Melanoma antigen gene protein MAGE-11 regulates androgen receptor function by modulating the interdomain interaction

Gene activation by steroid hormone receptors involves the recruitment of the steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs to activation function 2 (AF2) in the ligand binding domain. For the androgen receptor (AR), AF2 also serves as the interaction site for the AR NH(2)-terminal FXXLF motif in the androgen-dependent NH(2)-terminal and carboxyl-terminal (N/C) interaction. The relative importance of the AR AF2 site has been unclear, since the AR FXXLF motif interferes with coactivator recruitment by competitive inhibition of LXXLL motif binding. In this report, we identified the X chromosome-linked melanoma antigen gene product MAGE-11 as an AR coregulator that specifically binds the AR NH(2)-terminal FXXLF motif. Binding of MAGE-11 to the AR FXXLF alpha-helical region stabilizes the ligand-free AR and, in the presence of an agonist, increases exposure of AF2 to the recruitment and activation by the SRC/p160 coactivators. Intracellular association between AR and MAGE-11 is supported by their coimmunoprecipitation and colocalization in the absence and presence of hormone and by competitive inhibition of the N/C interaction. AR transactivation increases in response to MAGE-11 and the SRC/p160 coactivators through mechanisms that include but are not limited to the AF2 site. MAGE-11 is expressed in androgen-dependent tissues and in prostate cancer cell lines. The results suggest MAGE-11 is a unique AR coregulator that increases AR activity by modulating the AR interdomain interaction.     

GRIP1 mediates the interaction between the amino- and carboxyl-termini of the androgen receptor

The androgen receptor (AR) mediates transactivation of target genes by acting as a dimer in which its amino-terminal domain (AR-NTD) interacts with its carboxyl-terminal, ligand-binding domain (AR-LBD) (N/C interaction). Here we assessed if and how AR N/C interaction relates to AR transactivation activity and how the p160 coactivator GRIP1 participates in both processes. The concentration of dihydrotestosterone needed for half-maximal N/C interaction was approximately 10-fold higher than for half-maximal transactivation, indicating a disparity between the two processes. Although a mutation of an LXXLL-like motif, 23 FQNLF 27 --> 23 FQNAA 27 , in the AR-NTD abolished AR N/C interaction, it could be restored by the co-expression of the coactivator GRIP1. Co-expression of mutated forms of GRIP1, possessing alterations known to abolish either of the two AR interaction domains, could not restore AR N/C interaction, suggesting that wild-type GRIP1 normally bridges the two AR domains. Although AR transactivation activity can proceed without AR N/C interaction, we propose that part of the GRIP1 coactivation activity resides in its ability to bind both AR-NTD and -LBD, to stabilize the N/C complex and allow for secondary cofactors to be recruited more efficiently. Our results also indicate that AR N/C interaction enhances but is not necessary for AR transactivation activity.     

Mathematical modeling identifies inhibitors of apoptosis as mediators of positive feedback and bistability

The intrinsic, or mitochondrial, pathway of caspase activation is essential for apoptosis induction by various stimuli including cytotoxic stress. It depends on the cellular context, whether cytochrome c released from mitochondria induces caspase activation gradually or in an all-or-none fashion, and whether caspase activation irreversibly commits cells to apoptosis. By analyzing a quantitative kinetic model, we show that inhibition of caspase-3 (Casp3) and Casp9 by inhibitors of apoptosis (IAPs) results in an implicit positive feedback, since cleaved Casp3 augments its own activation by sequestering IAPs away from Casp9. We demonstrate that this positive feedback brings about bistability (i.e., all-or-none behaviour), and that it cooperates with Casp3-mediated feedback cleavage of Casp9 to generate irreversibility in caspase activation. Our calculations also unravel how cell-specific protein expression brings about the observed qualitative differences in caspase activation (gradual versus all-or-none and reversible versus irreversible). Finally, known regulators of the pathway are shown to efficiently shift the apoptotic threshold stimulus, suggesting that the bistable caspase cascade computes multiple inputs into an all-or-none caspase output. As cellular inhibitory proteins (e.g., IAPs) frequently inhibit consecutive intermediates in cellular signaling cascades (e.g., Casp3 and Casp9), the feedback mechanism described in this paper is likely to be a widespread principle on how cells achieve ultrasensitivity, bistability, and irreversibility.     

Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Bax- and Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells

Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.     

Dependence of selective gene activation on the androgen receptor NH2- and COOH-terminal interaction

The agonist-induced androgen receptor NH(2)- and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH(2)-terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH(2)-terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence (433)WHTLF(437) contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation.     

Novel FXXFF and FXXMF motifs in androgen receptor cofactors mediate high affinity and specific interactions with the ligand-binding domain

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for alpha-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.     

Parathyroid hormone-related protein signaling is necessary for sexual dimorphism during embryonic mammary development.

