Cancer cell metabolism differs significantly from the metabolism of non-transformed cells. This altered metabolic reprogramming mediates changes in the uptake and use of nutrients that permit high rates of proliferation, growth, and survival. The androgen receptor (AR) plays an essential role in the establishment and progression of prostate cancer (PCa), and in the metabolic adaptation that takes place during this progression. In its role as a transcription factor, the AR directly affects the expression of several effectors and regulators of essential catabolic and biosynthetic pathways. Indirectly, as a modulator of the one-carbon metabolism, the AR can affect epigenetic processes, DNA metabolism, and redox balance, all of which are important factors in tumorigenesis. In this review, we focus on the role of AR-signaling on one-carbon metabolism in tumorigenesis. Clinical implications of one-carbon metabolism and AR-targeted therapies for PCa are discussed in this context. Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and the fifth leading cause of cancer death in men worldwide . ClinicaIn PCa, the one-carbon metabolism pathway is modulated by the AR. This pathway is comprised of several connected pathways that promote the folate-mediated transfer of one-carbon units necessary for essential cellular processes including DNA synthesis and repair and the maintenance of redox status. Because one-carbon metabolism is also the major source of methyl groups, as a modulator of this pathway, the AR also plays critical roles in histone and DNA methylation and in epigenetic mechanisms that are known to be relevant in oncogenesis ,16,17. SIn this review, we will focus on the role of the AR on one-carbon metabolism and the implications for disease progression. The first two sections focus on the relevance of one-carbon metabolism and its link to cancer. The third section outlines how AR-signaling modulates the expression and activity of enzymes involved in one-carbon metabolism, and how it affects methylation-mediated epigenetic processes in PCa. The final section discusses targeting one-carbon metabolism in PCa, and the potential effects of current AR-targeting therapeutic modalities on one-carbon metabolism.S-adenosyl-methionine (SAM), the universal methyl donor for protein and DNA methyltransferase reactions. By donating a methyl group, SAM is converted to S-adenosyl-homocysteine (SAH), and subsequently to homocysteine to close the cycle [One-carbon metabolism involves a complex network with two central cycles: (1) the folate cycle; and (2) the methionine cycle . In the he cycle ,23,24,25he cycle ,27. Sinche cycle .N-methyltransferase (GNMT), the enzyme that catalyzes formation of sarcosine from glycine, which eventually donates methyl groups back to the THF in a reaction catalyzed by sarcosine dehydrogenase (SARDH) [Balance within the one-carbon metabolism network is maintained in part by interactions involving substrates and enzymes from these pathways . SAM inh (SARDH) ,30. SAM  (SARDH) ,32. Addi (SARDH) ,34. ThesCancer creates a demand and dependency on one-carbon metabolism. Proliferation of tumor cells not only requires increased DNA synthesis, but can also result in increased levels of reactive radical oxygen species (ROS), which are cytotoxic unless neutralized ,36. MethSeveral enzymes within the folate cycle are potentially oncogenic and are dysregulated in cancer. Serine hydroxymethyltransferase (SHMT) and glycine decarboxylase (GLDC) donate methyl groups to the folate pathway in sequential steps via the catabolism of serine and glycine, respectively . SHMT, iParadoxically, although folate is necessary for cancer cell proliferation, multiple studies have reported a positive correlation between folate deficiency and disease risk for multiple cancers, especially breast and colon cancers ,49,50,51In contrast, folate depletion blocked tumor progression in vivo and induced genetic instability in cells in vitro, in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model for PCa ,64. FurtEven in the presence of global DNA hypomethylation, many cancer cells contain gene specific hypermethylation, a silencing mechanism. The tumor suppressor Rb was the first gene found to be silenced by DNA hypermethylation during tumorigenesis . Since tHistone methylation is a SAM-dependent epigenetic process. Several methyl histone marks are dysregulated in many cancer types, and depending on the target residue, these methylated histones can contribute to gene activation or repression ,75,76,77SAM not only serves as a cofactor for methyltransferases, but it is also shunted from one-carbon metabolism and utilized in polyamine synthesis. Polyamines have been implicated in cancer, and their oncogenic function may be linked to their roles in protein synthesis and cell cycle regulation ,37,90. OAnother shunt from the methionine cycle is the transsulfuration pathway, which is important for cellular redox homeostasis. The high intracellular oxygen levels required for aerobic respiration create an environment that produces highly reactive ROS. While physiological levels of ROS are essential for cell survival, an excess of ROS can have a wide range of detrimental effects, including DNA and protein damage. To prevent damage, the cell tightly regulates a series of antioxidant systems to restore redox homeostasis. One of the major antioxidants made within cells is glutathione, which is a product of the transsulfuration shunt of one-carbon metabolism. Reduced glutathione acts a cofactor for redox and conjugation reactions catalyzed by glutathione peroxidases and glutathione transferases to reduce hydrogen peroxide, a reactive product of initial superoxide neutralization, and neutralize toxins and carcinogens. Interestingly, in multiple cancers, glutathione peroxidases and glutathione transferases are silenced by DNA hypermethylation suggesting that the reduced activity of the enzymes drives tumorigenesis, likely through increased DNA damage ,96,97,98Interestingly, elevated homocysteine may promote oxidative stress by inhibiting the expression and activity of glutathione peroxidases. Elevated plasma homocysteine levels, a condition that may also be associated with folate deficiency, is often seen in the setting of malignancy ,106,107.Taken together, alterations in one-carbon metabolism may contribute to tumorigenesis by fueling DNA synthesis, changing the DNA and histone methylomes, promoting protein translation, driving cell cycle progression and modulating redox balance. These changes can in turn promote sustained proliferation, induce tumorigenic gene expression changes, contribute to genomic instability, and promote survival—all important processes in tumorigenesis and cancer progression.In the prostate, androgens and the AR regulate the activity/expression of several enzymes involved in the one-carbon metabolism pathways, specifically enzymes involved in SAM homeostasis (GNMT and SARDH) and the entry into the transsulfuration (CBS) and polyamine synthesis (ODC) pathways . This suThe AR is a nuclear receptor that is essential for prostate differentiation and homeostasis and for PCa initiation and progression. Binding of androgen, its major ligand, triggers a conformational change that promotes AR homodimerization and translocation to the nucleus, where it binds to the regulatory regions of its target genes, affecting their transcription . StudiesGNMT catalyzes the transfer of a methyl group from SAM to glycine to form SAH and sarcosine. The reverse reaction involves the oxidative demethylation of sarcosine into glycine, and it is catalyzed by mitochondrial SARDH or peroxisomal PIPOX ,111. It N-methyltransferase (NNMT) [While controversy remains regarding whether the levels of GNMT in clinical PCa samples are downregulated  or uprege (NNMT) . NNMT, we (NNMT) . Similare (NNMT) . The aute (NNMT) ), overpre (NNMT) ,118 sugge (NNMT) .As discussed above, homocysteine can enter the transsulfuration pathway in a reaction that involves condensation with serine, resulting in cystathionine. In mammals, this first and committed step of the pathway is catalyzed by CBS. The second step, the hydrolysis of cystathionine to cysteine, is catalyzed by the enzyme γ-cystathionase . CysteinThe activity of CBS is stimulated by SAM binding ,32,129, S-transferase Pi (GSTP), an enzyme with a role in detoxification, by catalyzing the conjugation of many compounds to reduced glutathione [As we discussed earlier, the AR plays a role in regulation of GNMT. Thus, in modulating GNMT activity, the AR indirectly control homocysteine levels and the SAM:SAH ratio, critical to methylation reactions and to the level of CBS. H2S inhibits the activity of the AR  providintathione ,137,138.tathione .AMD1; The relevance of polyamines to cellular physiology is illustrated by the fact that knockout of several enzymes of the pathway are embryonic lethal in the mouse  and dysrThe prostate has exceptionally high levels of polyamines, which are synthesized in the epithelium for normal growth and for secretion into the seminal fluid ,147,148.The high polyamine requirements observed in the prostate, which are increased in PCa, sensitize the cells to folate levels . BlockinDNA and histone methylation are important epigenetic mechanisms that contribute to initiation and progression of PCa ,163,164.In PCa, changes in DNA methylation are detected before the cancer becomes invasive and are maintained throughout disease progression ,166. TheHistone methylation changes are also common in PCa. Studies using immunohistochemical methods have reported an overexpression of H3K27me3 global levels in metastatic prostate tumors compared with non-malignant prostate tissues . AlthougDNA and histone methyltransferases utilize SAM as substrate leading to SAH production, an inhibitor of methyltransferases . TherefoThe AR and androgen signaling play a role in controlling methylation, modulating the expression of methyltransferases and/or their activity. As discussed above, the AR has important roles in regulating GNMT and the metabolism of sarcosine and the enzymes involved in the diversion of methyl groups into the transsulfuration and polyamine synthesis pathways. Increased GNMT expression leads to increased levels of the methyltransferase inhibitor SAH . TherefoChanges in the one-carbon metabolism affecting methyltransferases (SAM:SAH ratio) also modulate DNA and chromatin methylation affecting the activity and/or expression of the AR. In addition, direct post-translational modification of the AR and/or co-activators by methyltransferases also occurs. The AR is directly methylated by the histone methyltransferase SET9 on lysine K632 resulting in enhanced transcriptional activity . InteresFinally, AR signaling can regulate expression of enzymes involved in histone methylation. It has been reported that androgens modulate expression of EZH2 in a concentration-dependent manner (EZH2 is repressed at 1 nM or higher). This effect requires a functional AR and is mediated by the binding of retinoblastoma (RB) and p130-associated proteins to the EZH2 promoter. While both mechanisms seem to be synergistic, their androgen dependence varies. RB-E2F1 are themselves regulated by androgens in PCa cells. p130 and its partner proteins bind to the EZH2 promoter in androgen-treated, but not in control treated cells ,209. FinIn summary, AR/androgen signaling has an important role in PCa epigenetics, both indirectly by controlling expression of key enzymes involved in one-carbon metabolism and associated pathways, and directly by controlling the expression and activity of DNA and histone methyltransferases. These effects can ultimately affect AR expression, which is also epigenetically controlled by DNA and histone methylation, or activity. These observations emphasize the precise link between the AR and one-carbon metabolism, and the potential effects that changes in AR signaling, that can occur with disease progression, may have on essential cellular processes .The accelerated proliferation of cancer cells places a robust demand on one-carbon metabolism, which can be exploited for anticancer therapies. The antifolate, aminopterin, originally used by Sydney Farber to treat pediatric patients with acute lymphoblastic leukemia, was the first successful anticancer chemotherapeutic agent . Today, Other branches of the one-carbon metabolism network have been explored as therapeutic targets. As we discussed previously, the natural polyamines, putrescine, spermine and spermidine are ubiquitous molecules; however, their requirements are particularly high in rapidly growing tissues during normal growth and development, and in tumors ,217,218.Changes in polyamine levels have been reported in PCa ,230,231.In addition to drugs targeting one-carbon metabolism itself, methyltransferase inhibitors are also used to treat a variety of cancers, and several inhibitors are currently being investigated for cancer therapy . 5′AzaciHistone methyltransferases are also prime targets in epigenetic cancer therapy. EZH2, MLL, and DOT1L are potentially attractive targets in PCa, as all three modulate the activity of the AR ,207,249.The one-carbon metabolism network integrates several pathways that, together, play central roles in the biosynthesis of nucleic acids and lipids, amino acid and vitamin metabolism, the maintenance of redox status, methylation reactions and polyamine biogenesis. Because of the relevance of these pathways to cell growth and proliferation, they are critical not only for cellular homeostasis but also for tumorigenesis, and are therefore significant therapeutic targets.The tight dependency among the pathways of the one-carbon metabolism network imposes an exquisite regulation to allow rapid responses to changes in cellular demands. The AR and androgen signaling regulate key enzymes involved in these pathways, including the ones that control the methylation potential of the cell and the entrance into the glutathione and polyamine biosynthetic pathways. Therefore, changes that occur in the AR levels or activity will have profound effects on the activity and output of the one-carbon metabolism network and downstream processes . ADT des


The androgen receptor (AR) is not only a ligand-dependent transcription factor, but also functions as a licensing factor, a component of DNA replication, which is degraded during mitosis. Furthermore, the deregulation of AR activity is involved in the initiation of prostate cancer and contributes to castration resistant prostate cancer (CRPC). While AR degradation is known to occur primarily through a proteasome-mediated pathway, very little is known about how this process is regulated, especially in M phase. PC-1 is an androgen-responsive factor and expresses specificity in prostate cancer, with higher expression noted at G2/M. In this study, PC-1 was shown to interact with AR and E3 ligase CHIP (Carboxy-terminus of Hsc70 Interacting Protein) and to enhance AR/CHIP interactions, thereby decreasing AR stability. Moreover, PC-1 was found to act in conjunction with CHIP in the decreasing of AR via ubiquitination, with the subsequent degradation predominantly occurring during M phase. PC-1 was also found to repress AR transcriptional activity in androgen-dependent and androgen-independent prostate cancer cells and attenuate the growth inhibition of AR. In conclusion, these findings should provide new clues regarding the modulation of AR turnover and activity via PC-1 and reveals an essential role of PC-1 in AR signaling. The androgen receptor (AR) is a ligand-dependent transcription factor belonging to the steroid hormone receptor superfamily and consists of three functional domains: the N-terminal transactivation domain, the C-terminal DNA-binding domain (DBD) and the ligand-binding domain (LBD) , 2. UponDeregulation of the AR signaling pathway, most likely through AR, is associated with the formation and development of primary prostate cancer as well as castration resistant prostate cancer (CRPC) progression –9. AbnorPC-1, also named PrLZ, belongs to the TPD52 gene family. PC-1 and TPD52 share homologous C-terminal domains, including a coiled-coil leucine zipper. Distinctively, PC-1 possesses a specific N-terminal 1-46 amino acid domain and its own transcription regulatory element. Moreover, PC-1 expression is predominantly prostate tissue specific and androgen responsive, whereas TPD52 expression can be detected in many tissue and cell types and is not affected by androgen , 21. PC-To investigate the relationship between PC-1 and AR in prostate cancer progression, we explored the possibility that PC-1 regulates AR protein stability. Because PC-1 was identified to be up-regulated in C4-2 in comparison with LNCaP parent cell line and did not express in PC-3 cell line, we overexpressed PC-1 in LNCaP cells and PC-3-AR cells and knocked down PC-1 in C4-2 cells. As shown in Figure in vivo. HEK293 cells were transfected with both PC-1 and AR expressing plasmids, with exogenous AR readily detected in exogenous PC-1 pull-down complexes ; The PSA reporter luciferase construct was a gift from Dr. Leland W.K. Chung ; The bacterial expression vectors encoding full-length GST-PC-1 or GST-CHIP fusion proteins were constructed by cloning the corresponding fragments into pGEX-4T1 vector.PC-1 plasmids was constructed by cloning PC-1 into pcDNA3.1-HA/Myc or pCMV-Flag 2B vector ; A series of AR expression plasmids encoding of AR (full length), AR-TAD (1-568) and DBD (484-651), LBD (652-918) and ARΔ629-634 were constructed by cloning corresponding AR fragments into pcDNA3.1-Myc or pCMV-Flag 2B. CHIP plasmids were constructed by cloning CHIP into pcDNA3.1-Myc or pCMV-Flag 2B vector; pARE2 in a humidified incubator. LNCaP-Zero and LNCaP-PC-1 were generated from LNCaP cells as described previously [HEK293, PC3 and DU145 cells were cultured in DMEM (Gibco) media with 10% fetal bovine serum  and 1% penicillin/streptomycin; LNCaP and C4-2 cell lines were cultured in RPMI 1640 (Gibco) with 10% FBS. All cells were cultured at 37°C with 5% COeviously . Human pTo study the effect of androgen, the cells were treated with a synthetic androgen R1881  in fresh phenol red-free RPMI 1640 with 5% charcoal stripped fetal bovine serum  for 12 h.To arrest prostate cancer cells in M phase, the cells were treated with 200 ng/ml nocodazole (Sigma-Aldrich) for 16 h or 2 μM or 5 μM 2-ME (Sigma-Aldrich) for 24 h before lysed.To test the AR turnover rate, the cells were treated with 10 uM CHX and were lysed at different time points for Western blotting.Proteasome chemical inhibitor MG132 was from Selleck; Protein synthesis inhibitor CHX was from Calbiochem; Anti-Flag M2 affinity gel was from Sigma-Aldrich; Glutathione Sepharose 4B was from Amersham Biosciences; Anti-AR (sc-7305), anti-CHIP (sc-133066), anti-Myc (sc-40), anti-HA (sc-805), anti-Flag (sc-807) and anti-β-tubulin (sc-5274) antibodies were from Santa Cruz; Anti-PC-1 antibody which is against PC-1 N-terminal 46 amino acids residues was made by our laboratory.PC-1 knockdown construct was created by inserting annealing oligos  into pLKO.1 vector. Lentiviral particles were generated by transfecting 293T cells with packaging vectors, psPAX2 and pMD2.g. Medium was changed every 24 hours, the 48 and 72 hour supernatants were pooled, filtered through a 0.45 μm filter. Lentiviral particles were applied to C4-2 cells with 8 μg/mL polybrene (Sigma Aldrich), and infected cells were isolated by 10 μg/mL puromycin (Sigma Aldrich). SiRNA against CHIP and scramble siRNA (Santa cruz) were transfected into LNCaP cells using Lipofectamine 2000 (Invitrogen).Cells were treated with lysis buffer containing 150 mM Tris base (PH 7.5), 50 mM NaCl, 1 mM EDTA (PH 8.0), 1%NP40, 1 mg/ml leupeptin and 1 mM PMSF. Protein concentrations were determined by BCA protein assay . Equal amounts of protein were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Bio-Rad). After blocking with 5% non-fat milk in tris-buffered saline with tween at room temperature for 30 min, the membranes were probed with indicated antibodies. After a series of washes the blots were further incubated with goat anti-mouse or anti-rabbit IgG antibody