Pristionchus (Nematoda: Diplogastridae), including Pristionchus pacificus, have been reported to be necromenic, i.e. to associate with beetles in their dauer diapause stage and wait until the death of their host to resume development and feed on microbes in the decomposing beetle corpse. We review the literature and suggest that the association of Pristionchus to beetles may be phoretic and not necessarily necromenic. The view that Pristionchus nematodes have a necromenic lifestyle is based on studies that have sought Pristionchus only by sampling live beetles. By surveying for nematode genera in different types of rotting vegetal matter, we found Pristionchus spp. at a similar high frequency as Caenorhabditis, often in large numbers and in feeding stages. Thus, these Pristionchus species may feed in decomposing vegetal matter. In addition, we report that one species of Panagrellus (Nematoda: Panagrolaimidae), Panagrellus redivivoides, is found in rotting fruits but not in rotting stems, with a likely association with Drosophila fruitflies. Based on our sampling and the observed distribution of feeding and dauer stages, we propose a life cycle for Pristionchus nematodes and Panagrellus redivivoides that is similar to that of C. elegans, whereby they feed on the microbial blooms on decomposing vegetal matter and are transported between food patches by coleopterans for Pristionchus spp., fruitflies for Panagrellus redivivoides and isopods and terrestrial molluscs for C. elegans.The lifestyle and feeding habits of nematodes are highly diverse. Several species of  Pristionchus (Nematoda: Diplogastridae), including the most studied Pristionchus pacificus, have been recently considered to be associated with beetles in a necromenic lifestyle, i.e. to associate with beetles in the nematode dauer diapause stage and wait until the death of the beetle to resume development and feed on microbes in the decomposing beetle corpse [Pristionchus nematodes now appears as a qualifier for the species, as in the "necromenic nematode P. pacificus" [Pristionchus have been considered to share some attributes of parasitic nematodes and to have a very different lifestyle from the free-living nematode C. elegans [The lifestyle and feeding habits of members of the nematode phylum are highly diverse. A single species may express diverse life cycles through production of free-living and host-associated stages. Many species of the nematode genus e corpse . The neccificus" ,3. Prist elegans .Pristionchus feeds on them. Recent field studies have looked for Pristionchus in live beetles, and to a lesser extent in soil [Pristionchus with beetles.Yet, to our knowledge, carcasses of naturally dead beetles have not been looked for and examined to assess whether  in soil –8. We dePristionchus species including P. pacificus can be found on beetles [Pristionchus species has recently been found in fresh tropical figs in association with their pollinating wasps [Caenorhabditis species are found in other specific substrates [Many  beetles ,8–14. , 466 other diplogastrids, 3927 Pelodera (Rhabditidae) and 508 parasitic Spirurida. Pelodera dauer larvae were present in 56 out of 114 Geotrupes beetles, with up to hundreds of Pelodera dauer larvae per beetle. Pristionchus was comparatively rarer: eight of 114 beetles (7%) yielded one Pristionchus individual, one carried two individuals and one had seven individuals, all in the dauer stage. P. pacificus is rare in Europe [Pristionchus were P. sp. 6, entomophagus, and lheritieri. In addition, a survey of 4242 beetles from Germany, France, Spain, and Switzerland again suggested that Pristionchus spp. are found in the dauer stage on beetles [er stage , based oPristionchus non-dauer stages feed on carcasses of beetles come from experiments where beetles were killed on a Petri dish containing E. coli in the laboratory , it is not possible to assess either the number of individuals or their developmental stage on the beetle. Similarly, if the beetles are killed by the experimenter and then placed in a soil environment, Pristionchus will proliferate in the vicinity of the dead beetle only after 7 days [Pristionchus may adopt a necromenic lifestyle by consuming the progeny of bacteria that were on the living host beetle, but they do not address whether this is an event occurring in the wild, and if so, how frequent it is for Pristionchus. The claim of a necromenic relationship does not take into account the possibility that the nematodes may disembark from their insect host well before it dies, should they encounter some food source. Necromeny may take place occasionally, but beetle corpses may be a very minor source of food for Pristionchus.Finally, the data supporting the conclusion that y . When aFig 1 in ). In theer stage . The deler stage . Becauser 7 days . These dPristionchus species had associated them with a number of substrates. Sudhaus and Fürst van Lieven [Pristionchus species were described from soil, humus, compost, moss, "diseased" stem, coffee berry, rotting bulbs of Allium vineale, damaged roots of coffee and garlic, around roots of several species, rotten potatoes, rotten wood, and decomposing fungi. Additionally, Pristionchus species were described to be associated with a wide range of insects: termites (two species), Ostrinia (Lepidoptera), "dead insects", and finally beetles (three species). The spectrum of habitats and associated insects thus appears much wider than the recent literature indicates.By contrast, older literature on n Lieven  reviewedPristionchus ecology with what is known of the ecology of the model organism Caenorhabditis elegans and other Caenorhabditis species. The history of collecting Caenorhabditis has been different, as are the conclusions. Caenorhabditis nematodes have been found to be rare in soil, but abundant in rotting vegetal matter, such as fruits and soft plant stems. In addition, C. elegans, C. briggsae and C. remanei have been found on terrestrial molluscs, isopods and millipedes [C. nouraguensis were found on cockroaches in a tropical forest and C. tropicalis once on a beetle [C. japonica, have an apparently specific carrier insect [Pristionchus spp.It is interesting to compare the recent history of research on llipedes –27. In lllipedes ,21,27–30a beetle . Some spr insect –34. Thesr insect . The posPristionchus and Panagrellus. By surveying different types of rotting vegetal and fungal substrates, we found diverse Pristionchus spp. at a similar frequency as Caenorhabditis, often in high numbers and in non-dauer feeding stages in rotting fruits and stems. In addition, we report that a single species of Panagrellus (Nematoda: Panagrolaimidae), which we identify as Panagrellus redivivoides, is found in rotting fruits but not in rotting stems and appears to be associated with Drosophila fruitflies.We here provide data on nematode genera found in rotting vegetal matter, focusing on the genera C. elegans Normal Growth Medium agar plates [Escherichia coli strain OP50 in the center of the plate. The samples were spread around the bacterial lawn. 1–2 ml water or M9 solution were added to humidify the samples [Collected samples were placed onto standard r plates  previous samples .Caenorhabditis, Pristionchus, Panagrellus, Oscheius, Mesorhabditis species were identified as described in [Caenorhabditis were systematically identified to the species level as indicated in [For surveys in ribed in ,37 , posterior vulva and viviparous reproduction , their gut color is generally greyish/black, rather than brownish. Our notes are too scarce to distinguish them here and we placed them in a single category, with the exception of the ones for which we report 18S sequences.Genera of other rhabditids can be Oscheius are very commonly found in rotting vegetal matter or soil and can be recognized by their greyish gut color, their lack of middle pharyngeal bulb, their thin female tail and their long and inflated rectum. The occurrence of Oscheius is likely underestimated as these animals are less striking, rarely reach large population sizes and develop more slowly than the above. They are here almost exclusively represented by small-size hermaphroditic Oscheius of the Tipulae and sometimes Dolichura subgroups .Panagrolaimus are long and quite thin nematodes, with an only very slightly posterior vulva.Mesorhabditis species are easier to find after one to two days on the plate and they may remain in the vicinity of the sample. They are recognized by their posterior vulva and dark body color. In our surveys, they were particularly looked for and isolated in 2015–17.Protorhabditis/Prodontorhabditis/Diploscapter clade [Animals of the er clade ,51 are sRhabditophanes has a black gut color and characteristically lay almost round eggs, instead of oval-shape ones. They were not systematically surveyed.Bunonema individuals are easy to identify by their very small, spindle-shaped body, and a left-right asymmetric cuticle. They grow slowly and can easily be missed if their population is overwhelmed by other genera.The "Other" category contains nematodes that are generally not bacterial eaters but fungi-eaters such as aphelenchs or parasitic nematodes that do not grow in our culture conditions.C. elegans Normal Growth Medium agar plates. Pristionchus species were frozen with the C. elegans freezing protocol [2 to the freezing solution. This protocol does not allow for a good retrieval of Pristionchus and Panagrellus nematodes. We recently adopted a DMSO-Dextran protocol that allows for better recovery (courtesy of Andre Pires da Silva), whereby a pellet of nematodes in M9 solution is resuspended in 7 ml of a mix of 1 g Dextran (Sigma D9260-500G), 1 ml DMSO and autoclaved H2O to 10 ml. The nematodes are incubated for 10 min at room temperature before being place in the -80°C freezer in styrofoam boxes.Strains were derived from single individuals in selfing species, or from a mated female or a male-female pair in male-female species, and cultured on standard protocol , sometimPristionchus, we crossed 5 hermaphrodites from the P. pacificus JU1102 strain [For Panagrellus, we crossed 3–5 L4 females with 3–5 males and monitored first and second generation progeny. A control with 5 L4 females of the tester strain JU385 without males did not yield any progeny (2 replicates).For Pristionchus isolates was amplified using primers SSU18A (5'-AAAGATTAAGCCATGCATG-3') and SSU26R (CATTCTTGGCAAATGCTTTCG), and sequenced using SSU18A or SSU9R (AGCTGGAATTACCGCGGCTG), as in [5’-TACAATGGAAGGCAGCAGGC) and RHAB1868R (5’-CCTCTGACTTTCGTTCTTGATTAA) were used.In some surveys indicated in ), as in ; alternaPanagrellus isolates was amplified using primers D2A (ACAAGTACCGTGGGGAAAGTTG) and D3B (TCGGAAGGAACCAGCTACTA) as in [The large subunit  