direct sampling of tumor tissue or tumor-derived cells. An additional challenge is disease heterogeneity, particularly in the metastatic setting, because different tumor cell populations are likely to have different responses to certain forms of ADT. Therefore, to obtain a clear picture of the somatic alteration landscape, it would be very informative to sample various tumor cell populations prior to therapy and also assess which of these populations persist during/after therapy. In the immediate future, prior to developing a systematic and prospectively annotated clinical database of tumor samples obtained from this stage, one possible way to sample the overall tumor landscape is through CTC capture. MYC amplification, TMPRSS2-Ets gene fusions, PTEN deletion, Her-2/NEU, EGFR, and insulin-like growth factor [CTC can provide a snapshot of the molecular composition of the cell populations driving the progression of the disease at a given time. Once identified, CTCs can be analyzed and can provide information that may direct therapy to appropriate targets in an individual patient at the time of treatment decision. Several molecular markers that may impact future therapies have been detected in CTCs, including h factor , 91, 92.CTC analysis is dependent on the detection technique used to identify them. CellSearch has been regarded as the standard technology, using magnetic separation based on epithelial cell adhesion molecule (EpCAM) antibody-coupled ferrofluid, and based on rigorous morphologic criteria and expression of cytokeratins, DAPI, and excluding CD45 staining , 93. NewHowever promising as a source of molecular profiling, CTCs may not reflect the entire molecular milieu explaining the advance of CRPC. Stromal epithelial interactions and conditions of the host tissue may not be adequately assessed in CTC molecular profiling. Obtaining metastatic tissue samples provides a richer material for molecular analysis. Samples usually include stromal and epithelial elements and are a better reflection of the microenvironment that allows metastatic implants to grow and expand. However, availability of metastatic material is limited because few clinical indications exist today for sampling of metastatic lesions. Sampling requires invasive procedures associated with significant morbidity, usually consisting of needle sampling to obtain the tissue. This results in small-sized samples, frequently barely sufficient for histopathologic diagnosis, with limited if any amount available to collect frozen tissue. While buffered formalin compatible with molecular testing is becoming the standard fixative in most pathology laboratories, there is still large variability regarding time to and total time of fixation, which may impact certain molecular techniques. Furthermore, since the vast majority of metastatic PCa involves bone, sampled tissue frequently requires decalcification processes for proper evaluation. Routine decalcification involves treatment of tissue with formic acid, resulting in significant degradation of DNA, and hence rendering samples unsuited for molecular biological testing. De Jong et al. have recently described a protocol for molecular genetic testing of decalcified formalin-fixed, paraffin-embedded tissue, with promising results . Another limitation of tissue sampling is the heterogeneity within a biopsy sample as well as the variable presence of stroma or normal tissue, which can be a confounding factor for genomic analysis. Depending on the ratio of tumoral versus nontumoral cells, results from molecular testing may yield impaired results. Particularly challenging is the presence of abundant blood, necrosis, and inflammatory reactions in the sample, which may overshadow the DNA from the tumor cells. Cell enrichment techniques such as microdissection and laser capture microdissection can improve the sample quality for molecular analysis but are labor intensive. It is also possible to separate distinct clonal cell populations within an individual biopsy, which may be useful for identifying the cell types that respond to specific therapeutic interventions. For example, a recent study used flow cytometry-based DNA content measures to collect diploid and aneuploid tumor cell populations from PCa biopsy specimens, followed by genomic analysis using array CGH . In thisKnowledge of predictive biomarkers for patients with CRPC may lead to identifying novel pathways of early failure, which may lead to new therapeutic targets. For example, more aggressive therapeutic strategies could be used for patients who are destined to have a limited period of clinical response to ADT. In contrast, patients with biomarkers associated with long-term responses to ADT could undergo intermittent rather than the usual continuous ADT exposure and avoid the many toxicities of chronic ADT administration, including osteoporosis, loss of sexual libido, increased risk of diabetes and coronary artery disease, and metabolic syndrome. Indeed, the efficacy of intermittent ADT is noninferior to continuous ADT  but is nWe now present examples of using emerging predictive biomarkers related to the androgen-AR axis as the basis of stratifying the metastatic PCa patient population for biomarker-based clinical trials. In