conjugated to horseradish peroxidase  and detected using the ECL kit (Pierce).HEK293 cells were transiently transfected with the expression plasmids which are shown in the figures. After transfection 48 h, cells were lysed in lysis buffer (RIPA buffer) and 500 μl of crude cell lysate containing 200–500 μg of total protein were incubated with 20 μl anti-FLAG-M2 agarose beads. The immunoprecipitates were washed and resuspended in lysis buffer. The proteins bound to the agarose beads were analysed with the indicated antibodies for Western blotting. To test the endogenous AR interaction with CHIP, LNCaP and LNCaP-PC-1 cell lysates were incubated with 2 μg AR antibody and protein A/G-Sepharose beads (Santa Cruz Biotechnology) at 4°C overnight. Sepharose beads were washed with lysis buffer three times and resuspended in SDS-PAGE loading buffer for Western blotting using anti-CHIP antibody.In vitro expression and purification of recombinant GST fusion proteins were performed according to the protocol. Briefly, the recombinant vector was introduced into BL21 cells. An overnight culture of a colony of the transformants was diluted 1: 100 and grown for 1–2 h at 37°C before induction for 4–6 h at 30°C with 0.1 mM IPTG. The cultures were centrifuged at 4000 rpm for 5 min at 4°C and the cells were resuspended in ice cold phosphate-buffered saline (PBS). Bacterial cells were lysed by sonication on ice for 15 consecutive 15s intervals. Following addition of Triton X-100 to a final concentration of 1% (v/v) and centrifugation, the supernatants were incubated with glutathione-agarose beads (Amersham Biosciences) for 1 h at 4°C. Beads were used directly in GST-pull down assay after extensive washing with buffer.For GST-pull down assay, HEK293 cells were transfected with the Myc-AR and AR deletion mutants or HA-PC-1 expression plasmid. The cells were rinsed with PBS and lysed in lysis buffer described above. Cell lysates were incubated with approximately equal amounts of GST or GST-PC-1/GST-CHIP fusion proteins at 4°C for 4–6 h with gentle rotation. The complexes were isolated by binding to glutathione-sepharose beads, and analyzed with the indicated antibodies for Western blotting.HEK293 cells were co-transfected Myc-ubiquitin, Flag-AR/Flag-ARΔ629-634 with HA-PC-1 or Myc-CHIP alone or together. After transfection 36 h, cell lysates were immunoprecipitated with Flag beads and analyzed by Western blotting.2-Luc, or the PSA reporter plasmid, 500 ng-1000 ng of pcDNA3.1 or PC-1 expression plasmids, 25 ng pRL-TK  with or without 100 ng pSG5-hAR with the Lipofectamine2000 (Invitrogen). Complete medium was replaced after 36 h with cFBS with or without 1 nM R1881. The cells were lysed 48 h after transfection. Luciferase activity was subsequently measured using a Dual-Luciferase Assay kit (Promega) according to the manufacturer's instructions. All transfection experiments were carried out in triplicate wells and repeated three times.LNCaP, PC-3 and DU145 cells were seeded in 24-well plates in complete media and then transiently transfected with 300 ng of pARECell growth and viability was measured using the MTT proliferation assay. Briefly, 2000 cells were seeded in 96-well plates in 200 μl RPMI 1640 containing 10% FBS with or without 2 nM R1881 treatment. Cell growth was examined 7 days later. Before testing, 20 μl of MTT reagent  was added and the cells were incubated for a further 4 h at 37°C. Then 250 μl of dissolving reagent DMSO (Amresco Inc.) was added to dissolve the formazan crystals. The optical density (OD) was measured at wavelength of 490 nm on a microplate reader.t-test. p < 0.05 was considered the threshold value for statistical significance.Statistics analysis was performed using two-tail Student's


FOXO factors are tumour suppressor proteins commonly inactivated in human tumours by posttranslational modifications. Furthermore, genetic variation within the FOXO3a gene is consistently associated with human longevity. Therefore, the pharmacological activation of FOXO proteins is considered as an attractive therapeutic approach to treat cancer and age-related diseases. In order to identify agents capable of activating FOXOs, we tested a collection of small chemical compounds using image-based high content screening technology. Here, we report the discovery of LOM612 (compound 1a), a newly synthesized isothiazolonaphthoquinone as a potent FOXO relocator. Compound 1a induces nuclear translocation of a FOXO3a reporter protein as well as endogenous FOXO3a and FOXO1 in U2OS cells in a dose-dependent manner. This activity does not affect the subcellular localization of other cellular proteins including NFkB or inhibit CRM1-mediated nuclear export. Furthermore, compound 1a shows a potent antiproliferative effect in human cancer cell lines. DAPI was used to counterstain the cell nuclei at a concentration of 1.43 μM. Samples were imaged at 22°C using 63× water immersion lenses on a Leica SPE confocal imaging system. Images were obtained using Leica software.U2OS cells were cultured as described above and exposed for 30 min at 1.5μM LOM612 (compound 1a). The cells were fixed with 100% methanol (5 min) and then permeablilized and blocked for 1hour with 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBSTween. The cells were then stained with FOXO3a antibody  kit and reverse transcribed using NZY First-strand cDNA Synthesis kit . qPCR was done in technical triplicate for each sample. qPCR reaction was carried out using Lumino Ct SYBR Green qPCR ReadyMix (Sigma). All qPCR results are representative of two separate experiments. Data analysis was carried out using the ΔCt method. Comparisons were made using the unpaired Student’s t test. Values represent the mean ± standard error of the mean (SEM) and are represented as error bars. Statistical significance as indicated.1a (LOM612), a newly synthesized isothiazolonaphthoquinone. The isothiazolequinones are an extremely rare class of compounds. To the best of our knowledge, only two natural products, pronqodine A [In order to identify specific FOXO relocator compounds we designed a sophisticated screening strategy . As the qodine A  and auloqodine A  and a feqodine A ,30 have 1a, together with its analogues 1b-c, were prepared according to the two-step procedure reported in 3a and 3b [N,N-dimethylurea or N,N-dibenzylurea urea and chlorocarbonylsulphenyl chloride respectively. Then, the 1,3-dipolar cycloaddition between the nitrile sulfide generated in situ by termal decarboxylation of 3a and 3b [1a and 4. The intermediate 4 was then treated with cerium ammonium nitrate (CAN) in acetonitrile–water [1b was obtained in high yield. Following the same synthetic strategy isothiazolonaphthoquinones 1c was obtained starting from 1,3,4-oxathiazol-2-ones 3c.Compound a and 3b  were syna and 3b  and the le–water  at room 1b (LOM621) which differs from compound 1a LOM612 in the amino group by the replacement of two methyl groups by two hydrogens  and was not further analyzed. These results suggest that the mode of action of LOM612 depends on stringent structural requirements.Interestingly, in our experiments, compound ydrogens  did not ydrogens . Similar50) value of LOM612 compound. The FOXO reporter cells were cultured as described previously and treated with twelve different concentrations of LOM612, LOM621 and LMB for 30 min maintaining the transferred volume constant. 50 value of 1.5μM. The EC50 value of the reference compound LMB was 2.3nM (data not shown) in these experiments. In contrast, LOM621 failed to induce the accumulation of GFP-FOXO in the nucleus . Accordingly, compounds which interfere with CRM-1-mediated nuclear export induce the accumulation of NES-bearing proteins including FOXO factors. In order to determine if LOM612 triggers nuclear localization of FOXO by inhibiting CRM1, we used a high content screening approach based on mammalian cells stably expressing green fluorescent protein (GFP)–labeled Rev protein, which contains a strong heterologous NES . We seedThe activity of many transcription factors such as FOXO, NFAT or NFkB is controlled via the regulation of their subcellular localization. NF-κB proteins shuttle constitutively between the cytoplasm and nucleus, and only accumulate in the nucleus and become activated upon cellular stimulation . IL-1 or50 values in the high nanomolar or low micromolar range 2 moiety with a NH2 (1b) or a phenyl group (1c) at position 3 renders the compound inactive in the FOXO translocation assay, suggests that the dimethylamino group plays an essential role in the interaction with the molecular target. In order to further explore the structural requirements, we are currently preparing a collection of chemical derivatives to perform structure activity relationship analysis. The functional readout for these experiments is based on FOXO subcellular translocation. However, because FOXO proteins are transcription factors that are considered poorly druggable targets [et al. showed that Cdc25 regulates the FOXO target gene Matrix Metalloprotein 1 (MMP1) via Cdk2 and FOXO1 [in vitro Cdc25 phosphatase assay. The subcellular localization of FOXO proteins is regulated by a variety of upstream proteins, most prominently serine/threonine kinases. Therefore among the primary target candidates of LOM612 are kinases that are known to phosphorylate FOXOs at sites involved in the regulation of its intracellular localization either directly, such as AKT1–3, SGK 1–3, MST-1, CK1, Cdk2, DKYR1a and IKKβ or indirectly by activating AKT, such as PI3K and PDK1 [Compounds capable of reactivating FOXO based on its tumor suppressor properties are considered a very attractive anti-cancer therapy ,12,35. A targets  it is un targets . The mod targets . Importa targets –46 which targets . Intrigu targets . In linend FOXO1 . Howeverand PDK1 ,37.S1 FigU2OS cells were treated either with DMSO or 5μM of compound LOM612 for 6 hours. qRT-PCR from U2OS cells of FOXO target genes, p27 (A) and FasL (B) whose LOM612-induced expression is induced. Expression relative to GAPDH. n = 2, *p < 0.05.(PPTX)Click here for additional data file.


KLF4 promoter and enhances KLF4 expression, which reciprocally targets the AR promoter, thus sustaining KLF4 activity. Ectopic KLF4 expression in androgen-independent prostate cancer cells induced AR expression and decreased cell proliferation, invasion and bone metastasis. We previously showed that increased microRNA (miR)-1 expression is associated with reduced bone metastasis of prostate cancer cells. Here we observed that KLF4 targets the primary miR-1-2 stem-loop promoter and stimulates miR-1 expression. In clinical prostate cancer specimens, KLF4 levels were positively correlated with miR-1 and AR levels. These data suggest that the loss of KLF4 expression is one mechanistic link between aggressive prostate cancer progression and low canonical AR output through miR-1 inactivation.In prostate cancer, Krüppel-like factor 4 (KLF4) depletion occurs frequently, suggesting a role as suppressor tumor. KLF4 is a transcription factor associated with androgen receptor (AR) expression; however, its cellular functions and signaling regulation mechanism remain largely unknown. In this study, we demonstrated that activated AR binds to the  Therefore, the resistance mechanisms and molecular pathways involved in changes of AR function as prostate cancer progresses are being increasingly investigated.AR is activated after the binding of androgenic ligands and has a pivotal role in prostate cancer development.14 Altered miR expression and the subsequent effects on target gene transcription have been frequently associated with metastatic trait acquisition.15 miR-1 has been proposed as a candidate tumor suppressor and prognostic marker for prostate cancer.18 We previously established that ectopic miR-1 expression negatively affects the growth ability of prostate cancer cells.19 Notably, we demonstrated that miR-1 expression suppresses experimental bone metastasis and that miR-1 is directly and positively modulated by AR signaling.20The microRNAs (miRs) represent a class of small, regulatory, noncoding RNA molecules that can become dysregulated in cancer.KLF4 expression is directly and transcriptionally upregulated by AR, and AR expression is reciprocally upregulated by KLF4. Moreover, in prostate cancer cell lines, KLF4 binds to the miR-1 promoter and induces its expression. Our findings also revealed that KLF4 is positively associated with AR and miR-1 levels in patient tissue samples. Here, we established a central role for KLF4 in tumor suppression by connecting its regulation by AR and modulation of miR-1 expression.In this study, we explored the regulatory mechanisms linking AR signaling with miR-1 expression in prostate cancer. We demonstrated that 21 the specific role of KLF4 in AR pathway-activated prostate cancer remains unclear. We analyzed the relationship between KLF4 and AR signaling and observed that AR-expressing cells LNCaP, LNCaP-AR and 22Rv1 had higher KLF4 expression than did cells without AR expression PC3, DU145 and RasB1  in the upstream region of the KLF4 promoter  analysis in LNCaP cells after a chromatin immunoprecipitation (ChIP) assay by using anti-AR and anti-FOXA1 antibodies. The binding of AR and FOXA1 was observed only at the ARE1 and ARE2 motifs, and the binding significantly increased after DHT treatment  region of AR. We noted significantly increased nuclear KLF4-binding signals at putative K1 and K4 sites in DHT-treated cells compared with those in untreated cells (AR promoter (K1 and K4) are functional, we conducted reporter assays by using a construct with a KLF4-RE at the K1 and K4 sites incorporated into an RFP reporter. The reporter activity increased in response to DHT . The samples were divided into two groups of low and high levels of both AR and miR-1 expression, as measured using qRT–PCR. Analysis of variance confirmed that KLF4 expression levels were significantly higher in the groups with high AR or miR-1 expression levels . Similar27 . We also studied the underlying mechanisms contributing to the resistance to androgen-deprivation therapy and poor AR antagonist efficacy in advanced prostate cancer. Our results may aid in understanding individual intervariability in drug responses to consequently improve drug therapies: by modulating KLF4 expression to repress cancer malignancy, an alternative target may be identified to improve the efficacy of current prostate cancer therapy modalities.A combination of androgen-deprivation therapy and surgical prostatectomy or radioablation has been used to treat therapy-naive patients with prostate cancer.KLF4 expression is directly and transcriptionally upregulated by AR, and AR expression is reciprocally upregulated by KLF4. This study highlights the role of the KLF4-AR axis in androgen deprivation and reveals pivotal mechanisms responsible for castration-resistant prostate cancer development. Despite the gaps in the understanding of KLF4 and its interplay with cell motility and proliferation pathways, we present a novel function for the KLF4-miR-1 axis—a new putative predictive and surveillance biomarker of antiandrogen therapy for advanced prostate cancer.In conclusion, 48 DU145, PC3, LNCaP and 22Rv1 cell lines were from the American Type Culture collection . All the cells were cultured in the RPMI 1640 medium supplemented with 10% FBS . After DHT treatment , cells were cultured for 24 h in a 10% charcoal-stripped FBS-containing medium. Some cells were treated with the AR antagonist MDV3100  at 10 μm for 24 h in a 10% FBS-containing medium. The cells with stable or transient AR, FOXA1, or KLF4 expression were established through transfection with AR, FOXA1 or KLF4 expression vectors, respectively; an empty vector, pCDH-CMV-MCS-EF1-Puro  with a puromycin-selectable marker, was used as a control. The siRNA (scramble and siKLF4) and shRNA vectors (shLuc and shKLF4) were purchased from Thermo Scientific  and the RNAi Core Lab . Transient transfection of the plasmids and siRNAs was performed using the X-tremeGENE HP DNA transfection reagent  and Lipofectamine RNAiMAX , respectively. By using GRCh37, KLF4 binding sites were located on human pri-miR-1-2 and AR on chromosomes 18 and X, respectively, and AR binding sites were located on human KLF4 on chromosome 9 . All the primers used for these constructs are listed in Androgen-dependent LNCaP-AR  and metastatic RasB1 (DU145 expressing a constitutively active Ras) cell lines were obtained from Dr Kathleen Kelly  and maintained as described previously.mosome 9 . The AR,6 cells/well) were lysed in 150 μl of RIPA buffer containing complete protease inhibitors (Roche) and phosphatase inhibitors (Roche), 25 mm β-glycerophosphate, 10 mm sodium fluoride and 1 mm sodium vanadate. Twenty micrograms of protein was separated per lane through SDS-gel electrophoresis. After transfer to polyvinylidene fluoride membrane, the blots were blocked with 5% bovine serum albumin in phosphate-buffered saline plus Tween 20. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies were incubated at room temperature for 1 h as indicated in The cells grown on six-well plates . For reverse transcription, 3 μg of total RNA was used with the SuperScript III kit (Invitrogen). In the amplification step, SYBR green PCR master mix  was used. For all primer pairs, the thermocycler was run for an initial 95 °C incubation for 10 min, followed by 40 cycles with 95 °C for 15 s and 60 °C for 1 min. All the reactions were normalized to human m) for 4 h. The cultured cells (107) were cross-linked with 1% formaldehyde at room temperature for 15 min. Fixation was quenched with glycine, and the cells were washed twice with cold phosphate-buffered saline containing a complete protease inhibitor (Roche). The cell pellets were resuspended in cell lysis buffer and incubated on ice for 15 min. Nuclei were collected by centrifugation at 104 r.p.m. and 4 °C for 10 min and resuspended in nuclear lysis buffer. Chromatin was sheared using a sonicator  with a microtip in a 20 s burst followed by 1 min of cooling on ice for a total sonication time of 5 min per sample. This procedure results in DNA fragment sizes of approximately 100 to 300 bp. Sheared chromatin was divided to perform immunoprecipitation with a rabbit IgG antibody  or primary antibody at 4 °C overnight. Immunoprecipitation, washing, elution, reverse cross-linking and DNA purification steps were performed according to Millipore's protocol. A qRT–PCR was performed in triplicate with 2 μl of eluted chromatin. ChIP antibodies and PCR primers are listed in The cells were treated with or without DHT , and relative median fluorescent intensity was measured from the first peak of fluorescence, as described previously.m sodium citrate to each well, and absorbance was measured at 550 nm on an ELISA reader .The PC3 and RasB1 cells were transfected with the KLF4 expression vector at a density of 2000 cells/well. Each day, the cells were stained with 0.5% crystal violet fixative solution for 15 min, washed with distilled water and allowed to air dry. At the end of the experiment, crystal violet was dissolved by adding 100 μl of 50% ethanol containing 0.1 4 cells/well. Single-cell suspensions of KLF4-transfected RasB1 cells or shKLF4 vector-transfected LNCaP-AR or DU145 cells were seeded in six-well plates in the RPMI 1640 medium supplemented with 10% FBS. The colonies were counted on day 21 after plating in triplicate and normalized with a control shLuc vector.Colony-formation assays were performed using a starting cell count of 5 × 105 cells/ml in a serum-free medium. We purchased Matrigel from BD Biosciences  for the invasion assay. Matrigel-coated transwell dishes were prepared by adding 200 μl of Matrigel diluted 10-fold with the serum-free medium. The cells were plated at 2.5 × 105 per well in the serum-free medium above the Matrigel. The lower chamber was filled with 600 μl of the serum-containing medium. The cells that had invaded the Matrigel-coated transwells after 12 h were fixed and stained with a 0.5% crystal violet fixative solution for 15 min. The invading cells on the underside of the membrane were counted and quantified in five medium-power fields for each replicate in triplicate. In the migration assay, we used transwells without Matrigel, and the cells were fixed and stained as described for the invasion assay.For invasion and migration assays, KLF4 or AR expression vector-transfected PC3 and RasB1 cells with a control siRNA or siKLF4 or siKLF4-transfected LNCaP-AR cells were resuspended at a concentration of 2.5 × 105 RasB1 human metastasis prostate cancer cells harboring a luciferase expression vector with a control empty or AR expression vector or 105 AR-transfected RasB1 cells with a control shLacZ or shKLF4 expression vector. Bioluminescence imaging was performed double blinding at 28 days after injection as described previously.46 In all cases, the same mice were followed for up to 10 weeks after injection to measure the accumulation of bone metastases through bioluminescence imaging as well as the survival percentage. For survival studies, mice were killed upon 10% body weight loss, paralysis or head tilting.Animal experiments were performed according to the protocol approved by the Taipei Medical University Animal Care and Use Committee (Taiwan). To analyze metastasis, 7-week-old male nude mice  were intracardially administered either 10We collected 22 independent primary prostate tumor samples from the Wan Fang Hospital, Taipei Medical University (Taiwan). The study was approved by the Wan Fang Hospital, Taipei Medical University Institutional Review Board  and performed according to the approved guidelines. Immunohistochemistry was performed using anti-KLF4 (Sigma) and anti-AR  antibodies at 1:500 and 1:250 dilutions, respectively, as described in t-test or one-way analysis of variance, followed by Bonferroni's post test for comparisons among three or more groups. The method for determining the cutoff points was predecided by considering half the number of patients. A P-value of <0.05 was considered statistically significant.All the data are presented as the mean±s.e.m. Statistical calculations were performed on GraphPad Prism analysis tools . Differences between individual groups were determined using the Student's


Population-based patterns of care studies are important for trauma care but conducting them is expensive and resource-intensive. Linkage of routinely collected administrative health data may provide an efficient alternative. The aims of this study are to describe the rehabilitation pathway for trauma survivors and to analyse the brain injury rehabilitation outcomes in the two care settings .This is an observational study using routinely collected registry data . The study cohort includes 268 road trauma patients who were admitted to trauma services between 2009 and 2012 and received inpatient rehabilitation because of a brain injury.n = 166) were admitted to specialist units with the remainder (n = 102) admitted to non-specialist units. Those admitted to a specialist units were younger (p < 0.001), had a lower cognitive FIM score (p = 0.003) on admission than those admitted to non-specialist units. Specialist units achieved better overall FIM score improvements from admission to discharge  but at a cost of longer length of stay . There were very few discharges to residential aged care facilities from rehabilitation . There was a long time lag between trauma and admission to inpatient rehabilitation with only a quarter of the patients admitted to a specialist unit by end of week four. Few older patients (19%) with brain injury were admitted to specialist units.Of those who need inpatient rehabilitation, 62% (It is feasible to use routinely collected registry data to monitor inpatient rehabilitation outcomes of trauma care. There were differences in characteristics and outcomes of patients with traumatic brain injury admitted to specialist units compared with non-specialist units. The proportion of patients surviving trauma has increased in recent years because of quicker and safer transportation to specialised trauma facilities, more effective emergency care and advances in acute surgical management , 2. RoadAbout half of road trauma survivors have a traumatic brain injury . While tCurrent models of brain injury care in New South Wales (NSW), Australia, have been described by the Agency for Clinical Innovation . The eleThere are two care settings for inpatient brain injury rehabilitation: (1) specialist public brain injury units (operating through the NSW Brain Injury Rehabilitation Program) and (2) non-specialist private and public general rehabilitation units. All residents have free and equal access to specialist units and access is based solely on clinical need and bed availability. However, people of working ages (i.e. below age 65 years) are prioritised for specialist units . The cutThere are differences between the two care settings. Generally, non-specialist units may not offer  clinical psychology, may not offer neuropsychology services and may not have ready access to transitional living units. In specialist units, staff caring for patients are provided with specialised training to develop competencies in behavioural change strategies . It can There are no published data on the proportions of patients admitted into the two care settings in NSW. Patient selection for the two care settings is often decided by a rehabilitation physician or geriatrician. However, non-clinical factors including health system factors (such as bed flow or bed pressures) and insurance status  can influence this decision .For optimal patient outcomes for those with traumatic spinal cord injury, expert consensus  recommenThe aims of this study were to investigate the demographic characteristics, define the rehabilitation pathways, and analyse the rehabilitation outcomes after road trauma of a cohort of patients. Prospectively collected trauma registry data were record linked with inpatient rehabilitation data to answer these questions. This is an efficient way of monitoring trauma care as it is less expensive and not as resource-intensive as prospective cohort studies. The state-wide datasets are both population-based so results are generalisable. Analysing the outcomes may also provide insight into any gaps in the delivery of optimal care and identify opportunities for improvement.The study used data from the NSW Trauma Registry which was linked to the Australasian Rehabilitation Outcome Centre (AROC) Inpatient Dataset. AROC collects data from public and private rehabilitation units with almost 100% coverage. NSW Trauma Registry collects data from all trauma centres and it is expected that < 1% of trauma admissions opt out of data collection.The two datasets were linked using probabilistic methods with the following variables: age, sex, residential postcode, and date of acute discharge = date of admission to rehabilitation. This resulted in a 72% linkage rate with the missed linkages being missed randomly with no obvious bias . Ethics The cohort for this dataset was all road trauma admissions in NSW for the period 2009–2012 who survived to hospital discharge . Categorical variables were analysed using chi-squared test. For continuous variables with normal distribution, the mean (SD) were described and the parametric Student’s t-test used. For variables that were not normally distributed, both the mean (SD) and median (IQR) were described and the non-parametric Mann-Whitney U or Kruskal-Wallis test used. A The linked dataset contained records of 667 multi-trauma patients who received inpatient rehabilitation after a road accident. In 268 cases (40%) the reason for rehabilitation (based on AROC admission impairment code) was for brain injury rehabilitation Table . Three qOf those receiving brain injury rehabilitation, 166 (62%) were admitted to a specialist brain injury rehabilitation unit. The remainder were admitted to non-specialist general rehabilitation units (with 75% in the public sector and 25% in the private sector). Of those who were 65 years and over, only 19% (8 out of 42) were admitted to a specialist unit. Of the patients accepted by specialist units, only 5% were older than 65 years.The mean age of the study population was 42 years ; 70% were men. Two-thirds of the study population (67%) were in employment prior to the accident. The rest were either unemployed (13%) or retired (20%). Nearly all (97%) were living in private residences prior to the accident with the remainder in boarding houses or transitional living units.Time from trauma hospital admission to inpatient brain injury unit admission was analysed in our cohort. The cumulative percentage of patients admitted inpatient rehabilitation is described in Table Rehabilitation outcomes as provided in the AROC dataset for patients in specialist units were compared with those in non-specialist units Table . Those tp = 0.009) and comparable discharge FIM scores (p = 0.49) to those of specialist units. There were no discharges to residential aged care facilities from specialist units. Specialist units achieved significantly greater FIM gain (p < 0.001) compared with non-specialist units but at a cost of longer length of stay (p < 0.001) and therefore lower FIM change per day of admission (FIM efficiency) (p = 0.001).The non-specialist units achieved a higher rate of discharge to private residence , 24% were left with moderate disability (FIM score 72–108) and 14% with severe disability (FIM score < 72).As trauma systems mature, it is vital to have rehabilitation data to ensure that processes are efficient, the access is equitable and timely and the rehabilitation is effective. This study has documented brain injury rehabilitation outcomes after road trauma in NSW and compared the discharge outcomes between those who received inpatient brain injury rehabilitation in a specialist unit or a non-specialist unit.In this cohort there appears to be a trend in NSW of younger and more severely injured patients being admitted to specialist units. The 40% of brain injured patients needing inpatient rehabilitation managed in non-specialist units tend to be older and less cognitively impaired. With this large volume managed in non-specialist units, further research into selection criteria may aid in ensuring triage processes provide access to optimal care and health outcomes for brain injury survivors.Older survivors of brain injury have the potential to improve, just slower and maybe less well than the younger survivors . In our The rate of discharge to aged care facilities needs to be explored further. Young people with brain injury, no matter how disabled, should not be discharged to aged care facilities . SpecialOnly 2% of patients were discharged to aged care facilities after receiving their rehabilitation in non-specialist units. Typical rates of discharge to aged care facilities after inpatient stroke rehabilitation in Australia are around 12–20% . This leThere are no published historical data on the outcomes of inpatient specialist or non-specialist brain injury rehabilitation in NSW for comparison. The only published Australian inpatient rehabilitation case series is from a single centre in Queensland in the 1990s but the quality of the data is questionable . There aThe FIM score has been criticised for being a poor measure of brain injury rehabilitation programs because it is a measure that is dominated by physical disability and only offers a crude assessment for cognitive and psychosocial disability . The UK As trauma systems mature, it is important that the increase in survival comes with no rise in severe disabilities. We believe the discharge FIM score is particularly useful for this purpose. Using a FIM score < 72 to describe severe disability, the 14% in the specialist units that were left with severe disability after inpatient rehabilitation is comparable to a cohort from Norway which was reported as 15% . This meThis study has shown that only one quarter of the patients are transferred to a specialist unit by end of week 4 after injury. The mean length of stay in acute care for those managed in specialist units was 44 (± 23) days. This is at least 2 weeks more than international large published cohorts managed in brain injury units from United States (20–21 days) , 26 and As patients in NSW with severe brain injury are spending an average of 6 weeks in acute care, it is important to review the amount of rehabilitation received during this period. Like stroke, recovery in brain injury is fastest in the initial few weeks . Stroke There are two innovative models of care that can be developed to deliver high intensity coordinated rehabilitation to this population group. The first is to use existing in-reach rehabilitation teams that are available in most NSW trauma hospitals . These tAn alternative model is to set up “early brain injury units” or “hyper-acute rehabilitation” in designated trauma hospitals. These units are a collaborative model of care between intensive care and neurosurgery and rehabilitation medicine. These units can care for patients with severe brain injury who are still requiring high levels of medical care but are adequately resourced to provide intensive rehabilitation until they are stable enough to be transferred to a rehabilitation unit. There are examples of this model of care in Norway , Sweden The strength of this study is that the two linked datasets are population-based, capturing nearly all patients admitted with traumatic brain injury. This minimises the possibility of selection factors having unintended consequences on the study results. This study has also provided a methodology for evaluating the impact of any changes to the NSW brain injury rehabilitation system in the future.A limitation of this data linkage study is that patients were not matched for severity of brain injury and as such cannot be used to compare relative effectiveness of the two care settings. Markers of severity such as Glasgow Coma Score and duration of post-traumatic amnesia were not available for analysis.Another limitation is that the study only provides inpatient rehabilitation outcomes and does not provide any data on outpatient rehabilitation outcomes which may be equally important as rehabilitation interventions in this population group extends over time and beyond hospital care to where the person lives and works. In the future, it may be feasible for AROC to conduct phone interviews at certain time points  to track this group of patients to obtain better data on recovery. A similar model to this has been used by the Victorian State Trauma Registry and they have concluded that the cost of collecting such data is low (10 min required per patient to collect outcome measures) and the follow-up rate was high .This study may have identified opportunities for improvement in the service processes and clinical outcomes for brain injury patients in the future and these include: reviewing the appropriateness of limiting access to specialist units based on age; funding more specialist rehabilitation beds for a trial period to see if this may lead to more timely access; and development of innovative models of care for delivery early neuro-rehabilitation in trauma hospitals.This study has demonstrated that routinely collected registry data may be used to monitor trauma rehabilitation outcomes. There are differences in characteristics and outcomes of patients admitted to specialist compared with non-specialist brain injury units. The trend in NSW is that younger and more severely injured patients are being managed in specialist units with the older, less cognitively impaired patients managed in non-specialist units. This study may have identified opportunities for improvement in the service processes and clinical outcomes for brain injury patients in NSW.