of ribosomal DNA of A) as in .The sequences were trimmed by visual inspection of the chromatograms, leaving the first four nucleotides of the downstream primer when present at the end of the sequence. A ‘N’ corresponds either to a low-quality sequence  or a putative polymorphism of rDNA repeats. The sequences are available at Genbank with accession # (SUBMITTED SUB4276706 (18S) and SUB4277191 (28S)).Panagrellus redivivoides 28S sequences  with default parameters. In case of the apparent polymorphism in our 2 to 103, 4 for 103 to 104, and 5 for > 104.The abundance index is defined on a Log10 scale as in . An indeStatistical analysis was performed in R version 3.4.1 .Caenorhabditis spp. Around 20 years ago, we first had sampled soil and found some Pristionchus  were sampled on many occasions while others have been sampled once.We sampled rotting vegetal matter, mostly fruits and stems from herbaceous plants , and occl matter . In 2015Caenorhabditis was found in 34% of rotting fruit samples (n = 556) and 41% of stem samples (n = 204) (n = 204) , Table 1Elegans supergroup [C. elegans, C. briggsae, C. remanei are commonly found in temperate areas. We also found C. elegans in samples from the Oku mountains (Cameroon) and South Yunnan (China) and C. elegans, C. briggsae and C. tropicalis in samples from São Tomé. Among the Caenorhabditis species outside the Elegans supergroup, we found C. virilis in rotting apples  and in Portugal  and Franodelphis  was founPristionchus was found in 31% of rotting fruit samples (n = 556) and 24% of stem samples , a Geophilus myriapod, a dead bee, a dead Helix aspersa snail . The three locations where Pristionchus was not found were those sampled for Mesorhabditis spp. (mostly humus/rotting leaves samples), where Caenorhabditis was also not found. The two locations where Pristionchus but not Caenorhabditis was found were at Northern latitudes (Lofoten Islands) and on mountains with a continental climate and temperatures below freezing in winter . Conversely, it would be interesting to establish whether Pristionchus may be less common than Caenorhabditis in equatorial regions  in one sample . Pristionchus was the predominant species in terms of numbers in some samples  and 0% of the rotting stem samples  . Rotting fruits contain bacteria and fungi and we observed that Panagrellus adults were often attracted to fungi (yeasts) on the culture plates.al 0–2%) . Thus, iDrosophila (various species). We sampled live Drosophila in the field and on two occasions found Panagrellus , but never from other substrates (except once in compost containing rotting fruits). Among the substrates we sampled, P. redivivoides thus appears specific to rotting fruits. It does not appear in every orchard or region where we have sampled, for example it was not found in Utah, while it was found in Oregon and Washington states (Panagrellus isolate (JU3343) in a compost heap of unclear composition but containing no fruits. As an exception that proves the rule, this isolate corresponds to a different Panagrellus species both through mating tests and morphology  was present in all types of samples. Although it was found frequently in soil [Rhabditella, Auanema, Pelodera, etc. Mesorhabditis was often present in small numbers, and also found in humus and rotting leaves in forests. Panagrolaimus was also found in soil but often present in rotting fruits and stems, even if quite dry. Rhabditophanes was particularly observed in cold weathers and climates. Fruit samples also commonly contained fungi-eating nematodes such as aphelenchs.Other nematode genera were found in rotting fruits and stems.  in soil , we had Caenorhabditis, Pristionchus and Panagrellus in fruits and stems. A weak positive correlation was found in rotting fruits for the co-occurrence of Pristionchus and Caenorhabditis . For example, in fruits, 41.0% of the samples with Caenorhabditis  also had Pristionchus and conversely, 44.5% of the samples with Pristionchus (n = 173) contain Caenorhabditis. No other correlations were found significant .Many of the different nematodes co-occurred in the same sample . Fig 3C Pristionchus was present in rotting apples on every tested date of the year, spanning July to January. Caenorhabditis was not found on the two January dates (see further data in [Panagrellus was not found in July (twice in different years) nor on three dates in December and January. data in ) and PanPristionchus nematodes are found in rotting vegetal matter frequently and abundantly, in both dauer and feeding stages. This includes P. pacificus and other "beetle-associated" Pristionchus species, such as P. uniformis, P. entomophagus, P. triformis, P. quartusdecimus or P. atlanticus [Pristionchus species feed in rotting vegetal matter and are thus not exclusively necromenic, as previously reported by only sampling beetles. These decomposing invertebrates are likely not their main source of food compared to the microbial blooms and other prey nematodes that Pristionchus may encounter in rotting vegetal substrates.Our results demonstrate that lanticus ,12,56,64Pristionchus have not been shown to be feeding in the wild on naturally decomposing beetle corpses. Thus, although it cannot be ruled out that Pristionchus may be necromenic on beetles in some instances, the relationship of Pristionchus species with beetles may be similar to the relationship of Caenorhabditis species with isopods or terrestrial molluscs. Indeed, when any of the carriers of Caenorhabditis are killed on a Petri dish as Pristionchus-bearing beetles have been, Caenorhabditis will exit the dauer stage and start reproducing. . Evible 1 in ). Note tlid wasp ). It is Pristionchus species (and possibly population) needs to be considered separately. Our survey found a variety of Pristionchus species covering several clades within the genus  are attracted to the oriental beetle Exomala orientalis. On the beetle, eggs and dauers are produced and developmentally arrest due to a beetle chemical studied in the article. Upon beetle death, P. pacificus exit the dauer stage and as adults may be attracted to another beetle. Alternatively, they adopt a "free" life cycle (likely meaning that they do not go through a dauer stage) and eventually may reenter the dauer stage or be attracted to a new beetle as adults. Presumably, the free life cycle is on the dead beetle.A representation of the proposed , based oPristionchus adults [Two features of this proposed life cycle are surprising: i. the stage that is attracted to the beetle is the adult nematode (which has not been observed so far on wild-caught beetles); ii. once on the beetle, the adult nematode is able to produce dauer progeny. This new view of the nematode life cycle was probably derived from the fact that most behavioral studies of chemotaxis towards beetles used s adults ,17,67,68s adults ,70, whicP. pacificus and other Pristionchus species that may be found associated with various beetles and other insects. This life cycle is similar to that proposed for C. elegans [Pristionchus spp., whose populations expand until the food is exhausted whereupon the young larvae enter the dauer stage. Beetles may transport dauer larvae between these patchy plant food sources. In this life cycle scenario, the association of dauer larvae with the larger invertebrate is phoretic. Necromeny may be occasional, but its occurrence remains to be demonstrated.We propose in  elegans ,71. In oP. pacificus and other Pristionchus spp. may feed on other nematodes as a food source in decomposing vegetal matter. The predatory behavior of many Pristionchus species towards other nematodes is rendered possible by the plasticity of development of the mouth form [Pristionchus species in rotting vegetal matter, including but not restricted to Caenorhabditis and Panagrellus species (Pristionchus. We found a weak positive correlation for the co-occurrence of Pristionchus and Caenorhabditis in rotting fruits. Rather than a specific attraction of Pristionchus to Caenorhabditis-containing substrates (or conversely), we propose that this correlation may be simply explained by the degree of decomposition, humidity or microbial fauna in the fruits, which needs to be such as to sustain these nematode species.In addition to bacteria and fungi, uth form ,72,73. F species . These oC. elegans was shown to avoid sulfolipids secreted by Pristionchus pacificus [C. elegans and P. pacificus in the same sample. It would be interesting to sample further in areas where both C. elegans and P. pacificus were found, such as Southern California, Hawaii, South Africa or La Réunion [C. elegans and P. pacificus were chosen as the two most studied species of each genus, and it is not known whether the sulfolipids and the reaction to them are specific for these two species. It is possible that the avoidance behavior of C. elegans has evolved as a response to several Pristionchus species, and that conversely, several Caenorhabditis species may show avoidance to Pristionchus pacificus or other Pristionchus species.acificus . Due to  Réunion ,75. C. ePristionchus spp. dauers phoretic on beetles, it must be assumed that the beetles visit the kinds of habitats where we found Pristionchus populations, i.e. rotting fruits and stems. Are the beetles that have been associated with Pristionchus spp. likely to visit fruits or a forest floor with rotting stems? Detailed studies would be needed to answer this important question, taking into account the seasonality of the beetle’s life cycle itself. Geotrupes stercorosus was found to be the most reliable beetle source of Pristionchus nematodes in Europe by [Geotrupes adults may feed on forest litter or fungi [P. pacificus-carrying Exomala orientalis adults emerge from soil [Pristionchus were found to feed as adults on soft, high-quality diets and the micro-organisms therein [Pristionchus spp. and test our hypothesized life cycle . Further work would be required to make sure that there are two different species. Because of differences in rDNA sequence with P. levitatus, abundance on several continents and priority, we identify the biological species we found in rotting fruits as Panagrellus redivivoides.Our mating tests show that all the vivoides ,58. Its in Italy  and botheevil in . A new sevitatus . This sprence to  for theiP. redivivoides may be associated with Drosophila fruit flies, in contrast to other Panagrellus sp. that appear associated with bark beetles. The fruit fly association relies on several reports. Early on, using Drosophila traps made of potato puree, Aubertot [Panagrellus silusiae", now synonymized with P. redivivus [P. redivivoides was not described yet). P. redivivoides was found several times in laboratory cultures that were visited by fruitflies, including