contrast to a prognostic biomarker that is associated with clinical outcome regardless of ADT, a predictive biomarker provides information about treatment effect of ADT . The traEnrichment design is suitable for established predictive biomarkers that are generally accepted with extensive evidence . It requMarker strategy design and marker stratified design are both biomarker-based clinical trial designs that can be utilized to validate somatic mutations of AR or germline SNPs as predictive biomarker(s) of ADT. The marker strategy design  randomizAdaptive signature design  unifies Adaptive randomization designs , 106 areThe recent advances in the medical management of advanced prostate cancer include successful translation and development of novel agents that target different components of the androgen-AR axis. This progress has taken considerable time considering that new agents for therapy of advanced prostate cancer still adhere to the basic principles established in the seminal work of Huggins and Hodges , 3. Desp


The decision to grant a disability pension is usually the end of a long process of medical examinations, treatment and rehabilitation attempts. This study investigates to what extent the time spent on rehabilitation time prior to disability pension is associated with characteristics of the individual or the local employment and welfare office, measured as municipality variance.A study of 2,533 40 to 42 year olds who received disability pension over a period of 18 years. The logarithm of the rehabilitation time before granting a disability pension was analysed with multilevel regression.The rehabilitation time before a disability pension was granted ranged from 30 to 5,508 days. Baseline health characteristics were only moderately associated with rehabilitation time. Younger people and people with unemployment periods had longer rehabilitation time before a disability pension was granted. There were only minor differences in rehabilitation time between men and women and between different levels of education. Approximately 2% of the total variance in rehabilitation time could be attributed to the municipality of residence.There is a higher threshold for granting a disability pension to younger persons and those who are expecting periods of unemployment, which is reflected in the extended rehabilitation requirements for these groups. The longer rehabilitation period for persons with psychiatric disorders might reflect a lack of common knowledge on the working capacity of and the fitted rehabilitation programs for people with psychiatric disorders. Disability benefits are important because they provide economical assurance to people who are marginalised from the labour market due to health impairments. The decision to grant a disability pension is in most cases the end of the line of a long process of medical examinations, treatment and rehabilitation attempts. This process is likely to be a substantial strain on the persons involved, and theAlthough the health of the participant is an important factor when people struggle returning to work after a rehabilitation process, other demographic factors can be important to whether this ends up in employment or receiving a disability pension. Studies have shown that the likelihood of returning to work after rehabilitation decreases with increasing age-4 and thhttp://www.nav.no). Furthermore, each municipality has the responsibility to provide primary health care to its citizens. Although the rules and regulations pertaining to rehabilitation and disability pension are uniform and valid throughout Norway, the legislation on vocational rehabilitation functions as a framework law. As a consequence, each employment and welfare office can exercise discretion in their decisions in the rehabilitation process. This discretion may lead to variations in the rehabilitation process between municipalities, where the employment and welfare offices put more effort in finding and providing more opportunities for rehabilitation for people with better prospects in the labour market, and where disability pensions are given sooner when labour market prospects indicates that a return to work is less likely. Another factor that may differ between municipalities is the quality of the healthcare and the medical rehabilitation for people who have temporarily left the labour market because of health problems.In Norway, each municipality has an employment and welfare office that organises social welfare decisions  approved this study.The data were derived from the National Health Screening Service in Norway. Between August 1988 and March 1989 all residents of Nordland County in Norway aged 40 to 42 years were invited to participate. Data were linked to the national insurance database via a personal identification number, created by Statistics Norway and the Norway National Insurance Service. Follow-up time was from January 1Nordland County is situated in the northern region of Norway. At the time of the health screening, Nordland had 45 municipalities and approximately 240,000 inhabitants. Nordland County has a diversity of industries where some municipalities are dominated by fishing, some by agriculture, some by manufacturing industry and some