Objectives: To advance development of rehabilitation robots, the conditions to induce appropriate brain activation during rehabilitation performed by robots should be optimized, based on the concept of brain plasticity. In this study, we examined differences in cortical activation according to the speed of passive wrist movements performed by a rehabilitation robot.Methods: Twenty three normal subjects participated in this study. Passive movements of the right wrist were performed by the wrist rehabilitation robot at three different speeds: 0.25 Hz; slow, 0.5 Hz; moderate and 0.75 Hz; fast. We used functional near-infrared spectroscopy to measure the brain activity accompanying the passive movements performed by a robot. The relative changes in oxy-hemoglobin (HbO) were measured in two regions of interest (ROI): the primary sensory-motor cortex (SM1) and premotor area (PMA).Results: In the left SM1 the HbO value was significantly higher at 0.5 Hz, compared with movements performed at 0.25 Hz and 0.75 Hz (p < 0.05), while no significant differences were observed in the left PMA (p > 0.05). In the group analysis, the left SM1 was activated during passive movements at three speeds (uncorrected p < 0.05) and the greatest activation in the SM1 was observed at 0.5 Hz.Conclusions: In conclusion, the contralateral SM1 showed the greatest activation by a moderate speed (0.5 Hz) rather than slow (0.25 Hz) and fast (0.75 Hz) speed. Our results suggest an ideal speed for execution of the wrist rehabilitation robot. Therefore, our results might provide useful data for more effective and empirically-based robot rehabilitation therapy. A number of rehabilitation robots have been developed in the past two decades to aid functional recovery of impaired limbs in patients with brain injury , Positron Emission Tomography (PET) and functional Near Infrared Spectroscopy (fNIRS) provide important information about the activation of the brain by external stimuli  with no history of neurological, psychiatric, or physical illness were recruited for this study. Handedness was evaluated using the Edinburg Handedness Inventory  kinematic model with one revolute joint  from 80° (flexion) to 75° (extension) when the degree of neutral wrist position is 0°, with the wrist in a flat position, with velocity of the wrist motions up to 2 Hz.Cortical activity was measured using the fNIRS system, which has less sensitivity to motion artifacts and metallic materials than other functional neuroimaging techniques  was the reference point for optode holder placement and the lines connecting T3-T4 and Nasion-Inion were used as reference lines for second row and fourth column of optodes each. The NIRS optodes was mounted on optode holder for fixation and the distance between pairs of light source-detector optodes was set at 3 cm. Using a Fastrak System , the coordinates of all probe positions and the anatomical landmark positions  of each subject were obtained after data collection. The coordinates of the anatomical landmark and optode positions were used to estimate the position of each channel in the Montreal Neurological Institute (MNI) standard brain space —task (20 s)—rest (20 s)) was used for performance of flexion and extension movements of the right wrist by the wrist rehabilitation robot.Three different speeds of passive wrist movements performed by the wrist rehabilitation robot  were examined and the performance sequence was assigned randomly by applying a random permutation function “randperm” in MATLABR2012b . During the experiment, the fNIRS system measured the cortical activity, while the wrist rehabilitation robot provided passive movement of right-wrist only during the task section in the block paradigm design.t test  was calculated in units of millimolar-millimeter. SPSS software  was used for statistical analyses. Null hypotheses of no difference were rejected if p-values were less than 0.05.To test the role of chance in differences for the HbO value in each ROI dependent on the speed of passive wrist movements, one-way ANOVA with a least significant difference p < 0.05); however, no significant difference was observed between 0.25 Hz and 0.75 Hz (p > 0.05). In the left PMA, no difference was observed among three Hzs  were analyzed. In the individual GLM analysis, during passive movement of the right wrist by the wrist rehabilitation robot, in the left SM1 and PMA, HbO values changed as follows: the left SM1 −0.25 Hz: 0.0001, 0.5 Hz: 0.0037, 0.75 Hz: 0.0004 and the left PMA −0.25 Hz: 0.0007, 0.5 Hz: −0.0008, 0.75 Hz: −0.0011. In the left SM1 the HbO value was significantly higher at 0.5 Hz, compared with the value due to wrist movements performed at 0.25 Hz and 0.75 Hz (p < 0.05). The greatest activation in the SM1 was observed at 0.5 Hz rather than 0.25 and 0.75 Hz , and greater activation in the PMA was observed at 0.75 Hz than 0.25 Hz  to 75° (extension) of the wrist joint and several subjects could not accommodate the movements at 1 Hz speed. A previous study also reported that 1 Hz was too fast for performance of precise movements of the wrist joint in stroke patients  rather than slow (0.25 Hz) and fast (0.75 Hz) speed. Our results suggest an optimal condition for execution of the wrist rehabilitation robot in terms of speed. Therefore, we believe that our results might provide useful data for more effective and empirically-based robot rehabilitation therapy. In addition, fNIRS appears to be a useful evaluation tool for research on the rehabilitation robot.SJB and SHJ contributed equally to this work and should be considered as co-first authors. SJB and JPS contributed to acquisition and analysis of data. SHJ and PHC contributed to conception and design and interpretation of data. PHC participated in drafting the article and gave the final approval of the version to be submitted.This work was supported by the DGIST R&D Program of the Ministry of Science, ICT and Future Planning (16-BD-0401).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.


Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in urothelial carcinogenesis as well as tumor growth. While the precise mechanisms of the functions of the androgen receptor in urothelial cells remain far from being fully understood, current evidence may offer chemopreventive or therapeutic options, using androgen deprivation therapy, in patients with bladder cancer. Urinary bladder cancer, mostly urothelial carcinoma, is the second most common genitourinary malignancy, with an estimate of 429,800 new cases and 165,100 deaths in 2012 worldwide . Of noteMen are at a significantly higher risk of bladder cancer than women in the US as well as virtually all countries/regions, while there is an approximately 10-fold variation in its incidence internationally . CigaretPrevious studies have thus demonstrated that AR activation generally correlates with the promotion of the development and growth of urothelial cancer. In this article, we review these available data and highlight underlying molecular mechanisms.Androgens, first discovered in 1936, are a class of steroid hormones, mainly secreted by the testis, ovary, and adrenal cortex. These include testosterone and its metabolite via 5α-reductase in certain tissues, dihydrotestosterone (DHT), as well as adrenal androgens, dehydroepiandrosterone, androstenediol, and androstenedione. In males, androgens can stimulate the differentiation and maturation of the sex organs and the development of secondary sex characteristics as well as maintain sexual activity and reproductive function . The phyAR gene locates on the X chromosome (Xq11–12) and contains eight exons and seven introns with the total length exceeding 90 kb. The AR encodes four distinct functional domains: the N-terminal transactivation domain, the DNA-binding domain (DBD), a hinge region, and the C-terminal ligand-binding domain (LBD) [The AR, a 110 kD protein composed of 919 amino acids, is a member of the nuclear receptor superfamily that functions as a ligand-inducible transcription factor and mediates the biological effects of androgens in a wide range of physiological and pathological processes ,13. The in (LBD) . An altein (LBD) ,16,17. Tin (LBD) .Male internal genitalia, including the prostate and bulbourethral gland as well as urothelium, are derived from the urogenital sinus endoderm. Simultaneously, it is well known that the differentiation of the prostate and its development require the induction of AR signaling . Thus, wAR gene in all 33 superficial bladder cancer specimens examined [Prior to its cloning, a binding assay suggested higher levels of AR content in bladder tumor (49.5 Fm/mg) than in normal bladder mucosa (17.2 Fm/mg), as well as in male (68.0 Fm/mg) or low-grade /27.7  Fm/mg) tumors than in female (27.7 Fm/mg) or high-grade 32.4 Fm/mg) tumors, respectively .4 Fm/mg ,42,43,44examined .The positive rates of AR expression immunohistochemically detected in bladder tumor tissues involving more than 40 cases range from 13% to 55%, which are significantly lower than those in non-neoplastic urothelial samples in some studies ,36,40. ISome of these studies have compared the rates of AR positivity in low-grade or non-muscle-invasive tumors versus high-grade or muscle-invasive tumors. Similar to the AR positivity in bladder tumors compared with non-neoplastic bladders, its significant or insignificant down-regulation is observed in high-grade and/or muscle-invasive tumors ,38,40,43Prognostic values of AR expression in bladder cancer patients have also been assessed, and the findings remain controversial. Two studies indicated a correlation between AR positivity and a lower risk of tumor recurrence ,43. MeanAR gene have been documented in bladder cancer. Loss of heterozygosity at the AR locus was identified in muscle-invasive tumors and concurrent lesions of carcinoma in situ from female patients [AR gene, which in general is inversely correlated with its transcriptional activity, between bladder tumors and controls or different grades/stages of bladder tumors. Men and women who had 23 (odds ratio = 2.09) and 44  CAG repeats were found to have a significantly elevated risk of urothelial carcinoma, compared to those with longer CAG [AR gene were found in 99 cases of bladder cancer [In addition to the differential expression of AR protein, genetic alterations involving the patients . In addinger CAG . A signinger CAG . Moreovenger CAG . Short Cnger CAG . Meanwhir cancer , a molecr cancer ,56 identr cancer , suggestThe gender-specific difference in the incidence of bladder cancer as well as AR expression in benign and cancerous urothelium suggests the involvement of AR signaling in urothelial tumorigenesis. High incidence of high-grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma—in the development of both of which, AR plays a critical role—in cystoprostatectomy specimens undergone for bladder urothelial carcinoma  may alsN-butyl-N-4-hydroxybutyl nitrosamine (BBN), which is known to induce a bladder tumor effectively in experimental rodents and more rapidly in male animals than in females [N-nitrosobis(2-oxopropyl)amine [AR gene was disrupted in the ARKO mice [A chemical carcinogen,  females , has bee females . In 1997 females . Subsequ females . Testostyl)amine . Thus, ayl)amine , suggestRKO mice , the andRKO mice . SimilarRKO mice . In addiRKO mice . These op = 0.0004) [Several recent retrospective cohort studies have supported these findings in animals indicating that AR activation correlates with the induction of bladder tumorigenesis. First, men undergoing androgen deprivation therapy (ADT) for their prostate cancer were shown to have a considerably lower risk of subsequent development of bladder cancer (0/266 (0%)), compared with those undergoing surgery alone (5/437 (1.1%)) or radiotherapy (14/631 (2.2%)) . In this 0.0004) , althoug 0.0004) .CYP4B1 gene expression in male mouse bladders were found to be higher than those in female mouse bladders, and castration in males resulted in a decrease in its expression [Androgens have been shown to modulate the activity and/or expression of certain enzymes via the AR pathway. These enzymes include cytochrome P450  and UDP-glucuronosyltransferase  that are known to involve the activation and detoxification, respectively, of bladder carcinogens, such as aromatic amines. The levels of pression . Similarpression . In addipression  or ovaripression  up- or dpression . Moreovepression .GATA3 is a zinc-finger transcription factor and is highly expressed in urothelial cells. Loss of GATA3 expression in a subset of bladder cancers, especially high-grade and/or muscle-invasive tumors , as wellIn addition to its involvement in urothelial carcinogenesis, there have been a variety of studies suggesting that androgens and/or AR promote bladder cancer progression. As seen in prostate cancer cells, androgens could induce AR expression and its nuclear translocation as well as ARE promoter activity in bladder cancer cells ,81,82,83Using cell viability or colony formation assays, androgens have been shown to induce the growth of AR-positive bladder cancer cells ,87,88,89In AR-positive bladder cancer cells, androgens are able to modulate the expression or activity of various molecules/pathways. Those known to involve bladder cancer cell proliferation/migration/invasion as well as angiogenesis/metastasis include β-catenin/Wnt signaling and its downstream targets c-myc/cyclin D1 ,81,84,86As seen in prostate cancer cells, non-androgens, such as epidermal growth factor (EGF), could increase AR transcriptional activity in bladder cancer cells, which was blocked by AR antagonists . EGF couRecent in vitro studies have suggested a correlation between AR activity in bladder cancer cells and chemosensitivity. AR-positive cell lines were more resistant to cisplatin than control AR-negative or AR knockdown cells cultured in the presence of androgens . FurtherAs aforementioned, androgen-mediated AR transcriptional activity can be further enhanced by co-activators. Indeed, several AR co-regulators have been implicated in the modulation of bladder cancer cell growth. A cross-talk between AR-co-regulators and other signaling pathways in bladder cancer cells may further promote urothelial tumorigenesis and tumor progression.Immunohistochemistry in tissue samples showed the expression of NCOA1, NCOA2, NCOA3, CREBBP, and EP300, in 85%–100% of bladder tumors—some of which even lacked AR expression . FurtherImmunohistochemistry in radical cystectomy specimens also showed strong correlations of the expression of JMJD2A and LSD1, both of which were shown to mediate AR transactivation via histone-lysine demethylation mechanisms, with that of AR . MoreoveAltered expression of β-catenin is well known to correlate with the progression of bladder cancer and poor patient outcomes ,81,97. ACurrent evidence indicating correlations of AR activation with the promotion of urothelial tumorigenesis and tumor progression supports that bladder cancer is a member of endocrine-related tumors. It is thus likely that at least AR and its associated signaling pathways, as depicted in Again, current non-surgical conventional treatments, such as intravesical pharmacotherapy and systemic chemotherapy, often fail to completely prevent the recurrence of superficial bladder tumors or significantly reduce the mortality rate in patients with advanced bladder cancer. Moreover, no approved targeted therapy for bladder cancer is available. As aforementioned, AR signals likely promote the development and progression of urothelial cancer. We therefore anticipate that AR inactivation—even via available options clinically used for the treatment of, for instance, prostate cancer—offers an effective chemopreventive or therapeutic approach for urothelial cancer. Indeed, two phase II clinical trials are being conducted to assess the preventive effects of enzalutamide on tumor recurrence in patients with non-muscle-invasive bladder cancer (NCT02605863) and the therapeutic effects of abiraterone—an androgen biosynthesis inhibitor prescribed in men with castration-resistant prostate cancer—in patients with advanced bladder cancer (NCT02788201). In addition, a phase I trial (NCT02300610) assessing the combination effects of enzalutamide with gemcitabine and cisplatin in patients with urothelial cancer is recruiting participants. Further prospective cohort studies of anti-AR treatment in patients with bladder cancer are thus encouraged.


Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer.Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest  on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms.Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells.CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity. Androgen receptor (AR) is a nuclear transcription factor in the steroid hormone receptor superfamily. AR activation by binding to ligand, dihydrotestosterone, induces a conformational change in AR, upon which the activated dimerized AR translocates from the cytoplasm to the nucleus, where along with co-activators, co-repressors and other transcriptional proteins, binds to an androgen-response element (AREs) in the promoter and enhancer regions, leading to transcription of key genes encoding proteins related to growth and proliferation , 2. The Recently, it has become clear that AR plays a critical role in normal and malignant breast tissue –10. AR eAR mRNA expression needs to be validated. One study showed that gene expression profiling might be superior to IHC for predicting AR-target therapy [AR expression in the blood over time in response to AR-targeting treatment, a strategy known as “liquid biopsy.”Currently, AR expression in breast cancer is defined on the basis of immunohistochemical (IHC) staining. However, there is no clear definition of AR positivity , 10, 18. therapy . Hierarc therapy , 24. FinLiquid biopsy assay platforms to assay analytes such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating RNA, proteomics, and metabolomics have been developing. Accumulating evidence suggests that CTCs may be tumor-specific biomarkers of response to therapy. CTCs have been reported to be a surrogate marker for tumor treatment response, and the presence of CTCs has been linked to shorter survival in patients with metastatic breast, prostate, colorectal, and lung cancer –31. CTCsAt present, the only Food and Drug Administration (FDA)-approved platform for the enumeration of CTCs is the CellSearch system, which uses cell-surface epithelial cell adhesion molecule (EpCAM) for CTC enrichment. To improve the sensitivity of CTC detection, Epic Sciences has developed a platform that uses a non-enrichment-based non-biased approach allowing for enumeration and characterization of all CTCs, including traditional CTCs , CK-negative (CK-) CTCs, apoptotic CTCs, CTC clusters (groups of 2 or more adjacent CTCs with shared cytoplasmic boundaries), and small CTCs . The Epic Sciences platform can be used to quantitatively measure protein expression via immunofluorescence, to differentiate protein subcellular localization, and to perform simultaneous detection of fluorescence in situ hybridization (FISH) on the same cells. Another benefit of this platform is its ability to detect and molecularly characterize CTCs from blood samples cryopreserved in liquid nitrogen, in which the relative abundance of CTCs and protein biomarker  expression remains largely consistent with the values in matched fresh samples. Morphology, biomarker expression, localization, and genetic alterations assessed via FISH are preserved in archived CTCs, enabling retrospective biomarker studies in larger cohorts with detailed clinical histories .Given that AR-targeted therapies are under development, there is an urgent need to better understand the biology of AR in breast cancer. Also, longitudinal follow-up of AR expression in CTCs might be a future predictive biomarker for AR-targeted therapy. In this preliminary study, we used the Epic Sciences platform to identify CTCs in patients with metastatic breast cancer and determine AR protein expression and genomic analysis of the identified CTCs.This study was approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center (protocol number PA14-0778). Patients with metastatic breast cancer treated at MD Anderson Cancer Center diagnosed during the period from February 2015 through January 2016 were eligible. All patients signed an informed consent form before blood draw.6 nucleated blood cells/slide were deposited on up to 12 glass slides per sample, and slides were stored at -80°C for long-term storage.For this study, a single blood sample was obtained from each patient. Blood (10 mL) was collected in cell-free preservative blood collection tubes . Blood samples were sent at ambient temperature to Epic Sciences and processed within 96 hours of blood draw using previously described methods –47. BrieFor CTC identification and biomarker staining in CTCs, 2 slides per patient sample were thawed and immunostained with an antibody cocktail to detect CK, CD45, DAPI, and biomarkers of interest . CK, CD45, and DAPI were used to detect and enumerate CTCs; AR, ER, and HER2 were biomarkers of interest. Stained slides were scanned and images were analyzed using a multiparametric digital pathology algorithm to determine CTC enumeration and biomarker expression on CTCs as previously described . A tyramAs we previously reported , fluoresThe