Male mice lack mammary glands due to the interaction of circulating androgens with local epithelial-mesenchymal signaling in the developing mammary bud. Mammary epithelial cells induce androgen receptor (AR) within the mammary mesenchyme and, in response to androgens, the mesenchyme condenses around the epithelial bud, destroying it. We show that this process involves apoptosis and that, in the absence of parathyroid hormone-related protein (PTHrP) or its receptor, the PTH/PTHrP receptor (PPR1), it fails due to a lack of mesenchymal AR expression. In addition, the expression of tenascin C, another marker of the mammary mesenchyme, is also dependent on PTHrP. PTHrP expression is initiated on E11 and, within the ventral epidermis, is restricted to the forming mammary epithelial bud. In contrast, PPR1 expression is not limited to the mammary bud, but is found generally within the subepidermal mesenchyme. Finally, transgenic overexpression of PTHrP within the basal epidermis induces AR and tenasin C expression within the ventral dermis, suggesting that ectopic expression of PTHrP can induce the ventral mesenchyme to express mammary mesenchyme markers. We propose that PTHrP expression specifically within the developing epithelial bud acts as a dominant signal participating in cell fate decisions leading to a specialized mammary mesenchyme.     

The HIV-1-specific protein Casp8p41 induces death of infected cells through Bax/Bak

Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated primary CD4 T cells, suggesting mitochondrial involvement. Therefore, we assessed the dependency of Casp8p41-induced death on Bax/Bak and caspase 9. In wild-type (WT) mouse embryonic fibroblast (MEF) cells, Casp8p41 causes rapid mitochondrial depolarization (P < 0.001), yet Casp8p41 expression in Bax/Bak double-knockout (DKO) MEF cells does not. Similarly, caspase 9-deficient T cells (JMR cells), which express Casp8p41, undergo minimal cell death, whereas reconstituting these cells with caspase 9 (F9 cells) restores Casp8p41 cytotoxicity (P < 0.01). The infection of caspase 9-deficient cells with a green fluorescent protein (GFP) HIV-1 reporter virus results in cell death in 32% of infected GFP-positive cells, while the restoration of caspase 9 expression in these cells restores infected-cell killing to 68% (P < 0.05), with similar levels of viral replication between infections. Our data demonstrate that Casp8p41 requires Bax/Bak to induce mitochondrial depolarization, which leads to caspase 9 activation following either Casp8p41 expression or HIV-1 infection. This understanding allows the design of strategies to interrupt this form of death of HIV-1-infected cells.     

Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP).

The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.     

Targeting the Lnc-OPHN1-5/androgen receptor/hnRNPA1 complex increases Enzalutamide sensitivity to better suppress prostate cancer progression

Long non-coding RNAs (lncRNAs) have been found to play critical roles in regulating gene expression, but their function in translational control is poorly understood. We found lnc-OPHN1-5, which lies close to the androgen receptor (AR) gene on chromosome X, increased prostate cancer (PCa) Enzalutamide (Enz) sensitivity via decreasing AR protein expression and associated activity. Mechanism dissection revealed that lnc-OPHN1-5 interacted with AR-mRNA to minimize its interaction with the RNA binding protein (RBP) hnRNPA1. Suppressing lnc-OPHN1-5 expression promoted the interaction between AR-mRNA and hnRNPA1, followed by an increase of ribosome association with AR-mRNA and translation. This effect was reversed by increasing lnc-OPHN1-5 expression. Consistently, the in vivo mice model confirmed that knocking down lnc-OPHN1-5 expression in tumors significantly increased the tumor formation rate and AR protein expression compared with the control group. Furthermore, knocking down hnRNPA1 blocked/reversed shlnc-OPHN1-5-increased AR protein expression and re-sensitized cells to Enz treatment efficacy. Evidence from Enz-resistant cell lines, patient-derived xenograft (PDX) models, clinical samples, and a human PCa study accordantly suggested that patients with low expression of lnc-OPHN1-5 likely have unfavorable prognoses and probably are less sensitive to Enz treatment. In summary, targeting this newly identified lnc-OPHN1-5/AR/hnRNPA1 complex may help develop novel therapies to increase Enz treatment sensitivity for suppressing the PCa at an advanced stage.Subject terms: Prostate cancer, Drug regulation     

Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.     

Isorhamnetin in Tsoong blocks Hsp70 expression to promote apoptosis of colon cancer cells

The roots of Codonopis bulleynana Forest ex diels (cbFed), locally known as Tsoong, have been used as a tonic food. Tsoong has wide range of pharmacological effects, including anticancer efficacy. In the present study, the anticancer activity of Tsoong and its potential molecular mechanisms were investigated. Isorhamnetin, a flavonol aglycone, is important compound and metabolite in Tsoong. It can promote apoptosis of colon cancer cells through up-regulating apoptosis-related genes (Apaf1, Casp3 and Casp9) because it blocks Hsp70 genes (Hspa1a, Hspa1b and Hspa8). These were verified by in vitro and in vivo experiments. In vitro, cell counting kit-8 (CCK-8) assays and flow cytometry in HCT116 and SW480 colon cancer cell were used to assess the anti-proliferation and apoptosis-promoting activities of Tsoong. In vivo, the antitumor effect of Tsoong was assessed in colon cancer-bearing nude mice as a xenograft model. These results show that Isorhamnetin is very critical in Tsoong because Tsoong can down-regulate Hsp70 genes and promote apoptosis of colon cancer cells by inhibiting Hsp70 largely due to the efficacy of Isorhamnetin. Our results may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease.