Furthermore, AKR1B10 silencing increased retinoic acid (RA) levels in the serum of tumor-bearing mice and RA treatment attenuated 14-3-3ε-induced HCC cell proliferation. We further examined 14-3-3ε and AKR1B10 expression and clinicopathological characteristics of HCC tumors. Although the expression of AKR1B10 was significantly correlated with 14-3-3ε, an increase of AKR1B10 expression in 14-3-3ε positive patients paradoxically had better overall survival and disease-free survival rates as well as lower metastatic incidence than those without an AKR1B10 increase. Finally, we found a loss of AKR1B10 expression in cells exhibiting a high capacity of invasiveness. Silencing of AKR1B10 resulted in inducing snail and vimentin expression in HCC cells. These results indicate that AKR1B10 may play a dual role during HCC tumor progression. Our results also indicate that 14-3-3ε regulates AKR1B10 expression by activating β-catenin signaling. A combination of 14-3-3ε with AKR1B10 is a potential therapeutic target and novel prognostic biomarker of HCC.14-3-3ε is overexpressed in hepatocellular carcinoma (HCC) and its expression significantly associates with a poor prognostic outcome. To uncover how 14-3-3ε contributes to the tumor progression of HCC, we investigated the potential downstream targets regulated by 14-3-3ε. We found that 14-3-3ε increases expression and nuclear translocation of β-catenin and that 14-3-3ε-induced cell proliferation is attenuated by β-catenin silencing in HCC cells. Moreover, 14-3-3ε induces aldo-keto reductase family 1 member B10 (AKR1B10) expression through the activation of β-catenin signaling. Knockdown of AKR1B10 by siRNAs abolished 14-3-3ε-induced  The 14-3-3 protein family comprises seven isoforms  in all mammals and is implicated in regulating multiple cellular and physiological functions . Some 14Aldo-keto-reductase family 1 member B10  is a NADPH-dependent oxidoreductase and is considered a detoxification enzyme , 20. AKRTo investigate whether 14-3-3ε regulates HCC cell proliferation, Huh-7 and HepG2 cells were transiently transfected with a 14-3-3ε overexpression construct (flag-tagged), followed by MTT analysis. Transient overexpression of 14-3-3ε was confirmed by Western blotting analysis of flag-Huh-7 and flag-HepG2 Figure . These rvia β-catenin signaling activation.Wnt/β-catenin signaling has been implicated in promoting tumor progression. We therefore investigated whether β-catenin is involved in 14-3-3ε-induced HCC cell proliferation. The silencing of β-catenin by siRNA significantly reduced cell proliferation in both the control and 14-3-3ε overexpressed cells Figure . We nextvia microarray analysis. A total of 724 transcripts were changed by 14-3-3ε overexpression and 567 transcripts were regulated by β-catenin siRNA compared to their controls. There are 36 genes , the expression of AKR1B10 was compared with other clinicopathological characteristics. AKR1B10 expression exhibited an association with free surgical margins (p = 0.011), early BCLC staging (p = 0.042) and a lack of subsequent extrahepatic metastasis (p = 0.047) (Table p = 0.015) and metastasis (p = 0.011) in 14-3-3ε positive HCC patients , 14-3-3ε positive HCC patients with increased AKR1B10 expression paradoxically had better overall survival  and highly invasive cells  were determined by Western blot analysis. The highly invasive cells significantly expressed higher amounts of vimentin and N-cadherin, but there was slightly decreased E-cadherin expression  cells were transiently transfected with AKR1B10 overexpression vectors for 48 hrs. The expression of overexpressed AKR1B10 was confirmed by Western blotting analysis  is one of the potential targets upregulated by 14-3-3ε/β-catenin signaling . IL-18 ivs. Upregulation of AKR1B10 results in depletion of RA –38. RA eWe determined the level of RA in the sera of mice that were injected with 14-3-3ε overexpressing cells and compared those levels with the control cells. We found RA levels in mice with 14-3-3ε overexpressing cells to be relatively higher than the control group although the statistics were not significant. This may due to insufficient sample numbers . HoweverMoreover, AKR1B10 has been reported to promote cell survival by regulating lipid synthesis and eliminating production of carbonyls . SilenciThe role of AKR1B10 during HCC tumor prognosis is paradoxical. AKR1B10 is overexpressed in HCC and the silencing of AKR1B10 suppresses HCC cell proliferation . Conversin vivo in a nude mice model. Although the tumors were initiated within two weeks after injection of control . Huh-7 and HepG2 human cells were maintained in DMEM  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 units/ml streptomycin, in a humidified incubator with 5% CO2 at 37°C.Prostaglandin A1 (PGA1), oleanolic acid (OA) and trans-retinoic acid (RA) were purchased from Sigma-Aldrich Chemical Company . The Human Retinoic Acid ELISA Kit was from The vector construction of the 14-3-3ε overexpression vector, the transient transfection procedure and the selection of 14-3-3ε stable cells were all carried out as previously described . To estaThe selection of highly invasive Huh-7 cells and the invasion assay were performed in a modified Boyden chamber using a cell culture insert  coated with 0.1% matrigel containing 0.1% bovine serum albumin (BSA) in DMEM medium. Huh-7 cells were incubated in the upper chamber and cells with invasive capacity were harvested from the bottom chamber which contained 100 μg/mL fibronectin (Becton Dickinson), 20 ng/ml epidermal growth factor (EGF) and 10% BSA. This two-chamber selection was performed multiple times to establish highly invasive HCC cells.RNA was extracted and prepared using TRIzol  and cDNA was synthesized by the SuperScript III First-Strand Synthesis Supermix Kit (Invitrogen). RNA expression was quantified using quantitative real-time PCR (RT-PCR). RT-PCR of cyclin A, cyclin D, cyclin E and AKR1B10 was performed using SYBR Green  with specific oligonucleotide primers  and analGene silencing was performed by using 14-3-3ε, β-catenin and AKR1B10 siRNAs  and scramble control (Invitrogen) with reported sequences . TransieCell proliferation was measured by a 3--2,5-diphenyl tetrazolium bromide (MTT) assay, as described previously , 17. AbsCells were lysed in ice-cold RIPA buffer  containing cocktail protease inhibitors . Cell lysates were centrifuged at 15,000 rpm for 20 minutes at 4°C, and protein concentrations were determined. 20 μg of protein from each sample were run through a gradient SDS-PAGE gel, followed by immunoblotting onto PVDF membranes. The membranes were blocked and probed with primary antibodies of actin (Sigma-Aldrich), AKR1B10 and Zeb-1 , cyclin A, cyclin D, cyclin E, snail and β-catenin , E-cadherin and N-cadherin  and vimentin (Millipore). The membranes were immersed in PBST containing horseradish peroxidase-conjugated secondary antibodies, and protein levels were determined by use of enhanced chemiluminescence reagents.® 488 secondary antibody (Invitrogen) in PBS containing 5% bovine serum albumin at room temperature for 2 hrs. Samples were mounted and images were analyzed using the Leica TCS SP5 Confocal Imaging System .Immunofluorescence staining was performed as described in our previous study , 17. CelGene expression regulated by 14-3-3ε overexpression or β-catenin silencing in Huh-7 stable cells was analyzed by microarray analysis. All RNA samples were extracted from control/14-3-3ε overexpressing as well as scramble/β-catenin siRNA transfected cells using Qiagen RNeasy Mini Kit  and the microarray analysis was processed according to the manufacturers' instructions .3 cells were seeded onto six-well plates with 1 ml DMEM containing 0.8% low-melting agarose  followed by overlaying with 2 ml DMEM containing 0.8% low-melting agarose. After 3 weeks, cells were stained with 0.005% crystal violet in 25% methanol and colony numbers were counted.Anchorage-independent growth was assessed by a soft agar assay. Briefly, 6 × 106 cells in 0.3 ml PBS w were injected subcutaneously into the right flank of nude mice. Tumor volume was determined by sequential caliper measurements of length (L) and width (W) and calculated as LW2/2. After five weeks, mice were scarified, tumors were removed and tumor weight was measured.BALB/c nu/nu nude mice (8 weeks old) were purchased from the National Laboratory Animal Center, National Science Council and housed in micro-isolator cages at a specific pathogen-free facility at the Laboratory Animal Center, National Health Research Institutes of Taiwan. Control and 14-3-3ε stable cells were trypsinized, washed and resuspended in PBS. For AKR1B10 silencing experiments, Huh-7 cells with stable 14-3-3ε overexpression were transfected with scramble or AKR1B10 siRNA for 48 hrs before harvesting the cells. A total of 2 × 10For determining serum RA levels, blood from sacrificed mice was collected by heart puncture. The serum was separated by centrifuging the blood for 10 min at 1500 × g and 4°C and storing the serum at − 80°C. The concentration of RA was measured by a human retinoic acid ELISA kit .109 primary HCC samples were retrospectively obtained from patients who had undergone surgery for tumor resection or biopsy at Taichung Veterans General Hospital from January 1999 to December 2001. Slides from paraffin-embedded surgical specimens of primary tumors with surrounding non-cancerous liver parenchyma were subjected to immunohistochemical (IHC) staining. The pathological features, IHC staining results, clinical parameters, including Barcelona-Clinic liver cancer (BCLC) staging , and disFor immunohistochemistry analysis, an automatic immunostaining device and an ultraView detection kit  were used to detect 14-3-3ε expressions in paraffin-embedded tissues by use of a primary antibody against 14-3-3ε and AKR1B10 (Santa Cruz Biotechnology). A negative control was prepared by the same staining procedure without primary antibodies. The intensity of 14-3-3ε and AKR1B10 protein staining was semiquantitatively scored by a Quick-score (Q-score) method based on intensity and heterogeneity , 53–55. 4 for migration and 2 × 104 for invasion assay). Cells remaining on the upper side were removed and migrated/invasive cells on the bottom side were fixed as described previously [Bio-coat cell migration Boyden chamber was used for cell migration assays and invasion assays -DMEM medium in the upper chambers). Cells were trypsinized, re-suspended and cultured in the upper chambers for 16 h (3 × 10eviously , 17. Celt-test was used to analyze differences between two groups. Kaplan-Meier curves were plotted and the log rank test was used to analyze time-related variables of probabilities for metastasis and overall survival. Cox-proportional hazard regression was used in multi-variate analysis for the impact of prognostic factors on overall survival, disease-free survival and metastasis. P value < 0.05 was considered statistically significant.The Student's


A detailed analysis of the electronic structure of LiAr and Li3Ar shows that Ar in these compounds attracts electrons and thus behaves as an oxidizing agent. This is markedly different from the hitherto established chemical reactivity of Ar. Moreover, we predict that the P4/mmm phase of Li3Ar has a superconducting transition temperature of 17.6 K at 120 GPa.High pressure can fundamentally alter the bonding patterns of chemical elements. Its effects include stimulating elements thought to be “inactive” to form unexpectedly stable compounds with unusual chemical and physical properties. Here, using an unbiased structure search method based on CALYPSO methodology and density functional total energy calculations, the phase stabilities and crystal structures of Li−Ar compounds are systematically investigated at high pressure up to 300 GPa. Two unexpected Li In the Mg–O system, only the NaCl type structure MgO was observed in experiment at pressures up to 227 GPa2 and Mg3O2 are theoretically predicted to become enthalpically stable at 116 GPa and 500 GPa, respectively2 and semiconducting Mg3O2 exhibits significant ionic character. Dong et al.2He, with a fluorite-type structure, and predicted to be stable above 160 GPa –well below the pressures required for compound formation of He with other elements. Other manifestations include unusual stoichiometries in the Na-Cl system37d states are expected to become more accessible under pressure relative to the occupied s-states of simple metals, and Ar could serve as an electron acceptor. Lithium (Li) itself is considered to be a “simple” metal as its electronic structure is well described by a free-electron model at ambient conditions4042High pressure, by reducing the atomic distances and thus emphasizing the repulsive part of atomic interaction potentials, can alter the bonding characteristics of the elements, ultimately leading to novel chemical phenomena. Interactions with electropositive alkali or alkaline earth elements are particularly promising in this respect. For example, theoretical calculations of the Li-B binary system2628Here, we investigate systematically the stabilization of compounds of Li and Ar under high pressure. We employ an unbiased structure searching method as implemented in the CALYPSO  package4435363738mArn = –; ; and ) compounds. In such calculations, the formation enthalpy of per atom in LimArn is defined as follows:Phase stabilities in the Li-Ar system are established at various pressures by judging the formation enthalpy of stoichiometric Lihf is the formation enthalpy per atom, and H is the calculated enthalpy of each compound. Here, we restrict ourselves to ground state calculations, i.e H (Li), we used the relevant structures across the entire pressure rangeH (Ar), the hexagonal close packed (hcp) structure of solid Ar is adopted47P4/mmm phase of LiAr, the hexagonal phase of Ar, and the Cmca-24 structure of Li at 100 GPa are calculated to be as 86, 8, and 86 meV/atom, respectively. The contribution of ZPE to hf is thus quite small in the case of LiAr, and it is valid to neglect the contribution of ZPE when discussing the relative stability of Li−Ar systems. However, to account for all possible “escape routes”, we construct the “convex hull” or “global stability line” of all considered binary phases. In such a phase diagram, where mArn phases is displayed in n , which indicates that other unexpected compounds LimArn are perhaps synthesizable experimentally under high-pressure conditions. Specifically, from inspecting the convex hull it is found that LiAr and Li3Ar are enthalpically the most stable in under high pressures. The enthalpies of other phases are only slightly higher (about 0.1 eV/atom) than those of the stable compounds. In particular, Li4Ar .As shown in is study . In orden curves . No dyna3Ar and Li5Ar adopt relatively simple layered structures that can be interpreted as stackings of square lattices comprised of the elements 5(Li)5] and [(Ar)6(Li)6] stacking orders; at 100 GPa, these are 0.019 eV/atom and 0.036 eV/atom higher in enthalpy than P4/mmm-LiAr , which is also a layered structure, becomes the most stable phase of Li3Ar. Its conventional cell is shown in 5Ar crystallizes in the pressure range of 109 GPa to 130 GPa in a monoclinic P21 phase, which is also a stacking variant, and shown in 5Ar is most stable in an orthorhombic Cmcm phase  position in the unit cell, with −0.54e in each pocket. These interstitial electrons are at the same position as oxygen anions in the CuO2 layers of HgBa2CuO4. Because the lithium atoms’ valence electrons are not completely localized, but also partially populate the Ar-3d states, the structure remains metallic, in contrast to the pure lithium “electride” phase. The localized nature of the interstitial electron is confirmed by the Electron Localization Function (ELF), which carries information about the bonding character and valence electron configurations of atoms in a compoundz = 1/2 plane of tetragonal LiAr at 150 GPa: besides the 1 s2 shell of lithium, there is significant interstitial electron localization visible between adjacent lithium cations .To further investigate the nature of the stabilization of these Li-Ar compounds, we analyzed their electronic structure. Due to their predicted stability over a wide pressure range, we will focus on the LiAr and Lii3Ar see . Moreovei3Ar see . Note thd states are lowered relative to Li-2s states. Bader’s analysis gives partial charges of + 0.67e for Li and −0.67e for Ar at P = 200 GPa, which reduce to ± 0.62e at P = 300 GPa. As could be expected, no interstitial charge density forms in this structure – see the plot of the ELF in the [110] plane at P = 200 GPa in P = 175 GPa, LiAr is predicted to form a simple intermetallic compound – with anionic character of Ar. The Li core does not contain p-orbitals and is thus quite compact, due to the absence of orthogonality constraints, which contributes to the stability of LiAr with respect to elemental Li and Ar.The CsCl phase of LiAr at 200 GPa is also found to be metallic and from 3Ar, the electronic band structure and PDOS of the tetragonal P4/mmm phase at 200 GPa and the orthorhombic Cmmm phase at 310 GPa are compiled in 3Ar under high pressure is metallic in either phase. As in LiAr, this is driven by partial occupation of the Ar-3d states. The ELF plots for Li3Ar at 200 GPa and 310 GPa , + 0.55e (+0.49e) for Li2, and −1.03e (−1.02e) for Ar. The interstitial site at 2f  has a partial charge of −0.46e (−0.37e). In the orthorhombic structure of Li3Ar at 310 GPa, the charge transfer numbers agree qualitatively with all other Li-Ar phases discussed here.For Li GPa see  show thaP4/mmm-Li3Ar, we find an electron-phonon coupling strength of λ = 0.721 and a superconducting temperature Tc = 17.6 K at 120 GPa, which reduce to λ = 0.454 and Tc = 6.5 K at 200 GPa. Superconductivity in an electride has been measured at ambient pressure: in 12CaO·7Al2O3, a superconducting phase was found below Tc = 0.4 KSince all predicted stable phases are metallic, we estimated their potential for phonon-mediated superconductivity, with electron-phonon coupling calculated with the Quantum ESPRESSO packagemAr compounds (LiAr and Li3Ar) might be experimentally synthesizable over a wide range of pressures. Our calculations indicate that LiAr and Li3Ar are enthalpically and dynamically stable above pressures of 112 GPa and 119 GPa, respectively, while Li5Ar is stable in a small pressure window of 109–140 GPa. We found that all stable phases are metallic.In summary, by crystal structure searches based on CALYPSO methodology and density functional total energy calculations, potentially stable Li−Ar phases are systematically investigated at high pressure up to 300 GPa. Two unexpected Li3Ar exhibits reasonable electron-phonon coupling, with predicted superconducting temperatures of 17.6 K at 120 GPa and 6.5 K at 200 GPa, thus adding this compound to an intriguingly short list of candidates of superconducting electride phases.High pressure can induce argon to become an electron acceptor, as evidenced here by its ability to form stable intermetallic compounds with Li. In this particular system, the formation of ionic compounds (involving charge transfer from Li-2 s to Ar-3d states) competes with lithium’s propensity to shed its valence electron into interstitial space. The first stable Li-Ar compounds are thus predicted to feature both electride and metallic behavior. With higher pressure, the tendency of electron localization decreases in favor of increased ionic character. The absolute value of electronic charge transferred gradually decreased for all stable Li-Ar compounds. Amongst the stable Li-Ar compounds, LimArn  = -; ; and ) were investigated at pressures of 100, 200 and 300 GPa. The underlying ab initio structural relaxations and electronic band structure calculations used density functional theory with the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation (GGA) of the exchange-correlation energy as implemented in the VASP code22 s1, and 3 s23p6 electrons of Li and Ar, respectively, in the valence space. A cutoff energy of 900 eV was used for the plane wave expansion of the wave functions, and fine regular k-point grids used for Brillouin zone integrationsThe crystal structural predictions were performed by CALYPSO methodology as implemented in the CALYPSO code43How to cite this article: Li, X. et al. Stable Lithium Argon compounds under high pressure. Sci. Rep.5, 16675; doi: 10.1038/srep16675 (2015).


AR) or pathways involved in androgen synthesis form a critical component of the standard-of-care in treating aggressive, non-localized prostate cancers. The major problem with these therapies is that castration resistant clones arise within 1–3 years of treatment initiation, leading to clinical relapse and eventual death. Previously reported mechanisms of castration resistance include amplification and mutation of AR  serum level of 4.8 μg/l. The tumor cells expressed high levels of ASCL1, CHGA, SYP, and HES6, four classical markers of neuroendocrine prostate cancer   and 1 nM , while LNCaP cells transfected with empty vector were unable to grow in DHT-depleted mediums (Fig HES6 overexpression phenotype reported by Ramos-Montoya et al.To study the TURP sample further, we used ChimeraScan (Iyer DOT1L-HES6 and HES6 expression based on qRT-PCR, indicating that the fusion gene had originated post-orchiectomy (Fig DOT1L-HES6 positive TURP sample did not represent a new and independent tumor, we used the sequencing data to search for vestigial evidence of the ERG fusion present in the original diagnostic biopsy. Whole genome sequencing revealed a characteristic three megabase deletion between the genes TMPRSS2 and ERG in chromosome 21 in the TURP sample (Fig TMPRSS2-ERG expression in the TURP sample, although expression was very weak due to minimal AR activity.The diagnostic biopsy of patient #2 was negative for et al proposed a model in which HES6 promotes androgen independence by modulating AR binding. The lack of AR activity in our DOT1L-HES6 fusion positive sample may indicate the existence of additional, AR-independent mechanisms. An alternative hypothesis is that the DOT1L-HES6 fusion in the TURP sample of patient #2 promoted castration resistance at an intermediate stage of tumor evolution, but was later subsumed by another mechanism that additionally resulted in complete loss of AR expression. Nonetheless, the lack of ASCL1, CHGA, and SYP overexpression distinguishes this tumor from classical neuroendocrine prostate cancers and highlights the role that HES6 plays in castration resistant and androgen independent tumors. This finding also calls for a more extensive search for HES6 genomic alterations in cohorts of AR-negative and castration resistant prostate cancers.In their publication, Ramos-Montoya


Prostate Cancer (PCa) is an important age-related disease being the most common cancer malignancy and the second leading cause of cancer mortality in men in Western countries. Initially, PCa progression is androgen receptor (AR)- and androgen-dependent. Eventually advanced PCa reaches the stage of Castration-Resistant Prostate Cancer (CRPC), but remains dependent on AR, which indicates the importance of AR activity also for CRPC. Here, we discuss various pathways that influence the AR activity in CRPC, which indicates an adaptation of the AR signaling in PCa to overcome the treatment of PCa. The adaptation pathways include interferences of the normal regulation of the AR protein level, the expression of AR variants, the crosstalk of the AR with cytokine tyrosine kinases, the Src-Akt-, the MAPK-signaling pathways and AR corepressors. Furthermore, we summarize the current treatment options with regard to the underlying molecular basis of the common adaptation processes of AR signaling that may arise after the treatment with AR antagonists, androgen deprivation therapy (ADT) as well as for CRPC, and point towards novel therapeutic strategies. The understanding of individualized adaptation processes in PCa will lead to individualized treatment options in the future. Prostate cancer (PCa) is the most common cancer type among men in Western countries and the second leading cause of cancer-related death in males . In moreLocal recurrent PCa after failure of primary surgery/radiotherapy and hormone-naive metastatic disease are treated with androgen deprivation therapy (ADT) . LuteiniThe underlying molecular basis how PCa cells escape from the growth control by exogenous androgens is still poorly understood. Adaptive mechanisms of PCa cells include molecular alterations in response to androgen ablation leading to re-activation of the AR despite low circulating androgens after initial response to ADT . Over thMechanisms mediating castration-resistance comprise the re-activation of AR signaling despite low levels of circulating androgens on the one hand, as well as the activation of alternative AR-independent pathways on the other hand.de novo steroidogenesis provides persistent intraprostatic androgen concentrations sufficient to activate the AR despite low serum testosterone [Maintained AR activity under androgen deprivation is based on genetic and functional aberrations affecting components of the AR signaling axis which have been observed in CRPC cells . Geneticosterone . Additioosterone , 17. Genosterone , 8.Admittedly, no treatment options with curative intent are available for castration-resistant prostate cancer (CRPC) to date . CurrentThis review will highlight the current knowledge about adaptive mechanisms of the AR signaling as well as the significance of its interaction partners contributing to the development of castration-resistance.An adaptive response in AR signaling may also occur at the level of mutations and splice variants of the AR that occur under the selective pressure of ADT. Structurally, the AR is organized in 4 different domains: the N-terminal transactivation domain (TAD), a central DNA-binding domain (DBD), a hinge region (HR) which connects the DBD to the carboxy-terminal ligand binding domain (LBD) Figure . Upon anhttp://androgendb.mcgill.ca, [AR mutations are very rare in early stages of PCa. However, approximately 10-30% of CRPC patients carry AR mutations, especially when treated with ADT, indicating an adaptation to ADT by changing AR function . In CRPCgill.ca,  was repegill.ca, -28. Funcgill.ca, . While Tgill.ca, . Most imin vitro studies showed a high constitutive transcriptional activity of AR constructs in which the LBD has been artificially deleted [Early functional  deleted . Due to  deleted . So far, deleted   are susceptible to taxane-mediated microtubule stabilization that is abrogating nuclear translocation and transcriptional activity of these receptors [in vitro [As AR∆LBD do not express a functional LBD, they are insensitive to all currently available hormonal therapies targeting directly (anti-androgens) or indirectly (inhibitors of androgen synthesis) the LBD , 36. The setting , 38. In  setting . Both co setting , 40. As eceptors , 42. Enzeceptors . Moreovein vitro , 42. As The occurrence and detection of AR point mutations and AR∆LBD are of prognostic and therapeutic significance. In order to guide initial treatment selection or sequential therapies, there is an urgent need for new markers. Serum biomarker studies may be difficult to compare because of different procedures in various laboratories. Current research is focusing on the non-invasive retrieval of tumor DNA from blood/serum samples, i.e. circulating tumor DNA (ctDNA) or DNA from circulating tumor cells (CTC) to analyze aberrant AR variants. AR mutants like F876L or T877A have already been successfully isolated from blood and CTC samples isolated from patients suffering from advanced CRPC , 43, 44.The discovery of constitutively active AR∆LBD that do not express a LBD has led to the development of promising novel experimental approaches targeting AR-signaling in a LBD-independent manner -51. ReceThe analysis of AR-point mutations and/or AR-splice variants has led to the discovery of new prognostic and therapeutic targets in CRPC. In summary, the combination of new prognostic parameters able to guide treatment selection along with novel therapeutic approaches will establish a new era of personalized, targeted therapies.The upregulation of AR protein is a hallmark of CRPC and seems to represent an adaptive response to ADT . PresumaAn UC-rich 3′UTR motif is also the target site of another AR post-transcriptional inhibitor, ErbB3 binding protein (EBP1) that in addition also binds to a RNA stem-loop formed by the CAG repeats encoding the poly-glutamine stretch in the AR N-terminus . WhereasThe nucleic acid binding protein called heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is an additional inhibitor of AR mRNA translation and an inhibitor of PCa cell proliferation via binding to several sites in the AR mRNA including sites in both untranslated terminal regions and in the coding region . FurtherA mechanism for enhancement of AR translation was identified recently and is based on a reciprocal link between the AR and the ribonucleoprotein transcriptional enhancer complex assembled by midline 1 (MID1), a protein that is mutated in the developmental syndrome Opitz G/BBB , 63. OveAR regulation by MID1 occurs via enhancement of AR translation. However, the interaction of the MID1 complex and the AR axis is not uni- but bi-directional. Whereas MID1 regulates AR protein through translation control, AR is a negative regulator of MID1 via several AR binding sites in the MID1 gene. This mutual interaction forms a classical regulatory feedback loop that was suggested as a fine-tuning mechanism for homeostasis of AR protein level. ADT of PCa would disrupt this mechanism and result in MID1 and subsequently AR protein upregulation. In line with this, MID1 is significantly overexpressed in PCa in a stage-dependent manner .Noteworthy, the assembly of the MID1-alpha4/PP2Ac protein complex is disrupted by the antidiabetic drug metformin as first reported by Kickstein et al. . BesidesThus, the inhibition of AR protein levels by metformin suggests its use in the treatment of hormone-naïve PCa and CRPC. In support of this, a metformin treatment study in 44 men with progressive metastatic CRPC who received metformin until disease progression resulted in disease stabilization and prolongation of PSA doubling time . Retrospin silico screen for miRNAs targeting the long 3′UTR of the AR performed in PCa cell lines identified 75 miRNAs that regulate AR protein level [Last but not least, a new class of post-transcriptional regulators also acting on AR, are micro-RNAs (miRNAs) that control gene expression by inhibition of protein translation or induction of mRNA cleavage. These small RNAs of about 22 bases in length are generated by processing of mostly untranslated RNA and each miRNA can regulate a variety of target mRNAs , 80. Sevin level . FifteenIn conclusion, the diverse mechanisms of AR modulation at the level of mRNA and translation into protein can result in adaptation of AR signaling. The variety of proteins and miRNAs involved define a complex regulatory network for fine-tuning of AR protein level in PCa that might be used as drug targets and calls for efforts to develop methods to interfere with post-transcriptional AR regulation for improving inhibition of the AR axis in PCa therapy.There are several levels of interaction between growth factor receptors and AR in PCa. Transcription activation function of the AR could be enhanced by growth factors and growth factor-related receptors in a synergistic and ligand-independent manner , 89.in vitro in conditions of androgen depletion and pointing to possible adaptive and compensatory effects of endocrine therapy [Mechanistically, growth factors and related receptors lead to activation of MAPK that phosphorylate specific amino acids in the N-terminal region of the AR . In cons therapy . Consist therapy , 96. In  therapy .MAPK are implicated in activation of the AR by interleukin-6, a cytokine whose expression is elevated in human PCa . It was Taken together, the results of several studies mentioned above have indicated that tyrosine kinases are particularly important in AR activation in conditions in which the levels of circulating androgen are diminished during therapy, indicating an adaptation response.There is also a link between AR mutations and growth factor receptors. Androgens and hormones that activate mutated AR in LNCaP cells increase expression of epidermal growth factor receptor expression . These fBecause of these findings one can propose the development of preclinical and clinical inhibitors of tyrosine kinases to inhibit the crosstalk between growth factor receptor and AR signaling pathways. Treatment of cells with an anti-androgen and the anti-HER2 receptor monoclonal antibody herceptin could open the way for novel PCa therapies. In line with these observations, the dual ErbB1/ErbB2 tyrosine kinase inhibitor PKI-166 was tested in series of human PCa xenografts . The effIn context of crosstalk between signaling pathways, further aspects of inhibition of tyrosine kinases by sorafenib, a multikinase inhibitor, have to be discussed. Sorafenib inhibits proliferation and induced apoptosis in several PCa cell lines . Sorafenin vitro findings could also have clinical implications. Although PCa is a heterogenous disease, patients with higher expression of AR and several other sorafenib targets may be good candidates for sorafenib treatment. Thus, preclinical results obtained with sorafenib may serve as a basis for the development of a more personalized approach for PCa patients.These In summary, there are multiple interactions between signaling pathways that include tyrosine kinase receptors and the AR. These interactions are basis for a rational therapeutic targeting of these interactions in clinics.The AR signaling is not only controlled by the ligands of AR but also by signal transduction pathways including the MAPK and non-receptor tyrosine kinases. Among tyrosine kinases, Src is particularly important for AR phosphorylation . In PCa,Intracellular kinases that mediate the signal transduction of membrane associated receptor tyrosine kinases are often linked to cellular growth and seem to play a critical role in cancer development and progression. The family of non-receptor tyrosine kinases, including Src, are activated by and interact with various cellular pathways and regulate a plethora of different pathways including cell proliferation, cell motility, invasion, epithelial-to-mesenchymal transition, resistance to apoptosis, and metastatic spread , the corepressor recruitment leads to recruitment of chromatin repressor complexes to AR target genes or to coactivator displacement on the AR. These mechanisms are the molecular basis for AR inactivation mediated by some AR antagonists -123. In To analyze the underlying molecular mechanism, a battery of various inhibitors of signaling pathways were used to treat CRPC cells. The results indicated that the LCoR silencing function is repressed by the Src-Akt pathway . InhibitThese findings and those from other groups strongly suggest that inhibitors of members of the Src tyrosine kinase family in combination therapy with antagonists may be a very useful tool to inactivate AR signaling and progression of CRPC, which has been nicely reviewed recently . In lineSimilarly to LCoR, the corepressor Silencing Mediator for Retinoid and Thyroid hormone receptors (SMRT) seems also to be inactivated by signaling pathways in PCa. An activated MAPK signaling was shown to inhibit the SMRT corepressor function . SMRT biThus, mitogenic signaling pathways activate the AR signaling. One underlying molecular basis is the inactivation of AR corepressors by the Src kinase family members and/or the MAPK signaling. Co-treatment with signal transduction inhibitors may be a useful tool and therapeutic approach to inhibit CRPC. Presumably tissue specific inhibitors of Src, PI3K, Akt and MAPK are more useful in combination with AR inhibitors to reduce side-effects.Taken together, multiple adaptation processes seem to exist that allow persistent AR signaling in PCa Figure . The det


Methods. LV gene expression profile was performed in rats after 9 months of AR and compared to sham-operated controls. LV glucose and fatty acid (FA) uptake was also evaluated in vivo by positron emission tomography in 8-week AR rats treated or not with fenofibrate, an activator of FA oxidation (FAO). Results. Many LV genes associated with mitochondrial function and metabolism were downregulated in AR rats. FA β-oxidation capacity was significantly impaired as early as two weeks after AR. Treatment with fenofibrate, a PPARα agonist, normalized both FA and glucose uptake while reducing LV dilation caused by AR. Conclusion. Myocardial energy substrate preference is affected early in the evolution of LV-VO cardiomyopathy. Maintaining a relatively normal FA utilization in the myocardium could translate into less glucose uptake and possibly lesser LV remodeling.Patients with left ventricle (LV) volume overload (VO) remain in a compensated state for many years although severe dilation is present. The myocardial capacity to fulfill its energetic demand may delay decompensation. We performed a gene expression profile, a model of chronic VO in rat LV with severe aortic valve regurgitation (AR) for 9 months, and focused on the study of genes associated with myocardial energetics.  Aortic regurgitation (AR) is associated with a long asymptomatic period during which the left ventricle (LV) progressively dilates and hypertrophies in response to chronic volume overload. This process is accompanied by a decrease in LV function, occurrence of symptoms, and eventually heart failure . No mediGene expression profiles have been established in several animal models of LV eccentric hypertrophy, including by us in a rat model after two weeks of severe AR, a period characterized with intense LV remodeling –8. A simα expression, a transcription factor controlling a number of genes implicated in this process -fluorodeoxyglucose ([18F]-FDG) or [18F]-fluorothioheptadecanoic acid ([18F]-FTHA)  was injected via the caudal vein over 30 s. A 45 min dynamic PET data acquisition followed by a 15 min static acquisition was done to determine glucose utilization [myocardial metabolic rate of glucose (MMRG)] using multicompartmental analysis as previously described [18F]-FTHA was used to determine myocardial nonesterified fatty acid (NEFA) uptake (Km). Myocardial NEFA fractional uptake (Ki) was determined by a Patlak graphical analysis of the [18F]-FTHA data.Adult male Wistar rats were divided into 3 groups as follows: (1) Sham-operated control animals .Results are presented as mean ± SEM unless specified otherwise. Intergroup comparisons were done using Student's After 9 months, eight of fifteen (8/15) AR animals were still alive whereas all sham-operated animals were alive. No differences in body weight were observed between the sham and AR groups. Overall growth was similar between groups . Indexed wet heart tissue weights were significantly increased in the AR group compared to controls .Systolic arterial pressure was similar between 9-month AR and sham-operated rats . As expedP/dt in AR after 9 months . Left ventricular end-diastolic pressure was significantly increased in AR rats.Invasive measurements showed a decrease in both negative (an index of diastolic function) and positive (systolic function) LV end-diastolic and end-systolic diameters were significantly increased in AR rats . Stroke p value below 0.01 in order to consider a gene as regulated. Three hundred and ninety-four transcripts met these criteria (230 were upregulated and 164 were downregulated). As listed in Tables α) is the principal regulator of the expression of fatty acid β-oxidation FAO enzymes and transporters [α, and its coactivator, PGC1α was strongly downregulated (α binds to sequence-specific target elements as a heterodimer with the retinoid X receptor (RXR). In our microarray, we identified the RXR gamma isoform as the most expressed in the rat myocardium and the most downregulated in AR (not shown). We confirmed this using quantitative RT-PCR  DNA was measured and compared to nucleus (n) DNA. The LV ratio of mtDNA to nDNA remained constant  suggesting a stable proportion of mitochondria.α and its activator PGC1α was downregulated at 14 days. On the other hand, at two days, PPARα expression was significantly increased. The expression of RXR gamma followed a similar trend as illustrated in We then studied the expression of the same set of genes tested in the 9-month AR animals in the LV of rats with acute AR (2 and 14 days). As illustrated in α agonist) can help reduce LV dilation in the AR rat model [ in vivo the impact of fenofibrate on free fatty acid and glucose uptake by μPET quantification as shown in We showed recently that fenofibrate . Here, we present a gene profiling study of the left ventricles from an aging model of eccentric LVH after 9 months of severe AR.We observed that many upregulated genes in the left ventricles of AR rats were linked to the extracellular matrix remodeling whereas those downregulated were often associated with myocardial metabolism. We had observed in a previous evaluation of the gene profile of left ventricles from rats after only 14 days of AR  that mandP/dtmin. This could increase the occurrence of arrhythmias which we believe is the main cause of mortality in the AR rat model [Eccentric LV hypertrophy is not normally associated with an accumulation of myocardial fibrosis at least during the early stages of VO. Myocardial collagen loss has even been observed , 31. In at model , 10, 13.We have summarized in The present microarray analysis showed that an important number of downregulated genes was associated with the mitochondrial compartment confirming alterations in myocardial energetics in 9-month AR rats . We had μPET study. We only noticed a slight decrease in FA uptake after 8 weeks in the LV lateral wall. At this compensated phase of the disease, FAO enzyme activity is also only slightly reduced [Heart FA uptake seemed to be maintained later in the disease as evidenced by the  reduced , 15. In  reduced , 15, 26.A balance between FA uptake and utilization has to be stricken to avoid the accumulation of unwanted lipids in the cardiac muscle cell causing lipotoxicity. This has been observed in another model of eccentric LVH  . We repoα and RXRγ gene expression mirrored the different observations we made on the state of myocardial FAO in AR rats at different times. PPARs dimerize with RXRs to bind to their sequence-specific target sequences. Our microarray and qRT-PCR data showed that RXRγ was the most highly expressed in the heart and that it was strongly downregulated in AR in parallel to PPARα and PGC1α expression. Interestingly, PPARα and RXRγ gene expression was upregulated early after AR induction again suggesting that FAO is first stimulated before glycolysis becomes more central in the energy production of the dilating heart.PPAR in vivo in 8-week AR rats with or without treatment with fenofibrate showed that glucose uptake is clearly elevated before FA uptake decreased. This is an interesting observation indicating that, during the early phases of eccentric LVH, the heart relies on glucose to sustain its additional energy needs before FAO starts to decrease. Interestingly, FAO gene downregulation is also clearly present at 8 weeks whereas fatty acid uptake and oxidation are still relatively normal . This could be explained by a possible decrease in the protein turnover of the enzymes implicated in FAO. This hypothesis remains to be confirmed, however.The evaluation of energy substrates uptakeμPET analysis did not contradict these previous observations and confirmed that fenofibrate can indeed limit LV dilation. If the fenofibrate effects on FA uptake can be associated with its PPARα agonist activity, it is less clear if its effects on LV remodeling are completely mediated via PPARα although some evidences in the literature point to this direction [α gene expression in AR rats [α null mice develop more hypertrophy, production of more reactive oxygen species as well as an exaggerated production of extracellular matrix components [α null mice with fenofibrate exacerbates LVH development in a pressure overload situation suggesting that the benefits observed here are mostly via PPARα activation [Fenofibrate treatment decreased LV dilation in our model. We had previously observed that LV weight was not reduced by fenofibrate treatment, but we observed that its remodeling tended to be more concentric with less chamber dilation and increased wall thickness . Our preirection , 34. We  AR rats . PPARα nmponents , 36. Tretivation . Fenofibtivation –40. We htivation . Interestivation . Our mictivation , 43.The results of this study have to be viewed in the light of some limitations. Rodent heart metabolism may differ in some aspects from humans. This study relied mainly on the evaluation of gene expression levels and more thorough analysis at the level of protein content, activity, and localization are needed. The role of various signaling pathways in controlling the energy substrate preference shift involved in the development of eccentric LVH and metabolic alterations will need to be explored more in detail.Our results clearly show that the myocardium with chronic VO sustains a significant metabolic stress and develops important energetics adaptations. These findings may improve our view of the dilated and hypertrophied hearts of patients with severe VO from valve disease. Clinicians currently follow those patients without any intervention for a good number of years, simply waiting for the LV to become too dilated, for the occurrence of symptoms or until systolic function begins to fall. Based on our findings, we suggest that those hearts develop severe metabolic abnormalities even when systolic function seems preserved and that intervention then can limit dilation and metabolic abnormalities. Focusing on myocardial metabolism by various interventions such as targeted drugs, specific diets, or exercise may help this metabolically stressed myocardium to improve its energy production and maybe prolong the preheart failure state significantly. Some of our previous studies support this view , 15, 26,Additional materials and data. PCR primers used for the study of the concordance between the data obtained in the array and those from qRT-PCR for 11 up-regulated genes in 9-month AR rats. List of Gene Annotation System categories associated with up- and down-regulated genes identified from the array study. Finally, impact of an 8-week fenofibrate treatment on sham-operated and AR rats on the expression of FAO genes.


Meanwhile, Lewis basic tricoordinate boranes have been developed in recent years. Here we report the synthesis of an annulated 1,4,2,5-diazadiborinine derivative featuring boron atoms that exhibit both Lewis acidic and basic properties. Experimental and computational studies confirmed that two boron atoms in this molecule are spectroscopically equivalent. Nevertheless, this molecule cleaves C–O, B–H, Si–H and P–H bonds heterolytically, and readily undergoes [4+2] cycloaddition reaction with non-activated unsaturated bonds such as C=O, C=C, C≡C and C≡N bonds. The result, thus, indicates that the indistinguishable boron atoms in 1,4,2,5-diazadiborinine act as both nucleophilic and electrophilic centres, demonstrating ambiphilic nature.Boranes have long been known as the archetypal Lewis acids owing to an empty  Boranes are archetypal Lewis acids owing to an empty p-orbital on the boron, although Lewis basic tricoordinate boranes have been developed too. Here, the authors report an annulated 1,4,2,5-diazadiborinine derivative with boron atoms that exhibit both Lewis acidic and basic properties. Thus, the bonding situation therein is reminiscent of the σ donation and π back donation between a metal and a ligand in conventional transition metal complexes182021As classical trivalent boranes inherently possess an unoccupied p-block elements featuring both strong electron-donor and electron-acceptor sites or small highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) gap, may be utilized for small-molecule activation2324252627et al.2930Recently, it has been demonstrated that various nonmetallic systems based on π-systemVery recently, we have synthesized a 1,3,2,5-diazadiborinine bearing two boron atoms that are spectroscopically inequivalent. We showed that these two boron centres in an aromatic ring cooperatively activate small molecules in which one of them behaves as a Lewis acid centre, whereas the other serves as a Lewis base centre351 with excess amounts of potassium graphite (KC8) in benzene slowly proceeded under ambient condition, and after work-up, 1,4,2,5-diazadiborinine derivative 2 was obtained as an orange powder in 52% yield  of 1. In the 1H NMR spectrum, a peak for the methyl groups on two nitrogen atoms was observed at δ=2.90 p.p.m., and two signals for the CH protons of the imidazole ring moieties appeared at δ=6.07 and 7.40 p.p.m. These data indicate the highly symmetric nature of the product 2, which was further confirmed by an X-ray diffraction study ° (the sum of the bond angles: B1=359.75°). Both the B1–C1′ (1.491(3) Å) and B1–N1 (1.458(2) Å) distances are significantly shorter than those (1.610(2) and (1.551(2) Å) in 1. The N1–C1 distance of 1.403(2) Å is only slightly longer than the C1–N2 bond (1.397(2) Å). The C2–C3 bond (1.346(2) Å) lies in the range of typical double-bond distances of carbon–carbon bonds. These structural properties suggest the delocalization of electrons over the π-system including the six-membered B2C2N2 ring, which can be represented by the average of the several canonical forms, including 2a–g . Meanwhile, Wiberg bond index values were >1.0 for the B1–C1′ bond (1.19), the C1–N1 bond (1.13) and the C1–N2 bond (1.10), thus suggesting the partial double-bond character of these bonds. The HOMO of 2 exhibits a π-system over the six-membered B2C2N2 ring with a node found between two CBN π-units and also has large amplitudes on the N atoms and the C=C moieties of the annulated imidazole rings  value of parent 1,4,2,5-diazadiborinine 2′′ estimated at the B3LYP/6-311+G level is 37.9 kcal mol−1 smaller than that of benzene . A stoichiometric amount of MeOTf was added to a benzene solution of 2 at ambient temperature. After removing the solvent under vacuum, 3 was obtained as a mixture of diastereomers (1:1) in 87% yield 302 and pinacolborane (HBpin) as well as arylsilane derivatives (PhSiH3 and Ph2SiH2) because the reactions proceeded cleanly, and after work-up, adducts 4 and 5 were isolated in good yields . In contrast to the case of 3, both products 4 and 5 were obtained as single diastereomers. 2 could readily activate the P–H bond of diarylphosphine [(p-FC6H4)2PH] as well to afford product 6 in 85% yield. Products 3, 4, 5a and 6 were fully characterized by standard spectroscopic methodologies and X-ray diffractometry 2P group and the H atom on the B atom in 6 point in opposite directions.Compound 7% yield . An X-ra7% yield , suggestrms 2e–f . Having ctometry . In the 2 gas was introduced into a benzene solution of 2 at 1 bar, a white precipitate was instantaneously formed at room temperature. After work-up, compound 7 was isolated in 76% yield. We also performed a 13C-labelling study employing 13CO2, which afforded 7-13C quantitatively. The 13C NMR spectrum of 7-13C showed a broad singlet at 195.6 p.p.m. In the 11B NMR spectrum, a set of new broad peaks was detected at δ=−1.0 and −10.8 p.p.m. An X-ray diffraction study revealed the bicyclo[2.2.2] structure involving two boron atoms at the bridgehead, which was formed via [4+2] cycloaddition between 2 and one of the two C=O double bonds of CO2. 2 was also able to activate styrene in a similar manner, to afford the corresponding bicyclo[2.2.2] derivative 8 stereoselectively as a single diastereomer in 83% yield. Analogously, the reaction of 2 with 1,1-diphenylpropa-1,2-diene (H2C=C=CPh2) proceeded smoothly, and clean formation of 9 through regioselective [4+2] cycloaddition was confirmed (86% yield), demonstrating a rare example of allene activation with a FLPp-BrC6H4C≡CH, PhC≡N and p-ClC6H4C≡N) instantly reacted with 2, to provide 10 (80% yield), 11a (73% yield) and 11b (70% yield), respectively. All of the cycloadducts 8–10 and 11b were thoroughly characterized by various spectrometric methods and X-ray crystallography (SCRF)/6-311+G//B3LYP/6-311+G  level of theory  for the formation of 4 is consistent with the fact that elevated temperature (50 °C) was required to accelerate the reaction. We compared the energies of 4 and 5 with their respective diastereomers 4* and 5* that possess the H atom on the B atom and Bpin group (4*) or Ph2HSi group (5*) on the opposite side of the B2C2N2 ring. Both 4 and 5 are only slightly more stable than 4*  and 5* , respectively, indicating that the reactions may not be thermodynamically controlled.To gain insight into the reaction mechanism, pathways for the diastereo-selective formation of f theory 414243444termined , suggest2 with styrene, we could determine two plausible concerted pathways, which revealed that the formation of 8 is favoured both thermodynamically and kinetically  could attack the tricoordinate boron centre of INT-3 from either the same side or the opposite side of the B2C2N2 ring, to form a B–O bond. The former attack would afford 3A, whereas the latter would give 3B. Compound 3A is estimated to be 0.2 kcal mol−1 more stable than 3B. Similarly, compound 6 may be formed through a stepwise mechanism because the (FH4C6)2P group and the H atom on the B atom in 6 point in opposite directions  would be disfavoured due to the instability of the boryl or silyl cation fragment, which could be, at least in part, the origin of different stereo-selectivity observed in the formation of these products 4, 5 and 6. Isolation of ionic species as INT-3 and INT-6 is a subject of our ongoing study.The formation of compound  pathway . Howeverhway  mass spectra were obtained at the Mass Spectrometry Laboratory at the Division of Chemistry and Biological Chemistry, Nanyang Technological University. Melting points were measured with an OpticMelt Stanford Research System. Infrared spectra were measured with the Bruker Alpha-FT-IR Spectrometer with an ECO-ATR module. Continuous wave X-band electron paramagnetic resonance (EPR) spectrum was checked using a Bruker ELEXSYS E500 EPR spectrometer.All reactions were performed under an atmosphere of argon or nitrogen using standard Schlenk or dry box techniques; solvents were dried over Na metal, K metal or CaHn-butyl lithium  was added dropwise at −40 °C. The reaction mixture was allowed to warm to room temperature over 60 min, and then transferred to a THF (40 ml) solution of dimethyl phenylboronate  at −78 °C. The mixture was warmed to room temperature and stirred overnight. Then, trimethylsilane chloride  was added to the mixture at −78 °C, and the solution was slowly warmed to room temperature and stirred for 5 h. After removal of all the volatiles, the residual solid was re-dissolved in dichloromethane . To the solution, boron trichloride  was added at 0 °C, and the reaction mixture was warmed to room temperature, and stirred for 1 h. After filtration, all volatiles were removed under vacuum, and then the residue was washed with hexane and dried under vacuum to afford compound 1 as a pale yellow powder . Melting point: 235 °C (dec). 1H NMR : δ=7.95 , 7.30 , 7.18 , 6.76 , 5.52  and 2.84 ; 13C{1H} NMR : δ=133.0 (Ar-CH), 128.3 (Ar-CH), 127.5 (Ar-CH), 123.0 (CH), 122.2 (CH) and 34.8 (N-CH3); and 11B{1H} NMR : δ=−1.9 (s). High resolution mass spectrometry (ESI): m/z calculated for C20H21B2Cl2N4: 409.1329 [(M+H)]+; found: 409.1338.To a tetrahydrofuran  solution of 1-methyl-1H-imidazole , 1  at room temperature, and the reaction mixture was stirred for 5 days. After removal of graphite and inorganic salt by filtration, the solvent was removed under vacuum to afford 2 as an orange solid . 2 decomposes at 155 °C without melting. 1H NMR : δ=7.79 , 7.40 , 7.32, 6.07  and 2.90 ; 13C{1H} NMR : δ=135.4 (Ar-CH), 127.9 (Ar-CH), 126.9 (Ar-CH), 124.1 (CH), 113.8 (CH) and 36.1 (N-CH3); and 11B{1H} NMR : δ=18.3 (s). Ultraviolet–visible : λ=496 nm , 468 nm , 354 nm  and 273 nm . HRMS (ESI): m/z calculated for C20H21B2N4: 339.1952 [(M+H)]+; found: 339.1962.Potassium graphite  was added into a benzene (80 ml) solution of 2  at room temperature, and the reaction mixture was stirred for 5 min. After removal of the solvent, the solid residue was washed with hexane and dried under vacuum to afford a yellow solid of 3 as a 1:1 mixture of diastereomers . Recrystallization of 3 from a mixture of dichloromethane and hexane solution afforded single crystals of one of the diastereomers, which was confirmed by an X-ray diffraction analysis. When the single crystals were re-dissolved in C6D6 and checked by NMR spectroscopy, two diastereomers were observed in 1:1 ratio, suggesting that these two diastereomers are in equilibrium in solution at room temperature : δ=7.80 , 7.64 , 7.54 , 7.48 , 7.37 , 7.31–7.18 , 6.73 , 6.64 , 6.50 , 6.42 , 5.75 , 5.68 , 3.00 , 2.90 , 2.59 , 2.57 , 0.80  and 0.61 ; 13C{1H} NMR : δ=134.0 (Ar-CH), 133.2 (Ar-CH), 131.7 (Ar-CH), 131.4 (Ar-CH), 128.6 (Ar-CH), 128.5 (Ar-CH), 128.3 (Ar-CH), 128.2 (Ar-CH), 128.0 (Ar-CH), 127.9 (Ar-CH), 126.8 (Ar-CH), 126.5 (Ar-CH), 123.0 (CH), 122.9 (CH), 122.8 (CH), 122.7 (CH), 122.5 (CH), 122.4 (CH), 121.4 (CH), 121.0 (CH), 34.7 (N-CH3), 34.5 (N-CH3), 34.4 (N-CH3) and 34.3 (N-CH3); 11B{1H} NMR : δ=1.0 (br) and −7.7 (br); and 19F{1H} NMR : δ=−77.7 and −77.9. HRMS (ESI): m/z calculated for C22H24B2F3N4O3S: 503.1707 [(M+H)]+; found: 503.1715.MeOTf  was added into a benzene (2.0 ml) solution of 2  at room temperature, and the reaction mixture was stirred for 2 h at 50 °C. After removal of the solvent, the solid residue was washed with hexane and dried under vacuum to afford 4 as a yellow powder . Melting point: 159 °C (dec). 1H NMR : δ=7.86 , 7.70 , 7.39 , 7.33 , 7.26–7.20 , 7.12 , 6.75 , 5.92 , 5.87 , 3.20 , 2.68  and 1.09 ; 13C{1H} NMR : δ=135.3 (Ar-CH), 135.2 (Ar-CH), 128.2 (Ar-CH), 128.1 (Ar-CH), 126.2 (Ar-CH), 126.1 (Ar-CH), 123.8 (CH), 123.2 (CH), 121.5 (CH), 120.5 (CH), 82.0 (C(CH3)2), 35.2 (N-CH3), 34.1 (N-CH3), 25.4 (CH3) and 25.3 (CH3); and 11B{1H} NMR : δ=22.7 (s), −10.1 (s) and −11.1 (s). HRMS (ESI): m/z calculated for C26H34B3N4O2: 467.2961 [(M+H)]+; found: 467.2937.Pinacolborane  was added to a benzene (2.0 ml) solution of 2 , and the reaction mixture stirred for 1 h at room temperature. After removal of the solvent, the residue was washed with hexane and dried under vacuum to afford 5a as a white powder . Melting point: 182 °C (dec). 1H NMR : δ=7.95 , 7.66 , 7.62 , 7.55–7.53 , 7.38–7.30 , 7.24–7.16 , δ=7.14–7.11 , 6.65 , 6.56 , 5.67 , 5.59 , 5.57 , 2.64  and 2.56 ; 13C{1H} NMR : δ=139.3 (SiqC), 139.0 (SiqC), 136.3 (Ar-CH), 136.0 (Ar-CH), 135.6 Ar-CH), 135.1 (Ar-CH), 128.4 (Ar-CH), 128.31 (Ar-CH), 128.25 (Ar-CH), 128.22 (Ar-CH), 127.9 (Ar-CH), 127.8 (Ar-CH), 126.6 (Ar-CH), 126.4 (Ar-CH), 123.5 (CH), 123.1 (CH), 121.8 (CH), 120.9 (CH), 35.2(N-CH3) and 34.2 (N-CH3); 11B{1H} NMR : δ=−9.2 . HRMS (ESI): m/z calculated for C32H33B2N4Si: 523.2661 [(M+H)]+; found: 523.2671.Diphenylsilane  was added to a benzene (3.0 ml) solution of 2 , and the reaction mixture stirred for 1 h at room temperature. After removal of the solvent, the residue was washed with hexane and dried under vacuum to afford 5b as a yellow powder . Melting point: 163 °C (dec). 1H NMR : δ=7.82 , 7.60 , 7.35–7.29 , 7.23–7.19 , 7.11–7.09 , 6.71 , 6.63 , 5.75 , 5.73 , 4.82 , 4.77 , 2.75  and 2.63 ; 13C{1H} NMR : δ=136.2 (SiqC), 136.0 (Ar-CH), 135.2 (Ar-CH), 135.0 (Ar-CH), 128.5 (Ar-CH), 128.3 (Ar-CH), 128.2 (Ar-CH), 127.8 (Ar-CH), 126.7 (Ar-CH), 126.3 (Ar-CH), 123.5 (CH), 122.6 (CH), 121.7 (CH), 121.4 (CH), 35.0 (N-CH3) and 34.2 (N-CH3); and 11B{1H} NMR : δ=−9.7 . HRMS (ESI): m/z calculated for C26H29B2N4Si: 447.2348 [(M+H)]+; found: 447.2368.Phenylsilane  was added to a benzene (3.0 ml) solution of 2 , and the reaction mixture stirred for 10 min at room temperature. After removal of the solvent, the residue was washed with hexane and dried under vacuum to afford 6 as a white powder . Melting point: 210 °C. 1H NMR : δ=7.83 , 7.47–7.43 , 7.35 , 7.29–7.23 , 7.19–7.09 , 7.04 , 6.76 , 6.67 , 6.60 , 5.72 , 5.69 , 3.25  and 2.63 ; 13C{1H} NMR : δ=136.6 , 136.4 , 135.5 (Ar-CH), 134.8 (Ar-CH), 134.6 (Ar-CH), 128.1 (Ar-CH), 126.8 (Ar-CH), 126.6 (Ar-CH), 123.5 (CH), 123.1 (CH), 122.4 (CH), 120.8 (CH), 115.5 , 115.3 , 36.3  and 34.7 (N-CH3); 11B{1H} NMR : δ=–5.1 (s) and −10.2 (s); 19F{1H} NMR : δ=–114.95  and −115.98 ; and 31P{1H} NMR : δ=–34.1. HRMS (ESI): m/z calculated for C32H30B2N4F2P: 561.2362 [(M+H)]+; found: 561.2370.Bis(4-fluorophenyl)phosphine  was added to a benzene (3.0 ml) and THF (0.5 ml) solution of 2  was degassed using a freeze–pump–thaw method, and then CO2 (1 bar) was introduced into the schlenk tube. After stirring for 5 min at room temperature, a white precipitate was collected by filtration and dried under vacuum to afford 7 as a white solid . Melting point: 223 °C (dec). 1H NMR : δ=8.23 , 7.90 , 7.47–7.35 , 6.99 , 6.95 , 6.57 , 3.27  and 3.13 ; 13C{1H} NMR : δ=135.1 (Ar-CH), 133.1 (Ar-CH), 128.1 (Ar-CH), 128.0 (Ar-CH), 127.8 (Ar-CH), 127.3 (Ar-CH), 121.9 (CH), 121.4 (CH), 121.2 (CH), 120.5 (CH), 35.51 (N-CH3) and 35.45 (N-CH3); and 11B{1H} NMR : δ=−1.0 (s) and −10.8 (s). Infrared v cm−1 (solid): 1667 (s). HRMS (ESI): m/z calculated for C21H21B2N4O: 383.1851 [(M+H)]+; found: 383.1858.A benzene (2.0 ml) solution of 7, the reaction employing 13CO2 afforded 7-13C . Melting point: 223 °C (dec). 13C{1H} NMR : δ=195.6 (C=O), 135.0 (Ar-CH), 133.0 (Ar-CH), 128.0 (Ar-CH), 127.9 (Ar-CH), 127.7 (Ar-CH), 127.2 (Ar-CH), 121.8 (CH), 121.3 (CH), 121.2 (CH), 120.6 (CH), 35.43 (N-CH3) and 35.36 (N-CH3); and 11B{1H} NMR : δ=−0.8 (s) and −10.8 (br). HRMS (ESI): m/z calculated for C2013CH21B2N4O: 384.1884 [(M+H)]+; found: 384.1903.By following the same procedure utilized for the synthesis of 2 , and the reaction mixture stirred for 2 min at room temperature. After removal of the solvent, the residue was washed with hexane and dried under vacuum to afford 8 as a yellow powder . Melting point: 238 °C. 1H NMR : δ=7.83–7.82 , 7.44 , 7.38–7.32 , 7.26 , 7.13 , 7.02 , 6.84 , 6.56 , 6.36 , 5.88 , 5.67 , 2.73 , 2.61 , 2.55 , 1.93  and 1.04 ; 13C{1H} NMR : δ=155.6 (Ar-qC), 136.4 (Ar-CH), 135.2 (Ar-CH), 128.4 (Ar-CH), 128.1 (Ar-CH), 128.0 (Ar-CH), 127.8 (Ar-CH), 126.8 (Ar-CH), 126.4 (Ar-CH), 123.2 (Ar-CH), 121.1 (CH), 120.9 (CH), 120.1 (CH), 119.3 (CH), 35.3 (N-CH3) and 34.7 (N-CH3); and 11B{1H} NMR : δ=−5.8 (s) and −6.7 (s). HRMS (ESI): m/z calculated for C28H29B2N4: 443.2578 [(M+H)]+; found: 443.2581.Styrene  was added to a benzene (3.0 ml) solution of 2 , and the reaction mixture stirred for 1 h at room temperature. After removal of the solvent, the residue was washed with hexane and dried under vacuum to afford 9 as a white powder . Melting point: 245 °C. 1H NMR : δ=8.12 , 7.56 , 7.46 , 7.36 , 7.21–7.17 , 7.12 , 7.06–6.99 , 6.92 , 6.50 , 6.26 , 6.16 , 5.68 , 5.45 , 5.14 , 2.70  and 2.43 ; 13C{1H} NMR : δ=153.6 (Ar-qC), 152.0 (Ar-qC), 136.6 (Ar-CH), 130.9 (Ar-CH), 130.2 (Ar-CH), 128.2 (Ar-CH), 127.4 (Ar-CH), 127.3 (Ar-CH), 126.9 (Ar-CH), 126.6 (Ar-CH), 124.1 (Ar-CH), 123.9 (CH), 122.8 (CH), 121.1 (CH), 119.8 (CH2), 119.7 (CH) and 35.2 ; 11B{1H} NMR : δ=−3.5 (s) and −6.2 (s). HRMS (ESI): m/z calculated for C35H33B2N4: 531.2891 [(M+H)]+; found: 531.2896.1,1-Diphenylpropa-1,2-diene  was added to a benzene (2.0 ml) solution of 2 , and the reaction mixture was stirred for 1 h at room temperature. After removal of the solvent, the residue was washed with hexane and dried under vacuum to afford 10 as a yellow solid . Melting point: 238 °C (dec). 1H NMR : δ=8.02 , 7.86 , 7.67 , 7.48 , 7.38 , 7.34 , 7.28–7.23, 7.13 , 6.83 , 6.68 , 5.54 , 5.50 , 2.53  and 2.49 ; 13C{1H} NMR : δ=149.1 (Ar-qC), 136.7 (Ar-CH), 135.3 (Ar-CH), 130.7 (Ar-CH), 129.4 (Ar-CH), 128.4 (Ar-CH), 127.9 (Ar-CH), 127.1 (Ar-CH), 126.7 (Ar-CH), 121.5 (CH), 121.3 (CH), 119.2 (CH), 118.7 (CH), 118.4 (Ar-qC), 34.8 (N-CH3) and 34.7 (N-CH3); and 11B{1H} NMR : δ=−6.4 . HRMS (ESI): m/z calculated for C28CH26B2BrN4: 519.1527 [(M+H)]+; found: 519.1549.1-Bromo-4-ethynylbenzene  was added to a benzene (2.0 ml) solution of 2 , and the reaction mixture was stirred for 5 min at room temperature. A white precipitate was collected by filtration and dried under vacuum to afford 11a as a white solid . Melting point: 156 °C (dec). 1H NMR : δ=8.33 , 7.61 , 7.50 , 7.35–7.22 , 6.97 , 6.95 , 6.43 , 3.27  and 3.08 ; 13C{1H} NMR : δ=136.1 (Ar-CH), 135.2 (Ar-CH), 128.5 (Ar-qC), 127.8 (Ar-CH), 127.6 (Ar-CH), 127.5 (Ar-CH), 127.4 (Ar-CH), 127.2 (Ar-CH), 127.0 (Ar-CH), 126.5 (Ar-CH), 121.2 (CH), 120.9 (CH), 120.0 (CH), 119.6 (CH), 35.6 (N-CH3) and 35.2 (N-CH3); and 11B{1H} NMR : δ=−3.4 (s) and −8.2 (s). HRMS (ESI): m/z calculated for C27H26B2N5:442.2374 [(M+H)]+; found: 442.2369.Benzonitrile  was added into a benzene (2.0 ml) solution of 2 , and the reaction mixture was stirred for 30 min at room temperature. A white precipitate was collected by filtration and dried under vacuum to afford 11b as a white solid . Melting point: 160 °C (dec). 1H NMR : δ=8.31 , 7.61 , 7.51 , 7.33–7.29 , 7.19 , 7.00 , 6.98 , 6.44 , 3.27  and 3.05 ; 13C{1H} NMR : δ=135.9 (Ar-CH), 135.1 (Ar-CH), 129.1 (Ar-CH), 128.5 (Ar-qC), 127.9 (Ar-CH), 127.8 (Ar-CH), 127.5 (Ar-CH), 127.1(Ar-CH), 126.7 (Ar-CH), 121.2 (CH), 120.9 (CH), 120.2 (CH), 119.7 (CH), 35.6 (N-CH3) and 35.2 (N-CH3); and 11B{1H} NMR : δ=−3.3 (s) and −8.5 (s). HRMS (ESI): m/z calculated for C27H25B2ClN5:476.1985 [(M+H)]+; found: 476.1984.4-Chlorobenzonitrile  was added into a benzene (2.0 ml) solution of The data that support the findings of this study are available from the authors upon request.Accession codes. CCDC 1442910–1442920 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.How to cite this article: Wang, B. et al. Ambiphilic boron in 1,4,2,5-diazadiborinine. Nat. Commun. 7:11871 doi: 10.1038/ncomms11871 (2016).Supplementary Figures 1-59, Supplementary Tables 1-8, Supplementary Methods and Supplementary References1Crystallographic information for compound 2Crystallographic information for compound 3Crystallographic information for compound 4Crystallographic information for compound 5aCrystallographic information for compound 6Crystallographic information for compound 7Crystallographic information for compound 8Crystallographic information for compound 9Crystallographic information for compound 10Crystallographic information for compound 11bCrystallographic information for compound


Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. Androgen-targeted therapy (ATT) is the primary treatment for metastatic prostate cancers. It either blocks androgen synthesis or prevents the androgen receptor (AR) from being transcriptionally active. Unfortunately, tumors eventually progress into incurable castration-resistant prostate cancer (CRPC) and are most often driven by re-activated AR signaling via mechanisms involving AR gene amplification, gain-of-function mutations and intratumoral steroidogenesis –3. WhileGeneration of AR-v7 mRNA is through an alternative RNA splicing process, which is coupled with AR gene transcription, enhanced by castration conditions, and facilitated by AR gene amplification/rearrangement , 13. HowGenetic heterogeneity of prostate cancer cells is a significant challenge for cancer therapy and may produce profound impacts on AR-v7 protein expression. For example, cells carrying AR gene amplification or rearrangement express high levels of AR-v7 mRNAs to be translated , 13. CelIn this study, we apply multiple prostate cancer lines with different genetic backgrounds to study differential activation of PP-1 and Akt by AR inhibition. Activated PP-1 and Akt in turn determine the extent of serine 213 phosphorylation, pSer(213), of AR-v7 and Mdm2-mediated AR-v7 protein degradation. These results link genetic backgrounds of prostate cancer cells with AR-v7 protein stability when cancer cells are under AR inhibition conditions.We chose LNCaP, LNCaP95, 22Rv1 and VCaP cells to study AR protein expression under castration conditions, because they have representative genetic aberrations in AR and PTEN genes in addition to many other mutations in their genome. LNCaP, 22Rv1 and VCaP cells were cultured in medium containing fetal bovine serum (FBS) and then switched to androgen deprived medium plus 5μM of ENZ for maximum androgen blockade (MAB) Figure , which iTo determine whether PP-1 and Akt were causal factors determining protein expression of AR-FL and AR-v7, we first depleted Akt expression by siRNA in PTEN-deficient LNCaP95 cells Figure . Akt depPP-1 was demonstrated to de-phosphorylate only serine 650 in AR LBD and regulate AR-FL protein stability . In thisGain- and loss-of-function of PP-1 did not alter AR gene transcription  as well Both PP-1 and Akt were reported to regulate Mdm2 phosphorylation, thus its E3 ligase activity , 22. To Although our results showed that AR-FL and AR-v7 proteins were regulated similarly by PP-1 and Akt activities, we focused on AR-v7, the major AR splice variant in CRPC tumors in the current study. Ligand-dependent transactivation of AR-FL is potently suppressed by MAB regardless of AR-FL protein expression. However, AR-v7 is constitutively active and its protein expression could drive CRPC progression.in vivo ubiquitination assays  protein expression did not respond to gain- and loss-of-function of PP-1 Figure  and 5B od Figure . Furtherd Figure  and 5G. Enhanced expression of AR splice variants had been demonstrated to be one of the key mechanisms contributing to CRPC , 23, 24.Heterogeneity of prostate tumors is challenging, because ATT could effectively destroy many, but not all, cancer cells within tumors. Enrichment of cell populations carrying a variety of genetic mutations are frequently detected in CRPC . Genome-AR inhibition has profound impacts on AR protein expression, and involves complex mechanisms Figure . At tranOur results indicate that AR-FL and AR-v7 share the same mechanism of Mdm2 mediated ubiquitin-proteasome degradation process. However, these studies highlight that under AR inhibition conditions where AR-FL function is potently suppressed, the protein expression of AR-v7 becomes significantly important due to its constitutively and transcriptionally active nature, and its effectiveness in driving CRPC progression. Furthermore, our results indicate that PP-1 can induce de-phosphorylation of Serine 213 of AR-v7 and AR-v7 protein stabilization, suggesting that similar PP-1 action may also apply to AR-FL. PP-1 may exert its function through either directly de-phosphorylating pSer213 of AR-v7 or indirectly through de-phosphorylating, thereby deactivating Akt. Together, these results suggest that through modulating PP-1 activity, we may control AR-v7 as well as AR-FL protein expression in prostate cancer cells.In summary, we define the molecular mechanisms by which PP-1 and Akt signaling can regulate serine 213 phosphorylation of AR-v7, and thereby AR-v7 protein expression in cell genetic background-dependent manners. These findings provide new insights into resistant mechanisms of CRPC. They also suggest that profiling PP-1 and Akt signaling in CRPC patients would be useful to develop personalized therapeutic strategy in order to demolish AR signaling more efficiently in prostate tumors.LNCaP, 22Rv1, VCaP, PC3 and 293T cell lines were purchased from ATCC . The LNCaP95 cell line was a generous gift from Dr. Alan Meeker of Johns Hopkins University. Cells were maintained in phenol red free RPMI1640 medium containing 5% charcoal stripped serum as we have shown , 29. LNCTotal RNA was extracted by TRIZOL reagent (Invitrogen), treated with deoxyribonuclease and reversely transcribed by random hexamers and superscript II (Invitrogen). Real-time qPCR was performed on the ABI PRISM 7900 HT system (Applied Biosystems) using the FastStart Universal SYBR Green Master mix (Roche) following standard protocol as we reported . All reaWhole cell lysates were extracted by the buffer containing 50mM of Tris pH8.0, 150mM of NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and proteinase and phosphatase inhibitors (Roche). A nuclear protein extraction kit (Sigma) was used to isolate cytoplasm and nuclear proteins according to the manufacturer's instruction. Immunoblotting assays follow the standard protocol as reported . Informain vivo ubiquitination assay was carried as described previously [An eviously  with a mCells were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100, and blocked with 1% BSA for 1 hour at room temperature. Cells were then incubated with the primary antibody, washed with PBS with 0.1% Triton X-100, and incubated with FITC-conjugated secondary antibody (1:1000 in PBST containing 1% BSA). Cell imaging was captured by a Zeiss fluorescent microscope .Cells were transfected with PSA-luciferase reporter plasmid with the renilla reporter as a control for transfection efficiency. Luciferase activities were determined using the luciferin reagent  according to the manufacturer's protocol. Transfection efficiency was normalized by renilla luciferase activity.


AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5′UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5′UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5’ untranslated region (5’-UTR) of the androgen receptor ( Post-transcriptional control of gene expression is considered an important mechanism for cells to quickly respond to internal and external stimuli. The untranslated regions (UTRs) at both ends of an mRNA play thereby an important role. While the 3′UTR has been extensively studied as a major place of regulation e.g. through microRNA mediated mRNA degradation and translational inhibition , translaAR gene, while this applies only to roughly one-third of PAIS cases . 18. 1814]. AR directly upstream of a green fluorescent protein (GFP) reporter gene and transfected the fusion constructs in HEK293 cells. Importantly, the newly generated uORF is out of frame with the primary ORF (pORF) of the AR as well as of the GFP in the AR5′UTR-GFP vector and therefore cannot create an AR-uORF-GFP fusion protein. Transcript analysis of the GFP reporter showed no difference in mRNA accumulation levels between the wild type and mutant AR5′UTR-GFP fusion construct  or the mutated (mut) onstruct . In ordesurement . In contonstruct . We alsoed cells . Therefoin vitro.We then wanted to investigate, if the mutation in the 5′UTR also reduces AR-protein function AR-CDS and transfected the corresponding plasmids into AR-negative HEK293 cells together with a plasmid containing an androgen response element (ARE) in front of a luciferase reporter gene. Firefly luciferase measurements were normalized for transfection efficiency by co-measuring Renilla luciferase expression. Cells transfected with the AR5′UTRwt-AR construct showed a mean 5.7 fold induction of AR mediated luciferase expression while this induction was strongly reduced in cells transfected with the AR5′UTRmut-AR plasmid  has been designed previously [AR5′UTRmut-AR plasmid, we found that AR-121 ran at the same height as the lower molecular weight band, indicating that this band is likely to be an AR-product initiating at the second translational start  showed a reproducible DHT dependent transcriptional induction. More recently we identified another AR-target gene in GF, the Phosphatidic Acid Phosphatase Type 2B (PPAP2B), and included it in this study. GF derived from male foreskin tissue served as control. We measured mRNA levels of APOD and PPAP2B in the presence and absence of DHT and calculated the ratio of induction. While control GF showed a three to four fold up-regulation of APOD mRNA, GF of the index patient failed to respond to DHT as measured by APOD-induction and behaved similarly to GF obtained from a CAIS patient with a documented AR frameshift mutation [PPAP2B compared to APOD, it also showed a significant induction in response to DHT in male control GF (p<0.01) but a failure of induction in GF derived from the index patient and the CAIS patient harboring the AR frameshift mutation APOD) shomutation . We concde novo ATG and therefore reach only insufficiently the AR-primary ORF (pORF). In the second option, a polypeptide is built from this ATG. The majority of ribosomes would fall off after completion of the polypeptide and only few would reach the AR-pORF due to leaky scanning [AR-5′UTR and replaced the tagged uORF version (uORF-HIS) with the untagged one in the above described AR5′UTR-GFP vector. This resulted in an AR5′UTRwt-HIS-GFP and an AR5′UTRmut-HIS-GFP vector with ATG as potential uORF start codon  . We obtaThe sequencing library was prepared from genomic DNA of both index patients as well as controls according to the Haloplex protocol (Agilent) with oligos spanning the chromosomal region ChrX:66,754,874–66,955,461 (hg19). Sequencing was performed on a MiSeq benchtop sequencer (Illumina). Alignment to the hg19 reference genome and single nucleotide polymorphism (SNP) was performed by the MiSeq-Reporter software (Illumina). Small insertion deletion (indel) calling was performed using the SureCall software 3.0.1.4 from Agilent.Sanger sequencing was performed on ABI 310 and ABI 3130 Genetic Analyzers (Applied Biosystems) according to standard protocols.AR-5’UTR on the expression of GFP we created a modified version of the pcDNA3.1 plasmid by removing all transcribed sequences between the putative transcription start site (TSS) and the polyadenylation signal [AR  and to the GFP gene were created with primers inserting an EcoRI and NcoI site to the wt and mutant AR-5’UTR, and a NcoI and BamHI site to GFP. The two fragments were ligated into the modified pcDNA3.1 vector and sequenced to verify the correct sequence. The uORF is out of frame related to the GFP gene, reflecting the in vivo situation of the AR. For cloning of the AR-5’UTR in front of the AR-CDS, we performed a PCR covering the AR-5’UTR inserting a NsiI and BamhHI site at the very 5′ end until the SexAI restriction site at the beginning of the AR-CDS. This PCR product was cut with NsiI and SexAI and cloned into a NsiI/ SexAI cut psvARO vector [AR-5′UTR and -CDS was then cloned into the above pcDNA3.1 derived vector and sequenced for verification. For the HIS tagged uORF an overlap extension PCR was performed in order to insert six histidines in front of the uORF stop codon in both the wt and mut AR-5′UTR. The HIS-tagged uORF containing AR-5′UTR was then cloned in the modified pcDNA3.1 vector and sequenced as described above. A schematic representation of the in vitro constructs is shown in In order to investigate the effect of the n signal . The vecO vector . A BamHIAR5′UTRwt-AR vector, AR5′UTRmut-AR vector or empty vector together with 80 ng Renilla-luciferase vector as well as 400 ng of the ARE-luciferase reporter vector. The next day, DHT was added to the cells at a final concentration of 10 nM in order to activate the AR-protein. After further 24h, Firefly luciferase and Renilla luciferase expressions were measured according to the Dual-Luciferase Reporter Assay protocol (Promega) using a Veritas microplate luminometer (Turner BioSystems). PC3 cells were grown in RPMI supplemented with 10% FBS. Typically 200,000 cells were plated in each well of a 6 well plate and transfected the day after using 2 μg of plasmid DNA/well and lipofectamine 2000 (Life Technologies). The next day, cells were washed twice in RPMI without FBS and then incubated in RPMI supplemented with charcoal treated FBS containing either 10nM DHT or 100% ethanol for 24 hours. Cells were lysed in RIPA buffer for protein extraction.HEK293T cells were grown in DMEM supplemented with 10% FBS. Typically 400,000 cells were plated in each well of a 6 well plate and transfected the day after using 1 μg of plasmid DNA/well and lipofectamine 2000 (Life Technologies). After 72h of transfection, cells were collected for concomitant protein and RNA extraction using the PARIS kit (Life Technologies) and for FACS analysis by resuspending the cells in phosphate buffered saline (PBS) supplemented with 2% FBS. For luciferase experiments 200,000 HEK293T cells were transfected with 10 ng of either 5 cells were resuspended in FACS buffer . Flow cytometry data were acquired on a BD Accuri C6 (BD Biosciences) and analyzed with the BDCSampler software (BD Biosciences).Roughly 1x10APOD, AR and SDHA. All primers were purchased from Qiagen and used following the manufacturer′s instructions. For HEK293T transfections, 500ng RNA extracted using the PARIS kit (Life Technologies) was treated twice with TURBO DNase (Life Technologies) to eliminate residual plasmid DNA and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The reverse transcription reaction was also performed in the absence of Reverse Transcriptase. Quantitative PCR was performed with the QuantiTect SYBR Green master mix (Qiagen) using primers against the AR-5′UTR and GFP . 500ng of total RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR was performed with the QuantiTect SYBR Green master mix (Qiagen) using primers against  and GFP  and the ® Novex® 4–12% Bis-Tris Protein gel (Life Technologies) and transferred to a nitrocellulose membrane . The membrane was blocked for 1-2h at room temperature with TBS containing 0.1% Tween and 5% non-fat dry milk. For AR and HIS detection F39.4.1 antibody (BioGenex) (1:100 dilution) and Anti-6xHIS tag antibody   was used respectively over night at 4°C, for actin detection Anti-Actin (A 2066) antibody (Sigma-Aldrich) was used for 1-2h at room temperature . Anti-GFP-HRP (MACS) was used for the duration of 1h at room temperature for GFP detection  and GAPDH antibody (GeneTex) was used for 45min at room temperature  for GAPDH detection. Secondary anti-rabbit and anti-mouse antibodies (Immuno Reagents Inc.) were incubated for 1-2h at room temperature . All antibodies were resuspended in TBS containing 0.1% Tween and 5% non-fat dry milk. Signals were detected on an Amersham Hyperfilm ECL  using the Luminata Forte Western HRP Substrate (Millipore). Expected molecular weights of polypeptides were calculated using EMBOSS GUI v.1.14: pepstats.Whole cell protein lysates were obtained by resuspending the cells in ice-cold RIPA buffer, supplemented with a cocktail of protease inhibitors . Protein extracts were separated by SDS-PAGE on a NuPAGES1 FigThe depicted Kozak consensus sequence is a sequence occurring on eukaryotic mRNA. Big letters correspond to high evolutionary conservation.(PDF)Click here for additional data file.S2 FigAR5′-UTRwt-GFP, AR5′-UTRmut-GFP or not transfected at all. After 72h of transfection, RNA was isolated and treated with DNAse in order to completely digest vector DNA. The expected size for neo is 95bp, that of GFP is 201bp. The minus reverse transcriptase control (-RT) shows no PCR-product, demonstrating that plasmid-DNA was completely digested from the samples.HEK293 cells were transfected with either empty vector, (PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets. Prostate cancer (PCa), which has lately emerged as one of the most frequently detected forms of cancer, remains the second leading cause of death amongst men . Though 629RKLKK633 by coactivator p300 and deacetylation by NAD-dependent histone deacetylases Sirtuin 1 (Sirt1) .,28.transoylation ,27,28,58tylation . Acetylatylation ,61. Mutatylation ,61.deacetylase activity and at the same time reversed the repression of AR signaling [Sirt1 was recently shown to bind directly to the AR and Sirt1 expression inhibited AR activity ,28,60,61ignaling ,62. Inteet al. showed that over expression of Cdc25B in cancer cell lines and human cancers can be correlated with histological grade of prostate tumor and frequently with more poorly differentiated tumors [PSA promoter [Published studies have shown that AR is differentially expressed in most of primary PCa ,64,65,66d tumors . Mutatiopromoter ,69. PSA deacetylation of MYST1 by Sirt1 [vs. AR regulate the acetylation of lysine 16 on histone H4. Notably, in AR lacking PC3 cells, as well as, in AR-depleted LNCaP cells, diminution of MYST1 activates the cleavage of PARP and Caspase 3 that leads to apoptosis [deacetyltransferases, including Sirts in the pathogenesis of androgen-independent PCa. One of the recent studies demonstrated that the level of BIRC6 is elevated in castration-resistant PCa . Interesby Sirt1 . Furtherpoptosis . In contpoptosis . Concomipoptosis . CollectPSA expression, cell proliferation, and survival in various cell-based models of androgen-independent PCa [Castration alone or in combination with AR antagonists is routinely used for treatment of PCa. Although, initial effects of this androgen ablation results in the inhibition of AR with concomitant reduction of PSA expression , PCa reldent PCa ,79,80,81dent PCa , FGF [83dent PCa  and VEGFdent PCa . Howeverdent PCa . This stet al. elucidated the molecular alterations during the progression to androgen independence by comparing the proteomes of multiple androgen-independent  and androgen-dependent (LNCaP and VCaP) and/or normal prostate epithelial (RWPE) cell lines using mass spectrometry [More recently, Saraon trometry . This stThe androgen-dependent lysine acetylated proteins were distributed between different biological processes, and pathways that include metabolism, spliceosome, and RNA processing . TogetheChemo- and radiotherapies, androgen ablation, vaccines and AR pathway antagonists are some of the most common approaches for treatment of PCa ,90. InhiOwing to the complex nature of the onset and progression of PCa, a diverse group of drugs with different targets have been tested in the recent past with varying therapeutic outcome. Therefore, a better understanding of the molecular mechanisms of androgen action combined with existing knowledge of AR signaling might offer avenues to formulate therapies against new targets leading to better therapeutic options, especially in the setting of metastatic and androgen independent PCa.deacetylation during advancement of PCa, it certainly needs more biochemical investigations. Taken, together, it is possible that targeting HAT(s) in androgen-dependent state or targeting HDAC(s) during androgen-independent state cold have therapeutic outcomes. Mann and his colleagues using high-resolution mass spectrometry discovered that at least 3600 acetylated lysine sites in 1750 cellular proteins suggesting that acetylation, like phosphorylation, is a cellular wide phenomenon ,15. Emerdeacetylation, which if perturbed by a small molecule has the potential to impact cell fate decisions that eventually has made them attractive targets for drug discovery program. The FDA-approved HDAC inhibitor, vorinostat, which induces the differentiation of tumor cells has been approved for treatment of T cell lymphoma [Particularly, the dynamism exhibited by epigenetic modifications, such as acetylation/lymphoma . Besideslymphoma ,107. Moslymphoma . ClearlyThe AR primary interactome was compiled using manual data curation and various software including Database of Interacting Proteins (DIP), Interologous Interaction (I2D) Database, InnateDB, IntAct, MatrixDB, The Molecular INTeraction Database (MINT), Molcon, The Microbial Protein Interaction Database (MPID), Uniprot, Simons Foundation Autism Research Initiative (SFARI).Cytoscape tool was used for creating AR interactome visualizations. Additionally, the Reactome FI plugin for cytoscape was used to enrich gene sets and visualize pathways ,110.Functional Enrichment Analysis was performed using the WebGestalt software (“WEB-based GEne SeT AnaLysis Toolkit”) . The Web