in the original description of the species by Goodey [Drosophila flies. Lees [P. silusiae"  and Drosophila funebris and showed that the latter could transport the former to new Petri dishes. "Panagrellus zymosiphilus", now synonymized with P. redivivoides [Drosophila cultures, the second time on Drosophila obscuroides recently isolated from nature [P. zymosiphilus" was also found in grapes, presumably brought by a Drosophila [Drosophila cultures with JU385, isolated in a Drosophila willistoni culture , the laP. redivivoides, is the most commonly associated with rotting fruits and Drosophila larvae, a distinguishing ecological feature compared to most Panagrellus species. Its life cycle may be similar to that depicted for C. elegans [Pristionchus , Pristionchus (yellow), Panagrellus (orange) are indicated first. The other groups are indicated roughly in order of ease of extraction from the sample and identification. On the right, in columns 'Caenorhabditis abundance log index’ and ‘Pristionchus abundance log index’, is indicated the abundance log index Click here for additional data file.S2 TablePristionchus. The table indicates the origin of the strains of Pristionchus (with JU or BRC standard strain names), and other samples that were noted to contain Pristionchus. The sample ID is a temporary ID for a given collection date. Other non-Pristionchus frozen strains from the same samples are indicated on the right. nd: not determined. Sheet B: Panagrellus. The table indicates the origin of Panagrellus strains. Sheet C: Other species of nematodes collected from rotten fruit and characterized by 18S rDNA sequencing.Sheet A: (XLSX)Click here for additional data file.


AR transcript variants. Moreover, inhibition of several splicing factors repressed tumor growth in vivo. Altered expression of splicing factors is correlated to biochemical recurrence in prostate cancer patients. Thus, these findings suggest that splicing factors would be a potential therapeutic target. This review focuses on the emerging roles of splicing factors in prostate cancer progression and AR signaling.Although inhibition of the androgen–androgen receptor (AR) axis effectively represses the growth of prostate cancer, most of all cases eventually become castration-resistant prostate cancers (CRPCs). Enhancement of the expression of AR and its variants along with the downstream signals is important for disease progression. AR-V7, a constitutive active form of AR, is generated as a result of RNA splicing. RNA splicing creates multiple transcript variants from one pre-messenger RNA (mRNA) by removing introns/exons to allow mRNA translation. The molecular mechanisms leading to marked increases of AR and generation of AR-V7 have been unclear. However, recent papers highlighted the roles of RNA splicing factors which promote AR expression and production of variants. Notably, a broad range of splicing components were aberrantly regulated in CRPC tissues. Interestingly, expression of various spliceosome genes is enhanced by RNA-binding protein splicing factor proline- and glutamine-rich (PSF/SFPQ), leading to changes in the expression of  Dysregulation of gene expression is a well-known feature of cancer cells . Of noteAR-V7 mRNA is spliced at the alternative 3’ splice site (3’SS) of a cryptic exon, exon 3B, rather than at the 3’SS of exon 4 [AR-V7 mRNA is produced. It has been established that AR-V7 expression is well correlated with CRPC development and resistance to androgen deprivation therapy [Because prostate cancer progression is dependent on androgen signaling, androgen depletion therapy (ADT) is the primary treatment for advanced prostate cancer ,15. Althf exon 4 . However therapy ,20,21,22 therapy . A recen therapy , these fTumors that express canonical AR signaling are highly sensitive to ADT . PatientProtein-coding exons are disrupted by non-coding introns . ThroughMechanistically, both the regulatory RNA sequences and their correlated RNA splicing proteins are important for determining splicing site. Some regulatory sequences are called exonic splicing enhancers (ESEs) or intronic splicing enhancers (ISEs) . MoreoveDuring transcription elongation, these protein complexes move along the gene. The RNA spliceosome complex screens the pre-mRNA to determine and excise the splice sites before the termination of transcription . Thus, tSpliceosome assembly needs to occur repeatedly every time an intron is removed from a pre-mRNA in a eukaryotic nucleus . An intrFive snRNP complexes  assemble and recognize on each intron to form a catalytically active spliceosome ,8,9,30. After the preassembled U4/U6.U5 tri-snRNP complex is recruited to the splicing site, the U1/U4 snRNPs are released to form the catalytically activated complex B (complex B*). Further conformational rearrangements lead to the formation of the catalytic step 1 spliceosome (complex C) .(APC) result in exon skipping or the creation of a new splice site [(TP53) were found in close proximity to splice sites, leading to intron retention or activation of a cryptic splice site to produce a frameshifted mRNA [SF3B1 [U2AF1 [SRSF2 [SF3B1 mutation) [SRSF2 mutation) [SF3B1, U2AF1, and SRSF2 were found as heterozygous point mutations at specific amino acids and occur in a mutually exclusive manner to one another [SF3B1 mutations in uveal melanoma (15–19%), pancreatic ductal adenocarcinoma (4%), and breast cancer (2–4%), as well as U2AF1 mutations in lung adenocarcinoma (3%) [Unexpected, recent genomic sequencing analysis has revealed that the dysregulation of pre-mRNA splicing is involved in cancer initiation, maintenance, and progression ,36. Geneice site ,41. Simited mRNA  and chroted mRNA . These mted mRNA ,47,48,49A [SF3B1 , U2AF1 [ another . Mutatiooma (3%) ,46,50. Ioma (3%) .SF3B1 (1.1%) and U2AF1 (0.5%), which are associated with the recognition of 3’ SS, were mutated. However, the frequency of mutation in these factors is relatively low compared with other types of cancer [AR splicing in prostate cancer progression [AR splicing process. AR-V7 mRNA synthesis requires the removal of intron sequences through an RNA recognition process. The spliceosomal complex excises the 3’ SS and ligates exon 3 with exon 3B by recognizing this exon [The alteration of splicing activity by mutations induces severely abnormal development and tumorigenesis . Howeverf cancer ,45,46,47gression . Alteredhis exon ,56. Notahis exon ,57. The PSF mRNA expression was significantly elevated in metastatic or advanced prostate cancer samples, suggesting that PSF expression was associated with prostate cancer progression. Using an in vivo model of CRPC, knockdown of PSF reduced the growth of tumors in mice castrated after tumor development. Notably, the study presented a new mechanistic insight, with the identification of global RNAs bound with PSF. Long non-coding (lnc) RNAs, such as androgen-regulated lnc RNA, C-terminal binding protein 1 antisense (CTBP1-AS) [(SchLAP1) [A recent study showed a role of a splicing factor, proline- and glutamine-rich (PSF/SFPQ), in the progression of prostate cancer by using the deep-sequence based approach of clinicopathological analysis . High PSTBP1-AS) , and a pSchLAP1) , are posSchLAP1) . FurtherSchLAP1) . These aSchLAP1) .AR transcript with PSF was observed in CRPC model cells compared with hormone-dependent cancer cells [AR splicing process and promotes production of AR and its variants at the mRNA level. PSF forms a complex with other RNA-binding proteins and non-POU domain-containing octamer-binding protein (NONO) [AR transcripts and AR-V7 production   to promooduction . Throughd HNRNPU ,64,65,66HNRNPH1 transcripts negatively regulated by miR-212 were associated with the expression of AR and AR-V7 in prostate cancer cells. HNRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3), and activates androgen-regulated genes in both a ligand-dependent and -independent manner. Silencing of HNRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumor growth in vivo [A genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen has identified the essential genes for prostate cancer growth . This st in vivo . These f in vivo . HNRNPL is also involved in circular RNA (circRNA) formation ,73. TranAR-V7 in prostate cancer cells [AR-V7 splicing. The expression of JMJD1A was significantly associated with that of AR-V7 in prostate cancer tissue. Thus, the level of JMJD1A or factors that regulate the JMJD1A–HNRNPF interaction would promote the alternative splicing of AR-V7 in prostate cancer. Because increased levels of AR and AR-V7 are important for CRPC progression [The histone demethylase, Jumonji domain containing 1A (JMJD1A), was originally found to function as a key coactivator for AR through epigenetic regulation of H3K9 methylation marks ,75. Moreer cells   box (DDX) RNA helicases such as DDX39A, B [(MALAT1) [MALAT1 is regulated by suppressive androgen responsive element (ARE) in the promoter. Therefore, treatment with enzalutamide, an AR antagonist, enhances the expression. Increased MALAT1 associates with SRSF1  to promote AR-V7 production. MALAT1 knockdown and the enhanced AR-V7 degradation suppressed enzalutamide-resistant prostate tumor growth in vivo [Another RNA-binding protein, Src-associated in mitosis 68 KDa protein (Sam68), is involved in an alternative splicing process by 3’-end formation of mRNA . Sam68 iDX39A, B , lncRNA (MALAT1) , and sev(MALAT1)  and AKT (MALAT1) , are als in vivo .A subtype of castrate-resistant prostate cancer, called treatment-induced neuroendocrine prostate cancer (NEPC), has occurred owing to the widespread use of AR blockers . NEPC haREST mRNA inactivates REST, implying that SRRM4 induced NEPC independently from the REST-mediated mechanism [It is of note that a splicing event is an essential part of this mechanism. Serine/arginine repetitive matrix 4 (SRRM4) strongly drives NEPC development . SRRM4 iechanism . SRRM4 iechanism . In addiechanism . One tarechanism ,95. Thesechanism .There is an abundance of evidence that the alternative splicing pathway is essential for gene regulation in cells. The dysregulation of splicing is involved in many types of cancer, including prostate cancer. In this review, a number of cancer-specific spliceosomal factors have been discussed, including PSF and SRRM4. From the analysis of clinical data, it has been demonstrated that a variety of splicing factors are upregulated in metastatic prostate cancer tissues, indicating the possibility that enhanced splicing ability induces the diversity of gene functions and drives the progression of prostate cancer into CRPC. There is a pressing need to expand research into alternative splicing in the context of many other important diseases. Moreover, understanding the regulation of alternative splicing should provide a new pathway for the development of novel therapeutic strategies.