specificity of AR, ER, and HER2 assays was evaluated using assay-specific positive and negative cell line controls. As shown in Panel A in As shown in Panel C and D in Panel E and F in To understand the genomic profile of various types of CTCs and the underlying cellular and potential genomic heterogeneity, we sequenced CTCs from a patient with AR+/HR+/HER2- tumor tissue on IHC staining. Single-cell CTC isolation and next-generation sequencing (NGS) library preparation were performed as previously described . The indArchived breast tumor tissues were obtained from the pathology files at MD Anderson Cancer Center. IHC staining for AR was performed using the polymeric biotin-free horseradish peroxidase method on the Leica Microsystems Bond Max autostainer . In each case, 1 unstained 4-μm-thick tissue section that had been prepared from a representative paraffin block of tumor was incubated at 60°C for 25 min. Following heat-induced epitope retrieval with citrate buffer for 25 min at 100°C, slides were incubated with mouse monoclonal antibody to AR . The Refine Polymer Detection kit was used to detect bound antibody, with 3,3-diaminobenzidine serving as the chromogen (Leica Microsystems). Slides were counterstained with Mayer’s hematoxylin. Results were evaluated with known positive and negative tissue controls. The percentage and intensity of any nuclear staining in the tumor cells were recorded. AR positivity was defined as at least 10% of cells expressing AR.Hormone receptor (HR) positivity was defined as positive if ≥ 10% of cells had positive IHC staining for ER and/or progesterone-receptor (PR). HER2 positivity was defined as a HER2/CEP17 fluorescence in situ hybridization (FISH) ratio of ≥2.0 and/or an immunohistochemical (IHC) staining score of 3+ .Data variables of interest were summarized using standard descriptive statistics. Associations between categorical variables were examined by using Fisher’s exact test. All statistical tests were 2-sided, and P < 0.05 was considered statistically significant. R software, version 3.2.0, and GraphPad PRISM 7 were used for statistical analysis.Seventy-four patients were enrolled in this study and had a blood sample collected. Of those 74 patients, 6 (8%) were excluded from the final analysis: 4 with low blood volume and low cell count whose samples were not processed for initial staining and 2 with low nucleated cells of less than 1.5 M after staining.The baseline characteristics of the 68 patients included in the final analysis are shown in The Epic Sciences platform identified CTCs in all 4 categories of interest . Of the Of the 26 patients with HR+/HER2- tumors on IHC staining, 24 (92%) had traditional CTCs detected, and 25 (96%) had all CTC candidates detected . Among pAR N-terminal (N-term) is referred to the region of the AR gene product targeted by the antibody. The significance of this is that this region is shared by all clinically relevant splice variants of the AR gene in addition to the full length AR. Results of AR analysis for all 68 patients in the study are shown in All 10 patients with AR+ CTCs (excluding 2 patients with only AR+ apoptotic CTCs) had CTCs stained to analyze ER expression. Three of the 10 patients had ER+ CTCs. All 3 patients had HR+ tumors by IHC staining. Two of the 3 patients had higher AR+ CTC counts and higher ER+ CTC counts than the other patient . ArchiveTo understand the genomic profiles of various types of CTCs, we sequenced CTCs from patient 11 (AR+/HR+/HER2- in tumor tissue and AR+/ER+ in CTCs). Before sequencing, we also stained the CTCs with HER2 immunofluorescence assay. Interestingly, although the tumor was HER2- by IHC staining, HER2+ CTCs were detected in this patient . A totalThe single-cell sequencing revealed 3 genomic patterns: pattern A, the dominant pattern, which included frequent copy number changes, among them amplification of chr8p11-p12 and chr11q13-q14; pattern B, which was similar to pattern A but had an additional partial deletion of chr6; and pattern C, which had no significant copy number changes . While tOur findings from this study show that the Epic Sciences CTC platform, which uses a non-enrichment-based non-biased approach, can be used to identify CTCs in patients with metastatic breast cancer and to identify AR expression in those CTCs. These preliminary results suggest that CTC AR expression identified using this approach has potential as a biomarker for identifying patients who might benefit from AR therapy and suggest that this approach should be clinically validated. We detected traditional CTCs (CK+ CTCs and CK+ clusters) in more than 50% of the 68 patients in this study and all CTC candidates in 75% of them. We detected CTCs in more than 90% of the patients with HR+/HER2- tumors and approximately 60% of the patients with the other tumor types .AR gene amplifications as well as other alterations that are consistent with prostate cancer. These data suggest that the identified CK- CTCs were real tumor cells in prostate cancer. Further evidence supporting the reproducibility of our current method is that in 21 samples from 20 healthy donors, no CTCs were detected [In terms of the reproducibility of the current non-enrichment-based non-biased approach to identify CTCs, CK- CTCs were determined by a combination of morphology and biomarker expression; for example, large cells with high nucleus-to-cytoplasm ratio, irregular nuclear contours, and high AR expression. We previously reported an association between higher detection of non-traditional CTCs, including apoptotic CTCs, CK- CTCs, and CTC clusters, and worse overall survival in metastatic prostate cancer . In a sedetected . These fTo further understand the difference of genomic profiles for each CTC with different biomarker positivity, we have sequenced 78 CTCs and 1 WBC from a patient who had HR+/HER2- disease on tissue biopsy. Our finding that this patient had not only ER+ and AR+ but also HER2+ CTCs even though IHC staining of the tissue biopsy specimen showed no HER2 expression suggests a potential discordance between the tissue and CTCs. A similar discordance was observed in a previous study wherein 33% of patients with HER2- primary tumors had HER2+ CTCs .KAT6A and NSD3 (previously known as WHSC1L1) in this region may be involved in driving tumor cell growth and carcinogenesis [PAK1 and SF1 genes are located on this region; PAK1 amplification may play a key role in MAPK activation, and SF1 amplification may predict therapeutic resistance to adjuvant tamoxifen therapy [Our genomic analysis of the CTCs in this 1 patient revealed 3 major genomic patterns: pattern A, which had amplification in chromosomal regions chr8p11-p12 and chr11q13-q14; pattern B, similar to pattern A but with an additional partial deletion of chr6; and pattern C, with no significant copy number changes. Amplification of chr8p11-p12 was previously reported to be common in breast cancer (occurring in 12%-15% of cases), and its presence correlated with poor patient outcome . Amplifiogenesis . Amplifiogenesis . The PAK therapy . While m therapy . These rOne of the limitations of our current study is that we were not able to co-stain multiple markers of interest on the same slides owing to technical and resource limitations and thus could not investigate the possibility of co-expression of AR, ER, and HER2 in each CTC. Another limitation relates to the threshold for biomarkers. The 95th percentile is a commonly acceptable threshold for development of FDA-approved immunofluorescence and IHC in vitro diagnosis assays. We applied the same rigor to the CTC-based immunofluorescence assay in the current study. However, because the study was preliminary, we did not have an appropriate clinical variable such as clinical outcome to use for sensitivity analysis to set the threshold for specific clinical questions. Another study is warranted to validate the threshold for biomarkers.In conclusion, these preliminary results suggest the need for clinical validation of CTC AR expression as a potential biomarker to identify patients who might benefit from AR therapy. The intrapatient heterogeneity of CTC AR expression leads to the hypothesis that patients with AR+ CTCs might have heterogeneous disease with multiple drivers. Further studies are warranted to serially monitor changes in AR and to investigate the clinical applicability of AR+ CTCs and their heterogeneity at the genomic level with full sequencing. In the future, we plan to apply this technology to make a model to predict patients who are more or less sensitive to AR signaling inhibitors such as enzalutamide in prospective clinical trials.S1 Fig(A) Scatter plot showing AR signal in AR-positive VCaP cells and absence of AR signal in AR-negative PC3 cells. The AR assay uses a signal amplification method, yielding a background signal near 11 (cutoff thresholds for positivity are drawn at the 95th percentile of the negative control cell line). (B) Representative images of AR-positive (VCaP) and AR-negative (PC3) cells showing nuclear-specific AR expression and no AR expression, respectively. Red = Cytokeratin (CK), Green = CD45, Blue = DAPI, and White = AR. (C) Scatter plot showing ER signal in ER-positive MCF-7 cells and absence of ER signal in the triple negative MDA-MB-231 cells. The ER assay uses a signal amplification method, yielding a background signal near 6 (cutoff thresholds for positivity are drawn at the 95th percentile of the negative control cell line). (D) Representative images of ER staining with MCF-7 and MDA-MB-231 cell lines showing ER expression and no ER expression, respectively. (E) Scatter plot showing HER2 expression in HER2-positive SKBR3 cells and HER2-negative MDA-MB-231 cells. The HER2 assay is a non-amplified assay, and the cutoff for positivity is drawn at 3 on the basis of the lower limit of detection of the platform. (F) Representative images of HER-positive SKBR3 and HER2-negative MDA-MD-231 cells showing HER2 expression and no HER2 expression, respectively.(TIFF)Click here for additional data file.S2 FigDot plot depicts AR expression in CTCs identified in patients with HR+/HER2-, HR+/HER2+, HR-/HER2+, and TNBC metastatic breast cancer. Each dot represents a single CTC, and the color indicates the subtype, defined as CK+, CK-, CK+ cluster, or apoptotic. CTCs were detected in 54 of 68 patients tested. Interpatient heterogeneity was observed in levels of AR expression and subtypes of CTCs. Although most CTCs were CK+, all subtypes were observed, often within the same patient. Twelve of the 68 patients had 1 or more CTCs that were above the threshold for AR positivity (indicated with dotted line). N-term, N-terminal region.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.