by services. This diversity in industries suggests that municipalities have been affected differently by business fluctuations during the follow-up period.st 1992, and covers all cases of disability pensions in Norway.Disability pension is granted to people whose earning ability is permanently impaired by at least 50% due to illness, injury or inborn defect. It is also a requirement that the illness or injury is the main reason for the impaired wage earning capacity. Data on new incidents of disability pensions were available from January 1The dependent variable in this study was the duration of the rehabilitation period before disability pension. The rehabilitation time in days was calculated as the time between the first date of work disability and the date for granting a disability pension. The first date of work disability represents the point in time when a person’s earning ability was permanently reduced – in most cases the first day of being sick-listed. The time for granted disability pension is always set to three months ahead of the date of application for disability pension. Both dates are registered at the time disability pension is granted. The rehabilitation period normally includes long-term sick leave, medical rehabilitation and vocational rehabilitation programmes which can deal with vocational assessment, work retraining, education, counselling, work guidance and other forms of preparation for returning to work..In this study, information on different aspects of health and disease were used to adjust for health impairment at baseline. A summarised index of the number of chronic illnesses was constructed including the following conditions: myocardial infarction, angina pectoris, stroke/cerebral infarction, Bechterew’s disease, cancer, diabetes, chronic bronchitis, arthritis, epilepsy, migraine and gastro-intestinal problems. Self-rated health was assessed by the question, “What is your health condition like?” with the four answer categories: “very good,” “good,” “fair” and “poor”. Depression was assessed by the question, “Have you been sad or depressed the last 14 days?” with the four answer categories “almost all the time,” “frequently,” “sometimes” and “never or rarely”. Headache and pains in the neck and shoulders were measured with a four-point scale, with answer categories ranging from “never/rarely” to “daily”. Smoking was assessed with a three-point scale with three answer categories “non-smoker,” “former smoker” and “smoker”. Consumption of alcohol was assessed with a four-point scale, with answer categories ranging from “non-drinker” to “daily drinker.”Although people can be caused by several diagnoses, the National Work and Welfare Administration codes one major diagnosis after disability pension has been granted. Musculoskeletal and psychiatric diseases are the most common medical diagnoses for being granted a disability pension in Norway, and theWith data obtained from the national insurance register, study participants with any periods of unemployment throughout the follow-up period were classified as having been unemployed.The age of the participants ranged between 40–42 years at baseline. To investigate whether the duration of the treatment period was different for different age groups; the participants’ ages at the first date of disability was recorded, which ranged from 44 to 61 years. The participants were divided into six age groups. Level of education was measured with the three categories: “primary school”, “high school” and “college/university”.A variable was created representing municipality size, reporting whether the respondents were living in a small , medium  or large municipality .Rates of people on vocational rehabilitation for each municipality for every year of the follow-up ranged from 0.24% to 6.43%. The rehabilitation rate was recorded the same year as the first date of work disability.Intra- class correlation coefficient (ICC) was calculated as an estimate of the relative importance of place of residence on the length of the rehabilitation period before receiving a disability pension. The main analyses were performed in a three-level model with individuals nested within years within municipality of residence. The diagnosis-specific analyses had no indication of year differences, and thus were performed as a two-level analysis.The distribution of the rehabilitation time in days was skewed. Accordingly, a log-transformation was performed to correct the skewed data. A linear multilevel regression analysis was applied to individuals nested by municipality of residence and year of start of rehabilitation. To explore the impact of place of residence, the The statistical analysis of the duration of the rehabilitation period was performed in three models. Model 1 was adjusted only for age, sex and unemployment. In model 2, baseline health status and health behaviour  were added. In model 3, education, municipality size and rehabilitation rate in the municipality were added to model 2’s parameters. The separate analyses for the different diagnoses were done with the same models. The precision of the estimates was presented using 95% confidence intervals (CI). The analyses were limited to the participants with complete information in all