Arabidopsis was identified as a positive regulator of systemic acquired resistance (SAR), paralogs NPR3 and NPR4 were later shown to be negative SAR regulators. The mechanisms involved have been the subject of extensive research and debate over the years, during which time a lot has been uncovered. The known roles of this protein family have extended to include influences over a broad range of systems including circadian rhythm, endoplasmic reticulum (ER) resident proteins and the development of lateral organs. Recently, important advances have been made in understanding the regulatory relationship between members of the NPR1-like protein family, providing new insight regarding their interactions, both with each other and other defense-related proteins. Most importantly the influence of salicylic acid (SA) on these interactions has become clearer with NPR1, NPR3, and NPR4 being considered bone fide SA receptors. Additionally, post-translational modification of NPR1 has garnered attention during the past years, adding to the growing regulatory complexity of this protein. Furthermore, growing interest in NPR1 overexpressing crops has provided new insights regarding the role of NPR1 in both biotic and abiotic stresses in several plant species. Given the wealth of information, this review aims to highlight and consolidate the most relevant and influential research in the field to date. In so doing, we attempt to provide insight into the mechanisms and interactions which underly the roles of the NPR1-like proteins in plant disease responses.The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and related NPR1-like proteins are a functionally similar, yet surprisingly diverse family of transcription co-factors. Initially, NPR1 in  NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), as well as PATHOGENESIS-RELATED (PR) genes, play a fundamental role in a plant’s response to pathogen challenge. NPR1 plays a significant role in the establishment of systemic acquired resistance (SAR) as well as induced systemic resistance (ISR)  gene expression and the overall role of NPR1 in pathogen defense are important topics of study.The ce (ISR) ; it actsse to SA . NPR1 is factors . The exaPR gene expression. Activation of various TGA transcription factors occurs under these conditions (PR gene expression (12–16 h after SA treatment)  . Expressathogens . Hence NNPR1-like genes have been found. This family of proteins contain ankyrin repeats and Broad Complex, Tramtrack and Bric a brac/Pox virus and Zinc finger (BTB/POZ) domains, two well documented protein–protein interaction domains  . In respSA induction-deficient 2 (sid2) or enhanced disease-susceptibility 5 (eds5) and plants expressing the salicylate hydrolase nahG gene, display compromised SAR induction , salicyloyl glucose ester (SGE), methyl salicylate (MeSA), methyl salicylate O-β-glucoside (MeSAG) and 2,5 dihydroxybenzoic acid (gentisic acid)  the cinnamic acid pathway which requires PHENYLALANINE AMMONIA LYASE (PAL) and (2) the isochorismate pathway requiring ISOCHORISMATE SYNTHASE (ICS) and ISOCHORISMATE PYRUVATE LYASE (IPL) . The isoc stress , a statepersicum . Severalic acid) . NotablyPR genes . In factPR genes . Most nod potato . Howeverelopment . In addielopment . Other celopment .Pseudomonas syringae pathovars produce the phytotoxin coronatine, which indirectly represses ICS1 and activates BENZOIC ACID/SALICYLIC ACID CARBOXYL METHYLTRANSFERASE 1 (BSMT1) expression, which converts SA into MeSA, to suppress SA accumulation , stimulates JA responsive pathways thereby suppressing SA signaling -like motif within the PR promoter  proteins . These ppression . Howeverpression . Moreovenduction . SimilarNIMIN-2a . In addi tobacco .nim1-4 mutant (Arg432Lys) severely impairs its potential to establish SAR  proteins , a gain-of-function mutation which renders the mutant protein insensitive to SA and constitutively represses SA-inducible defense genes  and BOP2 genes encode proteins with structure similar to other NPR1-like proteins, containing both N-terminal BTB/POZ and C-terminal ankyrin repeat domains  and PR1 , Sec61α, DEFENDER AGAINST APOPTOTIC DEATH 1 (DAD1), and CALRETICULIN 3 (CRT3) (cis-element (TL1) which is bound by TBF1 a heat shock factor-like transcription factor instrumental in the growth-defense transition  . These sansition . Increasansition . Supportsyringae . Similarapoplast . Furtherand CRT3 . Thus, tArabidopsis  promoting the existence of NPR1 in its oligomeric form, opposing the action of SA-induced thioredoxins , which is positively involved in SA-induced defense gene expression, interacts with and sumoylates NPR1 following SA treatment . This inoylation . Additiooylation . TogetheArabidopsis pks5 mutant as is with the S11A/S15Anpr1 mutant displays reduced expression of WRKY38 and WRKY62  interacts with and phosphorylates NPR1, specifically Ser589 and likely also Thr373, which are required for nuclear import of NPR1 and subsequent pression . Interespression . It is ppression . Furtherpression . The autpression . TherefoTIMING OF CAB2 EXPRESSION 1 (TOC1), an evening circadian clock gene, is upregulated by the application of SA. However, the timing of its expression does not change irrespective of whether SA is applied at dawn or dusk , a known antagonist of TOC1  in Arabidopsis is able to complement the npr1 mutant, however, several negative consequences are observed, including enhanced herbivore susceptibility  and MpNPR1  enhanced resistance to several important pathogens in Malus x domestica  increased salt tolerance (Arabidopsis npr1 mutants are complemented using BnNPR1 (Brassica napus) while overexpression in B. napus enhanced resistance to P. syringae without any obvious negative effects, emphasizing similarity between NPR1-dependant defense responses in these two species (NPR1 incompatibility between species is made apparent as apical dominance is affected in Arabidopsis overexpressing VvNPR1.1 (Overexpression of  studied . Apple Aomestica . Likewissyringae . In tobaolerance , 2014. Solerance . Additio species . HoweverVvNPR1.1 . These rNPR1-like genes across various tissues in Arabidopsis (NPR1-like genes exhibit unique spatial preferences (Arabidopsis npr3 knockout mutants which display increased resistance to P. syringae on developing flowers but not leaves (Since its discovery more than 20 years ago, NPR1 has been the focus of countless studies. During this time several NPR1-dependant pathways have been uncovered as have many of the complex mechanisms governing the regulation of NPR1 . Howeverbidopsis . Similarferences . The impt leaves .Important aspects of the regulation of NPR1 function and homeostasis which require further attention are that of post-translational modification and proteasome degradation of NPR1. Important questions have been raised regarding these processes, including whether NPR3 and NPR4 act as E3 ligases which lead to the ubiquitinylation of NPR1. Surprisingly, even though NPR2 is induced by biotic stress and was shown to play a significant role in the perception of SA (Arabidopsis, MAPK6, influences the activity and gene expression of NPR1 (Arabidopsis npr1-5 null mutants which are impaired in SA biosynthesis suppress precocious leaf senescence characteristic of pat14 mutants (Given that NPR1, NPR3, NPR4, BOP1, and BOP2 all function in the regulation of various transcriptional processes, it is hard to ignore the possibility that NPR2 may serve a similar yet undefined function. This is especially true since on of SA . Phytohoon of SA . Further of NPR1 . Therefo mutants . Togethein planta and across species. Instead emphasis should be placed on describing temporal, spatial and plant–pathogen interaction specific functions. Nonetheless, continued research on the NPR1-like protein family is warranted and will undoubtedly bring forth novel insights into the molecular pathways involved in plant stress responses and development.Given the complex nature of the NPR1-like protein family and uncertainty surrounding the aspects mentioned above, caution should be exercised regarding generalizing statements concerning functions RB conceptualized, drafted, and reviewed the manuscript. SN reviewed and assisted in drafting the manuscript. NvdB conceptualized, reviewed, and assisted in drafting the manuscript. All authors contributed to and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neuropilin-1 (NRP1) is an essential transmembrane receptor with a variety of cellular functions. Here, we identify two human NRP1 splice variants resulting from the skipping of exon 4 and 5, respectively, in colorectal cancer (CRC). Both NRP1 variants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes. This increased endocytic trafficking of the two NRP1 variants, upon HGF stimulation, is due to loss of N-glycosylation at the Asn150 or Asn261 site, respectively. Moreover, these NRP1 variants enhance interactions with the Met and β1-integrin receptors, resulting in Met/β1-integrin co-internalization and co-accumulation on endosomes. This provides persistent signals to activate the FAK/p130Cas pathway, thereby promoting CRC cell migration, invasion and metastasis. Blocking endocytosis or endosomal Met/β1-integrin/FAK signaling profoundly inhibits the oncogenic effects of both NRP1 variants. These findings reveal an important role for these NRP1 splice variants in the regulation of endocytic trafficking for cancer cell dissemination. O-glycosylation of neuropilin-1 is involved in cancer migration and invasion. Here, the authors show that defects in N-glycosylation of two neuropilin-1 splice variants enhance the co-internalisation of Met, it’s interaction with β1-integrin, and subsequent constitutive activation of FAK/p130Cas signalling in endosomes to promote colorectal cancer metastasis. NRP1) gene encoding a transmembrane protein is located on chromosome 10p12 and consists of 17 exons2. NRP1 acts as a co-receptor for several growth factors including VEGF, TGF-β, HGF, FGF and PDGF, and exhibits versatile functions for neuronal axon guidance, angiogenesis and cancer initiation, growth, and metastasis6. The multifunctional capacity of NRP1 is attributed to its four ligand-binding domains , a membrane proximal MAM domain (c), and a cytoplasmic C-terminal domain7. NRP1 interacts with several receptor tyrosine kinases  and other transmembrane proteins  to elicit a range of intracellular signaling cascades initiated by specific ligands14. Upon ligand binding, receptors are internalized by endocytosis and transported to early and late endosomes before either recycling back to the plasma membrane or selected for degradation15. Endosomal signaling activated by endocytosis of receptor tyrosine kinases such as Met and EGFR play a critical role in cellular functions including, organism development and cancer progression18.The human neuropilin-1  NRP121. NRP1 also undergoes post-translational glycosylation modifications including O-linked glycosylation at site Ser61223 and N-linked glycosylation at several putative asparagine residues25. The O-linked glycosylation of NRP1 at Ser612 plays an important role in the modulation of VEGF signaling, cell proliferation and migration and cancer invasion27. However, the role of N-linked glycosylation of NRP1 is poorly characterized and understood.Several human NRP1 variants generated by alternative splicing mechanisms have been reportedIn this study, we discover two human NRP1 splice variants from colorectal cancer (CRC) cell lines and tissue specimens. The two NRP1 variants are generated by skipping exon 4 and exon 5, resulting in defects in N-linked glycosylation at asparagine (N) positions N150 and N261, respectively. The altered N-linked glycosylation plays a critical role in regulation of the endocytic trafficking of NRP1 and its associated receptors, Met and β1-integrin, for CRC cell migration, invasion and dissemination. Furthermore, our work highlights that CRC expressing these NRP1 splice variants could potentially be targeted by blocking endocytosis of them and their binding receptors such as Met or β1-integrin, or their endosomal signals on activation of the FAK/p130Cas pathway.22. To explore the functional importance of NRP1 in CRC progression, we cloned NRP1 cDNA from an HCT116 CRC cell library by RT-PCR using primers at the 5′ and 3′ends of the full-length human WT NRP1 open reading frame (2772 bp cDNA encoding 923 amino acids). Surprisingly, sequence analysis of NRP1 cDNA clones led to characterization of two NRP1 alternatively spliced transcripts: one that skipped exon 4 with exon 3–5 splicing  sequence1 for generation of the NRP1-∆E4 and NRP1-∆E5 variants. To further validate the existence of these variants, RT-PCR of the region that includes exons 4 and 5 was performed with several human CRC cell lines and a subset of CRC tissues as well as the adjacent nonmalignant colonic mucosa using the forward primer in exon 3 and the reverse primer in exon 6. Three bands were visualized on gel electrophoresis in HCT116 cells: the expected NRP1-WT 532-bp, along with NRP1-ΔE4 376-bp and NRP1-Δ5 304-bp fragments  and the primary CRC and liver metastasis tumor cell lines  at an expression level equal to or above that found for NRP1-WT mRNA  and not significantly observed as tumors progressed through stages I–IV  compared with NRP1-WT in HT29 cells Fig. . NRP1-∆ETo determine the rates of NRP1 internalization and subsequent recycling, surface-biotinylated NRP1 isoforms were measured by streptavidin-agarose pulldown after different incubation periods. After 15 min incubation with regular culture medium, 50–73% of the two NRP1 splice variants were internalized versus 12% of the NRP1-WT Fig. . On re-i25 with the consensus motif, N-X-S/T31  and mature β-chain (p-145) forms of Met, as well as with β1-integrin  in HCT116 cells expressing NRP1-∆E4 or NRP1-∆E5, compared to the modest inhibition (~20%) observed in the cells expressing NRP1-WT Fig. . Given t15. To determine if endocytosis of the NRP1 variants, Met and β1-integrin are dependent on dynamin, HCT116 cells were treated with the dynamin inhibitor, dynasore37, or transfected with a dynamin dominant-negative construct (dynamin-2 K44A-GFP). Treatment with dynasore or expression of the dynamin-2 K44A mutant increased the levels of the NRP1 variants, Met and β1-integrin at the plasma membrane with a concomitant decrease in their intracellular pools  and Y1349 (docking site) over that seen for NRP1-WT Fig. , which smes Fig. , and silmes Fig. . Furthermes Fig. . CollectSilencing FAK using siRNA or treatment with the FAK specific inhibitor VS-6063 that inhibits phosphorylation of both FAK and p130Cas, showed a greater inhibition of cell migration and invasion in HCT116 cells expressing NRP1-∆E4 or NRP1-∆E5 than in those expressing NRP1-WT Fig. . Similar40. Characterization of the NRP1 variants generated by alternative splicing mechanisms and modification of NRP1 by O-linked glycosylation have provided profound insights into our understanding of the regulation of NRP1 function in cancer growth, angiogenesis, invasion, metastasis and modulation of therapeutic outcomes41. Here, our findings add two human NRP1 splice variants generated by the skipping of exon 4 and exon 5, respectively, in CRC. While the NRP1-ΔE4 is dominantly expressed in CRC associated with tumor progression, both NRP1 splice variants are defective in N-linked glycosylation modification, and exhibit increased endocytosis/recycling activity with decreased levels of degradation, which lead to their accumulation on endosomes and persistent activation of FAK/p130Cas signaling through interaction and co-internalization with Met and β1-integrin. This perturbed trafficking of the two NRP1 splice variants promotes CRC cell migration, invasion and metastasis, directly linking endocytosis and metastatic progression  or β1-integrin can remain active within endosomes to provide spatially and temporally restricting signals that contribute to pathway-specific tumor progression44. NRP1 lacks a typical kinase domain, primarily functioning as a co-receptor to form ligand-specific receptor complexes7. Our data are consistent with the view10 that NRP1-WT associates with the Met receptor and resides on the plasma membrane, and that they co-internalize upon stimulation by HGF, but not VEGF, followed by rapid degradation in CRC cells  NRP1-ΔE4 and NRP1-ΔE5 lack a part of a2 domain with defective N-glycosylation modification at the sites N150 and N261, respectively, and had modest internalization upon VEGF165 stimulation; and (2) the other three putative N-glycosylation-defective mutants, N300Q, N522Q and N842Q, or the O-linked glycosylation-deficient S612A mutant did not affect NRP1 localization on the plasma membrane and were less responsive to HGF stimulation, we propose that the partial deletion of a2 domain or defect in N150- or N261-linked glycosylation modification may reduce NRP1 binding to VEGF165, but enhance its binding to HGF, which results in the observed enhancement in HGF-stimulated endocytosis even in cells with depletion of Met or β1-integrin under basal conditions . All media were supplemented with 10% FBS (Sigma), streptomycin (100 µgml−1) and penicillin (100 unitsml−1). All cell lines were subjected to regular mycoplasma testing via PCR using e-Myco Plus kit (iNtRON Biotechnology) and underwent short tandem repeat (STR) profiling (Genetica). The 126 frozen fresh primary colorectal tumors including NF90, NF99, NF103, NF105, NF106, and NF110 and adjacent normal control tissues (N1-4) were collected from patients who had undergone surgery resections at the Nanfang Hospital, Southern Medical University, China and the Markey Cancer Center, University of Kentucky. Experiments were performed under protocols approved by the Southern Medical University Ethics Committee and the University of Kentucky Institutional Review Board. All CRC cases were confirmed by a senior pathologist and staged based on the 2011 Union for International Cancer Control TNM classification of malignant tumors. The pathological diagnoses of all enrolled patients were confirmed by two different pathologists according to the WHO grading system57. Data regarding the clinical characteristics of patients are listed in Supplementary Tables Human HCT116, HT29 and DLD-1 cell lines were obtained from American Type Culture Collection (ATCC). HCT116 and HT29 cells were cultured in McCoy’s medium (Sigma). DLD-1 cells were cultured in RPMI-1640 medium (Sigma). Primary human CRC cell lines, Pt93 and Pt130, as well as Pt2337 and its paired liver metastasis cell line, LM2377, were established from patient primary and metastatic tumors 165, EGF, and HGF were purchased from R&D Systems. LysoTracker Deep Red was purchased from ThemoFisher Scientific. Cycloheximide, tunicamycin, swainsonine, benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) and dynasore were obtained from Sigma. PHA-665752 was purchased from Selleckchem. VS-6063 was purchased from MedChem Express.VEGFTotal RNA was isolated from cells using the RNeasy plus mini kit (Qiagen), and from tissues using TRIzol reagent (ThermoFisher Scientific), according to the manufacturer’s instructions. Equal amounts of RNA were used as templates for all reactions. cDNA was generated with the SuperScript III First Strand Synthesis System (ThermoFisher Scientific). PCR was performed using the Phusion Hot Start II High-Fidelity DNA Polymerase kit (ThermoFisher Scientific). The primers 5′-TACGAAACACATGGTGCAGGA-3′ (forward) and 5′-CTGCAGACCAGTTGGTGCTAT-3′ (reverse) were used to amplify NRP1-WT, NRP1-∆E4 and NRP1-∆E5 in the human samples. The amplified PCR fragments were separated on an agarose gel and subjected to DNA sequencing. Primers 5′- ACAACTTTGGTATCGTGGAAGG -3′ (forward) and 5′-GCCATCACGCCACAGTTTC-3′ (reverse) were used to amplify human GAPDH.59. The NRP1 mutants N150Q, N261Q, N150Q/N261Q, N300Q, N522Q, N842Q and S612A were generated using a QuikChange XLII mutagenesis kit (Stratagene). All sequences were verified by automated DNA sequencing. To establish stable transfectants with specific protein expression, cells were infected with lentivirus using the indicated pLenti6.3 constructs followed by selection with puromycin (2 µgml−1) for 7–10 days58. Dynamin-2 K44A-GFP (#34687) and WT dynamin-2-GFP (#34686) were purchased from Addgene.Human NRP1-WT, NRP1-∆E4 and NRP1-∆E5 were amplified by PCR using a HCT116 cDNA library, and then subcloned into the pLenti6.3 or pCMV6 vector−1) for 7–10 days58.The siRNA against human NRP1 #79833, #79835), clathrin , FAK , p130Cas  and non-targeting control siRNA (#0207) were obtained from GenePharma. The ON-TARGETplus human GIPC1 siRNA pool (L-019997-00-0005) and non-targeting control siRNA pool (D-001810-10) were purchased from Dharmacon. Cells were transfected with 50 nM siRNA against the indicated genes or control siRNA using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (ThermoFisher Scientific). After 36–48 h transfection, cells were utilized for the indicated assays. The lentiviral shRNAs against human NRP1, Met, β1-integrin or FAK were cloned into the pLKO.1 vector (Sigma). The Non-Target Control shRNA (SHC002) was from Sigma. Both siRNA and shRNA sequences are listed in Supplementary Table 3, #798354) were grown on collagen-precoated coverslips in the regular growth medium containing 10% FBS, or serum starved overnight followed by stimulation with VEFG165 (50 ngml−1), EGF (50 ngml−1) or HGF (50 ngml−1) for 30 min. Subsequently, cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized in 0.2% Triton X-100 and 0.5% BSA in PBS for 5 min and then blocked with 4% BSA in PBS for 10 min. The cells were incubated overnight at 4 °C with the indicated primary antibody. After three washes with 0.05% Triton X-100 in PBS, cells were incubated with the indicated secondary antibody for 1 h. Cells were then washed three times, mounted with DAPI containing mounting medium , viewed, and photographed under a Nikon A1+-Ti2 confocal microscope. For image quantifications, picture fields were chosen arbitrarily on the basis of DAPI staining. For the NRP1 variant and mutant intracellular localization and co-localization experiments, ten pictures were taken per condition. The percentages of co-localizations were determined using Nikon NIS-Elements AR software. The primary and secondary antibodies used in this assay are listed in Supplementary Table Cells 5 × 10 were groCells were lysed in RIPA buffer . Protein concentrations were measured using the BCA protein assay reagent (Thermo Fisher Scientific). The cell lysates (500 µg protein) were immunoprecipitated with 2 µg of the indicated antibody overnight followed by incubation with a 50% slurry of protein G sepharose beads for 3 h at 4 °C. The beads were washed three times with the lysis buffer, and the immunoprecipitated protein complexes were resuspended in 2× Laemmli sample buffer followed by western blot analysis. For western blot analysis, equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, immunoblotted with specific primary and secondary antibodies, and detected using chemiluminescence . A list of all antibodies used in this work and dilutions can be found in Supplementary Table Cells were lysed in RIPA buffer, and the cell lysates were treated with PNGase F (peptide N-glycosidase F) for 3 h at 37 °C according to the manufacturer’s instructions . The reaction was stopped by the addition of 5 × loading buffer followed by western blot analysis using anti-NRP1 antibody .N-hydroxysulphosuccinimide (sulpho-NHS)-SS-biotin; ThermoFisher Scientific) according to the protocol described by Joffre et al.17. Briefly, cells were incubated with 0.15 mg ml−1 biotin for 10 min at 4 °C and the excess biotinylation reagent was quenched by washing with the cold buffer containing 25 mM Tris at pH 8, 137 mM NaCl, 5 mM KCl, 2.3 mM CaCl2, 0.5 mM MgCl2 and 1 mM Na2HPO4. Cells were lysed in RIPA buffer and centrifuged. A fraction of the supernatant  was collected. The residual supernatant was added to pre-washed streptavidin-agarose beads (ThermoFisher Scientific) and rotated at 4 °C for 2 h. Beads were collected by centrifugation and the supernatant  was collected. Beads were washed 3 times with the RIPA buffer at 4 °C and proteins (surface pool of NRP1 or Met = ‘bound’) were extracted by heating at 95 °C with sample buffer. Equivalent volumes were analyzed by western blot and densitometric analyses were carried out. The percentage of intracellular NRP1 or Met was calculated using the formula: intracellular NRP1 or Met receptor = (NRP1 or Met in unbound fraction)/ × 100.To measure the relative proportions of the cell surface and internal pools of NRP1, cell surface proteins were labeled covalently using a membrane-impermeable biotinylation reagent  in 50 mM Tris and 100 mM NaCl at pH 8.6 for 15 min. MesNa was quenched by the addition of 180 mM iodoacetamide  for 10 min.The trafficking assay for NRP1 and its variants was performed according to the method described by Joffre et al.To measure internalized NRP1 and its variants, the assay was terminated by lysing the cells. Lysates were passed three times through a 27-gauge needle and clarified by centrifugation . Equal protein amounts were incubated with streptavidin-agarose beads with agitation at 4 °C for 2 h. The beads were collected by centrifugation , washed in lysis buffer and proteins were extracted by heating at 95 °C with sample buffer.To measure the proportion of internalized NRP1 proteins and the variants that recycle back to the cell surface, the internalized fraction was returned to 37 °C for 15 min as a chase. Cells were then returned to ice and biotin was removed from recycled proteins by a second reduction with MesNa. Unreacted MesNa was quenched with IAA and cells were lysed and processed as before for measurements of internalized receptor.For each internalization or recycling assessment, two controls were carried out. To measure the total NRP1 or its variants at the surface, biotinylated cells at 4 °C were lysed without biotin reduction. To verify the efficiency of the surface biotin removal, the biotin reduction and MesNa quenching steps were carried out on cells that had remained on ice (time 0) and lysis was carried out.Equivalent volumes were analyzed by western blotting of NRP1 and densitometric analyses. The percentages of internalized or recycled NRP1 and its variants were calculated using the following formulae: internalized receptor = (NRP1 or its variant level after first incubation at 37 °C)  – (NRP1 or its variant level at time 0)/ × 100; recycled receptor = 100 – [(NRP1 or its variant level after 15 min of re-incubation at 37 °C)  – (NRP1 or its variant level at time 0)/(NRP1 or its variant level after first incubation at 37 °C)  – (NRP1 or its variant level at time 0) × 100].The concentration of HGF in FBS and the FBS neutralized with bovine HGF antibody  was determined in triplicate wells using the Bovine HGF ELISA Kit according to the manufacturer’s protocol (LifeSpan BioSciences).−1) and harvested at indicated time points. The cells were lysed in RIPA buffer and equal amounts of total protein were analyzed by western blotting.Cells were treated with cycloheximide (50 µgml4/well) were seeded in 6-well plates in triplicate. The number of viable cells was counted every day for 3 days using the Vi-CELL XR 2.03 (Beckman Coulter)60. Each experiment was performed in triplicate and repeated at least three times.Cells (5 × 10S-transferase (GST)-tagged p21 binding domain of PAK1 (GST–PBD) pulldown assay61. Briefly, cells (2.5 × 106) were grown on collagen-precoated 6 cm dishes to ~70% confluency in regular growth medium containing 10% FBS, or serum starved overnight followed by stimulation with HGF (50 ngml−1) for 5 min. Subsequently, the cells were lysed in lysis buffer , and soluble proteins were incubated with purified GST-PBD to pull down activated Rac1. The amount of total and active Rac1 was detected using Rac1 antibody . Briefly, cells were added to the upper chamber of the transwell insert. Complete medium containing 10% FBS, HGF (50 ngml−1) or VEGF165 (50 ngml−1) as a chemoattractant was added to the bottom chamber. To assess the effect of endocytosis and kinase inhibitors on cell migration and invasion, the inhibitors or their vehicle control DMSO were added to the bottom chamber medium. The plates were incubated at 37 °C in 5% CO2 for indicated time periods. After incubation, cells in the upper compartment were removed with a cotton swab, and cells that migrated or invaded to the filter surface facing the bottom chamber were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet. The number of migrated or invaded cells were counted in at least five areas at x 20 magnification using an inverted microscope.Migration and invasion assays were performed in Boyden chambers coated with collagen or Matrigel, respectively2 incubation chamber. Time-lapse phase images were taken every 30 min for 24 h. The movement of twenty individual cells was tracked and analyzed using Nikon Element AR software.In addition to the transwell migration assay, a time-lapse live cell imaging system was used to record the migration of live cells. Cells were placed on collagen-precoated glass-bottom culture dishes and monitored at 37 °C using a Nikon BioStation IMQ equipped with a CO6/mouse) of athymic nude mice (n = 6/group) as described60. To monitor metastasis, mice and lung tissues were imaged with luciferase signals using the IVIS Spectrum system and results were analyzed by the Living Image 3.0 software .Male athymic nude mice (6 weeks old) were purchased from Taconic  and maintained and treated under specific pathogen-free conditions. Experiments were carried out under a protocol approved by the University of Kentucky Institutional Animal Care and Use Committee. For the experimental lung metastasis assay, cells with co-expression of firefly luciferase and GFP were injected into the tail vein (1 × 10t-test, Mann–Whitney test or χ2-test. All data are presented as mean ± s.e.m. Differences between groups were considered statistically significant at p < 0.05. GraphPad Prism software was used for these analyses.Statistical analyses for each experiment were performed as described in the corresponding figure legends. Data between groups were compared using a two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Hadal trenches are among the most remote and least explored ecosystems on Earth and can support high benthic microbial standing stocks and activities. However, information on the role of viruses in such ecosystems and their interactions with prokaryotic hosts is very limited. Here, we investigated activities of benthic viruses and prokaryotes and their interactions in three hadal trenches  and in their nearby abyssal sites. Our findings reveal that these hadal trenches, compared with the surrounding abyssal sites, support higher abundances and biomasses of prokaryotes. In addition, the high prokaryotic biomasses of hadal trenches could favor high rates of viral infection and cell lysis, especially in the Japan Trench. Hadal viruses can release large amounts of highly labile and promptly available organic material by inducing cell lysis, which could contribute to sustain benthic prokaryotes and decrease their dependency on the enzymatic digestion of the more refractory fraction of sediment organic matter. Our results suggest that this process can contribute to explain the discrepancy between high prokaryote biomass and apparent low efficiency in the utilization of the sedimentary organic matter in the hadal ecosystems. Concluding, hadal trenches may be characterized by a highly dynamic viral component, which can boost prokaryotic biomass production, thereby profoundly influencing the functioning of these remote and extreme ecosystems. They arsediment , thus fasediment . Despitesediment , their d6 to 107 viruses cm–3  is the northernmost among the three investigated trenches and is subjected to high sedimentary inputs from both surface waters and land thanks to its proximity to the continent . The IzuIn this study, we analyzed distribution, activity and interactions between viruses and prokaryotes in the surface sediments at the bottom of the Japan, Izu-Ogasawara, and Mariana trenches, and in nearby abyssal plain sites. We also investigated the potential implications of viral infection and lysis of prokaryotic hosts on the functioning of these extreme systems in order to provide new insights into hadal microbial ecology.ABISMO at the Izu-Ogasawara Trench  and at the adjacent abyssal plain on the Pacific plate , respectively, during the JAMSTEC KR11-11 cruise (R/V Kairei) in Dec. 2011 (Kairei) in December 2012. Sediment cores  were obtained from the North Pacific abyssal plain  using a push corer during Shinkai 6500 dive 1394 of the JAMSTEC YK14-06 cruise (R/V Yokosuka) in 2014. Finally, during the JAMSTEC KR14-01 cruise (R/V Kairei) in January 2014, a sediment core (KR14-01 C1-2) was collected using the 11K lander system from the Challenger Deep of the Mariana Trench  and an adjacent abyssal sediment cores (KR14-01 E) were obtained using a multiple corer . Sub-aliquots of the surface 0 to 1 cm depth in sediment cores were used to carry out the analysis of the biochemical composition of the organic matter and microbiological parameters. Immediately after the sediments collection, the samples for the analyses of the biochemical composition of organic matter  were frozen and stored at −20°C. For the analyses of prokaryotic abundances, samples were fixed with buffered 2% formalin and stored at 4°C until further processing on land. Samples for determining viral production rates were processed on board immediately after the collection of the sediments. All analyses were carried out for three replicates.Surface sediment samples were collected from the three Northwest-Pacific hadal trenches  and also from adjacent abyssal sites . Sedimenec. 2011 . A sedimec. 2011  during ta and phaeopigments were extracted  in 5 ml of 90% acetone, and the extracts were analyzed fluorometrically to estimate chlorophyll-a, and after acidification with 0.1 N HCl to estimate the phaeopigments  was defined as biopolymeric carbon ; 4-methylumbelliferyl (MUF)-b-D-glucopyranoside (Glu-MUF), and 4-MUF-P-phosphate (MUF-P), respectively  under saturating substrate concentrations  and 365 nm excitation, 455 nm emission (for Glu-MUF and MUF-P), immediately after the addition of the substrate and after the incubation, checking for linearity of the increase in fluorescence during the incubation period  and incubated for 15 min at room temperature. The samples were properly diluted with 0.02-μm pre-filtered seawater, filtered onto 0.02-μm-pore-size Al2O3 filters  and then stained with 100 μl of SYBR Gold 2x  for viral counts were collected immediately after dilution of the sediments in sterile tubes (15 ml) and after 3, 6, and 12 h of incubation in the dark at in situ temperature. Subsamples were then analyzed as reported above for the determination of viral abundance. The viral production was assessed as the maximum increment of viral abundance observed during the incubations, divided by the incubation time-lapse (in hours)  by measuring the increase in viral abundance in the interval between the beginning of the experiment and 6–12 h later , 2015. Sn hours) .–1).The number of prokaryotes killed by viruses was estimated from the ratio between viral production and burst size previously determined for hadal sediments  was calculated by multiplying the cell size for the number of cells killed per hour .The concentration of organic carbon (C) released from the cells lysed by viruses in each sediment sample (expressed as mg C mp < 0.05). The analyses were carried out on Euclidean distances using 999 permutations with Monte Carlo simulation, considering all the factors as fixed and unrestricted permutation of raw data. Each statistical analysis was carried out by using the PRIMER v.6.1.12 & PERMANOVA + v.1.0.2 software.For each of the variable analyzed in this study, univariate distance-based permutational analyses of variance (PERMANOVA) was applied to assess differences between sampling stations (six levels). The analyses were followed by the pair-wise test when significant differences were encountered , while lipid concentrations from 0.11 ± 0.03 to 2.48 ± 0.55 mg g–1 .The results of the biochemical composition of organic matter in the surface sediments are shown in p < 0.01) when compared with data reported from the Izu-Ogasawara and Mariana trenches. The sediments of the Izu-Ogasawara Trench were characterized by the highest carbohydrate concentrations (2.63 ± 0.03 mg g–1) among all of the trenches investigated in the present study. Both protein and lipid concentrations in the bottom sediments from the three trenches were significantly higher than those from their related abyssal sites. Instead, carbohydrates concentration in the Japan Trench site was significantly lower than in the abyssal plain site , whereas no differences were observed between the Mariana Trench site and its related abyssal site. The Izu-Ogasawara Trench site, instead, showed significantly higher carbohydrates concentrations compared to its relative abyssal site (p < 0.01).The Japan Trench sediments contained significantly higher concentrations of proteins and lipids , being higher in hadal systems compared to their related abyssal sites. The concentrations of total phytopigments were significantly higher in the benthic site of the Japan Trench (269.32 ± 13.81 μg g–1) than in both the Mariana Trench (6.83 ± 0.25 μg g–1) and the related abyssal site .Biopolymeric carbon contents  ranged fp < 0.05 and p < 0.01) within the trench sites than in their related abyssal sites, with the highest values in the benthic sites from the Izu-Ogasawara Trench (2.31 ± 0.17 × 108 cells sediment g–1), followed by those within the Japan and the Mariana trenches . Prokaryotic abundance and cell size in the sediments of the three hadal trenches investigated were negatively related . Prokaryotic biomasses followed a pattern similar to that of the abundances, with values significantly higher (p < 0.01) within the trench sites compared to their abyssal sites. However, the highest value of prokaryotic biomass was found in the benthic site of the Japan Trench (5.39 ± 0.4 μg C g–1), followed by those from Izu-Ogasawara and Mariana trenches , while alkaline-phosphatase and β-glucosidase activity from 1.50 ± 0.19 to 109.6 ± 10.1 nmol h–1 g–1 and from 0.02 ± 0.01 to 0.89 ± 0.15 nmol h–1 g–1 in the abyssal sites surrounding the Izu-Ogasawara and Japan trenches (p < 0.01), with the exception of the β-glucosidase activity that exceeded only the value found in the Izu-Ogasawara Trench . The aminopeptidase activity normalized per prokaryotic cell (used here as a proxy of degradation efficiency) ranged from 2.49 ± 0.29 × 10–8 to 1.02 ± 0.13 × 10–5 nmol h–1 cell–1 in the Japan Trench and its control site, the β-glucosidase activity per cell between 6.1 ± 0.1 × 10–10 and 8.39 ± 0.06 × 10–8 nmol h–1 cell–1 in the Izu-Ogasawara Trench and the abyssal site related with the Mariana Trench, and the alkaline phosphatase activity per prokaryotic cell ranged between 1.23 ± 0.13 × 10–8 and 2.59 ± 0.24 × 10–6 nmol h–1 cell–1 in the site of Izu-Ogasawara Trench and in the abyssal site related with the Japan Trench (p < 0.01). In the site of the Izu-Ogasawara Trench, the aminopeptidase per prokaryotic cell was significantly higher than in the surrounding abyssal site (p < 0.01), whereas alkaline phosphatase and β-glucosidase activities per prokaryotic cell were not significantly different compared to the abyssal values.Aminopeptidase activity ranged from 2.0 ± 0.23 to 433.2 ± 54.7 nmol htrenches . The Marn Trench . The spe7 viral particles g–1 to 5.8 ± 0.11 × 108 viral particles g–1 in the bottom sites of the Mariana and Izu-Ogasawara trenches, respectively, and did not show a clear spatial pattern (p < 0.01). Conversely, the benthic site of the Mariana Trench showed significantly lower viral abundances than in the abyssal sites (p < 0.01). The virus to prokaryote abundance ratio (VPR) ranged from 0.9 ± 0.0 to 10.3 ± 3.3 . In the Japan Trench site, the ratio was not significantly different compared to the VPR found in the surrounding abyssal site, which, however, presented highly variable values . Overall, viral production was always higher in the sediments from the trench sites than in their adjacent abyssal sites  with higher rates within the trench sites, and significantly higher in the benthic sites of the Izu-Ogasawara and Mariana trenches compared to the related abyssal sites  than in the respective abyssal site . Overall, the C released from prokaryotes lysed by viruses was higher within the hadal sites of the trenches than in their related abyssal sites.Virus-induced prokaryotic mortality, obtained by the number of killed cells hn Trench . In the 7–109 cells g of sediment dry weight, 2 consumption rates in the hadal sediments exceeding those from surrounding abyssal sites  were lower than in the surrounding abyssal site. Since prokaryotic biomasses and biopolymeric C concentrations were high in the trench, with protein concentrations even higher than in benthic coastal sites , the reaViral abundances in the surface sediments of the investigated trenches were in the range of previously published values from hadal sediments  and otheViral abundances and VPR in the hadal and abyssal sites investigated did not showed a clear pattern of distribution. However, benthic viral abundance in the Izu-Ogasawara Trench was higher than in the other trenches, and only in this site VPR exceeded the value found in the respective abyssal site. VPR values have been used in previous studies to infer relationships between viruses and hosts . HoweverViral production in the hadal trench sediments was similar or even higher than in the respective abyssal sites. However, overall viral production rates were comparable to the values already obtained from other benthic deep-sea surface sediments worldwide . These fViruses in the sediments of the hadal trenches showed short turnover times (0.3–2.5 days), especially in the Izu-Ogasawara and Mariana trenches, where these values were 1.4–5.4 times shorter than in the respective abyssal sites and in other previously published deep-sea sediments worldwide , are expected to play a key role in microbial dynamics of trench sediments. The combined effect of the environmental factors needs to be further investigated to better understand the functioning of microbial food-webs and their implications on biogeochemical cycles in these extreme ecosystems.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.CC, AD, and RD conceived the study. ER provided samples for the study. EM, ER, and MT performed the laboratory analyses. EM, ER, MT, AD, and CC contributed to the data elaboration and interpretation. TN and HN allowed the oceanographic cruise and samples collection. EM, CC, and AD wrote the manuscript and all the authors contributed to the discussion and its finalization.