study variables . All analyses were conducted using STATA 11 software .Of the 10,497 invited to the health screening, 4,302 men and 4,310 women attended, resulting in an attendance rate of 78% and 86% for men and women, respectively. A totalRehabilitation time for all participants varied from 30 to 5,785 days with a mean of 805 days 2.2 years) and standard deviation of 608 days. In Figure.2 years TableThe results in model 1 were based on those having complete information on all study variables. A sensitivity analysis . The information in this study was obtained from a highly reliable source established by Statistics Norway and the Norway Social Insurance Service. Although numerous studies are published on rehabilitation and return to work, this is, to our knowledge, the first study that investigates variations in the duration of the rehabilitation period for a group of participants ultimately becoming disability pension recipients.The accuracy of the rehabilitation time period is presumably high as the information was obtained from a highly reliable source set up by Statistics Norway and the Norway Social Insurance Service.The questionnaire in this study did not contain formerly validated health scales. However, the study had comprehensive information on several diseases and complaints that are well known risk factors for disability pension. Furthermore, the study included self-rated health, a common measure for both physical and mental health and also an independent predictor for disability pension-21. The The number of participants was limited to those with complete information for all study variables  in the regression models. There might be selection effects in the study, meaning that the respondents who chose not to answer questions about their health or health behaviour may have experienced a different rehabilitation pattern and rehabilitation time than those included. The diagnosis-specific analysis was limited to the participants that were registered with a diagnosis at time end of the follow-up . The diagnosis for disability pension can be delayed for some persons, meaning that our data had missing information about diagnosis for some of the participants that received disability pension the last years of the follow-up.This study considered rehabilitation time only for those who eventually were granted disability pension, and the results of the rehabilitation process may have differed if we had included those succeeding return to work.The study did not have full information on disability pension and unemployment from 1990 and 1991. Hence, information from the participants starting their disability process before 1992 was not available.Age was associated with the length of the rehabilitation period. Several other studies has shown that the chances of job return after a rehabilitation period is attenuated with increasing age,22. ThisThe length of the rehabilitation process was approximately the same for different levels of education. Although a recent Norwegian study concludePeople who experienced unemployment in the follow-up period had a longer rehabilitation period before disability pension was granted. Previous studies have shown that having a job to return to is associated with returning to work after a rehabilitation period, compared with those without a job to return to,6. A lonOne would expect poor health to be associated with a shorter rehabilitation period, given that poor health is a premise for being granted a disability pension. However, in this study health measures were only marginally associated with the length of the rehabilitation period. Several studies have shown that people with more severe diseases are less likely to return to work,15, and The multilevel analysis showed that 2% of the variance could be attributed the municipality level. These results might indicate fairly equal practice between social service offices across municipalities. This is also in line with the results of a previous study based on the same material, assessing the risk of disability pension between the different municipalities where approximately 2% of the variance could be attributed to the municipality level.Previous studies have shown that subjects living in regions with a low level of unemployment were more likely to return to work,9, and tThe present study’s results indicated that people with psychiatric diagnoses were granted a disability pension sooner in the largest municipalities. This finding may be due to organisational characteristics or other characteristics of some employment and welfare offices in some large municipalities. Hence, this finding requires more research attention. One interpretation of this finding is that the employment and welfare offices in the smallest municipalities have less experience with people with psychiatric diagnoses, have more problems assessing their work capacity and has a lack of knowledge on suitable rehabilitation programmes for this diagnostic group.This study revealed a longer rehabilitation time for younger people and those who have experienced unemployment during the follow-up period. Higher thresholds for granting a disability pension to younger persons and for those having experienced unemployment can reflect a demand for extended rehabilitation measures for these groups. Baseline health characteristics were only moderately associated with rehabilitation time, and no substantial differences in rehabilitation time between men and women, or for different levels of education were found This result may be explained by the fact that the heterogeneity among employees is strongly reduced when we study only those that are granted disability pension. This sample is thus adjusted for all factors that affect the probability of being granted a disability pension . Place of residence had modest importance for the length of the rehabilitation time. Larger municipalities had a considerably shorter rehabilitation time before the granting of a disability pension. The longer rehabilitation period for persons with psychiatric disorders could reflect difficulties assessing their working capacity and a lack of knowledge on rehabilitation programs for this group.The authors declare that they have no competing interests.MS carried out the data processing, the epidemiological modeling and statistical analysis and wrote the manuscript. KP, RJ and JHB contributed to the epidemiological modeling, statistical analysis, data interpretation and drafting of the manuscript. NF, ES, SOS and BC participated in the design of the study and helped to write the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/375/prepubAppendix: Table 6. Multilevel linear regression of the logarithm of days  in rehabilitation time prior to disability pension award. Complete case. (DOC 44 kb)Click here for file


Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear.FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor–formation assays.We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn’t influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth.These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway. It indicated that FOXA1 and AR may serve as potential gene therapy in EC. Endometrial cancer (EC) is one of the most common gynecologic malignancies. The incidence of EC has markedly increased in recent years. EC is broadly classified into two groups ; type I Several studies have shown that the majority of ECs also express another hormone receptor, androgen receptor (AR) ,5. The rForkhead box A1 (FOXA1) is a transcription factor that belongs to the forkhead family consisting of the winged-helix DNA-binding domain and the N-terminal and C-terminal transcriptional domains. FOXA1 is expressed in various organs, including breast, liver, pancreas, and prostate, and can influence the expression of a large number of genes associated with metabolic processes, regulation of signaling, and the cell cycle ,8. FOXA1An aberrant Notch pathway has been documented in various cancer types and has been associated with tumorigenesis -15. The In the present study, we investigated the dependency of AR on FOXA1 expression in tissue paraffin sections, in multiple cellular contexts, and on tumor-bearing nude mice. Here we show, for the first time, that FOXA1 activates the Notch pathway through AR and that AR is required for FOXA1-enhanced cell proliferation in EC.A total of 57 normal endometrial samples, 11 atypical hyperplasias, and 76 EC specimens obtained from Chinese female patients who underwent surgical treatment from 2011 to 2013 at the Shanghai Jiao Tong University Affiliated International Peace Maternity & Child Health Hospital  were available for examination in this study. Tissues were embedded in paraffin. Two independent pathologists verified the histological diagnosis of all collected tissues. No patient had received neoadjuvant therapy or endocrine therapy before the surgery. The clinicopathological characteristics of EC patients are presented in Table Staining was performed on paraffin-embedded specimens using primary antibodies as follows: anti-FOXA1  and anti-AR . The percentage of positively stained cells was rated as follows: 0 point = 0%, 1 point = 1% to 25%, 2 points = 26% to 50%, 3 points = 51% to 75%, and 4 points = greater than 75%. The staining intensity was rated in the following manner: 0 points = negative staining, 1 point = weak intensity, 2 points = moderate intensity, and 3 points = strong intensity. Then, immunoreactivity scores for each case were obtained by multiplying the values of the two parameters described above. The average score for all of five random fields at 200× magnification was used as the histological score (HS). Tumors were categorized into two groups based on the HS: low-expression group (HS = 0–5) and high-expression group (HS = 6–12).2 at 37°C. The human endometrial cell line MFE-296 was purchased from Sigma . The MFE-296 cell line was grown in high-glucose DMEM (4.5 g/L glucose) (HyClone) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C.The human endometrial cell lines AN3CA, RL95-2, and HEC-1B were obtained from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. These three cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12  supplemented with 10% fetal bovine serum  in a humidified atmosphere of 5% CO−9 to 10−7 M for different periods (0–48 h). To block the activation of AR-mediated transcription, flutamide (10−6 M) was added into the media 30 min before DHT. Vehicle contained 0.1% absolute