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to  Interestingly, numerous studies have shown that the androgen receptor (AR) remains a crucial driver in CRPC3. The development of novel, highly potent second-generation anti-androgens such as enzalutamide4, apalutamide5, and darolutamide6 has been a major step towards more efficient inhibition of the AR. Enzalutamide is currently the most commonly used second-generation anti-androgen and confers a survival benefit in metastatic and non-metastatic CRPC patients9. However, the efficacy of enzalutamide and other anti-androgens is limited by the rapid development of acquired resistance and numerous in vitro and in vivo models have been developed16. These model systems together with corroborating clinical data from patients have led to the identification of a large variety of anti-androgen resistance mechanisms19. These involve AR re-activation by mutation of the AR, expression of constitutively active splice variants, or intra-tumoural androgen synthesis18. In addition, the glucocorticoid receptor may take over the function of the AR (AR bypass) and drive the activity of the AR pathway20. However, a subpopulation of patients display AR independent mechanisms of anti-androgen resistance that often involve neuroendocrine (NE) trans-differentiation and upregulation of MYCN 22. Interestingly, the use of the AR signalling inhibitors enzalutamide and abiraterone increased the number of non-neuroendocrine tumours that are independent of AR pathway activity to more than 20% of CRPC patients23. Typically, AR independent PCa is associated with loss of AR expression but the groups of Beltran and Sawyers have recently described anti-androgen resistance mechanisms through an AR indifferent state, in which the cells do not depend on AR activity despite continued AR expression25. The molecular adaptations that lead to such AR indifferent PCa cells remain elusive since the available AR indifferent models systems were generated by targeted methods  and not in response to anti-androgen treatment26.Androgen deprivation therapy is part of the first-line treatment for advanced prostate cancer, but it inevitably leads to the development of castration resistant prostate cancer (CRPC)In this study, we describe the independent generation of two distinct multi anti-androgen, castration, and chemotherapy resistant cell lines derived from LNCaP that show an androgen responsive, AR indifferent non-neuroendocrine phenotype that is driven by hyper-activation of the cell-cycle master regulator E2F due to multiple alterations in cyclin D/E, E2F1, RB1 and Myc signalling.6. Of note, the proliferation of ResB cells was stimulated at low concentrations of most anti-androgens. This demonstrates that ResA and ResB cells have acquired a highly resistant phenotype against the most common clinically used therapies targeting the AR pathway.We independently treated the AR reliant prostate cancer cell line LNCaP with enzalutamide (ResA) and RD-162 (ResB) to generate two distinct anti-androgen resistant cell lines. Both long-term anti-androgen treated cell lines were highly resistant against the anti-androgenic effects of enzalutamide, RD-162, apalutamide, darolutamide, and abiraterone acetate Fig.  as well 11, which may impact treatment sequencing in patients. Therefore, we measured the efficacy of paclitaxel and docetaxel on the proliferation of LNCaP, ResA, and ResB cells. Both anti-androgen resistant cell lines were partially cross-resistant against the growth inhibitory effects of both chemotherapeutic drugs, which was due to significantly reduced apoptosis induction in ResA and ResB cells . In addition, the spatial distribution and morphology  and low (27%) AR activity  in ResA cells and downregulation of CCND1 (cyclin D) in ResB cells  and cyclin E-CDK6 lls Fig. . Importalls Fig. . We did lls Fig. . Howeverlls Fig. . Since Mlls Fig.  may alsolls Fig. . Taken t23. However, the molecular adaptations that lead to this sub-type of CRPC remain elusive due to the lack of well-characterized models that faithfully recapitulate the development of this phenotype. In this study, we describe the independent generation of two distinct anti-androgen resistant cell lines with an “AR indifferent” phenotype25. These cells express the AR and are partially androgen responsive at the transcriptional and proliferative level  and the pan BCL-2 inhibitor obatoclax 43 and mycoplasma test were performed at least once every 6 months. All experiments were performed in normal growth medium  unless otherwise noted.The cell lines LNCaP-FGC and 22Rv1 were obtained from ATCC and cultured as recommended. The ResA cell line was generated by passaging of LNCaP cells with increasing concentrations of enzalutamide at the current IC44 by live cell analysis  using a nuclear mKate2 fluorescent label.The proliferation measurements were performed as described beforeFor proliferation experiments we used apalutamide (HY-16060), darolutamide (HY-16985), paclitaxel (HY-B0015), olaparib (HY-10162), and obatoclax (HY-10969) from MedChemExpress as well as enzalutamide , RD-162 (Sequoia Research Products), abiraterone acetate , R1881 , and docetaxel .3 the mice were treated with 10 mg/kg enzalutamide  or vehicle by oral gavage on a daily basis. Tumour volume was monitored by calliper measurements every 4 days and the specific doubling rate was calculated by linear regression. The Ethical Committee for Animal Experimentation (ECD) of the KU Leuven approved all in vivo experiments (project number P078/2014) and the experiments were carried out in accordance with the relevant guidelines and regulations.Nude athymic mice (8–10 weeks old) were anesthetized with ketamine/xylazine and injected subcutaneously into each flank with 7.5 million cells suspended in matrigel . Once the tumour size reached ~200 mmWe incubated the cells with the CellEvent Caspase-3/7 Green Detection Reagent  and measured the percentage of positive nuclei 48 hours after treatment with the IncuCyte ZOOM live cell imaging system. For UV induced apoptosis, we irradiated the cells suspended in PBS in an UVLink 1000 crosslinker .For siRNA mediated knockdown we transfected the cells with 30 pmol ON-TARGETplus SMARTpools  with Lipofectamine 3000  and measured the proliferation rate as described.Images from three independent biological replicates were acquired on an IncuCyte ZOOM live cell imaging system and the outline of each cell was manually marked. The aspect ratio and circularity of each cell was measured with ImageJ.Images from three independent biological replicates were acquired on an IncuCyte ZOOM live cell imaging system and the position of all cells was measured using a nuclear mKate2 fluorescent label. To determine the spatial distribution we determined how many cells are in close proximity (two nuclei diameters = 22 µm) to each cell. The data was summarized by calculating the average number of surrounding cells for each image and the total number of cells to correct for the cell density.www.catch-colonies.net), which was verified by manual classification of 170 colonies under a microscope.We incubated 1500 cells per T75 flask (coated with poly-L-lysine hydrobromide) for 14 days and stained the colonies with 0.5% crystal violet in 25% methanol. The flasks were filled with pure white starch and scanned on a flatbed scanner  at 4800 dpi. The colonies were classified into para-, mero-, and holoclones with the CATCH Colonies software  before addition of 0.1% Triton X-100 and incubation for 5 minutes on ice. The nuclei were collected by centrifugation at 1300 RCF for 4 minutes at 4 °C and separated from the cytoplasmic proteins in the supernatant. The nuclei were washed once with ice-cold cytoplasmic lysis buffer before addition of sample buffer and sonication. The proteins were separated on NuPAGE Novex 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred to 0.45 µm PVDF membranes . The following antibodies were used: AR (in-house made), AR-V7 specific , GAPDH , H2AX , MYC , E2F1 , and RB . Detection was performed with an ImageQuant Las4000 using the western lightning Plus-ECL reagent  and HRP conjugated secondary antibodies .The cells were lysed in RIPA buffer supplemented with HALT protease inhibitor on ice and cleared by centrifugation for whole cell protein extraction. For subcellular fractionation the cells were resuspended in ice cold cytoplasmic lysis buffer  database (GSE77930). Gene set activity scores were calculated in R with the GSVA package45. For GSEA and GSVA analysis the Hallmark gene sets collection from the Molecular Signatures Database (MSigDB) was used. Unless otherwise noted all comparisons between the cell lines are at conditions similar to normal growth conditions . All visualizations of the read coverage were performed with IGV46.Cells were seeded in DCC medium and after 24 hours they were treated with 10 nM DHT, 10 µM enzalutamide, a combination of the two, or vehicle (DMSO) for 18 hours. Total RNA was extracted with the RNAeasy plus mini kit  and RNA integrity was verified with an Agilent Bioanalyzer. The libraries for sequencing were generated with the TruSeq RNA Library Prep Kit according to the manufacturer’s recommendations and sequenced on a HiSeq 2500 System. The reads were mapped to the hg19 human reference genome using TopHat v1.3.3 with default settings. Differential gene expression and GSEA pathway analysis were performed with the Qlucore Omics Explorer v3.5. For analysis of mCRPC tissue samples a publicly available dataset generated by Kumar 47. GSEA analysis of the “C1: positional gene sets” from MSigDB was used to identify cytogenetic bands with significant deregulation (FDR < 0.05) in the anti-androgen resistant cell lines compared to untreated LNCaP.Genome-wide SNP genotyping was performed with Illumina CytoSNP arrays according to the manufactures recommendation and scanned on an iSCAN (Illumina). Genome-wide allele-specific copy number alterations were determined with ASCAT (v2.1)48 in R. All error bars represent the 95% confidence interval.R (v3.5.2) was used for the statistical analysis and all comparisons between two groups were done with Student’s t-Test. The Shapiro-Wilk Normality Test was used to confirm normal distribution of the samples in the xenograft experiment. P values lower than 0.05 were considered significant and indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001. All experiments have been performed in at least three biological replicates unless noted otherwise. Calculation of IC50 values was performed with the DRC packageSupplementary FiguresSupplementary Table 1