ethanol/phenol red–free DMEM/F12.To investigate the impact of FOXA1 on the AR-mediated transcription, the AR pathway agonist 5α-dihydrotestosterone (DHT)  and the AR pathway blocker flutamide (Sigma) were purchased and dissolved in 100% ethanol for storage. In this study they were diluted with phenol red–free DMEM/F12 (Gibco) immediately before each experiment, with the final concentration of ethanol at 0.1%. DHT was added into the cell culture media at concentrations of 10To stably knock down endogenous FOXA1 expression, MFE-296 cells were grown to 30% confluency in 6-well culture plates and then infected with lentivirus carrying an shRNA targeting FOXA1 (shFOXA1) or a negative control vector  at a multiplicity of infection of 70 in the presence of polybrene (8 μg/mL). After 48 h of infection at 37°C, the medium was replaced with fresh medium and incubated further for 72 h before analysis using quantitative RT-PCR (qRT-PCR) and western blotting for FOXA1 expression. The shRNA sequences used were 5′-GAGAGAAAAAAUCAACAGC-3′ (shFOXA1) and 5′-TTCTCCGAACGTGTCACGT-3′ (NC).The plasmid PWP1/GFP/Neo-AR containing transfection-ready AR cDNA (exAR) and its negative control PWP1/GFP/Neo were gifts from Doctor Yuyang Zhao at Shanghai First People’s Hospital. MFE-296 cells stably transfected with shFOXA1 or NC were transiently cotransfected with PWP1/GFP/Neo-AR (exAR) or its negative control (NC). The plasmid pCMV/3FLAG/Neo-FOXA1 containing transfection-ready FOXA1 cDNA (exFOXA1) (GenBank: BC033890) and a pure pCMV/3FLAG/Neo (NC) were purchased from Genechem . AN3CA cells were transiently transfected with exFOXA1 or NC or cotransfected with a siRNA targeting AR (siAR)  or its negative control (NC) in Opti-MEM  using Lipo2000 (Invitrogen). The siRNA targeting FOXA1 (siFOXA1) and its negative control (NC) were purchased from Genephama Biotech . AN3CA cells were transiently transfected with exAR or NC or cotransfected with siFOXA1 or NC in Opti-MEM (Invitrogen) using Lipo2000 (Invitrogen). The transfection solution was removed from the cells and replaced with standard medium after 8 h. The sequences of the siRNA oligos used were: siAR: sense: 5′-AUGUCAACUCCAGGAUGCUTT-3′, antisense: 5′-AGCAUCCUGGAGUUGACAUTT-3′; siFOXA1: sense: 5′-GAGAGAAAAAAUCAACAGC-3′, antisense: 5′-GCUGUUGAUUUUUUCUCUC-3′.Total RNA was extracted from cultured cells by Trizol Reagent (Invitrogen). RNA was converted to cDNA with the one-step Prime Script RT reagent kit , and the cDNA was analyzed by real-time PCR using SYBR Premix Ex Taq (TaKaRa) in an Eppendorf Mastercycler® realplex. Each sample was assayed in triplicate in each of three independent experiments. All values are expressed as mean ± standard deviation. The following primers were used: FOXA1: sense: 5′-AGGTGTGTATTCCAGACCCG-3′, antisense: 5′-TTGACGGTTTGGTTTGTGTG-3′; AR: sense: 5′-CCTGGCTTCCGCAACTTACAC-3′, antisense: 3′-GGACTTGTGCATGCGGTACTCA-5′; MYC: sense: 5′-AAAGGCCCCCAAGGTAGTTA-3′, antisense: 5′-TTTCCGCAACAAGTCCTCTT-3′; XBP1: sense: 5′-CCTTGTAGTTGAGAACCAGG-3′, antisense: 5′-GGGGCTTGGTATATATGTGG-3′; UHRF1: sense: 5′-AAGGTGGAGCCCTACAGTCTC-3′, antisense: 5′-CACTTTACTCAGGAACAACTGGAAC-3′; and ZBTB16: sense: 5′-CCAGCAGATTCTGGAGTATGCA-3′, antisense: 5′-GCATACAGCAGGTCATCCAAGTC-3′.Total protein was extracted using a RIPA kit  containing a 1% dilution of the protease inhibitor PMSF (Beyotime). Protein concentrations were determined by the enhanced BCA Protein Assay kit (Beyotime). Equal amounts of protein in each lane were separated by 8% SDS-PAGE and transferred to a PVDF membrane . After blocking the membrane in blocking buffer  Tween 20), the membrane was incubated with primary antibodies against FOXA1 , AR , Notch1 , Hes1 , and β-actin  at 4°C overnight. Peroxidase-linked secondary anti-rabbit or anti-mouse antibodies were used to detect the bound primary antibodies.−7 M DHT for 24 h . After protein quantification, 500 μg of each cell lysate was added to 10 μl of anti-FOXA1 (Epitomics) and shaken at 4°C overnight, then added to 30 μl of Protein A + G Agarose (Beyotime), shaken at 4°C for 4 h, centrifuged at 2500 × g for 5 min, and washed with a RIPA kit (Beyotime) to collect the immunoprecipitate-bound agarose beads. Each immunoprecipitate was denatured with 20 μl of 1× SDS-PAGE loading buffer at 100°C for 5 min. Each supernatant was subjected to SDS-PAGE (8% acrylamide). It is important to note that FOXA1 (51 kDa) is close in size to IgG (55 kDa). To avoid detecting IgG protein left from the immunoprecipitation process and FOXA1 protein from the same species in the western blot at the same time, we used anti-FOXA1 from mouse in western blotting, whereas anti-FOXA1 from rabbit was used for immunoprecipitation. Primary antibodies against AR (1:2000) and FOXA1  were used for western blotting. Other steps were as described in the Western blotting section.Total protein was extracted from cells treated or not treated with 10Chromatin immunoprecipitation (ChIP) assays were performed as previously described  using an3 cells/well) were plated in 96-well plates. Then, 20 μl of 3--2,5-diphenyltetrazolium bromide  was added to each well and subsequently incubated at 37°C for 4 h. The absorbance at 490 nm was then measured using a microplate reader. Cells incubated with culture medium were used as a control group. Each sample was assayed in triplicate.Cells  or 200 cells/well (AN3CA cells) were seeded into 6-well plates. Dishes were returned to the incubator for 14 days, and the colonies were fixed with methanol for 30 min at room temperature and then stained with 0.5% crystal violet for 1 h.5/well (for the migration assay) or 2 × 105/well (for the invasion assay) in invasion chambers  with or without matrigel coating for invasion and migration assays. Complete medium (600 μl) was added to the lower chamber as a chemoattractant. After incubation for 5 h (MFE-296) or 24 h (AN3CA) for the migration assay, or after incubation for 24 h (MFE-296) or 48 h (AN3CA) for the invasion assay, cells were fixed with 4% paraformaldehyde for 1 h. Cells on the apical side of each insert were removed by mechanical scraping. Cells that migrated to the basal side of the membrane were stained with 0.5% crystal violet and counted at 200× magnification. The migration and invasion assays were repeated at least three times.Cells were trypsinized, centrifuged, and resuspended in serum-free medium. Cells were then plated at a density of 1 × 10Female athymic mice of 4 weeks of age were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Science. Our animal research was carried out in strict accordance with the recommendations in the Guideline for the Care and Use of Laboratory Animals of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Obstetrical and Gynecological Hospital affiliated Fudan University (Permit Number: SYXK (hu) 2008–0064). All efforts were made to minimize animal suffering.7 cells. Tumor measurement began one week after injection and was conducted weekly using digital calipers. The tumors were removed and weighed after 42 days. Tumor volume was calculated as follows: tumor volume (cm3) = (the longest diameter) × (the shortest diameter)2 × 0.5.To establish a nude mouse model bearing EC, uninfected MFE-296 cells (MFE-296), stable MFE-296 cells infected with lentivirus carrying shFOXA1 (MFE-296/shFOXA1) or vector alone (MFE-296/NC) were used. All mice were randomly divided into three groups of four mice. Each mouse was given a unilateral subcutaneous injection of 1 × 10Tumor samples from xenografted mice were collected and fixed according to routine procedures. Histological staining was then performed on the tissue sections of the paraffin-embedded tumors using the streptavidin-biotin-peroxidase method. Primary antibodies were as follows: anti-FOXA1 , anti-AR , anti-Notch1 , anti-Hes1 , anti-Ki67 , and anti-PCNA . The sections were then counterstained with hematoxylin and eosin (H&E).t-test or one-way ANOVA for multiple comparisons, and χ2 test for 2 × 2 tables was used to compare the categorical data. p < 0.05 was considered significant.Measured data were assessed by unpaired Student’s We assessed relative FOXA1 and AR levels in EC samples, atypical hyperplasias, and normal endometrial tissue samples using immunohistochemistry. FOXA1 was higher in atypical hyperplasias and even higher in EC compared with normal endometrial tissues (p = 0.005) Figure . CorrelaWe used western blotting to examine FOXA1 and AR expression in EC cells. FOXA1 was upregulated in MFE-296 cells compared with KLE, HEC-1B, and AN3CA cells. Furthermore, the AR level was also markedly higher in MFE-296 cells than in the other three EC cell lines  or with control vector (AN3CA/NC). Moreover, to knock down FOXA1 expression, MFE-296 cells were stably transfected with FOXA1 shRNA (MFE-296/shFOXA1) or control vector (MFE-296/NC)  and time-response study (0–48 h) indicated that 10−7 M DHT and 24 h of incubation elicited the strongest expression of AR and its target genes  and MFE-296 cells transfected with shFOXA1 (MFE-296/shFOXA1), NC (MFE-296/NC), or shFOXA1 together with exAR (MFE-296/shFOXA1 + exAR). qRT-PCR and western blotting analysis confirmed that transfection of exAR resulted in overexpression of AR Figure F. qRT-PCTo investigate whether FOXA1 affects AR-mediated transcription through binding to AR, we performed co-immunoprecipitation experiments. We used nuclear lysates from MFE-296 cells to conduct immunoprecipitation with anti-FOXA1. FOXA1 co-immunoprecipitated with AR, whereas immunoprecipitation with the isotype IgG control did not pull down AR or FOXA1 Figure A, indicaWe further examined whether FOXA1 and AR could bind to the five putative FOXA1-AR binding regions, including the promoter and enhancer regions upstream of the TSS (transcription start sites) of AR target genes such as MYC Figure C. Our ChPathway analysis in liver cancer shows that FOXA1/AR dual target genes are most involved in the cellular growth/proliferation pathway . Notch pTo examine the role of FOXA1 in EC cell proliferation, we assessed the effect of FOXA1 in colony-forming and MTT assays. In the colony-forming assay, MFE-296 cell transfected with shFOXA1 showed significantly decreased colony-forming ability when compared with MFE-296 cells transfected with NC Figure A. MoreovTo directly address whether the effects of FOXA1 in promoting EC cell proliferation can be attributed to its activation of AR, a rescue experiment in MFE-296 cells was performed. In the colony-forming assay, cotransfection with exAR rescued the decreased rate of cell growth caused by FOXA1 downregulation in shFOXA1 cells Figure E. The MTOur immunohistochemistry results revealed that patients with myometrial invasion displayed higher FOXA1 expression. With this observation in mind, we hypothesized that functional expression of FOXA1 might induce tumor metastasis in EC. To explore the role of FOXA1 in the regulation of metastatic function and to determine whether AR is involved in FOXA1-mediated regulation of metastatic function, we examined the migration and invasion ability of MFE-296/shFOXA1 and AN3CA/exFOXA1 cells after exAR or siAR cotransfection using transwell migration and invasion assays. MFE-296/shFOXA1 cells displayed a decreased rate of migration compared to MFE-296/NC cells , stably transfected with shFOXA1 (MFE-296/shFOXA1) or NC (MFE-296/NC). These measurements indicated that tumors in the MFE-296/shFOXA1 group grew significantly slower than those in the MFE-296/NC group and the MFE-296 group Figure A. Six weKip1 in an ATC cell line [Over the past decade, FOXA1 expression has been examined in several human cancers, and oncogenic and tumor-suppressive roles have been proposed for FOXA1 depending on the cancer type and, in some cases, the subtype. In acute myelocytic leukemia, esophageal squamous cell carcinomas, lung adenocarcinomas, thyroid carcinoma, prostate cancer, and AR-positive molecular apocrine breast cancer ,23-25, Fell line . On the ell line . In addiell line . It is cell line ,12,28.A previous study has addressed the expression and function of FOXA1 in EC; immunohistochemical analysis by Abe et al. indicated that FOXA1 was negatively associated with lymph node status in EC immunohistochemical specimens in Japanese, and FOXA1 repressed proliferation and migration in one type of EC cells (Ishikawa) . HoweverMost researches on hormone receptors in EC have focused on ER and progesterone receptor (PR). However, the expression of AR in the human normal endometrium and its disorders is not well understood. Though higher serum androgen levels have been certified to exist in the utero-ovarian vein blood samples from women with EC , the detMost FOXA1 studies have focused on its role as a pioneer factor that binds to DNA packaged in chromatin and opens the chromatin for binding of additional transcription factors including AR ,40. AccoAlthough the underlying mechanisms governing the FOXA1-AR correlation in tumor progression are not fully understood, a pathway analysis showed that 187 FOXA1/AR dual target genes were involved in the cellular growth/proliferation pathway in liver cancer . The NotKip1[The specific mechanism of cell proliferation in EC reported so far has been limited, although several classical transcription factors related to proliferation have been identified, including cyclin D1, p53, IGFBP-1, PTEN, and p27Kip1-48. In tWe further investigated the effects of FOXA1 and AR on migration and invasion of EC cells, and found that neutralization of AR activity did not inhibit FOXA1-enhanced cancer cell migration or invasion. These observations indicate that the promoting effect of FOXA1 on migration and invasion is not dependent on AR. Our findings in migration and invasion assays are consistent with our findings in immunohistochemical staining, which showed that higher expression of FOXA1 but not AR is found in tumors that displayed a greater depth of myometrial invasion. These results suggest that AR is not the only downstream target of FOXA1 in EC. Future studies will be necessary to define which transcription factors or pathways are involved in FOXA1-enhanced cell migration and invasion in EC.The traditional endocrine treatment (mainly targeting ER and PR) is ineffective in most ER-negative and PR-negative ECs, and even in some ER-positive and PR-positive ECs . In our In summary, our results suggest a new mechanism for the development of EC, in which FOXA1 promotes tumor cell proliferation through AR and activates the Notch pathway by influencing AR expression. The newly identified FOXA1-AR interaction will help further elucidate the molecular mechanisms underlying EC progression and suggests that FOXA1 and AR are potential targets for EC treatment.The authors declare that they have no competing interests.MQ, WB, and JW carried out the design of the experiments, performed most of experiments, and drafted the manuscript. TY and XH participated in the molecular biology experiments and statistical analysis. YL made the figures. XW was involved in financial support, the design of the experiments, data analysis, and final approval of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/78/prepubStatistical difference between FOXA1 expression in normal endometrium and endometrial cancer.Click here for fileStatistical difference between AR expression in normal endometrium and endometrial cancer.Click here for fileAR is a necessary medium in FOXA1-enhanced Notch pathway activation. Protein levels of AR, FOXA1, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC), exAR (AN3CA/exAR), or exAR and siFOXA1 (AN3CA/exAR + siFOXA1) were measured by western blotting (Right), and were further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p < 0.05, ** p < 0.01, ***p < 0.001, and NS p > 0.05.Click here for file